トランスポーター科学に基づくホウ素中性子捕捉療法 BNCT 用新規増感化合物開発

平成２６年度〜平成２８年度
私立大学戦略的研究基盤形成支援事業
研 究 成 果 報 告 書

平成２９年５月
学校法人名 獨協学園
大 学 名

獨協医科大学

研究組織名 大学院医学研究科
研究代表者 加藤 広行（獨協医科大学医学部教授）

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し が き

獨協医科大学大学院医学研究科は基礎医学、臨床医学、社会医学の各分野から構成され、それぞれの領域から様々な研究が行われており、それに加え、最近では研究科内の共同研究が進んできている。本プロジェ
クトの基盤となった細胞膜の物質輸送を担うタンパク質であるトランス
ポーターの研究は、当初本プロジェクトの研究代表者を務めた薬理学講座主任教授の安西尚彦が、前任地の杏林大学にて約１０年に渡り研究を継続してきたものであり、平成２３年後に安西が杏林大学から獨協医科
大学に異動後、基礎医学では生化学講座と病理学講座、臨床医学では第一外科、呼吸器外科、泌尿器科、産婦人科とそれぞれ個別に学内の共同研究として進めていたものでもある。
本プロジェクトは安西を中心としたそれまでの個別の共同研究というベースのある「トランスポーター研究」を特色とし、基礎から臨床に渡る双方向性の学内外多講座融合研究の活性化を担うものとして企画された。具体的には、主に細胞の生存のために欠くことのできない必須アミノ酸を運ぶ L 型中性アミノ酸トランスポーターLAT (L-type neutralamino acid transporter)に着目し、同分子を現在国家プロジェクトの一つとして進められている院内設置型小型加速器を用いたホウ素中性子捕捉療法 BNCT（Boron Neutron Capture Therapy）の抗腫瘍効果発現に必須のホウ素化合物をトランスポーターの視点から検討を加えることで、より有効な新規化合物を同定することを目標とした。本プロジェクトの推進のため学内の基礎医学および臨床医学の複数の講座が参画し、さらに院内設置型小型加速器が設置される国立がん研究センター中央病院放射線治療部を初めとして、同センター研究所、ジェイファーマ株式会社、
米国 Colorado 大学などの参画を促し国外にまで及ぶ共同研究体制を構築して腫瘍型トランスポーター指向性新規ホウ素化合物の同定を目指す研究を実施した。本プロジェクトは近年の治療法の進歩で多くのがんが治るようになったと言われる中で、未だ治療法の無い難治性がんも存在し、それらに向けた新たながん治療法開発に道を開くための基となる機序の解明に大きく貢献した。
冒頭に、多くの研究者の方々のご協力に深い感謝を表したい。

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研

究 組 織

研究代表者：加藤 広行（獨協医科大学医学部・教授）
研究プロジェクトに参加する主な研究者：
杉本 博之（獨協医科大学医学部・教授）
林 啓太朗（獨協医科大学医学部・准教授）
中里 宜正（獨協医科大学医学部・准教授）
Jutabha P（獨協医科大学医学部・助教）
川又 均（獨協医科大学医学部・教授）
金 彪（獨協医科大学医学部・教授）
窪田 敬一（獨協医科大学医学部・教授）
千田 雅之（獨協医科大学医学部・教授）
川又 均（獨協医科大学医学部・教授）
金 彪（獨協医科大学医学部・教授）
窪田 敬一（獨協医科大学医学部・教授）
千田 雅之（獨協医科大学医学部・教授）
石井 芳樹（獨協医科大学医学部・教授）
深澤 一雄（獨協医科大学医学部・教授）
釜井 隆男（獨協医科大学医学部・教授）
植木 敬介（獨協医科大学医学部・教授）
平林 秀樹（獨協医科大学医学部・教授）
村上 昌雄（獨協医科大学附属病院・教授）
坂本 攝（獨協医科大学附属病院・教授）
越川 千秋（獨協医科大学附属病院・薬剤部長）
主な共同研究機関等：
安西 尚彦（千葉大学大学院医学研究院・教授／獨協医科大学医学部・特任教授）
伊丹 純（国立がん研究センター中央病院・科長）
上園 保仁（国立がん研究センター研究所・分野長）
遠藤 仁（ジェイファーマ株式会社・代表取締役）
Michel F. Wempe（米国 University of Colorado・准教授）
ポスドク研究員（PD）：
Hasaya Dokduang（獨協医科大学大学院医学研究科・平成 26・27 年度）

森田
野原

亜洲華
正勝

（獨協医科大学大学院医学研究科・平成 28 年度）（獨協医科大学大学院医学研究科・平成 28 年度）

4研 究 経 費
平成２６年度

63,873千円

平成２７年度

28,122千円

平成２８年度

32,205千円

合124,200

千円

計

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研究プロジェクトの目的・意義・及び計画の概要
ホウ素中性子捕捉療法 BNCT（Boron Neutron Capture Therapy、）とは、原子炉などからの中性子とガン組織に取り込まれた中性子との反応断面積が大きい元素（ホウ素）との核反応によって発生する粒子放射線によって、選択的にがん細胞を殺すという原理に基づく放射線療法である。
中性子発生には、これまでは原子炉を必要としたため、BNCT の利用は限定的であったが、最近原子炉に代る病院設置型小型加速器が開発され、都市型病院にも設置可能となり、平成２７年度より共同研究先である国立がん研究センターで BNCT の施行が可能となった。BNCT による治療は、
1．正常細胞を巻き込むこと無くがん細胞を超選択的に死滅させることができる、２．一度で十分な治療を行うことができるため長期入院が不要、
３．原発性はもちろん、現在の治療方法では治療が困難な、再発・転移・末期がんにも有効、４．放射線治療後、陽子線治療後、重粒子線治療後の再発がんにも治療可能、など今までの治療方法とは異なる利点を持つ。
この BNCT の抗腫瘍効果に大きな影響を与えるのががん細胞に入って中性子の受け手となるホウ素を如何にがん細胞に蓄積させるかである。これまで第一世代のホウ素化合物としてホウ素化フェニルアラニン
BPA(p-boronophenylalanine)が使用されているが、BPA に替わる有効な化合物は未だ生み出されておらず、その開発が期待されている。
がん細胞は、正常細胞に比較し亢進した増殖能を持つが、それを支えるのは、生命活動に必要なエネルギー源および細胞を構成するタンパク質の原料である糖やアミノ酸といった栄養素の細胞外からの取込みである。糖やアミノ酸などの栄養素は、「トランスポーター」と呼ばれる特殊な膜タンパク質を介して細胞内に取込まれる。糖と異なりアミノ酸には多様なトランスポーターが存在し、正常細胞とがん細胞でアミノ酸取込みに関与するタンパク質（アミノ酸トランスポーター）は量的だけでなく質的にも異なることを報告している。本プロジェクト開始時、本学医学部薬理学講座の主任教授として在籍していた安西尚彦が研究対象とする L 型アミノ酸トランスポーターLAT1 は必須アミノ酸取込みを担い、多くのがんで発現が亢進するが正常組織には殆ど発現しない。先の BPA も
LAT1 によって輸送されると当時推定されていて、LAT1 を高発現するがん細胞にのみ取込まれるため、BNCT に有効であると考えられる。
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そこで腫瘍型アミノ酸トランスポーター研究を行う本学がその特色を生かし、トランスポーター科学の視点から新規化合物合成と LAT 発現細胞を用いたスクリーニングにより高いがん集積性を示す新たなホウ素化合物を同定することで、BNCT という極めて有効な医用原子力技術の推進
を通じて、難治性がん治療成績改善に貢献するだけでなく、我が国の目指す医療イノベーションに寄与することが意義として挙げられる。
予定された計画の概要は以下の通りである。平成 26 年度：各臓器由来
がんにおける L 型アミノ酸トランスポーターLAT1 発現を、がん患者組織検体を対象にした抗 LAT1 抗体を用いた免疫染色、特異的オリゴを用いた
定量 PCR の点から、さらにがん由来培養細胞を対象に質量分析計による
低分子物質解析および RI トレーサー測定によりアミノ酸輸送特性の点か
ら詳細な検討を加える。
並行して BPA 構造を基にした化合物設計を行う。
平成 27 年度：LAT1 安定発現細胞を用いた候補化合物のスクリーニングを行う。選別された少数の候補化合物に対しその蛍光標識体合成を行い、坦癌動物モデルへの投与によりがん細胞への集積性と体内動態の解析を
蛍光を指標に評価する。平成 28 年度：集積性の確認された化合物を坦癌
動物モデルに投与して中性子照射を行い、in vivo での腫瘍細胞におけるin vitro での影響を検討する。さらに蓄積の認められた正常組織の培養細胞にて細胞毒性効果を検討する。
この中で、平成 27 年度までに、腫瘍型トランスポーターLAT1 選択的な阻害を示す化合物のホウ素化を実施したところ、LAT1 特異的な阻害を示さないという状況が出現した。そこでその解決策として、平成 28 年度には当初の予定の代わりに以下の検討を行った：１、化合物の異なる部位にホウ素化を行うことでの LAT1 特異的阻害効果が回復するか、２、化合物を溶解する溶媒の違いが LAT1 特異的阻害効果に影響するか。検討は現在も進行中である。

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研

究 組 織

本研究プロジェクトを代表する研究代表者は、獨協医科大学医学部第一外科学主任教授の加藤広行（申請時は薬理学講座主任教授の安西尚彦、
現千葉大学）であり、当大学院医学研究科の基礎から臨床にわたる重要なトランスレーショナル研究の一つとして本プロジェクトを位置付けた。
その観点から代表者は本プロジェクトを全てにわたり、研究の進展についての統括を行った。
本研究プロジェクトは、当初研究代表者を務めた当時本学薬理学講座主任教授であった安西らが行っている、国内外で評価の高い「トランスポーター」研究の成果を展開する形で行われた。そのため、本学薬理学講座が基礎研究の主体となり、合計 3 名のポスドク研究員（PD）を雇用し、生化学講座（タンパク質解析）と病理学講座（組織切片作成）との連携をとりながら体内各臓器由来のヒトがん培養細胞株におけるアミノ酸輸送の多角的解析を行う（基礎系研究者 5 名）ことと並行して、消化器、呼吸器、腎泌尿器、婦人科などの各臨床科からのがん患者の臨床検体の解析を行った（基礎系研究者 17 名）。
研究チーム間の連携は実際の研究室内にて個別の共同研究として組まれた他、それを全体としてまとめる体制は、薬理学講座において開催された毎週月曜日の朝 9 時からの定例セミナーにおいて保障された。
また、基礎臨床連携に関しては、3 月、7 月、12 月の年 3 回打合せを定期的に開催した他、薬理学（前代表者所属）、第一外科（現代表者所属）、呼吸器
外科、泌尿器科のコアとなる教室間では特に、1 月、4 月、7 月、10 月の
年 4 回の打合せを行い、進捗状況を確認した。本プロジェクトの遂行には膨大な費用が必要であるため、各研究室に対する大学からの講座研究費も一部導入され、研究の支援・実施体制が確立された。
他の共同研究機関として 新規ホウ素化合物群の設計・合成に関する共同研究をジェイファーマ株式会社とその連携先である米国 Colorado 大学と行った他、院内設置型小型加速器の設置される国立がん研究センター
中央病院・研究所とは、in vivo 薬効および毒性評価の共同研究体制をとるなど、外部施設も参画する学際的共同研究開発体制を構築した。

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研

究 施 設

・ 設 備

等

本プロジェクトを実施する研究施設としては、獨協医科大学医学部基、実験
礎医学棟（7,652 m2、5 名）および臨床医学棟（21,154 m2、12 名）
動物センター（1,916 m2、5 名）に所属する各研究室・実験室が用いられた。参画した計 22 名の研究者は全てフルタイムで連日上記施設を利用した。主な研究装置・設備としては、主たるものとして以下のものが用いられた。
（研究装置）
共焦点レーザー顕微鏡 LSM780
20 時間／週
イメージング質量顕微鏡 iMScope
6 時間／週質量分析装置 QTRAP 5500
12 時間／週
質量分析装置 TRIPLE TOF6600
9 時間／週（研究設備）
in vivo イメージング装置 Clairvivo Opt plus
1 時間／週

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研

究 成 果

の 概 要

本研究プロジェクトとしての計画は次の２点に要約できる。
①腫瘍指向性蓄積を示す新規ホウ素化合物同定②各種がん細胞における治療標的としての腫瘍特異的 L 型アミノ酸ト
ランスポーターの検証
そこで研究成果の概要をこれら２点に分けて記載する。
①腫瘍指向性蓄積を示す新規ホウ素化合物同定
本プロジェクトの当初の研究代表者である薬理学講座の安西らは、これまでに必須アミノ酸の細胞内取り込みに注目し、杏林大学の遠藤 仁
氏（現ジェイファーマ株式会社）、金井好克氏（現大阪大学教授）らが世界に先駆けて同定した L 型中性アミノ酸トランスポーターLAT (L-type
neutral amino acid transporter)が、ホウ素中性子捕捉療法 BNCT（Boron
Neutron Capture Therapy）の抗腫瘍効果発現に現在利用されているホウ素化フェニルアラニン BPA (boronophenylalanine)を輸送する可能性を着想した。
Chemical structure of BPA(a) LAT ファミリーによる BPA 輸送の可能性の検討
これまでに LAT ファミリーには４つのアイソフォーム（LAT1, LAT2,
LAT3, LAT4）があることが知られている。さらに BPA はラセミ体（光学異性体である L 体と D 体との等量混合物）であることが知られており、光学異性体であることがトランスポーター活性に影響する可能性を検討するため、LAT1 から LAT4 までに対する BPA のラセミ体、L 体、D 体の作用を検討した。必須のホウ素化合物をトランスポーターの視点から検討を加えることで、より有効な新規化合物を同定することを目標とした。
図１に示すように LAT1 および LAT2 は BPA のラセミ体によりともに濃度依存性に阻害されたが、LAT1 に対する阻害の方が親和性が高かった。
LAT3 と LAT4 に対する作用は見出されなかった。また光学異性体ごとの検討では L-BPA による LAT1 阻害が強力で（>LAT2）あったが、D-BPA は
LAT2 を阻害しない LAT1 選択性であることが明らかになり、化合物のキ
ラリティの考慮の有効性を見出した（論文投稿中）。
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[14C]L-Leucine uptake(% of control)

(A)

LAT1

120
100
80
60
40
20
0
1

10

100

D-BPA
L-BPA
DL-BPA

705.2 + 1.17
48.6 + 1.29
215.7 + 1.21

D-BPA
L-BPA
DL-BPA

120.7 + 1.11
499.8 + 1.151000 10000

Concentration (µµM)[14C]L-Leucine uptake(% of control)

(B)

LAT2

120
100
80
60
40
20
0
1

10

100

1000 10000(C)[ 14C]L-Leucine uptake(% of control)

Concentration (µ
µM)

D-BPA

LAT3

120
100
80
60
40
20
0

L-BPA
DL-BPA

1

10

100

1000

10000

Concentration (µµM)[ 14C]L-Leucine uptake(% of control)

(D)
D-BPA

LAT4

120
100
80
60
40
20
0

L-BPA
DL-BPA

1

10

100

1000

10000

Concentration (µµM)

図１．L 型アミノ酸トランスポーターに対する BPA の抑制効果(b) LAT を介して細胞内に蓄積する新規ホウ素化合物創製
安西らは既に腫瘍型の L 型アミノ酸トランスポーターLAT1 と正常型のL 型アミノ酸トランスポーターLAT2 の安定発現細胞を樹立し、その中性
12アミノ酸ロイシン輸送に対する阻害効果のプロファイルから LAT1 特異的阻害薬の基幹構造情報を取得し、現在臨床試験が行われている化合物
JPH203 創 製 の基礎 とし た経 験を持 つ（ Morimoto, Anzai et al. J
Pharmacol Sci 108(4):505-16, 2008）。そこで本プロジェクトでは BNCT
治療に必要な増感薬の開発を目指し、LAT を介して細胞内に蓄積する新規ホウ素化合物の基幹構造の取得を試みた。
細胞内への化合物の蓄積を検討するために、以前に樹立した LAT1 および LAT2 安定発現細胞に加え、もう一つの輸送実験系であるアフリカツメ
ガエル卵母細胞系を導入し、トランスポーターの交換輸送特性により細胞内からのロイシンの流出が化合物の蓄積の指標となることに着目して、実験を行った。
ジェイファーマ株式会社を通じて海外の共同研究先である米国
Colorado 大学に化合物合成を依頼し、まずは trial としてホウ素化されていない状態のアミノ酸類似構造を持つ１３種類の化合物（set 1）を得た。
初めに LAT1 および LAT2 安定発現細胞を用いた阻害実験を行った。
Inhibition test on S2-stably expressed LAT1 (S2-LAT1)
or LAT2 (S2-LAT2)

CompoundMolecular

IC50 (µµM)

Weight

S2-LAT1

S2-LAT2

S1

469.01

33.14

>100

S2

183.18

11.83

29.81

S3

199.18

18.65

49.44

S4

183.18

14.73

46.27

S5

215.63

16.17

38.11

S6

338.98

45.2

>100

S7

307.09

9.97

23.29

S8

216.66

17.11

48.27

S9

183.18

13.58

33.79

S10

244.09

11.04

30.36

S11

195.22

14.85

50.13

S12

179.22

26.6>>>100S13

291.09

14.54

32.07

13

上記の表に示すように、程度の差はあれ多くの set 1 化合物が LAT1
選択性を示した。そこで続いてアフリカツメガエル卵母細胞発現系を用いたロイシン排出実験を行った（図２）。
LAT1-oocytes[14C]L-Leu efflux(% radioactivity)

50
40
30
20
10
0
(-)

L-Leu

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

S7

S8

S9

S10

S11

S12

S13

LAT2-oocytes

40
30
20

14

[ C]L-Leu efflux(% radioactivity)

50

10
0
(-)

L-Leu

S1

S2

S3

S4

S5

S6

図２．Set 1 化合物に対する LAT1 および LAT2 を介したロイシン排出この中で、S1, S6, S12 が LAT2 に比し、LAT1 においてロイシン排出の増加（すなわち化合物の細胞内蓄積）を認めた。そこでこれらの３化合物について濃度依存性変化を確認した（図３）。その結果 S12 が LAT1>LAT2の顕著な変化を示したので、これを最初の基幹構造とした。
(B)
LAT1
60

0 µM
1 µM
10 µM
100 µM

50
40
30
20
10
0
L-Leucine

S1

S6

S12

[14C]L-Leu efflux (% radioactivity)[ 14C]L-Leu efflux (% radioactivity)

(A)

LAT2
50

0 µM
1 µM
10 µM
100 µM

40
30
20
10
0
L-Leucine

S1

S6

図３．Set 1 化合物 S1, S6, S12 の濃度依存性変化
14

S12

S12 の構造を元に８化合物（set 2）を合成し,LAT1 および LAT2 への阻害効果（図４）と LAT1 および LAT2 を介するロイシン排出（蓄積効果）（図５）の解析を行った。

(A)

Inhibition on LAT2

A
2

A3

A
4

A5

A
6

A
7

A
8

W

W

W

W

W

W

W

co
n

W

l
A1

110
100
90
80
70
60
50
40
30
20
10
0

tro

[14C]L-Leu uptake (% of control)

110
100
90
80
70
60
50
40
30
20
10
0
co
nt
ro
l
W
A
1
W
A
2
W
A
3
W
A
4
W
A
5
W
A
6
W
A
7
W
A
8

[ 14C]L-Leu uptake (% of control)(B)
Inhibition on LAT1

図４．Set 2 化合物に対する LAT1 および LAT2 の輸送阻害(A)

LAT1-oocytes[14C]L-Leu efflux(% radioactivity)

30
25
20
15
10
5
0
(-)

S12

WA-1

(B)

WA-2

WA-3

WA-4

WA-5

WA-6

WA-7

WA-8

WA-4

WA-5

WA-6

WA-7

WA-8

LAT2-oocytes

14[ C]L-Leu efflux(% radioactivity)

30
25
20
15
10
5
0
(-)

S12

WA-1

WA-2

WA-3

図５．Set 2 化合物に対する LAT1 および LAT2 を介したロイシン排出15

Set 2 化合物の中では WA-7 において顕著な LAT1 選択的阻害効果とロ
イシン排出を認め、これを第二の基幹構造とした。そこで S12 と WA-7を基幹構造としてホウ素を導入した 10 化合物（set 3）の合成を行い、LAT1 および LAT2 への阻害効果（図６）の解析を行った。
(B)

120
100
80
60
40
20
0

LAT2
120
100
80
60
40
20
0

co
nt
ro
N l
A
1
N
A
2
N
A
3
N
A
4
N
A5
N
A
6
N
A
7
N
A
8
N
A
N 9
A
1
L- 0
Le
u

[14C]L-Leu uptake (% of control)

LAT1

co
nt
ro
N l
A
1
N
A
2
N
A
3
N
A
4
N
A5
N
A
6
N
A
7
N
A
8
N
A
N 9
A
1
L- 0
Le
u

[14C]L-Leu uptake (% of control)(A)

図６．Set 3 化合物に対する LAT1 および LAT2 の輸送阻害
この中では NA1, NA7, NA10 が LAT1>LAT2 のプロファイルを示したが、これらは上述の S12 と WA-7 を超える阻害効果を示さなかった。
すなわち、化合物へのホウ素原子の導入がかえって LAT1 による認識の親和性が低下したことを意味する。理由としてはホウ素導入が化合物の物性を変化させた可能性、さらには化合物を溶かしている溶媒の影響などが考えられたため、その可能性をにらみつつ、化合物の合成を３度行い（set 4, 5,
and 6）、LAT1 および LAT2 への阻害効果（図７、８）の解析を行った。
LAT1
120
100
80
60
40
20

16

1 µM JPH203

100 µM L-Leu

100 µM #8

100 µM #7

100 µM #6

100 µM #5

100 µM #4

100 µM #3

100 µM #2

100 µM #1

100 µM #1-9

100 µM #1-8

100 µM #1-7

100 µM #1-6

100 µM #1-5

100 µM #1-4

100 µM #1-3

100 µM #1-2

100 µM #1-1

0
control

14[ C]L-Leu uptake (% control)

(A)

17
100 µM #25
100 µM #26
100 µM #27
100 µM #28
100 µM #29
100 µM L-Leu
1 µM JPH203

100 µM #25

100 µM #26

100 µM #27

100 µM #28

100 µM #29

100 µM L-Leu

1 µM JPH203

0100 µM #24

20

100 µM #24

40

100 µM #23

60

100 µM #23

80

100 µM #22

100

100 µM #22120

100 µM #21

140

100 µM #21

LAT2

100 µM #20

100 µM #19

0

100 µM #20

100 µM #19(B)
100 µM #18

100 µM #17

100 µM #16

100 µM #15

100 µM #14

100 µM #13

100 µM #12

100 µM #11

100 µM #10

100 µM #9

control

14[ C]L-Leu uptake (% control)(A)

100 µM #18

100 µM #17

100 µM #16

100 µM #15

100 µM #14

100 µM #13

100 µM #12

100 µM #11

100 µM #10

100 µM #9

control[14C]L-Leu uptake (% control)

図８．Set 4 & 5 化合物に対する LAT1 および LAT2 の輸送阻害
1 µM JPH203

100 µM L-Leu

100 µM #8

100 µM #7

100 µM #6

100 µM #5

100 µM #4

100 µM #3

100 µM #2

100 µM #1

100 µM #1-9

100 µM #1-8

100 µM #1-7

100 µM #1-6

100 µM #1-5

100 µM #1-4

100 µM #1-3

100 µM #1-2

100 µM #1-1

control

14[ C]L-Leu uptake (% control)

(B)
LAT2

140
120
100
80
60
40
20
0

図７．Set 4 & 5 化合物に対する LAT1 および LAT2 の輸送阻害
LAT1

120

100

80

60

40

20

図７、８に示すように set 4, 5, 6 の計３８化合物に関してその効果を検討したが、これらの中には LAT1 特異的な阻害を示す化合物を見出すことはできなかった。
＜優れた成果が上がった点＞
今回検討した６９化合物の中で、S12 と WA-7 という LAT1 選択性の高い細胞内蓄積性を示す化合物の構造情報を得られた。これは本プロジェ
クトの目的を超え、他の標識体、特に新しいがん診断のための PET プロ
ーブ創製や、α線照射性の細胞内治療薬作成の際に利用が可能となるこれまで世界中でも未だ報告のなされていない貴重な情報であると言える。
＜課題となった点＞
図６で示したように、ホウ素化されていない段階では高い細胞蓄積性、高い LAT1 特異的阻害効果を示す化合物（S12 と WA-7）が、ホウ素化によりその有用性を失うことを初めて確認した。科学的には意義のあるもの
ではあるが、本プロジェクトの達成という意味ではそれが障害となり、３年間での新規ホウ素化合物同定という点では達成に至らなかった。
＜自己評価の実施結果と対応状況＞
年 3 回（3 月、7 月、12 月）定期的に開催した基礎臨床連携打合せ会の 3 月の会合(2016 年、2017 年)、および本プロジェクトのコアとなる教室（薬理学、第一外科、呼吸器外科、泌尿器科）で年 4 回（1 月、4 月、7 月、10 月）定期的に開催したコア打合せ会の 1 月の会合(2016 年、2017年)で、各教室単位での研究の進捗状況の調査を行うとともに、論文公表
数、学会発表数、研究費使用状況などの点から評価を行い、貢献度の低い教室への研究費の再配分を行うことで対応した。
＜外部（第三者）評価の実施結果と対応状況＞
実施せず。
＜研究期間終了後の展望＞
LAT ファミリーによる BPA 輸送と LAT を介して細胞内に蓄積する新規ホウ素化合物創製の両課題は引き続き継続して実施する。ホウ素化によりその有用性を失うという障害に対し、平成 28 年度から始めた取り組み
18

である２点：１、化合物の異なる部位にホウ素化を行うことでの LAT1
特異的阻害効果が回復するか、２、化合物を溶解する溶媒の違いが LAT1
特異的阻害効果に影響するかは、本研究の成功の鍵であるため、精力的に検討を行う。
＜研究成果の副次的効果＞
特になし。
②各種がん細胞における治療標的としての腫瘍特異的 L 型アミノ酸トラ
ンスポーターの検証
本研究プロジェクトは、当初の研究代表者である当時本学薬理学講座主任教授の安西らの研究グループがこれまで行ってきた、がん細胞特異的に発現するアミノ酸トランスポーターLAT1 が、多くのがんで高発現を示し、その盛んな増殖能を支えているというストラテジーに基づき、本検討を行った。
呼吸器外科と薬理学の共同研究をベースにして、林らは世界で初めて
ヒト胸腺癌検体における L 型中性アミノ酸トランスポーターLAT1 の高発
現を見出すとともに、Ty82 ヒト胸腺癌由来細胞において代表的中性アミノ酸であるロイシンの腫瘍細胞への高い取り込みと、LAT1 選択的阻害薬
JPH203 の細胞増殖抑制効果を初めて明らかにし、これまでその治療法の確定していなかった胸腺癌での LAT1 の貢献を示唆するとともに、治療標的としての重要性を明らかにした（雑誌論文 36）。
また以前からの薬理学の共同研究先であるタイ国 Khon Kaen 大学で樹立されたヒト胆管癌由来培養細胞を用いた in vitro および in vivo の検討を行ったところ、文部科学省国費外国人留学生として薬理学に在籍した Yothaisong らはヒト胆管癌検体における L 型中性アミノ酸トランスポーターLAT1 の高発現を見出すとともに、ヒト胆管癌由来細胞 KKU-055,
KKU-213 および MMNK1 を用いた解析により、ロイシンの腫瘍細胞への高い取り込みと、LAT1 選択的阻害薬 JPH203 の in vitro および in vivo での細胞増殖抑制効果を初めて明らかにし、これまでその治療法の確定していなかった胆管癌での LAT1 の貢献を示唆するとともに、治療標的としての重要性を明らかにした（雑誌論文 11, 39）。
婦人科と薬理学の共同研究をベースにして、森田らはヒト卵巣癌由来細胞 JAR および JEG3 において L 型中性アミノ酸トランスポーターLAT119

の高発現を見出すとともに、ロイシンの腫瘍細胞への高い取り込みと、LAT1 選択的阻害薬 JPH203 による抑制効果を初めて明らかにし、ヒト卵巣癌での LAT1 の貢献を示唆するとともに、治療標的としての重要性を明らかにした（論文投稿準備中）。
＜優れた成果が上がった点＞
世界に先駆けてこれまで治療法の無いヒト胸腺癌と治療抵抗性の悪性腫瘍であるヒト胆管癌における L 型中性アミノ酸トランスポーターLAT1の重要性を明らかに出来た。この成果は評価の高い国際科学雑誌に公表することが出来た。これらの成果は本プロジェクトが開発を目指すホウ素化合物を用いた BNCT 治療の科学的根拠を与えることに貢献した。
＜課題となった点＞
特になし。
＜自己評価の実施結果と対応状況＞
年 3 回（3 月、7 月、12 月）定期的に開催した基礎臨床連携打合せ会の 3 月の会合(2016 年、2017 年)、および本プロジェクトのコアとなる教室（薬理学、第一外科、呼吸器外科、泌尿器科）で年 4 回（1 月、4 月、7 月、10 月）定期的に開催したコア打合せ会の 1 月の会合(2016 年、2017年)で、各教室単位での研究の進捗状況の調査を行うとともに、論文公表
数、学会発表数、研究費使用状況などの点から評価を行い、貢献度の低い教室への研究費の再配分を行うことで対応した。
＜外部（第三者）評価の実施結果と対応状況＞
実施せず。
＜研究期間終了後の展望＞
本プロジェクト期間中に成果の得られたヒト胸腺癌、胆管癌、卵巣癌に関しては、今後現行の BNCT 増感剤である BPA の腫瘍細胞への蓄積を引き続き解析するとともに現在進行中の新規ホウ素化合物の合成がなされた際には、BPA 同様に解析を進めていく。
また期間中には検討に至らなかったヒト食道癌、胃癌、大腸癌、腎癌、膀胱癌、等についても継続して解析を進めて行く。
20

＜研究成果の副次的効果＞
特になし。

21

研

究 発 表

の 状 況

＜雑誌論文＞
1. Kojima S, Tohei A, Kojima K, Anzai N. Evidence for tachykinin NK3
receptors-triggered peptide YY release from isolated guinea-pig
distal colon. Eur J Pharmacol. 740:121-6, 2014.
2. Hayashi K, Anzai N. Role of LAT1 in the promotion of amino acidincorporation in activated T cells. Crit Rev Immunol. 34(6):467-79,
2014.
3. Satomura H, Sasaki K, Nakajima M, Yamaguchi S, Onodera S, Otsuka
K, Takahashi M, Muroi H, Shida Y, Ogata H, Okamoto K, Kato H. Can
expression of CXCL12 and CXCR4 be used to predict survival of gastriccancer patients? Anticancer Res. 34(8):4051-7, 2014.
4. Sohda M, Miyazaki T, Honjyo H, Hara K, Ozawa D, Suzuki S, Tanaka
N, Sano A, Sakai M, Yokobori T, Nakajima M, Fukuchi M, Kato H, Higuchi
T, Tsushima Y, Kuwano H. L-[3-¹⁸F]-α-methyltyrosine uptake by lymph
node metastasis is a predictor of complete response to CRT in
esophageal cancer. Anticancer Res. 34(12):7473-7, 2014.
5. Kojima S, Tohei A, Ikeda M, Anzai N. An Endogenous Tachykinergic
NK2/NK3 Receptor Cascade System Controlling the Release of Serotoninfrom Colonic Mucosa. Curr Neuropharmacol. 13(6):830-5, 2015.
6. Lee EY, Kaneko S, Jutabha P, Zhang X, Seino S, Jomori T, Anzai
N, Miki T. Distinct action of the α-glucosidase inhibitor miglitol
on SGLT3, enteroendocrine cells, and GLP1 secretion. J Endocrinol.
224(3):205-14, 2015.
7. Ouchi M, Oba K, Saigusa T, Watanabe K, Ohara M, Matsumura N, Suzuki
T, Anzai N, Tsuruoka S, Yasutake M. Association between pulse wave
22

velocity
andamarkerofrenaltubulardamage(N-acetyl-β-D-glucosaminidase) in patients without diabetes. J Clin
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8. Takayanagi K, Shimizu T, Tayama Y, Ikari A, Anzai N, Iwashita T,
Asakura J, Hayashi K, Mitarai T, Hasegawa H. Downregulation oftransient receptor potential M6 channels as a cause of
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9. Bulacio RP, Anzai N, Ouchi M, Torres AM. Organic Anion Transporter
5 (Oat5) Urinary Excretion Is a Specific Biomarker of Kidney Injury:
Evaluation of Urinary Excretion of Exosomal Oat5 after
N-Acetylcysteine Prevention of Cisplatin Induced Nephrotoxicity.
Chem Res Toxicol. 28(8):1595-602, 2015.
10. Jaiyen C, Jutabha P, Anzai N, Lungkaphin A, Soodvilai S,
Srimaroeng C. Interaction of green tea catechins with renal organic
cation transporter 2. Xenobiotica. 46:641-650, 2015.
*11. Yothaisong S, Namwat N, Yongvanit P, Khuntikeo N, Puapairoj A,
Jutabha P, Anzai N, Tassaneeyakul W, Tangsucharit P, Loilome W.
Increase in L-type amino acid transporter 1 expression during
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2015.
12. Muroi H, Nakajima M, Satomura H, Takahashi M, Domeki Y, Murakami
M, Nakamura T, Takada A, Kato H. Effectiveness of proton beam therapy
on liver metastases of esophageal cancer: report of a case. Int Surg.
100(1):180-4, 2015.
13. Muroi H, Nakajima M, Satomura H, Takahashi M, Yamaguchi S, Sasaki
K, Yokobori T, Miyazaki T, Kuwano H, Kato H. Low PHLDA3 expression23

in oesophageal squamous cell carcinomas is associated with poor
prognosis. Anticancer Res. 2015 Feb;35(2):949-54.
14. Miyazaki T, Sohda M, Tanaka N, Suzuki S, Ieta K, Sakai M, Sano
A, Yokobori T, Inose T, Nakajima M, Fukuchi M, Ojima H, Kato H, Kuwano
H. Phase I/II study of docetaxel, cisplatin, and 5-fluorouracil
combination chemoradiotherapy in patients with advanced esophageal
cancer. Cancer Chemother Pharmacol. 2015 Mar;75(3):449-55.
15. Toyoda S, Inami S, Masawa T, Shibasaki I, Yamada Y, Fukuda H,
Kubo T, Nakajima M, Kato H, Inoue T. Hypopharyngeal perforation
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16. Sasaki K, Miyachi K, Yoda N, Onodera S, Satomura H, Otsuka K,
Nakajima M, Yamaguchi S, Sunagawa M, Kato H. Long-term comparisonof boomerang-shaped jejunal interposition and Billroth-I
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17. Okamoto K, Tani Y, Yamaguchi T, Ogino K, Tsuchioka T, Nakajima
M, Yamaguchi S, Sasaki K, Kato H, Ohya T. Asymptomatic Mesenchymal
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18. Satomura H, Nakajima M, Sasaki K, Yamaguchi S, Domeki Y,
Takahashi M, Muroi H, Kubo T, Kikuchi M, Otomo H, Ihara K, Kato H.
Phase I Dose-Escalation Study of Docetaxel, Cisplatin, and
5-Fluorouracil Combination Chemotherapy in Patients With Advanced
Esophageal Carcinoma. Int Surg. 100(6):1153-8, 2015.
19. Nakajima M, Kato H, Sakai M, Sano A, Miyazaki T, Sohda M, Inose
T, Tanaka N, Suzuki S, Masuda N, Fukuchi M, Kuwano H. Planned
Esophagectomy after Neoadjuvant Hyperthermo-Chemoradiotherapy
24using Weekly Low-Dose Docetaxel and Hyperthermia for Advanced
Esophageal Carcinomas. Hepatogastroenterology. 62(140):887-91,
2015.
20. Miyazaki T, Ojima H, Fukuchi M, Sakai M, Sohda M, Tanaka N, Suzuki
S, Ieta K, Saito K, Sano A, Yokobori T, Inose T, Nakajima M, Kato
H, Kuwano H. Phase II Study of Docetaxel, Nedaplatin, and
5-Fluorouracil Combined Chemotherapy for Advanced Esophageal Cancer.
Ann Surg Oncol. 22(11):3653-8, 2015.
21. Miyazaki T, Honjyo H, Sohda M, Sakai M, Hara K, Tanaka N, Yokobori
T, Nakajima M, Kato H, Kuwano H. Successful Tumor Navigation
Technique During Intrathoracoscopic Esophagectomy: Laparoscopic
Ultrasonography Using Endoscopically Placed Marking Clips. J Am Coll
Surg. 2015 Dec;221(6):e125-8.
22. Ljubojević M, Breljak D, Herak-Kramberger CM, Anzai N, Sabolić
I. Expression of basolateral organic anion and cation transporters
in experimental cadmium nephrotoxicity in rat kidney. Arch Toxicol.
90(3):525-41, 2016.
23. Mishima M, Hamada T, Maharani N, Ikeda N, Onohara T, Notsu T,
Ninomiya H, Miyazaki S, Mizuta E, Sugihara S, Kato M, Ogino K,
Kuwabara M, Hirota Y, Yoshida A, Otani N, Anzai N, Hisatome I. Effects
of Uric Acid on the NO Production of HUVECs and its Restoration by
Urate Lowering Agents. Drug Res (Stuttg). 66(5):270-4, 2016.
24. Hayashi K, Ouchi M, Endou H, Anzai N. HOXB9 acts as a negative
regulator of activated human T cells in response to amino aciddeficiency. Immunol Cell Biol. 94(6):612-7, 2016.
25. Breljak D, Ljubojević M, Hagos Y, Micek V, Balen Eror D, Vrhovac
Madunić I, Brzica H, Karaica D, Radović N, Kraus O, Anzai N, Koepsell
H, Burckhardt G, Burckhardt BC, Sabolić I. Distribution of organic
25

anion transporters NaDC3 and OAT1-3 along the human nephron. Am J
Physiol Renal Physiol. 311(1):F227-38, 2016.
26. Sakoh T, Nakayama M, Tsuchihashi T, Yoshitomi R, Tanaka S,
Katafuchi E, Fukui A, Shikuwa Y, Anzai N, Kitazono T, Tsuruya K.
Associations of fibroblast growth factor 23 with urate metabolism
inpatients
withchronic
kidneydisease.
Metabolism.
65(10):1498-507, 2016.
27. Kokura K, Kuromi Y, Endo T, Anzai N, Kazuki Y, Oshimura M,
Ohbayashi T. A kidney injury molecule-1 (Kim-1) gene reporter in amouse artificial chromosome: the responsiveness to cisplatin
toxicity in immortalized mouse kidney S3 cells. J Gene Med.
18(10):273-281, 2016.
28. Hayashi K, Jutabha P, Maeda S, Supak Y, Ouchi M, Endou H, Fujita
T, Chida M, Anzai N. LAT1 acts as a crucial transporter of amino acids
in human thymic carcinoma cells. J Pharmacol Sci. 132(3):201-204,
2016.
29. Tsuchiya G, Hori T, Onizawa N, Otani N, Tanaka-Nakadate S, Iseki
T, Ouchi M, Hayashi K, Jutabha P, Oba T, Fukuda H, Anzai N. Molecularmechanism of the urate-lowering effects of calcium channel blockers.
Dokkyo J Med Sci 43(1): 23-29, 201630. Hanada K, Nakata T, Ouchi M, Ohtsubo M, Isono M, Morita A, Okamoto
K, Otani N, Kobayashi Y, Anzai N. Identification of carnitinetransporter CT1 binding protein Lin-7 in nervous system. Dokkyo J
Med Sci 43(1): 31-38, 2016
31. Tani Y, Nakajima M, Kikuchi M, Ihara K, Muroi H, Takahashi M,
Domeki Y, Okamoto K, Yamaguchi S, Sasaki K, Tsuchioka T, Sakamoto
S, Kato H.18F-Fluorodeoxyglucose Positron Emission Tomography for
Evaluating the Response to Neoadjuvant Chemotherapy in Advanced26

Esophageal Cancer. Anticancer Res. 36(1):367-73, 2016.
32. Ihara K, Yamaguchi S, Ueno N, Tani Y, Shida Y, Ogata H, Domeki
Y, Okamoto K, Nakajima M, Sasaki K, Tsuchioka T, Mitomi H, Kato H.
Expression of DNA double-strand break repair proteins predicts the
response and prognosis of colorectal cancer patients undergoing
oxaliplatin-based chemotherapy. Oncol Rep. 35(3):1349-55, 2016.
33. Kikuchi M, Nakajima M, Muroi H, Takahashi M, Itoh J, Yamaguchi
S, Sasaki K, Kato H. Significance of Expression of Complement C4d
in Esophageal Squamous Cell Carcinoma. Anticancer Res. 36(9):4553-7,
2016.
34. Nakajima M, Takahashi M, Domeki Y, Satomura H, Muroi H, Kikuchi
M, Ogata H, Yamaguchi S, Sasaki K, Sakai M, Sohda M, Miyazaki T,
Kuwano H, Kato H. Effective Mediastinal Lymphadenectomy for
Esophageal Cancer Using Slender Tracheal Forceps in Prone Position
Thoracoscopic Esophagectomy. In Vivo. 30(6):893-898, 2016.
35. Miyazaki T, Sakai M, Sohda M, Tanaka N, Yokobori T, Motegi Y,
Nakajima M, Fukuchi M, Kato H, Kuwano H. Prognostic Significance of
Inflammatory and Nutritional Parameters in Patients with Esophageal
Cancer. Anticancer Res. 36(12):6557-6562, 2016.
*36. Hayashi K, Anzai N. Novel therapeutic approaches targeting
L-type amino acid transporters for cancer treatment. World J
Gastrointest Oncol. 9(1):21-29, 2017.
37. Furihata T, Morio H, Zhu M, Suzuki Y, Ide H, Tsubota A, Fu Z,
Anzai N, Chiba K. Human organic anion transporter 2 is an entecavir,
but not tenofovir, transporter. Drug Metab Pharmacokinet.
32(1):116-119, 2017.
38. Shimizu Y, Satou M, Hayashi K, Nakamura Y, Fujimaki M, Horibata
27

Y, Ando H, Watanabe T, Shiobara T, Chibana K, Takemasa A, Sugimoto
H, Anzai N, Ishii Y. Matrix-assisted laser desorption/ionization
imaging mass spectrometry reveals changes of phospholipid
distributionininducedpluripotent
stem
cell
colonydifferentiation. Anal Bioanal Chem. 409(4):1007-1016, 2017.
*39. Yothaisong S, Dokduang H, Anzai N, Hayashi K, Namwat N,
Yongvanit P, Sangkhamanon S, Jutabha P, Endou H, Loilome W.
Inhibition of l-type amino acid transporter 1 activity as a new
therapeutic target for cholangiocarcinoma treatment. Tumour Biol.
39(3):1010428317694545, 2017.
40. Otani N, Ouchi M, Hayashi K, Jutabha P, Anzai N. Roles of organic
anion transporters (OATs) in renal proximal tubules and theirlocalization. Anat Sci Int. 92(2):200-206, 2017.
41. Kiuchi K, Morita A, Ouchi M, Otani N, Jutabha P, Nohara M,
Fukasawa I, Fujita T, Anzai N. Characterization of urate transportsystem in JAR and JEG-3 cells, human trophoblast-derived cell lines.
Dokkyo J Med Sci 44(1): 31-38, 201742. 大内基司, 大谷直由, 安西尚彦. 【遺伝性腎疾患】 尿酸代謝異常.
日本腎臓学会誌 57(4): 766-773, 2015.
43. 鬼澤信之, 安西尚彦. さまざまな病態における栄養のポイント 高尿酸血症・痛風治療 栄養療法と薬物療法. Medicina 51(13): 2320-2323,
2014.
44. 大谷直由, 大内基司, 安西尚彦. 高血圧と高尿酸血症. 循環器内科
76(4): 403-409, 2014.
45. 堀 貴行, 安西尚彦. 尿酸トランスポーターの役割を教えてください. 尿酸と血糖 1(2): 88-89, 2015.

28

46. 安西尚彦, 大内基司. 腎尿細管トランスポーター 尿酸およびグル
コース再吸収 revisit. 腎と透析 79(1): 141-145, 2015.
47. 大内基司, 児嶋修一, 安西尚彦. 摘出モルモット大腸標本からのタ
キキニン NK3 受容体作動性 peptide YY 放出. 消化と吸収 37(3): 231-233,
2015.
48. 安西尚彦, 岡本和久, 花田健治, 阿部篤朗, 大谷直由, 大内基司.
ビタミン・バイオファクタートランスポーター研究の最前線 プロスタグ
ラ ン ジ ン と 有 機 ア ニ オ ン の ト ラ ン ス ポ ー タ ー . ビ タ ミ ン 89(9):441-445, 2015.
49. 安西尚彦, 大内基司. SGLT2 阻害薬を使用すると血清尿酸値が低下するのはなぜですか?. 尿酸と血糖 1(3): 150-151, 2015.
50. 岡本和久, 大内基司, 森田亜州華, 花田健治, 阿部篤朗, 大谷直由,
林啓太朗, Jutabha Promsuk, 安西尚彦. ヒト腎臓尿酸トランスポーター
URAT1 と水溶性ヨード系造影剤 iodipamide の相互作用.Dokkyo J Med Sci.
43(1): 73-78, 2016.
51. 安西尚彦, 大内基司, 森田亜州華. 尿酸トランスポーターと糖代謝.
尿酸と血糖 2(1): 6-9, 2016.
52. 大野雄太, 大塚裕介, 野原正勝, 安西尚彦. Hartnup 病. 腎臓内科・泌尿器科 4(5): 459-464, 2016
53. 安西尚彦, 大塚裕介, 大野雄太, 野原正勝. 尿酸関連トランスポー
ターとその異常. 内分泌・糖尿病・代謝内科 43(1): 8-12, 2016.
54. 大塚裕介, 森田亜州華, 安西尚彦. 代謝 Hartnup 病. 小児科診療
79(Suppl.): 274, 2016.
55. 安 西 尚 彦 , 若 新 英 史 . GLUT9 そ の 電 位 差 駆 動 性 尿 酸 輸 送 特 性(URATv1)とグルコースとの関連性. 尿酸と血糖 3(1): 15-17, 2017.
29

56. 大内基司, 大谷直由, Jutabha Promsuk, 安西尚彦. リン酸・有機酸
トランスポーターNPT 群の概要と尿酸輸送メカニズム. 尿酸と血糖
3(1):27-30, 2017
＜図書＞
1. 大内基司, 大谷直由, 安西尚彦. 内分泌 基礎分野での進歩 腎尿細管尿酸輸送の分子機序. Annual Review 糖尿病・代謝・内分泌 2015,
160-164, 2015.
2. 安西尚彦, 大谷直由, 降幡知巳. プリン・尿酸代謝の基礎 尿酸排泄機構. 日本臨床 74(Suppl.9) 高尿酸血症・低尿酸血症, 51-55, 2016.
3. 安西尚彦, 大内基司, 森田亜州華. SGLT2 阻害薬による尿酸排泄機序.
日本臨床 74(Suppl.9) 高尿酸血症・低尿酸血症, 431-434, 2016.
＜学会発表＞
1. 林啓太朗, Jutabha Promsuk, 遠藤 仁, 安西尚彦. 中性アミノ酸トラ
ンスポーターLAT1 阻害薬の胸腺がんに対する増殖抑制効果. 第 73 回日本癌学会総会 2014.09
2. 土屋 豪, 鬼澤信之, 堀 貴行, Promsuk Jutabha, 大谷直由, 長谷川
元, 福田宏嗣, 安西尚彦. Ca 拮抗薬による尿酸降下作用の解析. 第 37回日本高血圧学会総会 2014.10
3. 大内基司, 児嶋修一, 安西尚彦. 摘出モルモット大腸標本からのタ
キキニン NK3 受容体作動性 peptide YY 放出. 日本消化吸収学会総会
2014.10
4. 安西尚彦, 大内基司, 大谷直由, Promsuk Jutabha. 尿酸トランスポーター研究 最近の展開. 第 48 回日本痛風・核酸代謝学会総会 2015.02
5. 土 屋 豪 , 堀 貴 行 , 鬼 澤 信 之 , 大 谷 直 由 , 大 内 基 司 , Promsuk
Jutabha, 福田宏嗣, 安西尚彦. カルシウム拮抗薬による尿酸降下作用
30の解析. 第 48 回日本痛風・核酸代謝学会総会 2015.02
6. 鬼澤信之, 井関 賛, 大谷直由, 大内基司, 長谷川元, 安西尚彦.
zotepine、chlorprothixene による尿酸降下作用の解析. 第 48 回日本痛風・核酸代謝学会総会 2015.02
7. 大谷直由, 大内基司, 相馬良一, 井上晃男, 安西尚彦. ヒト臍帯静脈内皮細胞を用いた低酸素血管内皮障害に対する尿酸の影響. 第 48 回
日本痛風・核酸代謝学会総会 2015.02
8.佐藤元康, 沢登祥史, 安西尚彦, 杉本博之. 分子イメージングをもちいた新規消化管ペプチドの探索. 第 回獨協医学会 2015.07
９. 井関 賛, 鬼澤信之, 中舘佐和子, 林啓太朗, 安西尚彦, 堀 貴行.
Zotepine 及び Chlorprothixene が持ちうる血中尿酸濃度低下作用及びその機作に関する考察. D 第 回獨協医学会 2015.07
10. 鬼澤信之, 大谷直由, 大内基司, 長谷川元, 安西尚彦. Na+依存性
モノカルボン酸トランスポーターSMCT2 の結合タンパク質の解明．第 38回日本高血圧学会総会 2015.10
11. Jutabha Promsuk, 大谷直由, 大内基司, 井上晃男, 安西尚彦.
Functional Analysis of Monocarboxylate Transporter 9(MCT9) as a
Urate Transporter. 第 38 回日本高血圧学会総会 2015.10
12. Naohiko Anzai. 「Renal tubular urate transporters as targets for
new drug development」(シンポジウム) The 13th Asia Pacific Federationof Pharmacologists Meeting (APFP) 2016/2/1～3 バンコク（タイ）13. 堀 貴行、大谷直由、大野雄太、大内基司、福田宏嗣、安西尚彦. 「腎尿酸排泄低下型動物モデルの創製とカルシウム拮抗薬の尿酸効果作用の検討」第 89 回日本薬理学会年会 2016/3/9～11 パシフィコ横浜（横浜市）

31

14. Promsuk Jutabha、林啓太朗、安西尚彦.「ヒト膵臓癌由来細胞におけ る Na+ 非 依 存 性 中 性 ア ミ ノ 酸 ト ラ ン ス ポ ー タ ー に よ るboronophenylalanine 取込み」第 89 回日本薬理学会年会 2016/3/9～11
パシフィコ横浜（横浜市）
15. 林啓太朗、Promsuk Jutabha、遠藤 仁、安西尚彦. 「アミノ酸トラ
ンスポーターLAT1 阻害薬による破骨細胞分化の抑制」第 89 回日本薬理学会年会 2016/3/9～11 パシフィコ横浜（横浜市）
16.大谷直由、堀 貴行、大野雄太、大内基司、安西尚彦.「新規高尿酸血症動物とカルシウム拮抗薬の尿酸低下作用の分子機序の解明」第 93回日本生理学大会 2016/3/22～24 札幌コンベンションセンター（札幌市）
17. Jutabha Promsuk, 大谷直由, 大内基司, 井上晃男, 安西尚彦.「モノカルボン酸トランスポーター９は腎臓の新規尿酸トランスポーターである」第 93 回日本生理学大会 2016/3/22～24 札幌コンベンションセン
ター（札幌市）18. 安西尚彦「マウスモノカルボン酸トランスポーター9（mMct9）は尿酸トランスポーターである」第 89 回日本内分泌学会学術総会 2016/4/21～23 国立京都国際会館（京都市）
19. 森田亜州華、野原正勝、林啓太朗、安西尚彦.「ホウ素中性子捕捉療法 BNCT 増感物質 boronopheylalanine(BPA)の L 型アミノ酸トランスポー
ターによる輸送特性」第 20 回がん分子標的治療学会学術集会 2016/5/30～6/1 別府国際コンベンションセンター（別府市）
20. 大塚裕介、大野雄太、森田亜州華、大谷直由、Jutabha Promsuk、大内基司、鶴岡秀一、安西尚彦「アンセリンによる尿酸降下作用の分子機序解明」第 59 回日本腎臓学会学術総会 2016/6/17～19 パシフィコ横浜（横浜市）
21. 大野雄太、堀 貴行、大塚裕介、大谷直由、大内基司、伊藤善規、
32

安西尚彦.「腎尿酸排泄低下型の創製と Ca 拮抗薬およびロサルタンにより尿酸降下作用の検討」第 134 回日本薬理学会関東部会 2016/7/9 国際医療福祉大学（栃木県大田原市）
22. 安西尚彦「腎臓尿酸トランスポーターと高尿酸血症」（特別講演）
東京理科大学 薬理学特別セミナー 2016/8/5 東京理科大学野田キャンパス（千葉県野田市）
23. 安西尚彦「腎臓の薬物輸送の分子機序 revisit」第 38 回日本小児体液研究会 2016/8/27 大村進・美恵子 聖路加臨床学術センター
24. Naoyuki Otani, Motoshi Ouchi, Promsuk Jutabha, Naohiko Anzai.
"monocarboxylate transporter 9 is a novel urate transporter inkidney" APCN/ANZSN2016（15th Asian Pacific Congress of Nephrology＆ 52th Australian and New Zealand Society of Nephrology ASM)
2016/9/17～21
Perth Convention and Exhibition Centre
25. 安西尚彦「突発性腎性低尿酸血症と腎尿細管トランスポーター」（講演）2016 年度稀少疾患研究会 2016/9/3 立命館大学びわこ・くさつキ
ャンパス（草津市）
26. 安西尚彦「尿酸トランスポーターと高尿酸血症」（講演）河田町循環器セミナー 2016/9/26 東京女子医科大学（東京都）
27. 安西尚彦、大内基司、大谷直由、JUTABHA Promsuk「ヒトの血清尿酸値制御の分子機序」（教育講演）第 39 回日本高血圧学会総会 2016/9/30～10/2 仙台国際センター
28. 大谷直由、堀 貴行、森田亜州華、野原正勝、大野雄太、大塚裕介、
Promusuk Jutabha,大内基司、福田宏嗣、安西尚彦「新規尿酸排泄低下型
高尿酸血症モデルマウスの創製とカルシウム拮抗薬とアンジオテンシン
Ⅱ受容体拮抗薬による尿酸効果作用の検討」第 39 回日本高血圧学会総会
2016/9/30～10/2 仙台国際センター

3329. 若新英史、安西尚彦、Jeffery Kopp「変異型 APOL1 は細胞内炎症性シグナリングを促進しポドサイト障害を増悪させる」第 9 回トランスポーター研究会九州部会 2016/10/1～2 宮崎市民プラザ（宮崎市）
30. 安西尚彦、大内基司「腎尿細管尿酸輸送と糖輸送のクロストーク」（教育講演）第 46 回日本腎臓学会東部学術大会 2016/10/7～8 京王プ
ラザホテル（東京都）
31. 森尾花恵、降幡知巳、朱 美艶、鯉渕明良、井出秀行、澁谷実香、
上市敦子、千葉 寛、安西尚彦、秋田英万「In vitro 肝臓モデル構築を目指した新規ヒト不死化肝類洞内皮細胞の樹立およびその特性解明」日
本薬物動態学会第 31 回年会 2016/10/13～15 キッセイ文化ホール、松本総合体育館（松本市）
32. 安西尚彦.「腎尿細管トランスポーター分子標的創薬」（講演）第 38回日本薬学会九州支部コロキウム 2016/11/12 熊本大学薬学部（熊本市）
33. 安西尚彦.「腎尿細管トランスポーターと抗糖尿病薬作用」（特別講演）第 10 回氷川フォーラム 2016/11/26 大宮ソニックシティ（大宮市）
34. 若新英史、安西尚彦、Jeffery Kopp「変異型 APOL1 は細胞内炎症性シグナリングを促進し、ポドサイト障害を増悪させる」2016 年度生理研研究会 「上皮膜輸送調節蛋白の異常と病態生理学の融合」2016/11/24～25 自然科学研究機構 生理学研究所（岡崎市）
35.
Naohiko Anzai 「Uricosuric agents and renal tubular uratetransporters」（シンポジウム）第 6 回日中薬理学・臨床薬理学ジョイン
トミーティング 第 37 回日本臨床薬理学会学術総会 2016/12/1～3
米子コンベンションセンター、米子市文化ホール（米子市）

34

PARINT-01435; No of Pages 8
Parasitology International xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Parasitology International
journal homepage: www.elsevier.com/locate/parint

Increase in L-type amino acid transporter 1 expression during
cholangiocarcinogenesis caused by liver ﬂuke infection and its
prognostic signiﬁcanceSupak Yothaisong a,f,g, Nisana Namwat a,f,g, Puangrat Yongvanit a,f,g, Narong Khuntikeo b,f,g, Anucha Puapairoj c,f,
Promsuk Jutabha d, Naohiko Anzai d, Wichittra Tassaneeyakul e, Panot Tangsucharit e, Watcharin Loilome a,f,g,⁎a

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, ThailandcDepartment of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, ThailanddDepartment of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi 321-0293, Japane
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, ThailandfLiver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, ThailandgCholangiocarcinoma Screening and Care Program (CASCAP), Khon Kaen University, Khon Kaen 40002, Thailandb

a r t i c l e

i n f oArticle history:
Received 17 August 2015
Received in revised form 29 October 2015
Accepted 30 November 2015
Available online xxxx
Keywords:
Opisthorchis viverrini
Inﬂammation
Oxidative stress
L-type amino acid transporter 1
Cholangiocarcinoma
PI3K/AKT signalinga b s t r a c t
L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, including cholangiocarcinoma (CCA), the most common cancer in Northeast Thailand. Chronic inﬂammation and oxidative stress inducedby liver ﬂuke, Opisthorchis viverrini, infection has been recognized as the major cause of CCA in this area. We show
here that an increased expression of LAT1 and its co-functional protein CD98 are found during carcinogenesis
induced by Ov in hamster CCA tissues. We also demonstrate that oxidative stress induced by H2O2 is timedependent and dramatically activates LAT1 and CD98 expression in immortal cholangiocytes (MMNK1). In addition, H2O2 treatment increased LAT1 and CD98 expression, as well as an activated form of AKT and mTOR in
MMNK1 and CCA cell lines (KKU-M055 and KKU-M213). We also show that suppression of PI3K/AKT pathway
activity with a dual PI3K/mTOR inhibitor, BEZ235, causes a reduction in LAT1 and CD98 expression in KKUM055 and KKU-M213 in parallel with a reduction of activated AKT and mTOR. Interestingly, high expression of
LAT1 in human CCA tissues is a signiﬁcant prognostic factor for shorter survival. Taken together, our data showthat LAT1 expression is signiﬁcantly associated with CCA progression and cholangiocarcinogenesis induced byoxidative stress. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated by the PI3K/AKT signaling pathway.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Cholangiocarcinoma (CCA) is a devastating cancer arising from bile
duct epithelial cells. It is characterized as a very poor prognosis and apoor response to current therapies [1]. CCA is the most common cancer
in Northeastern Thailand [2]. Epidemiological and experimental evidence strongly implicate the carcinogenic liver ﬂuke, Opisthorchis
viverrini (Ov), as the etiological agent inducing CCA development inThailand [3–5]. An oxidative stress condition due to overproduction of
ROS and RNS during chronic inﬂammation caused by Ov infection is considered to play important roles in DNA, lipid, and protein damage leading
to cholangiocarcinogenesis [3,6–8]. In hamsters, Ov-induced CCA tissues
show altered gene expression. Among these genes, PRKAR1A, the type
⁎ Corresponding author at: Department of Biochemistry, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand.
E-mail address: watloi@yahoo.com (W. Loilome).

1A regulatory subunit of protein kinase A (PKA), is signiﬁcantly involvedin CCA carcinogenesis [9] and represents a target for CCA therapy [10].
Furthermore, Dokduang et al. have shown that multiple kinase signaling pathways, including the PI3K/AKT, Wnt/β-catenin, JAK/STAT,
and MAPK signaling pathways are predominately activated in CCA
tissues and cell lines [11]. Moreover, we recently demonstrated
that upregulation of the PI3K/AKT pathway occurred during
Ov-induced cholangiocarcinogenesis [12], and that suppressing
PI3K/AKT pathway activity with the speciﬁc inhibitor BEZ235 caninhibit CCA growth [13].
Amino acid transporters are commonly unregulated in tumor cells
for their supply of amino acids to support massive protein synthesis
for continuous growth and proliferation [14,15]. Among several aminoacid transporters expressed in tumor cells, the L-type amino acid transporter 1 (LAT1) is frequently observed [16]. LAT1 requires covalentassociation with co-functional protein CD98 for its functional expression on the plasma membrane [16,17]. Beside the plasma membrane,

http://dx.doi.org/10.1016/j.parint.2015.11.011
1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

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S. Yothaisong et al. / Parasitology International xxx (2015) xxx–xxx

many studies have demonstrated that LAT1 positive staining isobserved in cytoplasm of cancer cells, such as glioma [18,19], pancreaticcancer [20], and lung cancer [21]. This may represent an intracellularpool of LAT1 while LAT1 expression on plasma membranes may represent its function.
Recently, there have been reports which demonstrated that highexpression of LAT1 is a promising prognostic marker to predict patient
survival [22–26]. It is well known that LAT1 plays an important role
not only as a prognostic marker but also in cancer treatment. Furthermore, the LAT1-speciﬁc inhibitor JPH203 (KYT0353) inhibited tumor
growth both in vitro and in vivo models [27–29]. In addition, suppressed
LAT1 activity can inhibit CCA cell growth, migration, and invasion [23,30]. To date, however, there have been no reports about LAT1 incholangiocarcinogenesis. We therefore examined the expression of
LAT1 and its co-functional protein CD98 in Ov-induced CCA in a hamster
model, as well as in human CCA tissues. Moreover, the molecular mechanisms by which LAT1 and CD98 upregulated under oxidative stresswere explored.
2. Materials and methods
2.1. Animals and tumor induction
Experiments in the animal model used in this study were conducted
and performed according to the guidelines of the National Committee ofAnimal Ethics and the protocol was approved by the Animal Ethics
Committee of the Faculty of Medicine, Khon Kaen University, Thailand(#AE002/2002). The induction of CCA in male hamsters was performed
using a combination treatment with Ov metacercariae infection and Nnitrosodimethylamine (NDMA), as in previous studies [9]. Male Syrian
golden hamsters ranging from 6 to 8 weeks were used in this study.
The hamsters were divided into 2 groups: Group 1 was untreated andserved as the control group; Group 2 was fed 50 Ov metacercariae by
intragastric intubation combined with oral administration of 12.5 ppm
NDMA (Sigma, St. Louis, MO, USA) for 8 weeks. Hamsters were housed
under conventional conditions, fed stock diet, and were given water ad
libitum. Animals were sacriﬁced on days 14, 30, 90, and 180 after treatment. Hamster liver tissue was collected and ﬁxed in 10% neutral buffered formalin and embedded in parafﬁn according to standard
techniques. Samples were used for histological and immunohistochemistry staining.

2.4. Antibodies and inhibitor
Antibodies against AKT (#9272), phospho-AKT (#9271), and mTOR(#2983) were purchased from Cell Signaling Technology (Danvers, MA,
USA.). An antibody against phospho-mTOR (ab109268) was purchasedfrom Abcam (Abcam, Cambridge, UK) and antibody against CD98 waspurchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LAT1
antibody was kindly provided by Dr. H. Endou (J-Pharma Co., Ltd.,Tokyo, Japan). Anti-β-actin antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA).
The dual PI3K/mTOR inhibitor, NVP-BEZ235, was kindly supplied
by Novartis Pharma AG (Basel, Switzerland). The stock hydrogen
peroxide, H 2 O 2 (30%), was purchased from Merck (Darmstadt,
Germany).

2.5. Immunohistochemistry staining
Immunohistochemistry (IHC) was performed to identify the expression of LAT1 and CD98 in CCA samples. Brieﬂy, the sections of CCA
tissues were de-parafﬁnized and rehydrated through a gradient of
ethanol. Following antigen retrieval and elimination of endogenous peroxidase, slides were blocked with 10% skimmed milk in PBS for 1 h.
Samples were then incubated with the primary antibody against the designed target proteins at 4 °C. After washing with PBST, sections wereincubated with peroxidase conjugated Envision™ secondary antibody(DAKO, Denmark) for 1 h. After washing, peroxidase-labeled polymer,
0.1% diaminobenzidine tetrahydrochloride solution was used for thesignal development, followed by counterstaining with hematoxylin,
dried and mounted. The stained sections were observed under a lightmicroscope using high magniﬁcation × 200 and × 400 (Axioscope A1,
Carl Zeiss, Jena, Germany).
Expression of LAT1and CD98 in human CCA tissues was assessed as
described in a previous study [13]. Brieﬂy, the grading of staining
depended on staining intensity and frequency in the tumor area. The
staining intensity was scored as follows: 0, negative; +1, weak expression; +2, moderate expression; +3, strong expression. The frequencyof staining was divided as follows: 0, negative; + 1, 1–25%; + 2,
26–50%; +3, N 50%. Staining scores were calculated by multiplying intensities and frequencies in each case which were classiﬁed into twogroups: low expression levels with scores b 4 and high expression levels
with sores ≥4.

2.2. Human CCA tissue microarrays
The human CCA tissue microarrays contained a total 178 cases. The
samples studied were collected from CCA patients admitted to surgicalwards of Srinagarind Hospital, Khon Kaen University. The protocol forthe collection and study was approved by the Ethics Committee for
Human Research, Khon Kaen University (#HE521209). Informed consent was obtained from individual patients. The 4-μm-thick sections ofhuman CCA tissues used in tissue arrays (TMAs) were constructed by
the Department of Pathology, Faculty of Medicine, Khon Kaen University, Thailand.
2.3. Cell lines and culture
Human CCA cell lines including KKU-M055 and KKU-M213 were
established at Khon Kaen University Liver Fluke and Cholangiocarcinoma Research Center from CCA patients living in the O. viverrini endemic
area of northeast Thailand [31]. An immortalized human cholangiocyte
cell line (MMNK1) was used in this study as a representative of normal
cells as previously described [32]. All cell lines were cultured in Ham's F12 medium (Gibco/BRL, Grand Island, NY, USA) supplemented with
44 mM NaHCO3, penicillin (100 units/ml), streptomycin (100 mg/ml),
and 10 % fetal bovine serum in a humidiﬁed atmosphere containing
5% CO2.

2.6. Western blot analysis
To study the effect of H2O2 or NVP-BEZ235 on LAT1 and CD98 as
well as PI3K/AKT signaling pathway, the cells were treated with
H2 O2 or NVP-BEZ235 with untreated cells being used as controls.
The experiments have been done in the serum containing cultured
media. Cells were then lysed with the RIPA lysis buffer and the lysate
was centrifuged at 4 °C and 12,000 rpm for 15 min. Whole cell lysates
were electrophoresed on 10% SDS–PAGE. Proteins from the gel weretransferred to polyvinylidene diﬂuoride membranes (Millipore, Bedford, USA). Membranes were blocked in buffer 5% skimmed milk in
TBS at room temperature for 1 h and incubated overnight with primary antibody at 4 °C. After blocking, the membranes were washedthree times with TBST for 5 min and then incubated with horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) at room temperature for 1 h. Membranes were again
washed three times in TBST and developed using ECL Prime Western
blotting Detection System (GE Healthcare Bio-Science, UK). The immunoblot and intensity were analyzed by the ImageQuant™ analysis
system (GE Healthcare Bio-Science, UK). Human β-actin was used asa loading control. The western blotting for each cell line was done in
two independent experiments.

Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

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2.7. Statistical analysis
Statistical analyses were performed by using SPSS software version
17 (IBM Corporation, NY, USA). Pearson's correlation coefﬁcient wascalculated for the correlation between IHC scores of each protein in
CCA tissues. The cumulative survival after tumor removal was calculated according to the Kaplan–Meier method, with a log-rank test. Cox
proportional hazard analysis was used to assess survival data. Factors
signiﬁcant in univariate analyses were further examined by multivariable analysis. P b 0.05 was considered statistically signiﬁcant.
3. Results
3.1. Histopathological changes of OV-induced cholangicarcinogenesis inhamster liver tissues
The liver tissues of hamster were collected at different times following treatment with Ov plus NDMA. No pathological changes wereobserved in the control group, whereas hyperplastic lesions, precancerous lesions and carcinomas were observed at 30, 90, and 180 days postinfection (p.i.), respectively [9].
3.2. Increase expression of LAT1 and CD98 in hamster liver tissues during
CCA development
The expression patterns of LAT1 and CD98 during carcinogenesis of
Ov-induced hamster CCA are shown in Fig. 1A. Positively stained signalsof LAT1 and CD98 were observed in both cytoplasm and membrane ofbile duct epithelial cells. A faint positive staining for LAT1 and CD98
was also seen in normal bile ducts of the untreated group. In the Ov
plus NDMA-treated groups, the intensity of LAT1 and CD98 positively
stained cells increased in a time-dependent manner during
cholangiocarcinogenesis with the highest positive signal being seen
when CCA was fully developed (180 days p.i.). Moreover, our results
also revealed the expression of LAT1 and CD98 in the inﬂammatory
cells. Thus, oxidative stress caused by Ov infection can induce LAT1
and CD98 expression in cholangiocytes.
3.3. Increased LAT1 and CD98 expression induced by H2O2-induced
oxidative stress condition via a PI3K/AKT signaling pathway
To prove whether the increased expression of LAT1 and CD98 wasa response to oxidative stress, we next determined the time course of
the increase in LAT1 and CD98 expression in immortalized
cholangiocytes subjected to oxidative stress induced by H2O2 withincreasing incubation time. MMNK1 cells were exposed to 200 μM
of H2 O2 for 0–48 h. Our results reveal that the treatment of
MMNK1 cells with H2O2 at the indicated concentration can induce
LAT1 and CD98 expression in a time-dependent manner as shownin Fig. 1B. We then showed that upregulation of LAT1 and CD98, as
well as an increased activation of PI3K/AKT signaling pathway, demonstrated by an increase of p-AKT and p-mTOR levels, occurredunder the oxidative stress induced by H 2 O 2 in both MMNK1 and
CCA cells (Fig. 2). Moreover, a decrease in the expression of LAT1
and CD98 protein, as well as a reduction p-AKT and p-mTOR, wasseen in the BEZ235, the speciﬁc dual PI3K/mTOR inhibitor treated
CCA cell lines (Fig. 3).
3.4. High LAT1 and CD98 expression in human CCA is associated with a poor
prognosis
To determine whether the increased expression of LAT1 and CD98
has a signiﬁcant biological meaning in human cholangiocarcinoma, we
examined the expression of LAT1 and CD98 proteins in 178 human
CCA tissues. The age of the patients ranged from 26 to 76 years (median = 57 years). Among all patients, 69% (122 cases) were male andFig. 1. Expression of LAT1 and CD98 during cholangiocarcinogenesis.
(A) Immunohistochemical staining of LAT1 and CD98 in hamster liver tissues at 14,
30, 90, and 180 days post-treatment compared with an untreated group, magniﬁcation × 200 and × 400. (B) MMNK1 cells were treated with 200 μM of H 2 O 2 for 0, 6,
12, 24, and 48 h, then expression of LAT1 and CD98 was analyzed by Western blotting.
Bar graphs show mean + S.E.M. of densitometry values of LAT1 and CD98 and present
as % of control.

Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

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S. Yothaisong et al. / Parasitology International xxx (2015) xxx–xxx

Fig. 2. Oxidative stress induced by H2O2 activates LAT1and CD98 expression along with PI3K/AKT activation in CCA. (A) The indicated cells were treated with H2O2 at 0, 200, 300, 400, and500 μM for 24 h. Then total lysates were extracted and analyzed by Western blotting using the indicated antibodies. (B) Bar graphs show mean + S.E.M. of densitometry values of protein
bands and present as % of control.

31% (56 cases) were female, resulting in a male-to-female ratio of 2.2:1.
All patients had advanced-stage cancer with 58% (104 cases) presenting
with metastasis. The histological types were classiﬁed as papillary type
CCA with 35% (63 cases) and non-papillary type CCA with 65% (115
cases).
The results of immunohistochemical staining revealed the expression of LAT1 and CD98 in both the cytoplasm and plasma membranesof positively stained cells. Normal bile duct epithelia showed no or
weak positive staining of LAT1 and CD98, while an increased expression
was observed in CCA cells (Fig. 4A). We found that LAT1 was positive in
86% (153 cases). This could be divided into high expression in 56% (100cases) and low expression in 30% (53 cases). Negative LAT1 was foundin 14% (25 cases). In addition, CD98 was positive in 95% (170 cases),
divided into high expression in 62% (111 cases) and low expression in
33% (59 cases). Negative CD98 expression was found in 5% (8 cases).
Co-expression of LAT1 with CD98 was found in 83% (147 cases).
LAT1 positive and CD98 negative expressions were observed in 3% (6cases), whereas LAT1 negative and CD98 positive were identiﬁed in
13% (23 cases). Two cases (1%) were negative for both proteins. Highexpression of LAT1 with CD98 was observed in 39% (70 cases). Low
expression of LAT1 with CD98 was found in 20% (35 cases). Highexpression of LAT1 with low or negative CD98 was seen in 17% (30cases). High expression of CD98 with low or negative LAT1 was identiﬁed in 23% (41 cases). LAT1 expression score was positively correlatedwith CD98 (P = 0.017, Table 1).
3.5. The high LAT1 expression is correlated with a short survival of CCA
patients
Increased levels of LAT1 or CD98 were not associated with age,gender, histological type, and tumor metastasis (data not shown).

The log-rank analysis indicated that a positive and high expressionof LAT1 was signiﬁcantly correlated with shorter patient survival(P = 0.002 and P b 0.001, Fig. 4B and C). However, no difference wasfound between the expression of CD98 and survival (data not shown).
Interestingly, patients positive for both LAT1 and CD98 (147 cases,83%), as well as those having a high expression of both LAT1 andCD98 (70 cases, 39%), had a signiﬁcantly shorter survival time thanpatients without these characteristics (P = 0.001 and P b 0.001, Fig 4D
and E).
According to the results of our univariate analysis, LAT1 positive,
high LAT1 expression, positive both LAT1 and CD98, as well as highexpression both LAT1 and CD98 with gender were prognostic in CCA
patients (Table 2). The subsequent multivariable analysis conﬁrmedthat high LAT1 expression (P = 0.002) and gender (P = 0.048) wereindependently prognostic of survival in these patients (Table 2). CCA
patients who had a high LAT1 expression had a 2.1-fold higher risk ofdeath than the patients whose tumors were negative or had a low
expression of LAT1.
4. Discussion
Several studies have demonstrated that the expression of LAT1 isclosely linked to the several types of cancer [16,17]. Moreover, increased
LAT1 expression is involved in cancer cell proliferation and progression,leading to a poor prognosis for various cancers [22–26]. Interestingly,
there is strong evidence supporting the hypothesis that overexpressionof LAT1 in tumor cells might be used as a tumor diagnostic marker
detected by positron emission tomography (PET) imaging [33–35]. Previous studies have demonstrated that a novel LAT1 inhibitor, JPH203,
can inhibit tumor growth in human colon cancer cells [27] and leukemic
cells [29] in both in vitro and in vivo models. Moreover, JPH203 canPlease cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

S. Yothaisong et al. / Parasitology International xxx (2015) xxx–xxx

5Fig. 3. Suppression of the PI3K/AKT signaling pathway decreased LAT1 and CD98 expression. (A) The indicated cells were treated with BEZ235 for 48 h at 10, 100, and 1000 nM an 0.1%
DMSO was used as control. Results were analyzed by immunoblotting. (B) Bar graphs show mean + S.E.M. of densitometry values of protein bands and present as % of control.

induce apoptosis in a model of human oral cancer [28] and leukemia[29], indicating that targeting LAT1 may be clinically useful as a cancer
treatment.
Only three studies have reported roles of LAT1 in CCA. The ﬁrst two
demonstrated that increased LAT1 expression predicts a poor prognosis
in CCA patients [23,36]. The third showed that targeted knockdown of
LAT1can inhibit leucine uptake and is concomitant with reduced cell
migration and invasion in KKU-M213 CCA cell [30]. However, there
was no evidence of LAT1expression during CCA carcinogenesis.
In the current work, we demonstrated that the expression LAT1 andits co-functional protein CD98 were increased during carcinogenesis
processes in Ov-induced hamster CCA tissues. Thus, oxidative stresscaused by Ov infection might be responsible for this event. LAT1 expression is regulated by the c-Myc gene [37], which is a proto-oncogene andupregulated by ROS treatment in a model of H2O2-treated melanoma
cells [38]. In addition, c-Myc expression is upregulated in CCA andappears to be a tumor diagnostic marker [39]. Therefore, upregulationof LAT1 and CD98 caused by oxidative stress during chronic Ov infection
may be regulated by the c-Myc gene.
Additionally, we then demonstrated for the ﬁrst time that oxidative
stress induced by H2O2 dramatically activates LAT1 and CD98 expression in a time-dependent way in immortal cholangiocyte (MMNK1)
via the PI3K/AKT signaling pathway. These results provide more information that is helpful in explaining the molecular mechanisms bywhich Ov-induced CCA develops. Chronic inﬂammation caused by Ov
infection leads to the overproduction and accumulation of reactive oxygen and reactive nitrogen radicals in inﬂamed target cells [6,40].

Moreover, increased levels of these radicals have the potential to damage DNA, proteins, lipids, and alter gene expression, all of which cancontribute to cellular carcinogenesis [4,6–9]. Repeated cycles of cell
damage and compensatory cell proliferation promote the developmentof tumor cells. LAT1 and its partner CD98 can supply the amino acids
needed to support massive protein synthesis for continuous growthand proliferation [14,15]. Interestingly, the increase of LAT1 and CD98
is probably regulated by a PI3K/AKT signaling pathway which is also
overactivated during Ov-infection-associated CCA tumorigenesis [12].
Additionally, the expression of LAT1 is closely correlated with p-AKT
and p-mTOR, which is the key downstream elements in PI3K/AKT signaling [41,42]. Moreover, mTOR is an upstream regulator of LAT1 in amodel of insulin-treated mouse myoblast cell line [43].
Furthermore, we report the expression of LAT1 and CD98 in human
CCA tissues from endemic areas of Ov infection. The increase of LAT1
and CD98 expression in human CCA might be regulated by PI3K/AKT
signaling for which the overactivation of a particular pathway has
been previously reported [11,13]. Interestingly, high expression of
LAT1 in human CCA tissue was a signiﬁcant prognostic factor for shorter
survival. Our results conform to previous reports in which the highexpression of LAT1 was signiﬁcantly associated with a shorter survival
in cancer patients [23,36]. The results from multivariate analysis conﬁrmed that LAT1 expression is an independent prognostic factor forpredicting shorter survival. The result from multivariate analysis also
indicated that male gender is an independent risk factor for poor prognosis. The number of male patients is 2.2-fold that of female patients,and male gender is a risk factor for CCA [44].

Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

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S. Yothaisong et al. / Parasitology International xxx (2015) xxx–xxx

Fig. 4. Expression of LAT1 and CD98 in human CCA tissue. (A) Representative immunohistochemical staining of LAT1 and CD98 in normal bile duct, magniﬁcation × 400 andhuman CCA tissue microarray, magniﬁcation × 100 and × 400, respectively. (B) and (C) Kaplan–Meier curve of CCA patients with positive LAT1 and high LAT1 expression,respectively. (D) and (E) Kaplan–Meier curve of CCA patients with co-expression of positive LAT1 and CD98 and high expression of LAT1 and CD98 in human CCA patients,
respectively.

Table 1
Correlation coefﬁcient between IHC scores of LAT1 and CD98 in human CCA tissues microarray (TMA).
LAT1
CD98

Correlation coefﬁcientP⁎ Correlation is signiﬁcant at the 0.05 level (2-tailed).

0.179
0.017⁎In the present study, we also demonstrated the signiﬁcant positive
correlation between the expression levels of LAT1 with its functional
protein partner CD98, indicating that CD98 is essential for the functional
expression of LAT1. Unfortunately, there was no signiﬁcant correlation
between CD98 and patient survival as previously reported in some
types of cancer, such as lung cancer and oral cancer [45–47].
In summary, we demonstrate that oxidative stress during chronic Ov
infection can induce LAT1 and CD98 expression in a CCA model. This is

Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

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Table 2Multivariate analysis of clinicopathological variables for survival by a Cox proportional hazards regression model.
Univariate
Variable (No. patients)
LAT1
Negative (25)
Positive (153)
LAT1
Other (78)High (100)
CD98
Negative (8)Positive (170)
CD98
Other (67)High (111)
LAT1/CD98
Other (31)
Positive/Positive (147)
LAT1/CD98
Other (108)High/High (70)
Age (years)
b57 (89)
N57 (89)
GenderFemale (56)Male (122)Histological types
Papillary (63)Non-papillary (115)
Metastasis
No (74)
Yes (104)

Multivariate

Hazard ratio

95% CIP

Hazard ratio

95% CIP

1
1.909

1.245–2.926

0.003⁎

1
0.889

0.329–2.379

0.809

1
2.241

1.644–3.054

b0.001⁎1
2.103

1.329–3.327

0.002⁎

1
1.562

0.724–3.368

0.255

1
1.151

0.848–1.564

0.367

1
1.998

1.341–2.976

0.001⁎

1
1.542

0.612–3.884

0.358

1
1.854

1.359–2.531

b0.001⁎1
0.881

0.567–1.369

0.573

1
1.099

0.811–1.489

0.542

1
1.488

1.077–2.055

0.016⁎

1
1.400

1.003–1.955

0.048⁎

1
1.131

0.830–1.541

0.435

1
1.304

0.962–1.766

0.087⁎ P value equal to or less than 0.05 was considered statistically signiﬁcant.

likely to be regulated by the PI3K/AKT signaling pathway. Moreover,high LAT1 expression can be used as a prognostic marker for CCA
progression. Further study to explore whether LAT1 can be used as apotential target for the CCA therapy needs to be carried out.
Acknowledgements
This research was supported by The Invitation Research Grant (grant
number I57235 and I57305), the Liver Fluke and Cholangiocarcinoma
Research Center (LFCRC) (LFCRC04/2556) to SY; Faculty of Medicine
Khon Kaen University and the grant of the Higher Education Research
Promotion and National Research University Project of Thailand, Ofﬁceof the Higher Education Commission (NRU582004), through the Health
Cluster (SHeP-GMS), Khon Kaen University to WL. The authors are
deeply grateful to Dr. Hitoshi Endou (J-Pharma Co., Ltd.) for providing
us with the anti-LAT1 antibody. We are grateful to Novartis for providing us with NVP-BEZ235 to complete this work. We also thank Professor
Trevor N. Petney for editing the MS via Publication Clinic KKU, Thailand.
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Please cite this article as: S. Yothaisong, et al., Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused byliver ﬂuke infection and its prog..., Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2015.11.011

Immunology and Cell Biology (2016), 1–6& 2016 Australasian Society for Immunology Inc. All rights reserved 0818-9641/16
www.nature.com/icb

SHORT COMMUNICATIONHOXB9 acts as a negative regulator of activated human
T cells in response to amino acid deﬁciency
Keitaro Hayashi1, Motoshi Ouchi1, Hitoshi Endou2 and Naohiko Anzai1

Q1

T-cell activation is an energy expenditure process and should be properly controlled in accordance with the availability ofnutrients such as amino acids to eliminate wasteful energy consumption. However, the details of response to amino acids
insufﬁciency in activated T cells remain largely unknown. Here we show that homeobox B9 (HOXB9), a member of thehomeobox gene family that is known as a morphogenesis regulator, acts as a suppressor of activated human T cells to addressamino acid starvation. The expression of HOXB9 was triggered by amino acid deprivation as well as functional inhibition of
L-type amino acid transporter 1 (also known as SLC7A5) via activating transcription factor 4 in activated T cells. HOXB9
interfered the activities of NF-κB, NFAT and AP-1 but not retinoic acid receptor-related orphan receptor, resulting in attenuationof the production of selective cytokines in activated T cells. Thus, the morphogenetic gene plays an unexpected role in theregulation of cellular metabolism with changes in the nutrition status in human T cells.
Immunology and Cell Biology (2016) 00, 1–6. doi:10.1038/icb.2016.13

Recent studies have made it increasingly evident that many cells havecertain systems to cope with nutrient starvation.1,2 The availability ofnutrients is constantly being monitored and once cells have ﬁguredout that the amount of the source for cellular metabolism is less thanthat needed, they initiate a stress response to suppress energy
consumption processes, which is safeguard against nutrient deﬁciency.
Mammalian target of rapamycin, a serine/threonine kinase, is one of
the representative nutrient sensors, which promotes protein synthesis
and cell cycle progression and inhibits autophagy induction provided
that nutrients are plentiful.3–5 Another major sensor for intracellularnutrients is general control non-derepressible 2 (GCN2).6,7 Deprivation of amino acids activates GCN2, which promotes the expression ofactivating transcription factor 4 (ATF4),8 a transcription factor that
regulates the expression of special genes responsible for adaptation toan amino acid shortage.9,10
Activated T cells aggressively proliferate and produce various
cytokines, which require increased cellular metabolism. It is necessary
for activated T cells to take a greater amount of nutrients to satisfy theincreased cellular metabolism. Indeed, full activation of T-cells induces
the expression of L-type amino acid transporter 1 (LAT1, also known
as SLC7A5), a transporter with the capability to intake essential aminoacids efﬁciently in humans and mice.11–13 On the other hand, it is also
evident that T cells willingly block further activation when LAT1
function is inhibited or amino acids are deprived.12,14 However, it is
not entirely clear how the systems monitoring nutrient availability inactivated T cells ultimately suppress the immunological reaction whenamino acids become deprived.
In this study, we characterized HOXB9, a member of the homeobox
gene family, as a negative regulator of activated T cells in response to

Q8

LAT1 dysfunction and amino acid starvation. The HOX family
encodes a transcription factor that has a DNA binding motif called
homeodomain and has been demonstrated to be critical for normal
morphogenesis, especially for normal segment formation.15–17
Although recent studies have shown that some HOX genes including
HOXB9 have substantial roles in cancers,18–20 the involvement of
HOX genes in stress response for nutrient starvation has not been
investigated.
We demonstrated that HOX protein contributes to the optimizationof immune reactions when activated T cells encounter an amino acidinsufﬁciency. Our surprising ﬁnding that the morphogenetic geneplays an unrealized role in stress response to amino acid starvation in
activated T cells may provide a new insight into the mechanism of thenutrient sensing system to control intracellular metabolism depending
on nutrient conditions.
RESULTS
Induction of HOXB9 expression by amino acid deﬁciency
Our previous study using a microarray assay demonstrated that
inhibition of LAT1 in activated human primary T cells induces
expression of a number of genes.12 One of those genes is HOXB9.
By using real time PCR (RT-PCR), we initially conﬁrmed theinduction of HOXB9 expression by JPH203, a LAT1-speciﬁc inhibitor,
in puriﬁed human blood T-cells stimulated with anti-CD3, CD28
antibodies (Figure 1a). Facilitated expression of HOXB9 was also
observed by activation of human T cells cultured in a medium inwhich essential amino acids were deprived (Figure 1b). We also foundthat previously activated human T-cells promote HOXB9 expression
by removal of essential amino acids (Figure 1c), suggesting that both1
Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan and 2J-Pharma Co, Ltd, Kanagawa, Japan
Correspondence: Dr N Anzai, Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu 321-0293, Japan.
E-mail: anzai@dokkyomed.ac.jp
Received 25 August 2015; revised 22 January 2016; accepted 23 January 2016

Q2

Q3

HOXB9 responds to amino acid starvation in T cells
K Hayashi et al2

Figure 1 Induction of HOXB9 expression by amino acid starvation in activated T cells. (a and b) Freshly isolated human CD4+ T cells were activated by antiCD3/CD28 in the presence of JPH203 (5 μM) (a) or without essential amino acids (-EAA) (b) for 3 days. HOXB9 expression was measured by RT-PCR. The
relative copy number of HOXB9 to one copy of GAPDH was shown. (c) Freshly isolated human CD4+ T cells were activated by anti-CD3/CD28 in a normal
medium for 5 days and further cultured in the medium with or without essential amino acids for 3 days. HOXB9 expression was measured by RT-PCR. The
relative copy number of HOXB9 to one copy of GAPDH was shown. Data are representative of three separate experiments. Data are expressed as themean ± s.d. *Po0.01.

primary and effector T-cells trigger HOXB9 expression as a responseto amino acid starvation. In contrast, we could not detect the HOXB9
messenger RNA (mRNA) in ﬂeshly isolated T cells, suggesting that
HOXB9 is not expressed unless T cells are activated.
Taken together, these results indicate that HOXB9 expression isprimed in response to amino acid starvation in activated human
T cells.
HOXB9 inhibits T-cell function
We hypothesized that induction of HOXB9 expression by a LAT1
defect or deprivation of amino acids might have special signiﬁcance inregulation of T-cell metabolism as a stress response to amino acidstarvation. To test this hypothesis, we analyzed the function of HOXB9
in T cells. Human primary T cells were transfected with a HOXB9
expression vector and stimulated with anti-CD3 and anti-CD28
antibodies. The amounts of IFNγ, IL-2, IL-4 and IL-17 produced
were then determined. Overexpression of HOXB9 signiﬁcantly
reduced the productions of IFNγ, IL-2 and IL-4 (Figure 2a). Interestingly, however, IL-17 production was not signiﬁcantly changed
by HOXB9.
We also found that knockdown of HOXB9 by small interfering
RNA (siRNA) in T cells that were activated without essential aminoacids slightly increased the productions of IFNγ, IL-2 and IL-4,although deprivation of essential amino acids decreased IL-17
production below measurable limits (Figure 2b).
These results indicate that HOXB9 acts as a negative regulator of
production of selective cytokines in activated human T cells, which
could contribute to the repression of cellular metabolism in response
to a lack of amino acids.

Q4

HOXB9 inhibits AP-1, and NFAT and NF-κB activities but not ROR
activity
We next investigated in more detail the basis for interference ofcytokine production by HOXB9 in T cells. NFAT, AP-1 and NF-κB
are representative transcription factors that positively regulate the
Immunology and Cell Biology

production of cytokines after T-cell activation.21 We therefore
investigated the inﬂuence of HOXB9 on NFAT, AP-1 and NF-κB
functions. Construct harboring the promoter including the binding
sequence of each transcription factors in front of luciferase gene weretransfected with a HOXB9 expression vector into Jurkat T cells, andthe cells were activated by PMA/Ionomycin to activate the pathway
from TCR/co-stimulation. As shown in Figure 3, HOXB9 signiﬁcantly
inhibited NFAT, AP-1 and NF-κB activities.
As HOXB9 did not impair the production of IL-17 in human
T cells, we also examined the effect of HOXB9 on the activity ofretinoic acid receptor-related orphan receptor (ROR), which is a
critical transcription factor for IL-17 expression.22 HOXB9 increased
the expression of a luciferase gene placed behind the ROR binding
element to a small extent (Figure 3), suggesting that HOXB9 slightly
augments ROR activity, which might explain why HOXB9 does not
suppress the production of IL-17 in activated T cells.
Taken together, these results suggest that HOXB9 acts as a potent
negative regulator of activated human T cells in response to aminoacid starvation by selective inhibition of transcriptional factors that areimportant for cytokine production.
Induction of HOXB9 expression is mediated by ATF4
We further investigated the mechanism of the induction of HOXB9
expression. GCN2 is one of the major sensors of amino acid shortagein various cells. Amino acids starvation increases uncharged transfer
RNAs, which activate GCN2 and the downstream signal ATF4
transcription factor, leading to gene expression that is responsible
for amino acid starvation. Indeed, ATF4 phosphorylation, which
activates its transcription activity,23 was enhanced by JPH203 inT cells (Figure 4a). ATF4 recognizes the consensus DNA binding
sequence composed of (A/G/T)TT(G/T/A)CATCA.24 We located thecomplete ATF4 consensus binding sequence (5′-ATTGCATCA-3′) at
334 bp upstream of HOXB9 translation initiation site in the humangenome. This fact prompted us to examine the involvement of ATF4
in the regulation of HOXB9 expression. We transfected ATF4 siRNAQ5

HOXB9 responds to amino acid starvation in T cells
K Hayashi et al
3

Figure 2 Effect of HOXB9 on T-cell activation. (a) Freshly isolated human CD4+ T cells were transfected with a HOXB9 expression vector and activated for
3 days. The concentrations of cytokines in the culture medium were determined. (b) Effect of HOXB9 siRNA on cytokine production. Freshly isolated humanCD4+ T cells were transfected with HOXB9 siRNA or control siRNA and activated for 3 days with (EAA(+)) or without (EAA( − )) essential amino acids. The
concentrations of cytokines in the culture medium were determined. ND, not detected. Data are representative of two separate experiments. Data areexpressed as the mean ± s.d. *Po0.05, **Po0.01.
Immunology and Cell Biology

HOXB9 responds to amino acid starvation in T cells
K Hayashi et al
4

Figure 3 Effect of HOXB9 on activities of NFAT, AP-1 NF-κB and ROR.
NFAT-, AP1- NF-κB- or ROR-luc construct was transfected with empty vector(Vec) or the HOXB9 expression vector into Jurkat T cells. The cells weretreated with PMA and Ionomycin (P/I) for 7 h. Luciferase activity wasmeasured and normalized with activity of pRL-TK-luc. Data arerepresentative of two separate experiments. Data are expressed as themean ± s.d. *Po0.01.

into human primary T cells and stimulated the cells with anti-CD3/
CD28 antibody and analyzed the HOXB9 expression. ATF4 siRNAfunctioned successfully in a substantial decrease in the expression levelof ATF4 (Figure 4b). HOXB9 expression was drastically suppressed by
ATF4 siRNA in essential amino acid-starved T cells (Figure 4b). These
results indicate that the induction of HOXB9 expression on aminoacid starvation in activated T cells is mediated by ATF4.
DISCUSSION
Prompt attention to nutrient deﬁciency is crucial for the survival ofactivated T cells as, if T cells leave it untreated, they will synthesize
aberrant cellular components and exhaust their energy, resulting incell death. In this study, we identiﬁed a new mechanism to addressamino acid starvation in activated T cells wherein HOXB9, a factor
known to be a morphogenesis regulator, is upregulated by ATF4 anddown-modulates the activity of transcription factors that are crucial
for cytokine production. These processes enable activated T cells to
avoid wasteful consumption of energy and material for prolonged
survival under a condition of nutrient scarcity.
The HOX family is composed of 39 genes,25 but little is known
about their functions other than functions in morphogenesis andcancer. Our study revealed a novel role of HOX gene; HOXB9 acts as
one of the factors to overcome the difﬁculty following amino acidstarvation in T cells.
Immunology and Cell Biology

Figure 4 Effect of ATF4 on HOXB9 expression. (a) Promotion of ATF4
phosphorylaion by JPH203 in activated CD4+ T cells. (b) Freshly isolated
human CD4+ T cells were transfected with ATF4-speciﬁc siRNA or control
siRNA and activated in the presence or absence of essential amino acids for
3 days. The expression of HOXB9 was determined by RT-PCR. Data arerepresentative of two separate experiments. Data are expressed as themean ± s.d. *Po0.01.

We found that the induction of HOXB9 expression is mediated by
ATF4. Although ATF4 is activated by signals from the amino acidsensor GCN2,9,10 the target genes of ATF4 are not clear. Our study
demonstrating that HOXB9 triggered by ATF4 diminishes theactivities of transcription factors for cytokine production provides a
new insight into the machinery by which ATF4 enables cellularmaterial and energy to be saved by modulating its target genes inamino acid-deﬁcient T cells.
Of interest is that not all cytokine production was impaired by
HOXB9. In this regard, we assume that multiple factors are involved
in halting T-cell activity when amino acids are starved. Indeed, we
demonstrated that DNA-damage-inducible transcript 3 downshifts
global cytokine production in response to amino acid deprivation in
activated T cells.12 The use of a combination of several factors withdifferent characteristics probably provides T cells with most appropriate tuning of cellular metabolism that is suitable for survival in aminimum nutrient environment.
The regulation of preferential cytokine production by HOXB9 raises
the possibility about connection between helper T-cells differentiationand HOXB9. We evaluated the expression of HOXB9 in Th1, Th2 andTh17 cells. All cells expressed almost the same level of HOXB9,
although Th17 cells cultured with no essential amino acids had aslightly lower HOXB9 expression compared with other linages(K.H., unpublished data). We also differentiated T-cells transfectedwith HOXB9 overexpression vector. However, we could not detect any
effects of HOXB9 transfection on Th differentiation (K.H., unpublished data). Since T cells cannot maintain a high level of transfectedgene expression for long periods with the electroporation method,HOXB9 responds to amino acid starvation in T cells
K Hayashi et al5another transfection system such as virus infection will be required toresolve this issue.
In conclusion, we uncovered a previously unrecognized mechanism
for adaptation to amino acid starvation in activated T cells. Ourﬁndings may contribute to an understanding of less well-known
eventual targets of pathways monitoring nutrient availability for adownshift of intracellular metabolism in response to changes innutrient conditions.
METHODS
Cells

Q6
Q7

Human peripheral blood mononuclear cells were isolated from healthy
volunteers by the histpaque centrifugation method (Sigma, St. Louis, MO,
USA). The study was approved by the Dokkyo Medical University Bioethics
Committee. CD4-positive (CD4+) T cells were isolated by Dynabeads
untouched human CD4 T cells kit (Invitrogen, Carlsbad, CA, USA). 5 × 105
CD4+ T cells were activated by Dynabeads coated with anti-CD3/CD28(Invitrogen) for the time indicated in RPMI1640 with 10% fetal calf serum.
For experiment of essential amino acid starvation, HBSS with glucose(Invitrogen) including vitamin solution (Sigma), MEM non-essential aminoacid solution (Invitrogen) and 10% fetal calf serum with or without MEM
amino acid solution (Invitrogen) was used. JPH203 was described previously.26

Western blot
For analysis of ATF4 phosphorylation, puriﬁed CD4+ T cells were stimulated
for 1 day in the presence of JPH203 and lysed with 20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1% Nonidet P-40, 20 mM sodium phosphate, 5 mM sodium
pyrophosphate, 1 mM Na3VO4, 10 mM NaF, 3 mM β-glycerophosphate andprotease inhibitor. After centrifugation at 16 000 g (5 min, 4 °C), the supernatant was used for western blot. For detection of ATF4 in siRNA-transfected
T cells, 2 × 104 cells were lysed in sodium dodecyl sulfate sample buffer andboiled. Electrophoresis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting were performed with a standard protocol. The
anti-human phospho ATF4 (Ser245) and anti-human ATF4 (C-20) werepurchased from Bioss (Boston, MA, USA) and Santa Cruz Biotechnology(Dallas, TX, USA), respectively.

Statistical analysis

All statistical signiﬁcance was tested with Student's t-test.

CONFLICT OF INTEREST
The authors declare no conﬂict of interest.

ACKNOWLEDGEMENTS
We thank Dr Amnon Altman at La Jolla Institute for Allergy and Immunology
for providing plasmid of AP-1- and NFAT-luciferase reporter constructs.

Transfection of human T cells
Pre-designed ATF4-speciﬁc siRNA (SI03019345) and its negative control siRNA(1027280) were purchased from Qiagen (Valencia, CA, USA). Pre-designed
HOXB9-speciﬁc siRNA (s6813) and its control siRNA (negative control) werepurchased from Ambion (Austin, TX, USA). siRNA (200 pmol) or plasmid(5 μg) was transfected into peripheral blood mononuclear cells using a
nucleofector (Lonza, Basel, Switzerland) with a human T-cell nucleofection
kit according to the manufacturer’s instructions. CD4+ T cells were thenpuriﬁed and activated for 3 days as described above. For cytokine production
analysis, the culture supernatant was measured by a cytometric bead array(Becton Dickinson, Franklin Lakes, NZ, USA).

Reporter assay
Jurkat cells were provided by RIKEN Bio-Resource Center (Tsukuba, Japan).
The cells were transfected by electroporation as previously described.27
ROR-luciferase (luc) was generated by insertion of ROR element sequence of
IL-17 enhancer (5′-GAAAGTTTTCTGACCCACTTTAAATCAATTT-3′)22 into
multi cloning site of pGL4.27 (Promega, Madison, WI, USA). AP-1- and
NFAT-luc reporter constructs were described previously.27 NF-kB-luc reporter
construct was obtained by inserting NF-kB consensus binding sequence(TTTCCGGGGA) into multi cloning site of pGL4.27 (Promega). Fireﬂy
luciferase activity was assessed using a Dual Glo lusiferase assay system(Promega) and normalized by the activity of renilla luciferase derived fromcotransfected pRL-TK (Promega).

Quantitative RT-PCR
Total RNA was extracted using an RNeasy mini kit (Qiagen). Complementary
DNA (cDNA) was synthesized from total RNA using a prime-script RT reagent
kit (Takara Bio, Shiga, Japan). RT-PCR was performed with SYBR Premix Ex
Taq (Takara Bio). Pre-designed primers for RT-PCR (HA162384 for HOXB9
and HA067812 for GAPDH) were purchased from Takara Bio. The sequencesof primers for detection of ATF4 are 5′-CGAATGGCTGGCTGTGGATG-3′
and 5′-AGGGCATCCAAGTCGAACTC-3′.
Copy numbers of HOXB9 and GAPDH mRNA were determined using
plasmid as a standard.

Plasmid construct
Human HOXB9 cDNA was obtained by the PCR method using cDNA
synthesized from human T-cell mRNA as a template. The cDNA fragment
was inserted into pcDNA3.

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Keeping the eIF2 alpha kinase Gcn2 in check. Biophys Acta 2014; 1843:
1948–1968.
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pathway is critical for tumour cell survival and proliferation in response to nutrient
deprivation. EMBO J 2010; 29: 2082–2096.
11 Morimoto E, Kanai Y, Kim K, Chairoungdua A, Choi HW, Wempe MF et al. Establishment and characterization of mammalian cell lines stably expressing human L-typeamino acid transporters. J Pharmacol Sci 2008; 108: 505–516.
12 Hayashi K, Jutabha P, Endou H, Sagara H, Anzai N. LAT1 is a critical transporter of
essential amino acids for immune reactions in activated human T cells. J Immunol
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cell differentiation. Nat Immunol 2013; 14: 500–508.
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activated T cells. Crit Rev Immunol 2014; 34: 467–479.
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257–278.
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1997; 3: 115–131.
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and malignant hematopoiesis. Blood 2012; 120: 519–527.
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2010; 10: 361–371.
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signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell
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Journal of Pharmacological Sciences xxx (2016) 1e4

Contents lists available at ScienceDirect

Journal of Pharmacological Sciences
journal homepage: www.elsevier.com/locate/jphs

Short communication

LAT1 acts as a crucial transporter of amino acids in human thymic
carcinoma cells
Keitaro Hayashi a, Promsuk Jutabha a, Sumiko Maeda b, Yothaisong Supak a,
Motoshi Ouchi a, Hitoshi Endou c, Tomoe Fujita a, Masayuki Chida b, Naohiko Anzai a, d, *a

Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Shimotsuga, Tochigi 321-0293, Japan
Department of General Thoracic Surgery, Dokkyo Medical University School of Medicine, Shimotsuga, Tochigi 321-0293, Japan
J-Pharma Co., Ltd., Yokohama, Kanagawa 230-0046, Japand
Department of Pharmacology, Chiba University Graduate School of Medicine, Chuo, Chiba 260-8670, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 March 2016
Received in revised form
27 June 2016
Accepted 11 July 2016
Available online xxx

L-type amino acid transporter 1 (LAT1, SLC7A5) incorporates essential amino acids into cells. Recent
studies have shown that LAT1 is a predominant transporter in various human cancers. However, thefunction of LAT1 in thymic carcinoma remains unknown. Here we demonstrate that LAT1 is a criticaltransporter for human thymic carcinoma cells. LAT1 was strongly expressed in human thymic carcinoma
tissues. LAT1-speciﬁc inhibitor signiﬁcantly suppressed leucine uptake and growth of Ty82 humanthymic carcinoma cell lines, suggesting that thymic carcinoma takes advantage of LAT1 as a qualitytransporter and that LAT1-speciﬁc inhibitor might be clinically beneﬁcial in therapy for thymic
carcinoma.
© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Keywords:
Thymic carcinoma
Anticancer drug
Transporter

Thymic carcinomas are rare malignant tumors and are often in
an advanced stage when detected (1). Although chemotherapy is
the mainstay of treatment for thymic carcinomas, patientsfrequently show resistance to drugs and more efﬁcacious treatment
should therefore be established.
LAT1 is a transporter that incorporates essential amino acids
into cells. A unique feature of LAT1 is its extremely high expression
in many human cancers (2,3), whereas only a small amount of LAT1
is detected in a healthy body (4,5), though LAT1 still has a clear rolein normal tissues (6e8).
A LAT1-speciﬁc inhibitor has shown powerful suppressive effects on many cancer cell lines in vitro and in vivo (9) and iscurrently under evaluation in a clinical trial of cancer patients.
Although a wide range of human cancers express LAT1, little is
known about LAT1 in thymic carcinomas. In this study, we investigated the role of LAT1 in human thymic carcinoma.

* Corresponding author. Department of Pharmacology, Chiba University Graduate
School of Medicine, 1-8-1, Inohana, Chuo, Chiba 260-8670, Japan. FAX: þ81 43 2262052.
E-mail address: anzai-path@umin.ac.jp (N. Anzai).
Peer review under responsibility of Japanese Pharmacological Society.

Ty82 human thymic carcinoma cells were purchased from Japanese Collection of Research Bioresources Cell Bank (Ibaraki). The
cells were cultured in RPMI1640 containing 10% FCS. S2 cells stablytransfected with empty vector, human LAT1 and LAT2 were
described previously (9).
Anti-LAT1 antibody (mouse monoclonal) was kindly provided
by J-Pharma (Tokyo).
Anti-b-actin antibody (clone C4) was purchased from Merck
Millipore (Darmstadt, Germany).
For human tissue staining, surgically excised thymic carcinoma
or thymoma was ﬁxed with formalin, embedded with parafﬁn, andsliced. The samples were stained with anti-LAT1 antibody anddetected by DAB. The study using human tissue was approved by
Dokkyo Medical University Bioethics Committee.
For western blot analysis, cells were lysed with lysis buffer(50 mM TriseHCl pH 7.8, 150 mM NaCl, 5 mM EDTA, 1% Tween-20,
10 mM NaF, 1 mM Na3VO4, 3 mM b-glycerophosphate, 5 mM pyrophosphate, protease inhibitor cocktail (Roche Diagnostics, Basel,
Switzerland)). Protein amount was determined using BCA Protein
Assay Kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA). Western
blot was performed with 3.5 mg of total protein as described previously (10).
JPH203 was described previously (9).

http://dx.doi.org/10.1016/j.jphs.2016.07.006
1347-8613/© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Hayashi K, et al., LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells, Journal of
Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.07.006

2

K. Hayashi et al. / Journal of Pharmacological Sciences xxx (2016) 1e4[14C]-L-leucine uptake was initiated by incubating the cells in
HBSS containing 1.0 mM [14C]-L-leucine (Moravek, Brea, CA, USA)and JPH203 at 37  C for 1 min. Uptake was terminated by washing
the cells 3 times with ice cold HBSS. Cells were lysed with 0.1N
NaOH and radioactivity was measured using an LSC-5100 b-scintillation counter (Aloka, Tokyo).
For apoptosis analysis, the cells were stained with annexin VFITC and propidium iodide (Medical & Biological Laboratories,
Nagoya) and analyzed by FACS (Becton Dickinson, Franklin Lakes,
New Zealand).
For cell cycle analysis, the cells were initially ﬁxed with 70%
ethanol. After washing the cells with PBS, cells were treated with
RNase A (100 mg/ml) for 30 min at 37  C and further incubated with
2 mg/ml of propidium iodide for 10 min at room temperature. Cell
cycle was analyzed with FACS.
All statistical signiﬁcance was tested with Student's t-test bycomparing JPH203-treated samples with control sample.
To understand the role of LAT1 in thymic carcinoma, we initiallyanalyzed the expression of LAT1 in human thymic carcinoma tissues. Strong expression of LAT1 was detected in thymic carcinoma(Fig. 1A). On the other hand, LAT1 was hardly found in thymoma(non-cancer) tissue (Fig. 1A). We next examined LAT1 expression in
Ty82 human thymic carcinoma cell line. Ty82 cells expressed LAT1
at a high level (Fig. 1B). These results indicate that LAT1 expression
is facilitated in human thymic carcinoma cells.
To investigate the functional signiﬁcance of LAT1 in thymic
carcinoma, we assessed the effects of JPH203, a LAT1-speciﬁc inhibitor, on the incorporation of amino acids in Ty82 cells. The cells
were incubated with or without JPH203 for 1 min and uptake of[14C]-labeled L-leucine was analyzed. JPH203 impaired the uptakeof leucine in a dose-dependent manner (Fig. 2). These results
indicate that LAT1 is crucial transporter of amino acids inTy82 cells.
We next examined the effect of a LAT1-speciﬁc inhibitor on
growth of Ty82 cells. The cells were cultured in the presence orabsence of JPH203 and the number of cells was counted. The
number of Ty82 cells was clearly reduced by JPH203 (Fig. 3A). To
determine whether the reduction of Ty82 cells by JPH203 arises
from aberrance of the cell cycle or cell death, we ﬁrst analyzed thesurvival rate of Ty82 cells treated with JPH203 by annexin V and

Fig. 1. LAT1 is a critical transporter of amino acids in thymic carcinoma cells. (A) Tissue staining of human thymic carcinoma (left) and thymoma (right) with anti-LAT1 antibody. (B)Ty82 cells were lysed and LAT1 protein was detected by western blot. As controls, lysates from S2 cells stably transfected with an empty vector (negative control), LAT1 (positive
control), and LAT2 (negative control) were also used.

Please cite this article in press as: Hayashi K, et al., LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells, Journal of
Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.07.006

K. Hayashi et al. / Journal of Pharmacological Sciences xxx (2016) 1e4

Fig. 2. Impaired incorporation of leucine by JPH203. [14C]-L-leucine and JPH203 wereadded to Ty82 cells and leucine uptake was analyzed by measuring radioactivity. Data
expressed as the mean ± S.D.

propidium iodide (PI) staining. 100 mM JPH203 increased apoptotic
cells (annexin V single positive cells) as well as total dead cells(annexin V positive cells plus PI positive cells) (Fig. 3B), indicating

3

that a high concentration of JPH203 induces cell death of Ty-82
that is derived from apoptosis. We also found that JPH203
modestly decreased the population of Ty82 cells in S phase andincreased the population of cells in G1 phase (Fig. 3C), suggestingthat JPH203 prevents progression of the cell cycle from G1 phase
to S phase. These results indicate that JPH203 has an antiproliferative effect on Ty-82 cells that is achieved by both cell
death and G1 arrest.
Here we demonstrated that a LAT1-speciﬁc inhibitor has theability to exert anticancer effects against thymic carcinoma cells.
Since thymic carcinoma is a relatively infrequent disorder andthere has not been sufﬁcient progress in its therapeutic approach,the results of our study could offer an additional and effective
treatment option for thymic carcinoma using a LAT1 inhibitor.
However, in our study, high concentration of JPH203 was required
for the sufﬁcient suppression of Ty-82 cells. In experiments withmice, it appears that the concentration of JPH203 in blood canexceed 100 mM at the point of administration, but immediately
drops into single digit (9). This result suggests that it is difﬁcult tomaintain a high concentration of JPH203 in vivo. Therefore, it isrecommended combining JPH203 at low concentration with otheranti-cancer drugs to maximize the therapeutic efﬁcacy andminimize adverse effects when JPH203 is used for clinicalapplication.

Fig. 3. Effect of JPH203 on growth of Ty82 cells. (A) Cells were cultured with JPH203 for the indicated days. The number of the cells was counted, and relative cell number to day 0 isshown. Data expressed as the mean ± S.D. *P < 0.01. (B) Cells were cultured with JPH203 for 3 days and stained with annexinV and Propium Iodide (PI). Data expressed as themean ± S.D. *P < 0.01. **P ¼ 0.011. (C) Cells were cultured with JPH203 for 4 days and the cell cycle was analyzed by staining the cells with PI. The numbers on the top of thehistogram indicate the percentages of cells in G1, S and G2 phase from the left.

Please cite this article in press as: Hayashi K, et al., LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells, Journal of
Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.07.006

4

K. Hayashi et al. / Journal of Pharmacological Sciences xxx (2016) 1e4

Conﬂicts of interest
The authors indicated no potential conﬂicts of interest.
Acknowledgments
This study was supported by JSPS KAKENHI Grant Number
26670631 and MEXT Program for the Strategic Research Foundation at Private Universities Grant Number S1412001.
References(1) Ahmad U, Yao X, Detterbeck F, Huang J, Antonicelli A, Filosso PL, et al. Thymic
carcinoma outcomes and prognosis: results of an international analysis.
J Thorac Cardiovasc Surg. 2015;149:95e100.
(2) Yanagida O, Kanai Y, Chairoungdua A, Kim DK, Segawa H, Nii T, et al. Human L-type amino acid transporter 1 (LAT1): characterization of functionand expression in tumor cell lines. Biochim Biophys Acta. 2001;1514:
291e302.

(3) Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H. Expression cloning
and characterization of a transporter for large neutral amino acids activated by
the heavy chain of 4F2 antigen (CD98). J Biol Chem. 1998;273:23629e23632.
(4) Kanai Y, Endou H. Heterodimeric amino acid transporters: molecular biology
and pathological and pharmacological relevance. Curr Drug Metab. 2001;2:
339e354.
(5) Matsuo H, Tsukada S, Nakata T, Chairoungdua A, Kim DK, Cha SH, et al.
Expression of a system L neutral amino acid transporter at the blood-brain
barrier. Neuroreport. 2000;11:3507e3511.
(6) Hayashi K, Jutabha P, Kamai T, Endou H, Anzai N. LAT1 is a central transporterof essential amino acids in human umbilical vein endothelial cells. J Pharmacol
Sci. 2014;124:511e513.
(7) Hayashi K, Ouchi M, Endou H, Anzai N. HOXB9 acts as a negative regulator of
activated human T cells in response to amino acid deﬁciency. Immunol Cell
Biol. 2016;94:612e617.
(8) Hayashi K, Anzai N. Role of LAT1 in the promotion of amino acid incorporationin activated T cells. Crit Rev Immunol. 2014;34:467e479.
(9) Oda K, Hosoda N, Endo H, Saito K, Tsujihara K, Yamamura M, et al. L-typeamino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci.
2010;101:173e179.
(10) Hayashi K, Jutabha P, Endou H, Anzai N. c-Myc is crucial for the expression of
LAT1 in MIA Paca-2 human pancreatic cancer cells. Oncol Rep. 2012;28:862e866.

Please cite this article in press as: Hayashi K, et al., LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells, Journal of
Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.07.006

World J Gastrointest Oncol 2017 January 15; 9(1): 21-29
ISSN 1948-5204 (online)

Submit a Manuscript: http://www.wjgnet.com/esps/
Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx
DOI: 10.4251/wjgo.v9.i1.21© 2017 Baishideng Publishing Group Inc. All rights reserved.

REVIEW

Novel therapeutic approaches targeting L-type amino acidtransporters for cancer treatment
Keitaro Hayashi, Naohiko Anzai
uptake of neutral amino acids into cells. Four LATs (LAT1,
LAT2, LAT3 and LAT4) have so far been identified. LAT1
(SLC7A5) has been attracting much attention in the fieldof cancer research since it is commonly up-regulated invarious cancers. Basic research has made it increasingly
clear that LAT1 plays a predominant role in malignancy.
The functional significance of LAT1 in cancer and thepotential therapeutic application of the features of LAT1
to cancer management are described in this review.

Keitaro Hayashi, Naohiko Anzai, Department of Pharmacology
and Toxicology, Dokkyo Medical University School of Medicine,
Shimotsuga, Tochigi 321-0293, Japan
Naohiko Anzai, Department of Pharmacology, Chiba University
Graduate School of Medicine, Chuo, Chiba 260-8670, Japan
Author contributions: Hayashi K wrote the manuscript; Anzai
N reviewed the manuscript.
Conflict-of-interest statement: There is no conflict of interest
associated with any of the senior author or other coauthors
contributed their efforts in this manuscript.

Key words: LAT1; Amino acid transporter; Moleculartarget drug; Amino acid starvation response; Signaltransduction

Open-Access: This article is an open-access article which wasselected by an in-house editor and fully peer-reviewed by external
reviewers. It is distributed in accordance with the Creative Commons
Attribution Non Commercial (CC BY-NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms,
provided the original work is properly cited and the use is noncommercial. See: http://creativecommons.org/licenses/by-nc/4.0/© The Author(s) 2017. Published by Baishideng PublishingGroup Inc. All rights reserved.

Core tip: The discovery of molecules preferentiallyexpressed in cancer cells is extremely valuable forthe development of molecular target drugs in cancer
therapy. Amino acid transporters have been receivinga great amount of attention as a candidate of suchmolecular targets. This review summarizes new
initiatives for clinical applications of the basic research
relative to L-type amino acid transporters, which arecommonly expressed in cancers.

Manuscript source: Invited manuscript
Correspondence to: Keitaro Hayashi, PhD, Associate
Professor, Department of Pharmacology and Toxicology, Dokkyo
Medical University School of Medicine, 880 Kitakobayashi,Mibu, Shimotsuga, Tochigi 321-0293,
Japan. khayashi@dokkyomed.ac.jp
Telephone: +81-282-872128
Fax: +81-282-862915

Hayashi K, Anzai N. Novel therapeutic approaches targeting
L-type amino acid transporters for cancer treatment. World J
Gastrointest Oncol 2017; 9(1): 21-29 Available from: URL: http://
www.wjgnet.com/1948-5204/full/v9/i1/21.htm DOI: http://dx.doi.
org/10.4251/wjgo.v9.i1.21

Received: August 15, 2016
Peer-review started: August 16, 2016
First decision: September 28, 2016
Revised: October 8, 2016
Accepted: November 1, 2016
Article in press: November 2, 2016
Published online: January 15, 2017

INTRODUCTION
Cancers consume a huge amount of materials forbiochemical reactions, and a continuous supply of
sufficient nutrients is essential for their survival. Hydro­
philic nutrients are delivered into cells by trans­porters.

Abstract
L-type amino acid transporters (LATs) mainly assist the

WJGO|www.wjgnet.com

21

January 15, 2017|Volume 9|Issue 1|

Hayashi K et al . Targeting LATs in cancer management
COOH

4F2hc

LAT1

S S

NH2
COOH

NH2

Figure 1 Structure of LAT1. LAT1 is composed of 12 transmembrane helices that are predicted to form a cylindrical conformation penetrating the cellular membrane.
LAT1 associates with 4F2hc for stable localization at the cellular membrane. LAT2 is similar in structure to LAT1, whereas LAT3 and LAT4 function independently of
4F2hc.
[2,3]

Recent studies have revealed several trans­porters
preferentially expressed in cancers. Inhibition of cancer
specific-nutrient transporters would be a good strategy
for cancer management with minimal side effects.
Indeed, a therapeutic approach using transporter
inhibitors for cancer prevention has been proven to beefficacious in cell lines and animal experiments and is
now under evaluation in a clinical trial.

two groups in 1998 . The major substrate of LAT1
is large neutral amino acids as typified by leucine.
The expression of LAT1 in normal adults is detected inproliferative zones of gastrointestinal mucosa, testicular
sertoli cells, ovarian follicular cells, pancreatic islet
cells, and some endothelial cells that serve as a barrier
between tissues (blood-brain, blood-retinal and blood[4]follicle barrier) . Recent studies revealed a crucial role
[5,6]
of LAT1 in activated T cells . As described below,
LAT1 expression is commonly up-regulated in various
cancers.
LAT2 (SLC7A8) was subsequently isolated on the[7-9]basis of sequence similarity to LAT1
. LAT2 has
broader specificity of its substrates including polar
uncharged and small neutral amino acids than that[8]of LAT1 . LAT2 is ubiquitously expressed in normal[4]
body , though LAT2 knockout mice show a mild
phenotype and almost no visible symptoms except[10]aminoaciduria . Both LAT1 and LAT2 are composedof 12 transmembrane domains that form the pathway[11]of their substrates(Figure 1). They associate withthe heavy glycoprotein subunit 4F2hc (SLC3A2) by[11]
sulfur bond . Although 4F2hc does not seem to havea function to directly transfer the substrates, it makes
the localization of its partner LATs more stable at the[12]plasma membrane .
LAT3 (SLC43A1) was isolated by expression cloning[13]
from hepatocarcinoma cells . Sequence analysis
revealed that LAT3 was identical to POV1, which was[14,15]originally identified as a cancer-up-regulated gene.
The substrate selectivity of LAT3 was similar to thatof LAT1. LAT3 mRNA is expressed in the liver, skeletal[16]
muscle, and pancreas . The physiological role of LAT3
in normal individuals of mammals remains unknown,

L-TYPE AMINO ACID TRANSPORTERS
Many cells take advantage of transporters to incorporate
what is necessary at the time of need. Transporters fall
into two broad categories based on ATP dependency[1]
for their transport form . ATP-dependent transporters,
known as ATP-binding cassette, hydrolyze ATP toobtain the energy for translocation of their substrates
across the membrane (active transport). Transporters
with no ATPase, called solute carriers (SLCs), facilitate
diffusive transport. Each SLC transporter is named
in combination with the family numeral based on thesequence similarity and individual number with letter A
between them (e.g., SLC3A2), with a few exceptions.
Most of the amino acid transporters were formerly
categorized into several groups (“System”) on thebasis of their substrates and sodium dependency (e.g.,
System L, which incorporates neutral amino acids
without sodium), but they are currently classified into
SLCs according to their protein homology.
L-type amino acid transporters (LATs) are categorized
as system L transporters. LATs mainly deliver neutralamino acids into cells in a sodium-independent manner.
So far, four LATs have been identified.
LAT1 (SLC7A5) was identified as the first LAT by

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Hayashi K et al . Targeting LATs in cancer management
but it was shown that LAT3 functions for podocyte[17]development in zebrafish .
LAT3 appears to behave as a critical transporter inseveral cancers. LAT3 is up-regulated in response to
androgen and knockdown of LAT3 expression by RNA
interference (RNAi) significantly inhibits the leucine
uptake and cell proliferation in human prostate cancer[18]
cell lines in vitro . Furthermore, high expressionof LAT3 is detected in prostate cancer patients, andstably knockdown of LAT3 by RNAi in human prostatecancer cell lines results in decrease of their growth andmetastatic potential with alteration of cell cycle gene
[19]
expression after xenografts into mice .
LAT4 (SLC43A2) was identified by searching for[20]
sequence homology to LAT3 . LAT4 is expressed in
the basolateral membrane of the small intestine, kidney
proximal tubule and thick ascending limb epithelial cells.
LAT4 knockout mice are smaller than their controls anddie within 9 d, presumably because of defective amino[21]acid absorption . Unlike LAT1 and LAT2, LAT3 as well
as LAT4 functions independently of heavy chain.

the affinity of LAT1 for leucine is much higher than that[30]of LAT2 , although LAT2 is ubiquitously expressed in[4]
the normal body . Cancers may therefore be more
dependent on LAT1 for rapid uptake of sufficient aminoacids, whereas normal cells need less amino aciddelivery that can be supported by LAT2.
The definite effect of LAT1 on the growth of various
cancer cell lines prompted researchers to apply the LAT1
inhibitor in a clinical setting. However, the concentrationof BCH required for suppression of cancer growth isextremely high (usually around 10 mmol/L). Moreover,the unselective effect of BCH that inhibits all LATs
is another problem, since LATs other than LAT1 areconsidered to have functions in the normal body. It has
been necessary to develop drugs that act on just LAT1
but not other transporters at a low concentration. In
2010, Endo and colleagues designed a new compound
named JPH203 ((S)-2-amino-3-(4-((5-amino-2phenylbenzo[d]oxazol-7-yl) methoxy)-3,5-dichloropheyl)[31]
propanoic acid) . JPH203 has structural analogy totyrosine, but it inhibits only LAT1 without affecting anyother LATs. JPH203 displayed potent suppressive effects[12,32,33]
on the growth of cancers in vitro. Moreover,this compound has the ability to powerfully inhibit the
proliferation of tumor cell lines of the colon and leukemia[31,33]
injected into nude mice
. Following improvements
in its specificity and pharmacological effect, JPH203
is under evaluation in a phase I clinical trial of cancer
patients.

LAT1
LAT1 is the most extensively studied transporter among
LATs. The interest in LAT1 is because of its extremely
high expression in diverse human cancers. LAT1 was[2]originally cloned from mRNA of C6 glioma cells .
Subsequent studies have shown that LAT1 is highly
expressed in many cancer cell lines. Histological analysis
with qualitatively enhanced antibodies confirmed potent
expression of LAT1 in human cancers in a broad range oftissues. The number of cancer types that were reported
to express a high level of LAT1 is well above twenty (Table1). LAT1 is thus a commonly up-regulated amino acidtransporter in multiple human cancers. Furthermore,
LAT1 expression level appears to be associated withprognosis of cancer patients. For example, elevatedexpression of LAT1 correlates with an adverse prognosis
[22]
[23]
[24]
in prostate , gastric , and pancreatic cancers ,suggesting that higher-grade tumors are more depen­
dent on LAT1. Not only the expression of LAT1 but also
the functional significance of LAT1 in cancers has beenverified by use of its inhibitors, by knockdown with RNAiand by gene disruption. 2-Aminobicyclo (2,2,1) heptane2-carboxylic acid (BCH) is an inhibitor of system L
transporters. BCH inhibits leucine uptake and strongly
suppresses the proliferation of many cancer cells (Table
1). Genetic manipulation confirmed the functionalsignificance of LAT1 in cancer cells. Knockdown of LAT1
[25-29]with RNAi
as well as genetic disruption of LAT1 by[12]zinc fingers nucleases-mediated gene knockout
incancer cells reduces leucine uptake and cell proliferation,
indicating that LAT1 is a predominant transporter thatis essential for growth of cancers. The reason that somany cancers use LAT1 despite the presence of manyother amino acid transporters might be that LAT1 hasa prominent capability for substrate transport. Indeed,

WJGO|www.wjgnet.com

CLINICAL APPLICATION OF LAT1
Positron emission tomography

By exploiting the characteristics of LAT1 expression,
an approach for the diagnosis of cancers throughradiolabeled substrates of LAT1 has been attempted.
1811[ F] or [ C]-labeled compound administered into thebody can be visualized by positron emission tomography[34](PET) . Cancers incorporating an isotopically labeledprobe can be located by tracing the body with PET. In
18
18
the past, 2- F-fluoro-2-deoxy-d-glucose ([ F]FDG)
was one of the most commonly used probe fordiagnosis of cancer with PET. This strategy exploits thecharacteristic of cancers consuming a huge amount of
glucose compared to that consumed by normal cells.
18
Although [ F]FDG has been of assistance in the clinicaldiagnosis of many cancers, it sometimes showed
false positive results, especially in brain, because evennormal brain cells take up a relatively large amount of
glucose. To overcome this problem, amino acids haveattracted attention as alternative probes to glucose.
Representative amino acids or their analogs developed18
as probes of PET are L-3-[ F]-fluoro-α-methyl tyrosine
18
18([ F]FAMT), 6- F-fluoro-L-3,4-dihydroxy-phenylalanine
18
1111( F-DOPA), l-[ C-methyl] methionine ([ C]MET)
18
18
and O-(2-[ F]fluoroethyl)-l-tyrosine ([ F]FET). If thecompounds are delivered into cells specifically through
LAT1, those cells are likely to be cancers. Indeed,

23

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Hayashi K et al . Targeting LATs in cancer management
Table 1 Summary of studies for expression and functions of LAT1 in cancers
CancerBiliary tract
Bladder
Bone
Brain
BreastColon
Esophagus
Hepatocyte
Gastrointestine
Laryngeal
Leukemia
Lung
Melanoma
Myeloma
Neuroendocrine
OvarianOral
Pancreas
PleuraProstate
TongueThymusUrinary tract

Expression (method of detection)

Inhibition of aminoacid uptake by

Growth inhibition by

Ref.

ImmunohistochemistryNorthernblot (cell line)
Immunohistochemistry
Immunohistochemistry, RT-PCR (cell line), Western blot (tissue,
cell line)
Immunohistochemistry, RT-PCR (cell line)
Western blot (cell line)

BCH
BCH

BCH

BCH

BCH

[71]
[72]
[73]
[74,75]BCH
Knockout (cell line)

RNAi, BCH
Knockout (cell line)
JPH203

Immunohistochemistry
Immunohistochemistry
Immunohistochemistry, Western blot (cell line)
Immunohistochemistry
RT-PCR (cell line)
Immunohistochemistry
Immunohistochemistry, Microarray (tissue),Western blot (cell line)
RT-PCR (cell line)
Immunohistochemistry, RT-PCR (tissue),Western blot (tissue)
Immunohistochemistry, RT-PCR (cell line),
Western blot (tissue, cell line)
RT-PCR (cell line)
ImmunohistochemistryWestern blot (cell line)
Immunohistochemistry
Immunohistochemistry, Western blot (cell line)
Immunohistochemistry
Immunohistochemistry, Western blot (cell line)
Immunohistochemistry

RNAi
BCH, JPH203
BCH
RNAi

[29,76-78][12][79,80][81][23,45][82][33][41,83-85][86,87]
[88]
[89]

BCH

BCH

[47,65,90]

RNAi
RNAi

RNAi
RNAi

[25]
[24,27,91]

RNAi, BCH

RNAi, BCH

JPH203

JPH203[92][18,19,22,28][93][94,95][96]

RT-PCR: Reverse transcription polymerase chain reaction; BCH: 2-aminobicyclo (2,2,1) heptane-2-carboxylic acid.

18

[35]

[ F]FAMT images accord well with LAT1 distribution .
Moreover, FAMT is incorporated by LAT1 but not by other[35]
amino acid transporters . Although there is still room
for improvement in its specificity, this method is powerfultool for diagnosis of cancers including microcarcinoma.

past was that it required a large-scale nuclear reactor to
generate neutrons. However, a compact accelerator has
been developed as an alternative to a nuclear reactor
and it can be installed in a hospital, making BNCT easierto perform. Such technology will expand the applicationsof BNCT in the future.

Boron neutron capture therapy

LAT1 is an attractive molecular target for boron neutron
capture therapy (BNCT). BNCT is an anticancer therapy
that utilizes high linear energy transfer alpha particles.
Particle radiation is produced by fission reaction
when irradiated thermal neutrons collide with boron
incorporated by a malignant tumor. The traveling
distance of particle radiation is limited (5-9 μm), and it
therefore disrupts only cancer cells incorporating boron[36,37]
without damage to other cells around target cells.
A key component of BNCT success is accumulationof boron specifically in cancer cells. This difficult
task could be achieved by the synthesis of a boron
compound that is selectively delivered by LAT1. Indeed,p-boronophenylalanine (BPA), a boron compound[38-40]commonly used in BNCT, is incorporated by LAT1,
suggesting that LAT1 is an optimal mediator for delivery
of boron in BNCT. However, since we cannot still
completely rule out the possibility of BPA uptake by othertransporters, it is necessary to develop compounds thatexhibit strict selectivity to LAT1. BNCT has accomplishedcertain clinical outcomes so far, but the problem in the

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LAT1 AND METASTASISIt has been suggested that LAT1 is involved in cancer
metastasis. A number of studies have shown a corre­
lation of increase in LAT1 expression with metastasisof multiple cancers. Lymph node metastasis-positivesquamous cell carcinomas express LAT1 whereas there is[41]
no positive signal of LAT1 in metastasis-negative cells .
LAT1 mRNA level was significantly higher in renal cell
[42]
carcinoma with metastasis . A group of cells with high
LAT1 expression showed a larger size of the metastatic[43]
lesion of gastric carcinoma . LAT1 expression inneuroendocrine tumors was significantly associated with[44]
lymph node metastasis . The potency of the functionalsignificance of LAT1 in metastasis has been shown.
Knockdown of LAT1 by RNAi inhibited the migration and[45]
invasion of gastric cancer and a cholangiocarcinoma cell[46]
line . BCH inhibited the proliferation and migration of a[47]
human epithelial ovarian cancer cell line . On the basisof these findings, inhibition of LAT1 will be good strategy
to prevent metastasis of cancer. However, it remains to24

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Hayashi K et al . Targeting LATs in cancer management
Amino acids

LAT1

mTOR

GCN2

c-Myc
ATF4

CHOP

LAT1

Cell proliferation

Apoptosis

Autophagy

Figure 2 Schematic model of acquisition and monitoring of amino acids in cancer. c-Myc promotes expression of LAT1, which supplies amino acids necessary
for growth of cancers. The availability of amino acids is constantly monitored by factors such as mTOR and GCN2. Once amino acid deficiency is detected, cancers
suppress their proliferation and, as occasion demands, induce apoptosis. mTOR: Mechanistic target of rapamycin; GCN2: General control non-derepressible 2; ATF4:
Activating transcription factor 4; CHOP: C/EBP homologous protein.
[54]

lines . Artificial manipulation to elevate Hif2a activity
induces LAT1 expression in lung and liver tissues,[54]
in which LAT1 expression is usually low . Arylhydrocarbon receptor (AHR) is a transcription factor
that is activated by interaction with its ligands such as[55]
dioxin, and it promotes tumorigenesis . AHR binds to
its consensus binding sequence in LAT1 and drives LAT1
[56]expression in breast cancer cell lines , suggestingthat LAT1 contributes to tumorigenesis induced by anenvironmental carcinogen. As described previously,[5,6]
T cell activation induces LAT1 expression . Nuclear
factor kappa B, AP-1 and nuclear factor of activatedT-cells are critical transcription factors that are activated
by T cell stimulation and enhance immunologicalreactions. The expression of LAT1 is prevented by[5,6]
inhibitors of these transcription factors . This meansthat LAT1 expression is induced by the common
regulators that also boost immunological reaction in T
cells.

be determined whether the metastasis defect is derived
from amino acid starvation or from other factors such as
an aberrance of adhesion molecules. It would thus bevaluable to investigate the relevance of LAT1 and integrin[48]
in metastasis, since they form a complex .

MECHANISM OF LAT1 EXPRESSION
Although it remains unknown how LAT1 expression
is facilitated in cancers, some possible molecularmechanisms have been proposed. c-Myc, a protooncogenic transcription factor, has been demonstrated
to be an upstream of LAT1. The expression of c-Myc in[49]
normal adults is generally low , but overexpressionof c-Myc triggered by some cues such as gene amplifi­[50]
cation, gene translocation or other gene mutations
is responsible for malignant transformation. Numerous
human cancer tissues strongly express c-Myc. Target
genes of c-Myc include many factors involved in pro­
[51]
gression of the cell cycle . On the other hand, theconsensus binding sequence of c-Myc is also located at[27]
the LAT1 promoter . Moreover, knockdown of c-Myc
leads to reduction of LAT1 expression in cancer cell
[27]
lines . These results suggest that up-regulation of LAT1
is mediated, at least in part, by c-Myc (Figure 2). Of noteis that c-Myc also enhances the metabolic reprogramin cancers by promoting the expression of enzymes[52,53]of glycolysis and glucose transporter. This is aningenious strategy of cancers since they can coordinate
multiple events required for cell growth by just one
factor.
Some other factors appear to regulate LAT1
expression. Hypoxia-inducible factor (Hif) is a criticalregulator in response to hypoxia. Hif2a, an isoform
of the Hif family, binds to the LAT1 promoter andenhances LAT1 expression in renal carcinoma cell

WJGO|www.wjgnet.com

DOWNSTREAM OF LAT1
Ensuring a sufficient supply of nutrients is an issue ofvital importance for cancers. The majority of cancers
are thought to constantly monitor the availability ofamino acids. Starvation of amino acids rapidly induces astress response that puts a brake on cellular biochemical
reactions to avoid wasting energy and materials. The
most extensively studied system for monitoring theamino acid availability is mechanistic target of rapamycin[57](mTOR) , a serine-threonine kinase. Plenty of aminoacids maintains mTOR kinase activity, resulting inprogression of the cell cycle, protein synthesis, or inhibi­tion of autophagy induction (Figure 2). Some mTOR
[58-60]regulators such as SLC38A9, Cellular arginine
[61]
[62]
sensor for mTORC1 (CASTOR1) and Sestrin2 have25

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Hayashi K et al . Targeting LATs in cancer management
been demonstrated to associate with amino acids todictate mTOR activity. Dissociation of those interactions
caused by amino acid deficiency inactivates mTOR andinverses the reaction of its downstream, resulting in a
halt of cancer growth. Growing evidence suggests that
LAT1 disruption leads to the inhibition of mTOR. LAT1
inhibition decreases mTOR activity in many cancer cell
[28,33,63-65]
lines. These findings suggest that the arrest ofcell growth of cancers by a defect of LAT1 is derived frominactivation of mTOR (Figure 2). mTOR inhibitors arebeing used in practical trials for therapeutic management[66]
of several cancers . Application of JPH203 together
with an mTOR inhibitor probably creates a synergistic
effect and might be useful for maximizing the benefitof treatment with a low-dose drug, which would help tominimize adverse effects.
General control non-derepressible 2 (GCN2) is[67]
another factor for detection of amino acid starvation .
GCN2 is a serine-threonine kinase that is activated byamino acid deficiency. Uncharged tRNAs caused by adecline of amino acid concentration activates GCN2,
which eventually induces activity of activating trans­
cription factor 4 (ATF4). ATF4 regulates the expres­sion of genes responsible for coping with amino acid[68]
deficiency . Several studies have shown that dysfunc­tion of LAT1 initiates the GCN2 signal. JPH203 pro­motes
the expression of C/EBP homologous protein [CHOP, also
known as DNA damage inducible transcript 3 (DDIT3)],[68]
which is up-regulated by ATF4
and probably takes[33]
part in apoptosis in leukemia . Gene disruption of LAT1
[12]in cancer cell lines activates the GCN2-ATF4 cascade .
Activation of ATF4 by LAT1 defect was also shown in cells
other than cancer. JPH203 triggers the expression of[5,69][70]
CHOP
and homeobox B9 , a novel target of ATF4,
in human T cells to repress cytokine production. Thesefindings suggest that GCN2-ATF4 is another criticalsystem for detecting amino acid deficiency evoked by
LAT1 inhibition (Figure 2).

for their growth and development of a LAT3-specificinhibitors is also encouraged. Advances in technologies
are expected to resolve such issues.

CONCLUSION

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2

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8

9

After the importance of LAT1 in cancer cells had been
established, basic studies on LAT1 have progressed withremarkable speed. Better still, research achievements
are potentially capable of technical developments forthe use of LAT1 as a molecular target in clinical practice.
However, although JPH203 is more effective andspecific than BCH, it still requires a high concentration
for sufficient suppression of the growth of cancers,
and wariness of adverse effect persists. Nevertheless,such concerns might be overcome, at least for thetime being, by virtue of the proper combinational use
of multiple drugs with different action points in cellular
metabolism (e.g., mTOR inhibitor). However, further
improvements in selectivity of the inhibitor, boron donorof BNCT and PET probe to LAT1 will raise the quality ofcancer treatment. Besides, although not to the extent
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L- Editor: A E- Editor: Wu HL

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Anal Bioanal Chem (2017) 409:1007–1016
DOI 10.1007/s00216-016-0015-x

RESEARCH PAPER

Matrix-assisted laser desorption/ionization imaging mass
spectrometry reveals changes of phospholipid distribution
in induced pluripotent stem cell colony differentiation
Yasuo Shimizu 1 & Motoyasu Satou 2 & Keitaro Hayashi 3 & Yusuke Nakamura 1 &Mio Fujimaki 1 & Yasuhiro Horibata 2 & Hiromi Ando 2 & Taiji Watanabe 1 &Taichi Shiobara 1 & Kazuyuki Chibana 1 & Akihiro Takemasa 1 & Hiroyuki Sugimoto 2 &Naohiko Anzai 3,4 & Yoshiki Ishii 1

Received: 8 July 2016 / Revised: 29 September 2016 / Accepted: 6 October 2016 / Published online: 4 November 2016
# Springer-Verlag Berlin Heidelberg 2016

Abstract Induced pluripotent stem cells (iPSCs) are opening
up new possibilities for medicine. Understanding the regulation of iPSC biology is important when attempting to apply
these cells to disease models or therapy. Changes of lipid
metabolism in iPSCs were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS). Analysis revealed changes of
the intensity and distribution of peaks at m/z 782.5 and 798.5
in iPSC colonies during spontaneous differentiation. Two
phosphatidylcholines (PCs) were identified: C44H81NO8P,
PC(36:4)[M+H]+ at m/z 782.5 and C 4 2 H 8 2 NO 8 P,
PC(34:1)[M+K]+ at m/z 798.5. The intensity of PC(36:4)
showed an inverse relation between undifferentiated anddifferentiated iPSC colonies. PC(34:1) displayed a diffuse distribution in undifferentiated iPSC colonies, while it showed aconcentric distribution in differentiated iPSC colonies, andwas localized at the border of the differentiated and undifferentiated areas or the border between undifferentiated iPSC andfeeder cells. These findings suggested that the distribution oflipids changes during the growth and differentiation of iPSCs
and that MALDI-TOF-IMS was useful for analyzing thesechanges. PC(36:4) might play a role in maintaining
pluripotency, while PC(34:1) might play a role in the differentiation and spread of iPSCs.
Keywords iPS . Phospholipids . Phosphatidylcholine .
MALDI . Imaging . Differentiation

Introduction
Electronic supplementary material The online version of this article(doi:10.1007/s00216-016-0015-x) contains supplementary material,
which is available to authorized users.
* Yasuo Shimizu
yasuo-s@dokkyomed.ac.jp

1

Department of Pulmonary Medicine and Clinical Immunology,
Dokkyo Medical University School of Medicine, 880 Kitakobayashi,Mibu, Tochigi 321-0293, Japan2

Department of Biochemistry, Dokkyo Medical University School of
Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan3

Department of Pharmacology and Toxicology, Dokkyo Medical
University School of Medicine, 880 Kitakobayashi,Mibu, Tochigi 321-0293, Japan

4

Department of Pharmacology, Chiba University Graduate School of
Medicine, 1-8-1 Inohana, Chuo, Chiba 260-8670, Japan

Induced pluripotent stem cells (iPSCs) are expected to havevarious medical uses in the future, such as for the constructionof disease models and for cell therapy [1]. For medical application, however, several points need to be considered before
medical application can be achieved, including methods ofregulating iPSC functions such as differentiation, cell purity,and the biological products [2, 3]. Therefore, to apply andoptimize iPSC technology for medical uses, better elucidationof iPSC biology is required.
Lipid metabolism influences numerous cellular processes,
including growth, proliferation, differentiation, and motility[4]. Phosphocholines (Pcho), which were precursors of phosphatidylcholine (PC) or polyunsaturated PCs, have been reported to be abundant in embryonic stem cells and iPSCs
compared to mature cells [5, 6].

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Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a technique that can be used
to identify substances by their molecular signatures. In particular, MALDI with time-of-flight/time-of-flight IMS (MALDITOF/TOF-IMS) is useful for the analysis of numerous biomolecules by collecting hundreds of mass peaks [7].
Imaging analysis enables in situ analysis that distinguishes
the undifferentiated from the differentiated regions of cell
cultures.
As an iPSC colony grows larger, spontaneous differentiation is initiated from the central region of the colony. In colonies of pluripotent stem cells, the differentiated cells show
obvious morphological changes and form typical flat squamous epithelium [8], so the differentiated area is apparently
different from the surrounding undifferentiated area of thecolony.
In the present study, an IMS method was developed toenable simultaneous mass spectrometry in situ analysis ofdifferentiated and undifferentiated iPSC colonies in culture.
Previously, preparation of different cell culture dishes with
undifferentiated or differentiated iPSCs and extraction oflipids from each culture were required. However, we considered that it might not be necessary to prepare separate differentiated and undifferentiated cell cultures and also might not
be necessary to add cytokines or special media to obtain differentiated cells. Therefore, we employed MALDI-TOF-IMS
to identify changes of lipids during spontaneous differentiation of iPSC colonies in the present study.

Materials and methods
Cell culture
The mouse embryonic fibroblast cell line SNL 76/7 (SNL
cells; ECACC) was cultured on gelatin-coated culture dishes
in D-MEM (Nacalai Tesque, Japan) with 10 % fetal bovine
serum and 1 % penicillin/streptomycin (Invitrogen, USA) as
feeder cells. A human iPS cell line (409B2) was obtained fromRIKEN BRC through the Project for Realization ofRegenerative Medicine and National Bio-Resource Projectof NEXT, Japan. The iPSCs were established with episomal
plasmid vectors using pCXLE-hOCT3/4-shp53, pCXLEhSox2-Klf4, and pCLXE-hLmyc-Lin28, as reported previously [9]. The iPSCs were transferred onto SNL cell feeder layers
and cultured in standard human embryonic stem cell medium(ReproCELL, Japan) containing 4 ng/ml basic fibroblast
growth factor (Wako, Japan). For MALDI analysis, SNL cells
were cultured on gelatin- and indium/tin oxide-coated (ITO)
glass slides (Sigma-Aldrich, USA) in a 10-cm culture dish.
Then, iPSCs were transferred onto the SNL cell layers andcultured for 6 days. On day 6, the culture dish was placed on
ice and the slides were removed from the dish and washed

Y. Shimizu et al.

three in times cold saline. Then, the slides were placed in an
ITO-coated glass cassette for MALDI analysis. Differentiatedand undifferentiated areas of the colony were identified by
microscopic examination (Olympus CK30, Japan) andmarked on the reverse side of the glass slide, which was immediately frozen at −80 °C.
Immunocytochemistry
Immunocytochemistry was performed according to the method reported previously [10]. iPSCs grown on ITO-coatedglass slides were fixed for 10 min at room temperature with
PBS containing 4 % paraformaldehyde. After washing withpure water, the cells were stained for alkaline phosphatase(ALP) by using a Leukocyte Alkaline Phosphatase kit(Sigma, England). To stain cells for stage-specific embryonicantigen-4 (SSEA-4), iPSCs were first treated with PBS containing 5 % normal goat serum (Funakoshi, Japan), followed
by incubation with mouse anti-human SSEA-4 antibody(Santa Cruz, USA) as the primary antibody and Alexa488conjugated goat anti-mouse IgG (Invitrogen) as the secondary
antibody. Then, the nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI; Invitrogen).
Matrix-assisted laser desorption/ionization time-of-flight
imaging mass spectrometry
MALDI-TOF/TOF-IMS was performed with an
iMScopeTRIO® (Shimadzu, Japan) equipped with a 355nm Nd:YAG laser [7]. ITO-coated glass slides with cultured
cells were thawed to room temperature just before MS analysis, and dried samples were automatically and homogenously
coated with a matrix (2-hydroxy-5-methoxybenzoic acid,
DHB) using an iMLayer® (Shimadzu). Mass spectrometry
data were acquired in positive ion mode in the mass range
m/z 500–1000. The laser beam diameter was set at 5 μm,
and the mass resolving power was 10,000 at m/z 1000 usedin MS and MS/MS scans. During culture, parts of the iPSC
colonies show spontaneous differentiation. To find lipids witha different distribution between the differentiated and undifferentiated areas of the iPSC colonies, screening of various m/z distributions was performed by IMS analysis. The steps in
the analysis were as follows: First, MS analysis was performed for the entire iPSC colony (both differentiated andundifferentiated colonies) on the ITO glass slide. Principal
component analysis (PCA) was performed as unsupervised
multivariate analysis, and hierarchical clustering analysis(HCA) of image patterns was done with iMScopeTRIO® software. Then, IMS analysis was performed on undifferentiatedareas in the undifferentiated iPSC colonies and on differentiated areas in the differentiated iPSC colonies. A total of 16
areas (n = 16) in each group from another four cell culture
samples were analyzed; the data were processed with

Phospholipid distributions in iPS colonies

iMScopeTRIO® software. Next, comparing the overall andfocal analyses, common target m/z in both differentiated andundifferentiated iPSC colonies were determined, followed by
MS/MS analysis of the peaks at target m/z values. The target
m/z was determined after elimination of m/z peaks derived
from DHB (matrix). Target m/z was further confirmed byreversed-phase ultra-high-pressure liquid chromatography(UHPLC) MS/MS analysis. The IMS and MS/MS analysis
data were compared with the Lipid MAP (http://www.
lipidmaps.org/) and LipidBlast and NIST MS/MS libraries(http://fiehnlab.ucdavis.edu/projects/LipidBlast), as
previously reported [11, 12]. Mass accuracy tolerance usedin database searches was 0.3 Da. To compare the intensity ofthe target lipids in the differentiated and undifferentiated areasof the differentiated iPSC colonies, imaging analysis was
performed after setting regions of interest (ROIs).
Reversed-phase ultra-high-pressure liquid
chromatography (UHPLC)-MS/MS analysis
Lipids were analyzed by reversed-phase UHPLC using an
Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm;
Waters, Milford, MA, USA) coupled to a 5500 QTRAP mass
spectrometer (Sciex Inc., Framingham, MA, USA). A binary
gradient consisting of solvent A (1:1:3 acetonitorile/methanol/
water containing 5 mM ammonium acetate) and solvent B (2propanol containing 5 mM ammonium acetate) was used. The
gradient profile was as follows: 0–1 min, 95 % A; 1–9 min, 5–
95 % B linear gradient; and 9–13 min, 95 % B. The flow rate
was 0.3 ml/min and the column temperature was 40 °C. The
instrument was operated with the following settings: source
voltage of 5000 kV, GS1 40, GS2 40, CUR 30, TEM 300, and
CAD gas HIGH. Phosphocholine-containing phospholipids
were monitored by precursor ion scan of m/z 184.
Extraction of lipids
After washing three times in cold saline, iPSC colonies grownon feeder cells were harvested with a cell scraper, and all of thecells were collected (including differentiated and undifferentiated iPSC and SNL cells). After sonication of the cells, lipids
were extracted by the Bligh–Dyer method [13]. Samples wereimmediately stored at −80 °C after sealing under nitrogen. To
confirm the lipids identified in the cell samples, lipid extracts
with DHB solution (DHB dissolved in 50 % CH3CN and 1 %trifluoroacetic acid) were spotted onto stainless steel slides for
MS/MS analysis by MALDI-TOF/TOF.
Statistical analysis
PCA was done for the cumulative contribution ratio of
PC > 80 % in each image and the contribution of PC value
to each group > mean PC value of each image. HCA was

1009

performed on the basis of the Euclidean distance and themethod of Ward. PCA, HCA, and generation of dendrograms were performed by using iMscopeTrio® software.
Differences in the relative intensity of the target m/z
values between the differentiated and undifferentiatedareas of colonies were determined by using Statview software (version 5.0; Abacus Concepts, Inc., Berkeley, CA).

Results and discussion
Confirmation of undifferentiated and differentiated areasin iPSC colonies by immunocytochemistry
Because the iPSC colonies grew outwards, the central
zone of each colony underwent differentiation before theperipheral region. To distinguish the differentiated andundifferentiated areas of the iPSC colonies, ALP and
SSEA-4 staining was performed on day 6 after passaging
the cells. ALP and SSEA-4 were positive in the undifferentiated iPSC colonies and in the undifferentiated areas ofthe differentiated iPSC colonies (Fig. 1a–e). In the central
zone of the differentiated iPSC colonies, there were only a
few SSEA-4-positive cells. DAPI staining was densely positive in the undifferentiated iPSC colonies and the undifferentiated areas of the differentiated iPSC colonies, but was also
positive in the differentiated areas (Fig. 1f–h). From thesefindings, the differentiated and undifferentiated iPSC colonies
were confirmed as reported previously [10].
MS analysis of entire colonies
MS analysis of entire colonies showed differences of therelative intensity of various m/z peaks between the differentiated and undifferentiated iPSC colonies (Fig. 1i–l).
The signal intensity of the top 100 peaks was screened
for entire colonies. Representative spectra are shown in
Fig. 1, including peaks derived from DHB and from
SNL cells located at the periphery of the iPSC colonies.
Thus, peaks showing high intensities did not necessarilyreflect the distribution of lipids in the differentiated or
undifferentiated iPSC colonies. PCA based on twodimensional image patterns identified 28 groups in theundifferentiated iPSC colonies and 30 groups in the differentiated iPSC colonies (see Electronic supplementarymaterial (ESM) Figs. S1 and S2 and Tables S1 and S2).
Although several peaks overlapped in several PCA
groups, most of the groups identified in undifferentiated
iPSCs also showed a high intensity in SNL cells localized
at the borders of the iPSC colonies, while several PCA
groups showed a higher intensity in the undifferentiated
iPSC areas than in the SNL cell areas. In differentiated
iPSC colonies, most of the PCA groups showed a similar

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Y. Shimizu et al.

Und-iPSC
colonya

Dif-iPSC
colony

b

ALP
1.0mm

c

d

e

f

g

h

SSEA-4DAPI(x100)

j
i

660 m

628.6

0.75

R.I.

Und-iPSC
colony

1.00

0.50
0.25

552.6
503.0
578.6

0.00
500

l
k

660 m

600

710.5

794.5

700

800

m/z

942.4
900

1000

(x100)
1.00

503.0

0.75

R.I.

Dif-iPSC
colony

666.5
646.6

601.0
628.6

0.50

0.25

798.5

545.1

0.00
500

782.5
666.5

600

741.5
725.5

824.5

700

800

m/z

846.5

896.6
900

1000

Fig. 1 Confirmation of undifferentiated (und-) and differentiated (dif-)
iPSC colonies by immunocytochemistry and MS analysis of the entire
iPSC colonies. Alkaline phosphatase (ALP) and stage-specific embryonicantigen-4 (SSEA-4) were positive in both undifferentiated iPSC colonies
and the undifferentiated zone of differentiated iPSC colonies (a–d). Scalebar indicates 1.0 mm. At higher magnification, there were several SSEA4-positive cells in the central zone of differentiated iPSC colonies (e).
Staining for 4′,6-diamidino-2-phenylindole (DAPI) was strongly positivein undifferentiated areas (f, g), but also positive in the central zones ofdifferentiated iPSC colonies at higher magnification (h). Squamous epithelial cells differentiated from iPSC were observed at the center of differentiated iPSC colonies (h). Microscopic images and spectra obtainedby MS analysis of entire colonies are shown for undifferentiated iPSC
colonies (i, j) and differentiated iPSC colonies (k, l). Scale bar indicates
660 μm (i, k). The spectra of differentiated and undifferentiated iPSC
colonies (j, l) showed different m/z peak patterns. R.I. relative intensity

high intensity in both the SNL cell areas and differentiated areas, but several PCA groups formed concentric circles in the differentiated iPSC colonies. In the next step,
HCA showed four clusters in the undifferentiated iPSC
colonies (Fig. 2a) and nine clusters in the differentiated
iPSC colonies (Fig. 2b). In the undifferentiated iPSC colonies, the high-intensity area of cluster 1 correspondedwith the undifferentiated cells. Cluster 1 clearly showeda different distribution from cluster 4, which correspondedwith the SNL cells. In the differentiated iPSC colonies,
cluster 1 showed a high intensity in the differentiatedzone, while cluster 9 showed a high intensity in the undifferentiated area (the m/z peaks belonging to cluster 4 inthe undifferentiated iPSCs and cluster 9 in the differentiated iPSC colonies are shown in ESM Fig. S3 andTable S3, plus Fig. S4 and Table S4, respectively). Bothcluster 1 at m/z 798.5 in the undifferentiated iPSC colonies (Fig. 2a) and cluster 9 at m/z 798.5 (Fig. 2b) had thesame m/z value, but different distributions in the undifferentiated area, and both areas were morphologically andimmunologically associated with undifferentiated features. Further, dendrograms of the HCA clusters

Phospholipid distributions in iPS colonies

a

1011

Cluster 1

Cluster 2

n=5

m/z 798.5bCluster 3

n=14

782.5

741.5

Cluster 4

n=9

n=72

504.3

799.5

n
1
2
3
4
5
6
•
•
72

m/z
628.6
588.7
552.7
550.7
570.7
552.6
•
•
610.7

1
2
3
4
5
•
•
19

m/z
798.5
782.5
824.5
504.3
799.5
•
•
784.5

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6

n=5

n=4

n=1

n=11

n=1

n=8

Cluster 7 Cluster 8 Cluster 9

n=7

n=44

n=19

Fig. 2 HCA analysis showed four clusters in undifferentiated iPSC
colonies (a). The top 100 high-intensity m/z peaks were assigned to
HCA clusters. Cluster 1 showed high-intensity m/z peaks in undifferentiated iPSC (n = 4), while cluster 4 showed high-intensity m/z peaks in SNL
cells (n = 72). Note that SNL cells were localized around the undifferentiated iPSC colonies. Images and m/z peaks of cluster 4 are shown in ESM

Fig. S3 and Table S3. HCA analysis showed nine clusters in differentiated
iPSC colonies (b). Cluster 1 had high-intensity m/z peaks in the differentiated zone and SNL cell area (n = 5), while cluster 9 showed highintensity m/z peaks in the undifferentiated rather than the differentiatedareas (n = 19). Images and m/z peaks of cluster 9 are shown in ESM
Fig. S4 and Table S4

demonstrated that the m/z peaks of clusters 1 and 4 in theundifferentiated iPSC colonies (Fig. 3a) or clusters 1 and
9 in the differentiated iPSC colonies (Fig. 3b) formed
separate trees.

m/z peaks based on intensity in order to find major abundant
lipids. MS analysis on focused areas showed high intensitiesof the peaks, such as m/z 503.0, 601.0, 638.9, 772.5, 782.5,
798.5, and 824.5 in the undifferentiated iPSC colonies (n =16), while the peaks such as m/z 503.0, 580.1, 601.0, and
681.0 were high, and m/z 782.5 and 798.5 also appeared in
the differentiated iPSC colonies (n = 16; Fig. 4b, d). Highintensity peaks derived from DHB were found at m/z 545.0,
580.1, 585.0, 681.0, and 721.0 (Fig. 4f). From the results of
the analysis of the entire colonies and the localized analysis,
candidate target peaks were selected as follows. First, the m/z
peaks derived from DHB were excluded from the peaks of theundifferentiated and differentiated iPSC colonies. Second,peaks common to the undifferentiated and differentiated
iPSC colonies in the localized analysis were selected (ESM
Fig. S5). Third, peaks common to the analysis of entire colonies and to localized analysis were determined. Finally, six
peaks at m/z 503.0, 578.6, 623.0, 638.9, 782.5, and 798.5 were

Localized IMS and MS/MS analyses
Analysis of the entire of colonies demonstrated the distribution of various lipids, but this analysis included data for DHB
and SNL cells. Therefore, to exclude these background data,
analysis of localized areas was done as the next step (Fig. 4a–f). Focused MS analysis was done by focusing on the undifferentiated areas in the undifferentiated iPSC colonies and on
the differentiated areas in the differentiated iPSC colonies. In
the previous step of screening entire colonies, the peaks of
DHB and SNL cells had been included, so the range had beenbroadly set as the top 100 m/z peaks based on intensity. In thestep of focused analysis, the range had been set as the top 50

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Y. Shimizu et al.

aE.D

Fig. 3 Dendrograms of HCA
clusters showed that the m/z peaks
in clusters 1 and 4 fromundifferentiated iPSC colonies (a)and the m/z peaks in clusters 1 and9 from differentiated iPSC
colonies (b) belonged to separate
trees. E.D Euclidian distance

2.8

1.4

0
Cluster 4
0.45

Cluster 4
0.45

Cluster 1
1.0

0.5

0

0

0

b

E.D

6.0

3.0

0
Cluster 1

Cluster 9

0.8

0.9

0

0

selected (Fig. 5a, b). When the six peaks were subjected to
MS/MS analysis by MALDI-TOF-IMS, two precursor ions ofm/z 782.5 and 798.5 were matched to be PCs by lipid databasesearch (Figs. 5a, b and 6a–h). Fragment ions of m/z 782.5(Fig. 6b, f) and 798.5 (Fig. 6d, h) also matched to eachPC(36:4) or PC(34:1) for m/z 782.5 and PC(34:1) for m/z

Phospholipid distributions in iPS colonies

a

660 m

b

798.5

782.5

(x100)

798.5

1.00
503.0
0.75

R.I

Und

782.5
0.50
601.0 638.9
739.4
577.5
688.3 725.5
660.3

0.25
0.00
500

c

660 m

550

600

650

700
m/z

1.00

772.5

824.5

899.7
897.6
846.5

750

800

782.5

(x100)

d

850

900

798.5

545.0

0.75

R.I

Fig. 4 MS analysis of localized
areas (a–f). MS analysis focusedon the area inside the black square(a, c, and e). Scale bar indicates
660 μm (a, c, and e). Highintensity peaks at m/z 503.0,
601.0, 638.9, 772.5, 782.5, 798.5,
and 824.5 were found in
undifferentiated iPSC colonies (b)(n = 16), while peaks at m/z 503.0,
580.1, 601.0, and 681.0 werehigh, and also m/z 782.5 and798.5, and were in differentiated
iPSC colonies (d) (n = 16). Highintensity peaks for DHB weredetected at m/z 545.0, 580.1,
585.0, 681.0, and 721.0 (f)

1013

Dif

0.50
0.25

e

660 m

716.1
737.0
698.0

605.5

522.9
0.00
500

681.0

601.0
580.1

503.0

550

600

650

700
m/z

817.0
798.5

899.7
897.7

800

850

817.0

857.0

800

850

750

900

(x100)

f

585.0

1.00
545.0

R.I

0.75

DHB

0.50

580.1

681.0

0.25 522.9
0.00
500

656.6
550

600

650

721.0

703.0 739.0
700

750

m/z

Fig. 5 a Selection of candidate
m/z peaks. Six peaks werecommon to the analysis of entire
colonies and to localized analysis,
and two peaks of m/z 782.5 and798.5 were matched to database.
b Summary of candidate target m/z peaks for the analysis of entire
colonies and for localized
analysis. Images ofundifferentiated and differentiated
iPSC colonies corresponding withthe target peaks are showna

MS forentire of colonies

MS for focused areas

m/z
503.0
578.6
623.0
638.9
782.5
798.5

MALDI TOF-IMS MS/MS and database search
m/z
782.5
798.5

b

m/z
Und

Dif

503.0

578.6

623.0

638.9

782.5

798.5

900

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Y. Shimizu et al.

798.5, according to previous reports [11, 12]. The identity ofthe other four m/z peaks could not be determined because
parental ions could not be found, although several fragment
ions were obtained by MS/MS analysis. Precursor ions m/z
782.5 was more abundant in the undifferentiated iPSC colonies than in SNL cells localized at around the colonies(Fig. 6a) and was also more abundant in the undifferentiatedareas than in the differentiated areas of the differentiated iPSC
colonies (Fig. 6e). Precursor ion m/z 798.5 was also moreabundant in the undifferentiated iPSC colonies than in SNL
cells (Fig. 6c), while it was distributed in concentric circles inthe differentiated iPS colonies (Fig. 6g). To confirm PCs obtained from MALDI-TOF-IMS, MS/MS analysis was performed using lipid extract in the next steps.
MS/MS analysis of lipid extracts from cell culture dishes,
including undifferentiated iPSC colonies, differentiated
iPSC colonies, and SNL feeder cells
To confirm the existence of the m/z fragments, MS/MS
by MALDI-TOF analysis was performed again using
droplets of lipid extracts from the whole culture dishof a day 6 iPSC culture. The differentiated iPSC colonies accounted for 49.9 ± 11.7 % of all iPSC colonies in
6-cm culture dishes (n = 4). Fragment m/z peaks of thepeaks at m/z 782.5 and 798.5 could be obtained, butthere were numerous background peaks (see ESM
Fig. S6; a representative m/z peak list is shown in
Table S5). From these results, UHPLC-MS/MS analysis
was done on lipid extracts on the next further step.
Lipid extracts from the whole culture dish of a day 3
iPSC culture (undifferentiated extracts) and day 8 iPSC
culture (differentiated extracts) were subjected to thisanalysis. Extracts from undifferentiated iPSC colonies
accounting for less than about 10 % of the undifferentiated colonies of all iPSC cultures (undifferentiated extracts) and extracts from differentiated colonies accounting for more than 80 % of the differentiated colonies of
the iPSC culture (differentiated extracts) were subjected.
Phosphocholine-containing phospholipids were monitored by precursor ion scan of m/z 184. From both
samples, the peak at m /z 184.1, which was aphosphocholine ion, was obtained by UHPLC MS/MS
analysis on precursor ions m/z 782.5 (ESM Fig. S7a, c)and 798.5 (ESM Fig. S7b, d). To identify target PCs,
product ion data obtained by both MALDI-TOF-IMS
and UHPLC were collected. From the precursor ion atm/z 798.5, the product ions at m/z 739.4, 615.5, and184.1 were detected. The difference between the m/z
values represents neutral losses (NLs) corresponding to
59 Da from m/z 798.5 to 739.5 and NLs corresponding
to 124 Da from m/z 739.5 to 615.5. NLs of 59 and
124 Da are known PC species of trimethylamine[N(CH3)3] and cyclophosphane ring [(CH2)2PO4H], respectively [12, 14, 15]. According to a database research, the peak at m/z 798.5 was identified as(C42H82NO8P, PC(34:1)[M+K]+). For the precursor ion
at m/z 782.5, the product ions at m/z 723.4, 599.5, and184.1 were detected. The difference between the m/z
values represents NLs corresponding to 59 Da from m/z 782.5 to 723.4 and NLS corresponding to 124 Dafrom m/z 723.4 to 599.5. According to a database research, peaks at m/z 782.5 were (C 4 4 H 8 1 NO 8 P,
PC(36:4)[M+H]+) or PC(C 42 H 82 NO 8 P, PC(34:1)[M+Na]+). Therefore, images for differentiated colonies
were compared between m/z peaks 782.5 and 798.5

m/z 782.5

m/z 798.5
599.5 723.4b(x10,000)

a
A.Int

0.0

599.5

200

300

400

500

600

700

(x10,000)
739.5

2.0

Und
2.5

615.5 739.4

d
c

723.5

5.0

Und

A.Int

A.Int

A.Int

1.0
0.0

800

615.5

200

300

400

m/z

g

5.0

Dif
599.5

2.5

782.6

0.0

200

700

800

300

400

500
m/z

600

700

Fig. 6 Lipids showing different distributions in undifferentiated anddifferentiated iPSC colonies. When the six peaks were subjected to MS/
MS analysis by MALDI-TOF-IMS, two precursor ions of m/z 782.5 and798.5 were matched to be PCs by database search. The peak at m/z 782.5
showed an inverse pattern between undifferentiated and differentiated
iPSC colonies (a, e). The peak at m/z 798.5 showed a diffuse distributionin undifferentiated iPSC colonies, but a concentric pattern in

800

739.445

7.5

A.Int

723.5

A.Int

Dif

600

h (x1,000)

f (x1,000)

e

500
m/z

5.0
2.5
0.0

615.450
200

300

400

500
m/z

600

798.645700

800

differentiated iPSC colonies (c, g). Fragment ion spectra obtained by
localized MS/MS analysis are shown (b, d, f, h). Fragment ion peaks
obtained by MS/MS analysis of the peak at m/z 782.5 were found at m/
z 599.5 and 723.4 in both undifferentiated (b) and differentiated (f) iPSCs,
while fragment ion peaks obtained from the peak at m/z 798.5 weredetected at m/z 615.4 and 739.4 in both undifferentiated (d) and differentiated (h) iPSCs

Phospholipid distributions in iPS colonies
Table 1

1015

Summary of the identified lipids

Molecular formula

C44H81NO8P
C42H82NO8P

Matched lipids

PC(36: 4)[M + H]+
PC(34: 1)[M + K]+

MS-observed m/z
Entire colony

Focused area

782.5
798.5

782.5
798.5

(Fig. 6e, g). These images indicated different PC distribution patterns between 782.5 and 798.5; nevertheless,the analysis was done on the same differentiated iPSC
colony. If m/z 782.5 is PC(34:1)[M+Na]+, the images ofm/z 782.5 and 798.5 (PC(34:1)[M+K]+) are possibly thesame; however, the images indicated different patterns
between m/z 782.5 and 798.5 in the same differentiatedcolony. Then, we concluded that m/z 782.5 was(C44H81NO8P, PC(36:4)[M+H]+). Taken together, thesefindings confirmed the presence of two distinct lipids, whichwere at m/z 782.5 (C44H81NO8P, PC(36:4)[M+H]+) and at m/z
798.5 (C42H82NO8P, PC(34:1)[M+K]+) (Table 1). The relative
intensity of PC(36:4), which was m/z 782.5, was significantly
higher in the undifferentiated areas (77.5 ± 18.2) than in the
differentiated areas (32.4 ± 14.7) of the differentiated iPSC colonies shown in Fig. 6e (n = 6, p = 0.0008).
It has been reported that PC(36:4) was the major lipid
showing elevation in murine embryonic stem cells and murine
iPSCs compared with murine fibroblasts [6]. An increase of
unsaturated PCs, such as PC(36:4), PC(38:6), and PC(38:4),
was reported previously, and it was speculated that changes of
PC saturation might alter cell membrane fluidity in relation tophenotypic changes of stem cells [5, 6]. PC(34:1) has beenreported in various human cancers, such as breast, colorectal,
esophageal, lung, gastric, thyroid, and liver cancer [11, 16].
PC(34:1) is also widely distributed in the rat brain [7], while
its level is reduced in ischemic areas [14]. For regenerating anorgan, MALDI-IMS revealed that multiple PCs were distributed in regenerating mouse liver. Interestingly, most of thePCs increased, such as PC(1-acyl 34:1)[M+Na]+, PC(1-acyl34:1)[M+K]+, PC(1-acyl 34:2)[M+Na]+, and PC(1-acyl34:1)[M+K]+, but PC(1-acyl 36:4) was not changed. PC(1acyl 40:6) and PC(1-acyl 40:8) decreased in the regenerating
liver [17]. From these reports, PC(34:1) may be abundant incells with high proliferative activity and metabolism, while itis decreased in low or stable metabolic states. PC localization
during cell differentiation was reported. MALDI imaging
analysis for the characterization of lipid markers ofchondrogenic differentiation by mesenchymal stem cells(MSCs) revealed that PCs and Pcho were more abundant in
the peripheral zone of micromasses composed by MSCs on
day 14 of differentiation, while those lipids were distributed
diffusely in micromasses in the early stage of differentiation
on day 2 [18]. In the present study, PC(34:1) was distributed in

Theoretical m/z

MS/MS fragments

782.569
798.541

184/599.5/723.4/782.5
184/615.5/739.4/798.5

concentric circles, with a high intensity at the border between
the differentiated area (center of the colony) and the undifferentiated area of iPSC colonies, as well as at the border between the undifferentiated area of iPSCs and the SNL cells(Fig. 6g). Therefore, PC(34:1) may play a role in the differentiation of iPSCs or the invasion of these cells into tissues.

Conclusion
Lipid metabolism changes during the differentiation of iPS
cells. This is the first report about the spatial distribution oflipids in iPSC colonies. MALDI-TOF-IMS revealed differences in the distribution of PCs in iPSC colonies, and the
distribution of peaks at m/z 782.5 and 798.5 changed during
the differentiation of iPS colonies. PC(36:4) and PC(34:1)
may be associated with iPS cell differentiation, but further
analysis is needed to elucidate the functional significance of
these lipids.
Acknowledgments This work was partly supported by a grant-in-aid
for the MEXT KAKENHI Grant, Japan (S1412001) and JSPS
KAKENHI Grant, Japan (16K09553). The authors are grateful to Isao
Asaka [Center for iPS Cell Research and Application (CiRA), Kyoto
University] for advice on iPSC culture and to Satomi Tateno, Mio
Maekawa, and Sachiko Nakadate (Department of Pharmacology and
Toxicology, Dokkyo Medical University) for assistance with iMscope
analysis, as well as Akiko Horigane (Department of Pulmonary
Medicine and Clinical Immunology) for assistance with cell culture.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterests.

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694545
research-article2017

TUB0010.1177/1010428317694545Tumor BiologyYothaisong et al.

Original Article

Inhibition of l-type amino acid transporter
1 activity as a new therapeutic target forcholangiocarcinoma treatment

Tumor Biology
March 2017: 1­–14© The Author(s) 2017
Reprints and permissions:sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/1010428317694545
https://doi.org/10.1177/1010428317694545
journals.sagepub.com/home/tub

Supak Yothaisong1,2,3, Hasaya Dokduang2,3, Naohiko Anzai4,
Keitaro Hayashi5, Nisana Namwat1,2,3, Puangrat Yongvanit1,2,3,
Sakkarn Sangkhamanon6, Promsuk Jutabha5, Hitoshi Endou7
and Watcharin Loilome1,2,3

Abstract
Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive
amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on
the plasma membrane, l-type amino acid transporter-1, a Na+-independent neutral amino acid transporter, is highly
expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type aminoacid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma
progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-typeamino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramaticallysuppressed [14C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expressionof l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203
induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of theproteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins,
P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase intrypan blue–stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma
cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice modelin a dose-dependent manner with no statistically significant change in the animal’s body weight and with no differencesin the histology and appearance of the internal organs compared with the control group. Our study demonstrates thatsuppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy forcholangiocarcinoma treatment.
Keywordsl-type amino acid transporter 1, cholangiocarcinoma, JPH203, cell cycle arrest, apoptosis, in vivo model
Date received: 26 May 2016; accepted: 23 December 2016

1Department of Biochemistry, Faculty of Medicine, Khon Kaen

6Department of Pathology, Faculty of Medicine, Khon Kaen University,

University, Khon Kaen, Thailand
2Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen
University, Khon Kaen, Thailand
3Cholangiocarcinoma Screening and Care Program (CASCAP), Khon
Kaen University, Khon Kaen, Thailand
4Department of Pharmacology, Graduate School of Medicine, Chiba
University, Chiba, Japan
5Department of Pharmacology and Toxicology, School of Medicine,
Dokkyo Medical University, Mibu, Japan

Khon Kaen, Thailand
7J-Pharma Co., Ltd., Tokyo, Japan
Corresponding author:
Watcharin Loilome, Department of Biochemistry, Faculty of Medicine,
Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002,
Thailand.
Email: watloi@yahoo.com

Creative Commons Non Commercial CC-BY-NC: This article is distributed under the terms of the Creative Commons
Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial
use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE andOpen Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

2

Introduction
Increased essential nutrient uptake, such as glucose andamino acids, is required for proliferating cells, especiallyin cancer cells which have lost their normal control of proliferation.1 Many studies have revealed that numerous
nutrient transporters are upregulated in cancer cells to support their massive growth. Among the known amino acidtransport systems, system L is a major Na+-independenttransport agency responsible for the transport of neutralamino acids, including several essential amino acids.2
System L has four isoforms: l-type amino acid transporter
1 (LAT1), LAT2, LAT3, and LAT4. While LAT1 and LAT2
require CD98, a heavy chained cell surface antigen forming a heterodimer for the obligatory amino acid exchangeon the plasma membrane, LAT3 and LAT4 are facilitateddiffusers of amino acid substrates.3–7
LAT1 is one of the most actively studied amino acidtransporters in basic research and drug development in
human cancers. Many studies have demonstrated that
LAT1 is overexpressed and plays a critical role in various
human cancers, including cholangiocarcinoma (CCA).8–12
CCA is a usually fatal cancer arising from the epithelial
cells of the biliary tract. The highest incidence of CCA has
been reported from northeast Thailand,13 where it is associated with the highest prevalence of liver fluke(Opisthorchis viverrini) infection.14,15 There is strong evidence indicating that chronic inflammation during liver
fluke infection is a key event of CCA carcinogenesis.16
The lack of effective medical treatment makes radical surgical resection the only chance of cure;17 however, patientswith CCA typically present at an advanced stage of thedisease with non-resectable tumors, resulting in a very
poor prognosis and only limited therapeutic possibilities.18
Thus, new approaches to therapy are required to improve
patient survival.
JPH203 (also known as KYT0353) is a novel tyrosine
analog that selectively inhibits LAT1 transport activity.19,20
To date, only three studies have shown that JPH203 hasanti-tumor activities in human cancers. JPH203 was highly
effective against [14C]l-leucine uptake and cell growth inhuman colon cancer cells,19 human oral cancer cells,21 andleukemic cells.22 Moreover, this specific LAT1 inhibitor
showed statistically significant growth inhibition against
xenografted human colon cancer cells19 and murine
T-lymphoma cells with tPTEN−/− xenografts in the nudemouse model.22 Furthermore, JPH203 was nontoxic tonormal murine thymocytes and human peripheral blood
lymphocytes,22 suggesting that inhibition of LAT1 activity
using JPH203 might be used as a novel therapeutic strategy for cancer treatment.
Our previous study has shown that the expression of
LAT1 and CD98 was increased in CCA development during oxidative stress due to O. viverrini infection, whichmight be regulated by the oncogenic signaling pathway,

Tumor Biology﻿
phosphatidylinositol-3-kinase (PI3K)/AKT. We also
showed that CCA tissues exhibited strong LAT1 immunostaining compared to normal bile duct tissues.
Furthermore, LAT1 plays an important role as a tumor
prognostic factor for CCA patients.11
Based on our previous report, we hypothesized that
LAT1 is the main system l-amino acid transporter in CCA,
potentially making it a target for CCA cell inhibition. In
this study, we investigated the expression of systeml-amino acid transporters and CD98 in CCA cell lines.
Moreover, we determined, for the first time, the effects ofa novel selective LAT1 inhibitor, JPH203, on cell growthand its mechanism for cell growth inhibition in the CCA
model. Our results from the in vitro model clearly demonstrated that LAT1 is the main system l-amino acid transporter in CCA cells and that it is expressed together with
CD98. Inhibition of LAT1 transport activity using JPH203
could dramatically suppress amino acid uptake and CCA
cell growth through altering the cell cycle distribution patterns as well as in inducing apoptosis. Furthermore,
JPH203 has anti-tumor activity against CCA cell growth in
the in vivo model without general toxicity. This result indicates the possibility of using JPH203 for CCA treatment.

Materials and methods
Cell lines and cell culture
The human CCA cell lines, KKU-055, KKU-213, andKKU-100, were obtained from CCA patients and established at the Liver Fluke and Cholangiocarcinoma ResearchCenter, Khon Kaen University. All of the cell lines were cultured in Ham’s F-12 medium (Sigma-Aldrich, St Louis,
MO, USA) supplemented with inactivated 10% fetal bovine
serum (FBS), 1% penicillin–streptomycin, and NaHCO3 ina humidified atmosphere containing 5% CO2.

Antibodies and inhibitor
Antibodies used in this study were as follows: anti-cyclin
D1 (#2926D), CDK4 (#2906P), CDK6 (#3136P), P21(#2946), and P27 (#2552P), purchased from Cell Signaling
Technology (Danvers, MA, USA). Antibody against CD98
was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Anti-caspase-3 (ab32351) and anti-Ki67
were purchased from Abcam (Cambridge, UK). AntiLAT1 antibody and JPH203 was kindly supplied by
J-Pharma (Tokyo, Japan).

Reverse transcription polymerase chain
reaction analysis
Reverse transcription polymerase chain reaction (RT-PCR)
analysis was performed to determine the expression of the
LATs and CD98 at the messenger RNA (mRNA) level.

3

Yothaisong et al.
Total RNAs were prepared from the CCA cells maintainedin the growth medium at 37°C in 10 cm Petri dishes using
an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) inaccordance with the manufacturer’s instruction. RT-PCR
analysis was performed with the PrimeScript® RT reagent
Kit (Takara Bio Inc., Shiga, Japan) under the conditions
recommended by the manufacturer and used as a templatefor PCR amplification. PCR amplification was performed
using the Promega PCR Master Mix (Promega, Madison,
WI, USA) following the protocol: &#57;5°C for 2 min;followed by 20 cycles of 95°C for 30 s, 55°C for 30 s, and72°C for 30 s; with a final extension step of 72°C for 5 min.
For LAT1, the forward and reverse primers used were
5′-TGCCTGTGTTCTTCATCCTG-3′ and 5′-CCTCCTGGCTATGTCTCCTG-3′, respectively. For LAT2, the forward and reverse primers were 5′-GCCCTCACCTTCTCCAACTA-3′ and 5′-AATGCATTCTTTGGCTCCAG-3′, respectively. For LAT3, the forward and reverse
primers were 5′-CACGCTACTGCAAGATCCAA-3′ and5′-AGAAGGGCTCTCCTTTCAGG-3′, respectively; andfor LAT4, the forward and reverse primers were 5′-AAATTTGGCCTTCACTGTGG-3′ and 5′-ACGACGATGAAGGAGACACC-3′, respectively. For CD98, the forward
and reverse primers were 5′-CAGAAGGATGATGTCGCTCA-3′ and 5′-CCAGTGGCGGATATAGGAGA-3′,
respectively. A pair of primers, 5′-GCTG CTTT TAAC TCTG
GTAA-3′ and 5′-CGCGGCCATCAC GCCACAGT-3′,
was used for the PCR amplification of glyceraldehyde
3-phosphate dehydrogenase (GAPDH).

Quantitative western blotting
To confirm the expression of LAT1 and CD98 in CCA celllines, the cells were grown in 10 cm Petri dishes before
harvesting and total protein extraction. To investigate theeffects of JPH203, CCA cell lines were plated at 4 × 105
cells in 10 cm Petri dishes, cultured overnight, and thentreated with JPH203 at 1–100 µM and 0.1% dimethyl sulfoxide (DMSO) for 48 h. After incubation, the cells were
harvested and subjected to protein extraction and western
blotting. Briefly, total proteins were isolated by ice-cold
radioimmunoprecipitation assay (RIPA) buffer (50 mM
Tris–HCl, pH 7.4, 1% Triton X-100, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),
1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM
sodium orthovanadate, and 5 mM sodium fluoride) supplied as Completed Protease Inhibitor Cocktails (1 tablet/
10 mL of buffer; Roche, Basel, Switzerland). Protein concentrations were calculated using a Pierce® BCA Protein
Assay Kit (Thermo Scientific, Rockford, IL, USA). A volume of 40 µg of proteins were then loaded onto the gels,separated by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore

Corp., Bedford, MA, USA) using the Bio-Rad western blot
system (Bio-Rad Laboratories, Berkeley, CA, USA). After
blocking with 5% skim milk in Tris-buffered saline (TBS),the membranes were incubated overnight at 4°C withappropriate primary antibody diluted in blocking buffer.
The specific horseradish peroxidase–conjugated secondary antibody incubation was performed for 1 h at room
temperature with gentle shaking. The signal was visualized using Pierce ECL Plus Western Blotting Substrate(Thermo Scientific), and the protein densitometric values
were analyzed by Multi Gauge software (Fujifilm, Tokyo,
Japan). The experiment was replicated three times, anddensitometry values for each protein were normalized withthe densitometry value of β-actin, which was used as internal control.

Amino acid uptake
To determine the inhibitory effect of JPH203 on aminoacid transport in CCA cells, [14C]l-leucine (Moravek
Biochemicals Inc., Brea, CA, USA) was used as the prototypical LAT1 substrate in uptake experiments evaluated
via radioactivity. Briefly, the indicated cells were seeded at
2.5 × 104 cells/well in 24-well plates and incubated at 37°C
in 5% CO2 for 2 days. After incubation, the adherent cells
were washed three times and pre-incubated for 10 min at
37°C with 0.5 mL of sodium free Hanks’ balanced salt
solution (Na+-free HBSS). Then, the uptake of 1 µM
[14C]l-leucine was measured in the presence of JPH203(0.01, 0.1,1, 3, and 10 µM) or 0.1% DMSO in Na+-free
HBSS at 37°C for 1 min and was terminated by washingwith cold Na+-free HBSS. Next, the cells were solubilizedwith 0.5 mL of 0.1 N NaOH for 20 min, and the cell lysate
mixed with 3 mL of scintillation liquid and radioactivity
was measured using an LSC-7200 β-scintillation counter(Hitachi Aloka Medical, Tokyo, Japan). An aliquot of thecell lysate was used to determine the protein concentration
using a Pierce BCA Protein Assay kit (Thermo Scientific).
All experiments were replicated three times. The IC50 values of [14C]l-leucine uptake were calculated using
GraphPad Prism5 (GraphPad Software Inc., San Diego,
CA, USA).

Cell growth inhibition assay
A sulforhodamine B (SRB; Sigma-Aldrich) assay was
performed to determine the effect of JPH203 on cell
growth inhibition. The CCA cells were seeded at a density of 2 × 103 cells in 100 µL of medium/well in 96-wellplates and maintained at 37°C in 5% CO2 overnight.
Then, the cells were treated with different concentrationsof JPH203 (0.1–100 µM) in 100 µL of medium, and 0.1%
DMSO was used as a control at 24, 48, and 72 h. Later,
the cell numbers were estimated using an SRB assay, as
previously described.23 In brief, the adherent cells were4
fixed with 100 µL of 10% trichloroacetic acid (TCA,
Wako Pure Chemical Industries Ltd., Tokyo, Japan) for
1 h at 4°C. Then, they were washed with deionized waterand stained with 0.4% SRB dye in 1% acetic acid for
30 min. After removal of the SRB dye, the cells were
washed with 1% acetic acid and were allowed to air dry.
Thereafter, SRB-stained cellular proteins were dissolvedin 200 µL of 10 mM Tris base (pH 10.5). The optical densities were measured at 540 nm in an enzyme-linked
immunosorbent assay (ELISA) plate reader, Varioskan
Flash (Thermo Fisher Scientific, Vantaa, Finland). The
percent viability of the cells in each well was calculated.
The experiments were replicated three times, and theIC50 values were calculated using GraphPad Prism 5(GraphPad Software Inc.).

Cell cycle analysis
CCA cells were seeded and treated with JPH203 as
described in section “Quantitative western blotting.” Afterincubation, 1 × 106 cells per experimental condition were
harvested and washed with cold phosphate-buffered saline(PBS). Thereafter, the cells were fixed with 70% cold ethanol and kept at −20°C until analysis. After fixation, thecells were washed with cold PBS and incubated with
100 µg/mL RNaseA (Applichem Inc., Cheshire, CT, USA),and the cell cycle distribution was determined by staining
the DNA with 40 µg/mL of propidium iodide (PI; Invitrogen,
Paisley, UK) for 30 min. The results were analyzed by flow
cytometry (FACS Calibur, BD Biosciences, San Jose, CA,
USA). All experiments were done in triplicate.

Trypan blue dye exclusion assay
Six-well culture plates were used for cell culture. After plating the CCA cells at 1 × 105 cells/well and culturing for overnight, designed concentrations of JPH203 were added for
48 h. After the treatment period, the cells were trypsinized
and washed with PBS. The cells were then mixed thoroughly and 10 µL of cell suspension was added to 10 µL of
0.4% trypan blue dye (Bio-Rad Laboratories, Inc., Watford,UK) solution. Dead cells were characterized by the uptakeof the dye and expressed as the percentage of cells staining
blue. The results were detected using a TC20™ Automated
Cell Counter (Bio-Rad Laboratories, Inc., Berkeley, CA,
USA). All experiments were done in triplicate.

Experimental animals
Six-week-old male athymic BALB⁄c nude mice (CleaJapan, Inc., Tokyo, Japan) were housed and monitored
under pathogen-free conditions in accordance with institutional principals. Food and tap water were provided ad
libitum. Mice were subcutaneously injected with 2 × 106
KKU-213 cells in 0.5 mL of medium into the dorsal area.

Tumor Biology﻿
After 3 days, the mice were randomly divided into fourgroups: a control group (n = 5), JPH203 6.3 mg/kg treatment group (n = 5), JPH203 12.5 mg/kg treatment group(n = 5), and JPH203 25 mg/kg treatment group (n = 5). On
the day of grouping (day 0), body weights and diameters of
the tumor (length and width) were measured. The tumor
volumes were calculated using the standard formula: tumor
volume (mm3) = longer diameter × (shorter ­diameter)2/2.
After grouping, the mice were intravenously injected with
0.2 mL of normal saline solution (NSS) daily for the control group and JPH203 for the treatment groups for 20 days.
Tumor volumes and body weights were assessed twice aweek. The tumor volumes were expressed relative to theinitial tumor volume (day 0). After treatment, all mice
were sacrificed for the collection of tumor samples as wellas the liver, lungs, spleen, and kidneys. Formalin-fixedparaffin-embedded sections from the internal organs wereanalyzed by hematoxylin and eosin (H&E) staining
according to standard methods. All slides were reviewed
by a pathologist and were photographed using an Olympus
CKX41 Microscope (Olympus, Tokyo, Japan) with
CellSens standard software. The in vivo study wasapproved by the local animal welfare committee and was
carried out in accordance with the local regulations.

Immunohistochemistry
The formalin-fixed and paraffin-embedded tumors weresliced into 4 µm sections and were deparaffinized in xylene
and then rehydrated in graded alcohol. Antigen retrieval
was accomplished by incubating the slides in Tris–EDTA
Buffer (10 mM Tris base, 1 mM EDTA solution, and 0.05%
Tween 20; pH 9.0) for 3 min in a pressure cooker. Then, theslides were incubated in 0.3% H2O2 for 30 min to suppress
endogenous peroxidase activity and then washed with
PBS. Next, the slides were blocked with 10% skim milk in
PBS for 30 min. The rabbit polyclonal Ki67 antibody wasused as primary antibody at a 1:300 dilution in PBS overnight at 4°C. The following day, after several washes with
PBS, the slides were incubated with peroxidase-conjugated EnVision™ secondary antibody (Dako, Glostrup,
Denmark) for 90 min, and a peroxidase-labeled polymer,
Diaminobenzidine tetrahydrochloride (DAB) solution,
was used for signal development for 5 min. The sections
were counterstained with hematoxylin followed by dehydrating and mounting. Ki67 positive cells of each tumor
sections were counted in least five of ×400 power fields.

Statistical analysis
Statistical analysis was performed using GraphPad Prism
version 5 (GraphPad Software Inc.). The data are expressed
as mean ± standard deviation (SD) or mean ± standard errorof the mean (SEM). The results of the western blotting
were analyzed with one-way analysis of variance (ANOVA)5

Yothaisong et al.
followed by a Tukey’s multiple-comparison test. The
results of cell cycle analysis, dye exclusion assay, and percent Ki67 positive cells were analyzed by Student’s t test.
The results of the relative tumor volume were analyzed
using a two-way ANOVA. A value of p < 0.05 was considered to indicate a statistically significant difference.

KKU-055 CCA cell proliferation compared with KKU213 and KKU-100 (Figure 1(g) and (h)). Consistent withthe result for amino acid uptake, KKU-213 was more sensitive to JPH203 than KKU-055 and KKU-100 (IC50 at
24–72 h for KKU-213 was lower than that of KKU-055
and KKU-100; Table 1).

Results

JPH203 altered LAT1 and CD98 expression

Expression of the system l-amino acidtransporters and CD98 in CCA cells

A previous study demonstrated that inhibition system L
activity in KKU-213 CCA cells using a broad system L
inhibitor,
2-aminobicyclo-(2,2,1)-heptane-2-carboxylic
acid (BCH), can upregulate the expression of LAT1 andCD98.9 Likewise, upregulated amino acid transporters,
including LAT1, CD98, xCT, ASCT1, and ASCT2, werereported in human prostate cancer cell lines treated with
BCH and leucine-free media.24 We, therefore, investigated
the impact of JPH203 on LAT1 and CD98 expression. In
accordance with previous reports, after the treatment of
KKU-055 and KKU-213 cell lines with JPH203 (0–100 µM) for 48 h, both LAT1 and CD98 levels increased ina dose-dependent manner as detected by quantitative western blotting (Figure 2(a)–(f)). These results indicate that
upregulated LAT1 and CD98 expression is a feedback
effect related to amino acid deprivation caused by the suppression of LAT1 activity using JPH203.

The PCR products for LAT1 and CD98 were clearly
detected in all cells studied, whereas LAT2, LAT3, and
LAT4 were hardly detected (Figure 1(a)). The presence of
LAT1 and CD98 in all cells studied was further confirmed
by quantitative western blotting. Consistent with the RTPCR analysis, the CCA cells clearly expressed LAT1 andCD98 (Figure 1(b)). For the functional expression at theplasma membrane, LAT1, a non-glycosylated light chain
with an apparent molecular mass of 40 kDa, forms a heterodimeric complex via disulfide bond with a glycosylated
heavy chain or CD98 with an apparent molecular massaround 85 kDa. The heterodimeric complex of LAT1–
CD98 is approximately 125 kDa. The results from the
RT-PCR analysis and western blotting indicate that LAT1
is the main system l-transporter in our CCA cell lines andoccurs together with CD98.

JPH203 inhibits [14C]l-leucine uptake and cell
growth in CCA cell lines
The inhibitory effects of JPH203 on [14C]l-leucine uptake
and cell growth in CCA cells are summarized in Figure 1
and Table 1. At the lowest concentration of JPH203(0.01 µM), [14C]l-leucine uptake in all CCA cells was
slightly increased when compared to the untreated control.
However, 0.1–10 µM JPH203 dramatically inhibited[14C]l-leucine uptake in a dose-dependent manner in all
CCA cells studied with low IC50 values. The KKU-213 cellline was more sensitive to JPH203 than KKU-055 andKKU-100 cell lines (Figure 1(c)–(e) and Table 1). The IC50
values (mean ± SD) were 0.20 ± 0.03 µM for KKU-055,
0.12 ± 0.02 µM for KKU-213 cells, and 0.25 ± 0.04 µM for
KKU-100 (Table 1).
To determine whether blocking [14C]l-leucine uptake
using JPH203 suppresses CCA cell growth, an SRB assay
was performed after treatment of the CCA cells with
JPH203 at various concentrations for 24, 48, and 72 h in
96-well plates. As shown in Figure 1 and Table 1, JPH203
could inhibit the proliferation of CCA cells and IC50 values were decreased in a time-dependent manner. However,the drug response for each CCA cell line to JPH203
showed different patterns, as indicated in Figure 1(f). At
0.1 and 1 µM JPH203, there were hardly any inhibition of

JPH203 induced cell cycle arrest throughregulating the cell cycle regulators
To determine the mechanism of the anti-proliferative effectof JPH203, cell cycle analysis was performed using flow
cytometric analysis after staining the cells with PI. As
shown in Figure 3(a), exposure of the KKU-055 cells to
10–100 µM JPH203 for 48 h resulted in a statistically significant increase in G2/M-phase (p < 0.01) cells comparedwith the control. This was accompanied by a significant
decrease in G0/G1 phase cells at 10 µM (p < 0.05) and asignificant decrease in S-phase cells at 100 µM (p < 0.05).
Unlike KKU-055, the results for KKU-213 showed thatthe number of cells in the G0/G1 phase was significantly
increased in response to 10–100 µM JPH203 (p < 0.01 at
10 µM and p < 0.001 at 100 µM). Concomitantly, 10–
100 µM of JPH203 significantly decreased the number ofS-phase cells compared to the control (p < 0.001). However,a slight decrease of G2/M-phase cells was detected in adose-dependent manner, but this was not statistically significant compared with the control (Figure 4(a)).
To gain insight into the mechanism of cell cycle arrest
on treatment with JPH203, we further investigated theexpression levels of cell cycle–regulator proteins whichfunction in the G0/G1 phase, including cyclin D1, CDK4,
and CDK6.25 The expression of the inhibitors of CDKs,
P21, and P27, which affect both G1/S and the G2/M,26–28
was also investigated in this experiment. The CCA cells

6

Tumor Biology﻿

Figure 1. Expression of LAT1 and CD98 and the functional effects of LAT1 inhibition on [14C]l-leucine uptake and cell growthin CCA cells. (a) Detection of system l-amino acid transporters (LAT1-LAT4) and CD98 by RT-PCR in CCA cells (KKU-055,
KKU-213, and KKU-100). (b) Expression of LAT1 and CD98 levels in the indicated cells detected by quantitative western blotting.
The densitometry value of LAT1 and CD98 was normalized to β-actin. The bar graphs show the average densitometry value ± SEM
from three independent experiments. (c–e) Inhibition of [14C]l-leucine uptake by JPH203 in CCA cells. [14C]l-leucine uptake wasmeasured for 1 min in the presence of various concentrations of JPH203. (f–h) JPH203 suppressed cell growth in CCA cells. The
indicated cells were treated with various concentrations of JPH203 for 24, 48, and 72 h. The cell viabilities were determined by SRB
assays and the growth curves are presented as percentage of untreated control. Each data point represents the mean ± SEM fromthree independent experiments.

7

Yothaisong et al.
Table 1. Inhibitory effects of JPH203 on [14C]l-leucine uptake and cell growth inhibition in CCA cells.
Cell line

KKU-055
KKU-213
KKU-100

IC50 (µM) value for[14C]l-leucine uptake
inhibition

IC50 (µM) value for growth inhibition
24 h

48 h

72 h

0.20 ± 0.03
0.12 ± 0.02
0.25 ± 0.04

31.95 ± 1.15
32.95 ± 1.16
48.74 ± 1.22

6.16 ± 1.17
5.98 ± 1.16
21.51 ± 1.25

5.78 ± 1.15
2.47 ± 1.19
3.00 ± 1.28

Figure 2. The effect of JPH203 on LAT1 and CD98 expression in CCA cells detected by quantitative immunoblot. (a–c) LAT1 andCD98 expressions were increased in a dose-dependent manner following treatment with JPH203 for 48 h in KKU-055 cells. (d–f)
LAT1 and CD98 expressions were increased in a dose-dependent manner following treatment with JPH203 for 48 h in KKU-213
cells. The results show a representative western blot for LAT1 and CD98 expression. Bar graphs show the densitometry values
normalized to β-actin and were presented as the percentage compared to the untreated control: They show the mean ± SEM fromthree independent experiments.

were exposed to JPH203 for 48 h and prepared for western blotting. The results in KKU-055 showed that JPH203
increased the levels of cyclin D1 and CDK4, except thatthe level of CDK4 at 100 µM was decreased by JPH203(Figure 3(b)–(d)). In addition, 10–100 µM JPH203 could
strongly suppress CDK6 levels (p < 0.001) as well as
induce higher P21 levels when compared with control(Figure 3(b), (e), and (f)). Moreover, 100 µM of JPH203
markedly increased P27 expression as shown in
Figure 3(g). This result indicates that the induction of
growth inhibition by JPH203 in KKU-055 cells wascaused by G2/M arrest mainly through elevated P21 levels. However, inhibition of KKU-055 cell growth at thehighest JPH203 concentration (100 µM) is caused byboth G2/M arrest and reduced S-phase cells due toincreasing P21 and P27 levels and decreasing CDK4 and
CDK6 levels.
As expected, the results for KKU-213 showed that thelevels of cyclin D1, CDK4, and CDK6 which are required
for G1/S phase transition were remarkably decreased in adose-dependent manner when compared to the control,with significant differences being observed at 10–100 µM
JPH203 for CDK4 levels (p < 0.05 and p < 0.01, respectively; Figure 4(b)–(e)). In addition, the levels of P21
increased in a dose-dependent manner, but this was not thecase for the CDK inhibitor P27 (Figure 4(b), (f), and (g)).
These results indicate that, in KKU-213 cells, the growth
inhibition of JPH203 caused by cell cycle arrest at the G0/
G1-phase and reduced S phase occurs via an increase in
the CDK inhibitor P21. This may further inhibit the8Tumor Biology﻿

Figure 3. JPH203 induced cell cycle arrest in KKU-055 cells by regulating cell cycle–related proteins. (a) A volume of 10–100 µM
JPH203 significantly increased G2/M-phase cells (p < 0.01) which was accompanied by significantly decreased G0/G1 phase at 10 µM(p < 0.05) and decreased S phase at 100 µM (p < 0.01) after treatment for 48 h, detected by flow cytometry. Data are expressed as themean ± SD of the percentage of cells in each cycle phase from three independent experiments. Results were analyzed by Student’s
t test. (b) A representative western blot for the expression of cell cycle–related proteins, including cyclin D1, CDK4, CDK6, P21,
and P27 in KKU-055 cells after treatment with JPH203 for 48 h. (c–g) The densitometry values of each protein were normalized
to β-actin and were presented as the percentage compared to the untreated control. The mean ± SEM from three independent
experiments are presented (***p < 0.001 versus control, one-way ANOVA followed by a Tukey’s multiple-comparison test).

activity of cyclin D1–CDK4/CDK6, which is downregulated by this selective LAT1 inhibitor.

Apoptosis induction in response to
JPH203 in CCA cells
Since JPH203-induced apoptosis has been demonstrated inhuman oral cancer21 and leukemic cells,22 we investigatedthe effect of this specific LAT1 inhibitor on apoptosis

induction in our CCA model. To validate the induction of
apoptosis in KKU-055 and KKU-213 CCA cells, a trypan
blue dye exclusion assay was conducted to measure thepercentage of cell death after 48 h of JPH203 (0–100 µM)
treatment. In addition, the apoptotic-related protein, caspase-3, and its cleaved form were assessed by immunoblot.
Our results showed that JPH203 could induce cell death inboth CCA cell lines, as shown in Figure 5(a) and (b).
Cell death was less than 15% in KKU-055 and 30% inYothaisong et al.

9

Figure 4. JPH203 induced cell cycle arrest in KKU-213 cells by regulating cell cycle–related proteins. (a) A volume of 10–100 µM
JPH203 increased G0/G1-phase cells (p < 0.01 and p < 0.001 at the dose of 10 and 100 µM, respectively) and decreased S-phase cells(p < 0.001) after treatment for 48 h, detected by flow cytometry. Data are expressed as the mean ± SD of the percentage of cells ineach cycle phase from three independent experiments. Results were analyzed by Student’s t test. (b) A representative western blot
for the expression of cell cycle–related proteins, including cyclin D1, CDK4, CDK6, P21, and P27 in KKU-213 cells after treatment
with JPH203 for 48 h. (c–g) The densitometry values of each protein were normalized to β-actin and were present as percentage
compared to the untreated control. The mean ± SEM from three independent experiments are presented (*p < 0.05, **p < 0.01 vscontrol, one-way ANOVA followed by a Tukey’s multiple-comparison test).

KKU-213 after treatment with 100 µM JPH203. This was asignificant change, p < 0.001, compared with the control.
The percentage of cell death decreased slightly at 1 µM
and increased slightly at 10 µM of JPH203 in both CCA
cell lines compared to the control. The results from quantitative western blotting showed that the treatment of the
CCA cells with JPH203 at 100 µM for KKU-055 and10–100 µM for KKU-213 increased the cleaved caspase-3/
caspase-3 ratio when compared to the control (Figure 5(c)–(e)). A statistically significant difference in the cleaved
caspase-3/caspase-3 ratio was observed between KKU213 treated with 100 µM JPH303 and the control (p < 0.05).
These results suggest that JPH203-induced apoptosis isregulated by the activation of the caspase cascade under10

Tumor Biology﻿

Figure 5. The effects of JPH203 on apoptosis induction in CCA cells. (a and b) CCA cells were treated with various
concentrations of JPH203 (0–100 µM) for 48 h. The percentage of cell death was measured by trypan blue staining. The data
represent the mean ± SEM from three independent experiments (***p < 0.001 by using a Student’s t test in comparison withthe control). (c) Expression and activation of the apoptotic protein caspase-3 by JPH203 in CCA cells. The indicated cells were
stimulated with JPH203 (0–100 µM) for 48 h. The cell lysate was prepared and analyzed by quantitative immunoblot. (d and e)
Densitometry values of caspase-3 and its cleaved form were normalized to β-actin and expressed as ratio of the cleaved caspase-3/
caspase-3. The mean ± SEM are shown from three independent experiments and analyzed by one-way ANOVA followed by aTukey’s multiple-comparison test (*p < 0.05 compared to the untreated control).

the highest concentration tested (100 µM), and that the
KKU-213 cell line was more sensitive to JPH203 than the
KKU-055 cell line.

JPH203 suppressed CCA cell growthin the in vivo model
Our in vitro models demonstrated that the KKU-213 cellline was the most sensitive to JPH203 when compared tothe other cell lines. Thus, we evaluated the anti-tumor
activity of JPH203 in a nude mouse xenograft model
derived from KKU-213 CCA cells. JPH203 was administered intravenously daily for 20 days at three different
doses (6.3, 12.5, and 25.0 mg⁄kg) starting at day 3 after the
injection of cancer cells. On the days 18 and 21, JPH203
showed dose-dependent inhibition on tumor growth withsignificantly inhibited tumor growth in the groups of
JPH203 at 12.5 mg/kg (on day 18, p < 0.05, and on day 21,
p < 0.01) and 25 mg/kg (on day 18 and day 21, p < 0.001)
when compared to the control group (Figure 6(a) and (b)).
To confirm our findings on this growth suppression mechanism, immunohistochemical analysis was conducted to
identify the expression of a proliferation marker Ki67 in
the tumor tissues. Reduction of the cells positive for Ki67
was found in JPH203 treatment groups at 12.5 and 25 mg/
kg (p < 0.01) compared with control group (Figure 6(c) and(d)). In contrast to the effects on tumor growth, the animals
did not show any clinical signs of toxicity, changes in general behavior, or changes in physical activity in the
JPH203-treated animals compared to the controls. Micetreated with JPH203 were healthy and had similar bodyweights to the control mice (Supplementary Figure 1). In

Yothaisong et al.

11

Figure 6. Anti-tumor activity of JPH203 in CCA-inoculated athymic BALB⁄c nude mice. (a) CCA tumor tissues were obtained
from vehicle and JPH203-treated mice (n = 5/group). (b) The tumor volume was expressed relative to the initial tumor volume. The
data were analyzed by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group). (c) IHC to identify
expression of Ki67 in tumor tissues, magnification ×400. (d) Bar graphs illustrate the proportion of Ki67 positive cells. The data ofKi67 positive cells were expressed as mean ± SEM (**p < 0.01 analyzed by Student’s t test).

addition, a histopathological review of the internal organs,including in the liver, lungs, spleen, and kidneys, analyzed
by H&E staining showed no discernible toxicity of JPH203(Supplementary Figure 1). There were no differences in
the weight or appearance of the internal organs of mice
between the control and treated groups. These results indicate that JPH203 was safe for the animals studied and thatit should be considered for use in CCA patients.

Discussion
Although suppressed LAT1 activity using BCH has beenreported in CCA,8,9 BCH is a broad system L inhibitor thattargets all members of LAT family. Therefore, BCH lacks
selectivity for both LAT1 and cancer cells, which results inlow cytotoxicity for several cancer cell lines, which isundesirable in clinical trials.29,30 Our previous study11
showed that LAT1 expression is significantly associatedwith O. viverrini-induced cholangiocarcinogenesis and ashorter survival time in CCA patients. Moreover, theexpression of LAT1 and CD98 in CCA is possibly regulated through the oncogenic PI3K/AKT signaling pathway.
Collectively, these data led us to investigate, for the firsttime, the effects of a selective LAT1 inhibitor in our CCA
model.
Our results show that LAT1 is the main system l-transporter, along with CD98, in CCA cells, including the
KKU-055, KKU-213, and KKU-100 cell lines, which
conforms to a previous study from Janpipatkul et al.9 who
reported a lower expression of LAT2, LAT3, and LAT4
than LAT1 in KKU-213 cells. Interestingly, our results
indicate that JPH203 possesses a higher efficacy forinhibiting CCA cell proliferation than for BCH as shownin previous reports.9,8 This phenomenon is possible due tothe specificity of JPH203 to LAT1 in CCA cells being
much higher than BCH.

12
The treatment of the CCA cells with JPH203 showedthat the IC50 values for [14C]l-leucine uptake inhibition
were lower than those for cell growth inhibition. This isconsistent with two previous studies reporting the effect of
JPH203 on human colon cancer19 and human oral cancer.21
Oda et al.19 suggested that JPH203 inhibits LAT1 by competing with its substrates; therefore, the differences in the
IC50 values would be caused by the concentrations of
LAT1 substrates (neutral amino acids) in the incubation
medium. Moreover, we also demonstrated that amino aciddeprivation caused by JPH203 treatment for 48 h results inincreasing LAT1 and CD98 expressions. This is likely torequire a higher concentration of JPH203 to target LAT1 in
the cell culture experiments that were performed for 24–
74 h when compared to amino acid uptake experimentsthat were performed with a short incubation period (1 min).
Furthermore, the increase in LAT1 and CD98 expressions
may function as a compensatory survival mechanism
under conditions of amino acid starvation. This result suggests that LAT1 and CD98 expression are regulated byintracellular amino acid levels, which is supported by previous studies in prostate cancer24 and also CCA.9
Previous studies have also shown that amino aciddepletion mediated by BCH contributes to cell cycle arrestat the G1 phase. This is regulated by cell cycle–related
proteins such as cyclin D3, CDK6, and P27 in human oral
cancer,31 and cyclin D3 along with P27 in human prostatecancer.32 These data are supported by our results; we showthat JPH203 clearly induced cell cycle arrest at the G0/G1
phase and reduced S-phase progression in KKU-213 cells
via the regulation of cell cycle–related proteins, including
cyclin D1, CDK4, CDK6, and P21. Our results also reveal
the different growth inhibition mechanism of JPH203 in
KKU-055. This specific LAT1 inhibitor induced G2/M
cell cycle arrest and reduced S-phase cells via increasing
P21 and P27 concentrations in KKU-055. The increase in
P21 and P27 during G2/M arrest by anti-cancer agents has
been reported in many types of human cancer.33–36 For
example, gallic acid can induce cell cycle arrest at theG2/M phase through increased P21 and P27 expression inbreast cancer.36 However, from our results, the different
pattern of cell cycle distribution caused by JPH203 treatment in our CCA cells indicates that JPH203 can selectively target several components of the cell cycle machineryto arrest the cell cycle. This is dependent on both cell typeand drug concentration.
Caspase proteins play a critical role in apoptosis andare responsible for many of the biochemical and morphological changes associated with this phenomenon.
Therefore, an increased level of activated caspase proteins is one of the most common apoptosis markers thathas been used to indicate apoptosis phenotype of cell.37,38
Two previous studies reported that JPH203 can induce
caspase-dependent apoptosis in cancer cells.21,22 The
previous study demonstrated that JPH203 can induce

Tumor Biology﻿
apoptosis of human oral cancer revealed by an increaseof apoptosis markers included the cleaved form of caspase-3, caspase-7, and caspase-9, as well as cleaved poly
(ADP-ribose) polymerase (PARP) together with 16.5%
of Annexin V–FITC-positive cells. These were observed
after treatment of the cells with 3 mM of JPH203 for
24 h.21 The next study on leukemia reported that JPH203
induced type ІІ cell death, autophagy, followed by caspase-3-dependent apoptosis at 48 h after treatment.22
Consistent with these studies, our data showed that thetreatment of CCA cells with JPH203 (100 µM) for 48 h
could increase the ratio of cleaved caspase-3/preform
along with an increased number of dead cells, more than
10% and 20% for KKU-055 and KKU-213, respectively.
These results indicate that JPH203 induced apoptosis in
CCA cells via caspase-3 activation. However, the molecular mechanisms underlying apoptosis caused by JPH203
in the CCA model require more study.
To date, there is only one study on the effect of a LAT1
inhibitor using the CCA xenograft model. Kaira et al.8
demonstrated the anti-tumor efficacy of BCH in HuCCT1
CCA xenograft nude mice; however, as indicated above,
BCH does not specifically target LAT1 but also other system l-amino acid transporters. Kaira et al.8 reported thatdaily intravenous administration of BCH, up to 200 mg/kg
for 14 days, caused a statistically significant delay in tumor
growth for 3 weeks after treatment. Compared to Kaira etal.’s study, daily intravenous administration of JPH203 at
amounts substantially lower than for BCH (16-fold,
12.5 mg/kg; 8-fold, 25 mg/kg) for 20 days could significantly inhibit KKU-213 CCA cell growth in nude micestarting at day 18. This was confirmed by the decreasing
percent of Ki67 positive cells found. Our results indicatethat JPH203 is considerably more effective than BCH
against CCA cell growth, conforming with the IC50 values
in inhibiting [14C]l-leucine uptake and cell growth in the invitro model.8,9 Furthermore, our results support the studyof Oda et al., the first report of JPH203 in an in vivo model,using intravenously administered JPH203 (12.5 and25.0 mg/kg). This showed a statistically significant inhibition of growth of HT-29 tumors transplanted to nude micewith only a slight decrease in body weight.19 However, in
our study, there was no statistically significant change that
could be attributed to general toxicity, such as changes in
the animals’ body weight, or the histology or appearanceof the internal organs.
Taken together, this study demonstrates that the inhibition of LAT1 activity using JPH203 in CCA cells whichshow high LAT1 and CD98 expression leads to an intracellular decrease in essential neutral amino acids. This results
in an altered expression of LAT1 and CD98, inhibiting cell
growth and inducing cell cycle arrest and apoptosis in the invitro model. Moreover, JPH203 shows anti-tumor efficacy
in nude mice bearing human CCA cell xenografts without
general toxicity. This study is the first to demonstrate theYothaisong et al.
effects of a selective LAT1 inhibitor in the CCA model,thus providing useful information for the development of
JPH203 as a therapeutic strategy for CCA patients.
Acknowledgements
The authors thank the research technicians (Department of
Pathology, School of Medicine, Dokkyo Medical University)
who kindly assisted in the processes of paraffin-embedded tissue
sections and H&E staining. They also thank Professor Trevor N
Petney for editing the article via Publication Clinic KKU,
Thailand.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Funding
This study was supported by the Invitation Research Grant (grant
numbers I57235 and I57305), The Liver fluke and Cholangiocarcinoma
Research Center (LFCRC04/2556), as well as a scholarship from theJapanese Government (The Ministry of Education, Culture, Sports,
Science and Technology (MEXT)) to S.Y.

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Anat Sci Int (2017) 92:200–206
DOI 10.1007/s12565-016-0369-3

REVIEW ARTICLE

Roles of organic anion transporters (OATs) in renal proximal
tubules and their localization
Naoyuki Otani1 • Motoshi Ouchi1 • Keitaro Hayashi1 • Promsuk Jutabha1 •Naohiko Anzai1,2

Received: 28 March 2016 / Accepted: 27 August 2016 / Published online: 10 September 2016
Ó Japanese Association of Anatomists 2016

Abstract Organic anions (OAs) are secreted in renal
proximal tubules in two steps. In the first step, OAs aretransported from the blood through basolateral membranes
into proximal tubular cells. The prototypical substrate forrenal organic anion transport systems, para-aminohippurate (PAH), is transported across basolateral membranes ofproximal tubular cells via OAT1 (SLC22A6) and OAT3
(SLC22A8) against an electrochemical gradient inexchange for intracellular dicarboxylates. In the second
step, OAs exit into urine through apical membranes ofproximal tubules. This step is thought to be performed bymultidrug efflux transporters and a voltage-driven organic
anion transporter. However, the molecular nature andprecise functional properties of these efflux systems arelargely unknown. Recently, we characterized an orphantransporter known as human type I sodium-phosphatetransporter 4, hNPT4 (SLC17A3), using the Xenopus
oocyte expression system. hNPT4 acts as a voltage-driven
efflux transporter (‘‘human OATv1’’) for several OAs suchas PAH, estrone sulfate, diuretic drugs, and urate. Here, we
describe a model for an OA secretory pathway in renal
tubular cells in which OAs exit cells and enter the tubular
lumen via hOATv1 (hNPT4). Additionally, hOATv1
functions as a common renal secretory pathway for bothNaoyuki Otani and Motoshi Ouchi contributed equally to this work.
& Naohiko Anzai
anzai@chiba-u.jp
1

Department of Pharmacology and Toxicology, Dokkyo
Medical University, 880 Kitakobayashi, Mibu, Shimotsuga,
Tochigi 321-0293, Japan2

Department of Pharmacology, Chiba University Graduate
School of Medicine, 1-8-1 Inohana, Chuo-ku,
Chiba 260-8670, Japan123

urate and drugs, indicating that hOATv1 may be a leakpathway for excess urate that is reabsorbed via apical
URAT1 to control the intracellular urate levels. Therefore,
we propose a molecular mechanism for the induction of
hyperuricemia by diuretics: the diuretics enter proximal
tubular cells via basolateral OAT1 and/or OAT3 and may
then interfere with the NPT4-mediated apical urate efflux
in the renal proximal tubule.
Keywords Organic anion  Transporter  Uric acid  Drugs

Introduction
The kidney plays an important role in homeostasis, as itcleanses the body of harmful metabolites, medications, andxenobiotics. The primary site in the kidney at whichorganic anions (OAs) are removed from the blood anddischarged into the urine is the proximal tubule. Renal
secretion of OAs, including exogenous substrates (e.g.,
drugs) and endogenous substrates (e.g., urate), is carriedout in at least two steps in proximal tubular cells (Møller
and Sheikh 1982). In the first step, OAs in the blood crossbasolateral membranes and are transported into proximal
tubular cells. When they are transported in this direction,
which is opposite to that of the electrochemical gradient,
OAs are exchanged for a-ketoglutarate and other intracellular dicarboxylates (Shimada et al. 1987). In the second
step, OAs cross the apical membranes of tubular epithelial
cells and are excreted into the urine. This energeticallyfavorable process for OAs is thought to be mediated bymembrane transporters. Because most OAs are bound toproteins, such as albumin in blood, they are not filteredthrough the glomerulus. Instead, they are transported fromthe blood across basolateral membranes into proximal

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization

tubules, and are then exported across apical membranes oftubular epithelial cells into urine.
In tubular cells, there are specific transporters foressential nutrients (e.g., glucose and amino acids) that are
filtered by glomerulus to keep them in the blood (Wright1985). In contrast, the transport of various endogenous andexogenous substances that are harmful to the body is carried out by transport systems that are characterized as‘‘multiselective;’’ i.e., one transporter recognizes many
types of substances as transport substrates. Because suchcompounds can be harmful, the body is protected by organs(such as the kidneys, liver, and intestines) that biotransformthe compounds into less active metabolites and excretethem. Eventually, drugs and environmental toxicants are
excreted—particularly from the kidney—into the urine.
Transporters of OAs, known as organic anion transporters(OATs), play a major role in determining several propertiesof drugs, such as their pharmacokinetics andpharmacodynamics.
The reabsorption pathways for urate—one of the clinically important OAs—are mediated by the apical humanurate/anion transporter, which was identified in 2002
(Enomoto et al. 2002) and designated urate transporter 1(URAT1)/SLC22A12, and also by the basolateral voltagedriven urate transporter 1 (URATv1)/glucose transporter 9(GLUT9)/SLC2A9, which was reported by our group andothers (Vitart et al. 2008; Anzai et al. 2008; Caulfield et al.
2008). The urate secretory transporters in renal proximal
tubular cells are not as specific as the reabsorptive ones.
They have been identified as multispecific organic anionicdrug transporters OAT1 and OAT3, located at the basolateral membrane (Hosoyamada et al. 1999; Cha et al.
2001). The voltage-driven OAT 1 (OATv1), identified by
us in pig kidneys in 2003, functions as an excretion pathway for OAs, and is found on the luminal membranes ofproximal tubules. One of the substrates transported byporcine OATv1 (pOATv1) is urate (Jutabha et al. 2003).
Thus, the pOATv1 protein is considered to be involved in
the voltage-sensitive pathway for apical urate secretion.
Human Na?-phosphate cotransporter 4 (hNPT4), which
belongs to the type 1 sodium-phosphate cotransporterfamily classified as SLC17A3, is a multispecific OA efflux
transporter that has also been characterized as a novel uratetransporter (Jutabha et al. 2010).

Organic anion transporter systems in the kidney
Many classes of OAs have been shown to be eliminated by
the kidney (Anzai et al. 2006), including endogenous
substances, such as urate and prostaglandins, and essential
drugs, such as antibiotics, furosemide, nonsteroidal antiinflammatory drugs, angiotensin-converting enzyme

201

inhibitors, and sulfate and glucuronide conjugates of drugs.
Molecular cloning has identified several families of elimination-involved OA transporters, including the OAT
family (Sekine et al. 1997; Sweet et al. 1997), the organic
anion transporting polypeptide (OATP) family (Jacquemin
et al. 1994), and the type 1 sodium-phosphate cotransporter(NPT) family (Reimer and Edwards 2004). ATP-dependent
OATs, such as multidrug resistance-associated proteins(MRPs), act as active efflux pumps (Giacomini et al. 2010)(Fig. 1).
OAT family SLC22
Substrates of the para-aminohippurate (PAH) transporter
have been suspected to include endogenous OAs, uremic
substances, drugs, and environmental compounds. Because
the PAH transporter is multispecific, it is suitable for
eliminating various endogenous metabolites and xenobiotics. In the kidney, the PAH transport system is believed
to participate in ‘‘tubular secretion.’’ After being isolated
by expression cloning methods, the PAH transporter was
designated OAT1 (Sekine et al. 1997; Sweet et al. 1997),
and OAT 2, 3, and 4 were subsequently identified (Anzai
and Endou 2007). In the kidney, the transport of OAs into
cells at the basolateral membrane is a tertiary active
transport process. The first step in this process is thecountertransport of an OA (e.g., PAH) against its electrochemical gradient in exchange for the movement of an
intracellular dicarboxylate (e.g., a-ketoglutarate) in thesame direction as its electrochemical gradient (Anzai et al.
2006). After the gradient of decarboxylate has been
directed outward, it is then maintained by being metabolized and transported across the basolateral membrane into
cells via the Na?/dicarboxylate cotransport system. The
Na? gradient, which is directed inward and drives thisprocess, is maintained by a transport step that requires
energy, i.e., the extracellular transport of Na? is performedat the basolateral membrane by Na?/K?-ATPase. Humanproteins OAT1–3 are localized on the basolateral membranes of proximal tubular cells. The OAT4 mRNA isexpressed in the kidneys, and the OAT4 protein is foundon the apical membranes of proximal tubular cells. The
OAT4 protein comprises 550 amino acids and shares its
secondary structure with other OAT proteins (Cha et al.
2000). The OAT1 protein has extremely broad selectivity
for substrates, which include endogenous substrates suchas urate, dicarboxylates (Shimada et al. 1987), cyclic
nucleotides, and prostaglandins, as well as exogenous
substrates such as glucuronides, sulfate conjugates, environmental compounds, and anionic drugs (e.g., b-lactam
antibiotics, diuretics, nonsteroidal anti-inflammatory drugs,
angiotensin-converting
enzymeinhibitors,
andmethotrexate).

123

202
Fig. 1 Proposed model oftranscellular urate transport in
the renal proximal tubule (Anzaiet al. 2010). URAT1 at theapical membrane participates inproximal tubular urate
reabsorption, and the reabsorbed
urate exits cells via basolateral
URATv1. The secretory
pathway for urate is known to
be mediated by OAT1 andOAT3, located on thebasolateral side, and the exit
pathway on the apical side
involves a long-hypothesizedvoltage-driven transporter thatis compatible with NPT4/
OATv1. The latter is also
proposed to be the exit path fordiuretics

N. Otani et al.

Lumen

Reabsorpon

URATv1

URAT1

Urate

Voltage-driven

NPT4/OATv1

Secreon

Voltage-driven

Loop
diurecs

OAT1

OAT3

OAs

OATP1aOrganic anions : OAs(drugs, urate, etc.)

OATP family SLC21/SLCOThe OATP family was first identified in the rat liver by theexpression cloning method as a family of sodium-independent bile acid transporters (Jacquemin et al. 1994).
Several OATPs are selectively involved in the hepatic
uptake of bulky and hydrophobic organic anions; however,most OATPs are expressed in various types of tissue andvarious locations, such as the blood–brain barrier, choroid
plexus, heart, lungs, intestines, kidneys, placenta, andtestes. The members of the OATP family are divided into
six subfamilies (OATP1–6). Of the OATPs found in
humans, the only one that is expressed mainly in the kidneys is OATP4C1 (Mikkaichi et al. 2004). Several substances that chiefly undergo renal excretion are substrates
of OATP family transporters. For example, digoxin,
methotrexate, and thyroid hormones such as triiodothyronine are transported with high affinity by OATP4C1,
which is expressed only in the basolateral membranes ofthe proximal tubular cells.
NPT family SLC17The SLC17 proteins, which were first considered to be phosphate carriers, have since been shown by molecular studies tobe expressed at the apical membranes of renal proximal tubularcells and to mediate the transport of OAs (Reimer and Edwards2004). Various OAs are transported via mouse NPT1 andhNPT1 in a chloride-dependent manner. Furthermore, because
hNPT1 tends to combine with PAH, as earlier studies withbrush-border membrane vesicles have shown, it is also believed
to be a classical voltage-dependent transporter of PAH.

123

Blood

MRP2

OATP4c

MRP4

MRP6

However, whether the transport of PAH is affected by membrane potential is unclear (Uchino et al. 2000).
MRP family ABCCThe ATP-dependent excretion of both drugs and xenobiotics is mediated by P-glycoprotein (P-gp). This protein isexpressed in the luminal membranes of the small intestineand the blood–brain barrier and in the apical membranes of
excretory cells, such as hepatocytes and the epithelial cellsof kidney proximal tubules (Giacomini et al. 2010).
The multidrug resistance-associated protein (MRP)
family primarily comprises active transporters with an
ATP-binding cassette motif. The protein P-gp is this family’s prototype (Ford and Hait 1990) and extrudes varioushydrophobic molecules, in particular antineoplastics suchas adriamycin, daunorubicin, vinblastine, and vincristine.
In addition, multidrug resistance is conferred by P-gp
(Gottesman et al. 2002). In proximal tubular cells, themembers of the MRP family are believed to function as
extrusion pumps on the apical membrane for OAs, especially those that are large and hydrophobic. Two isoformsof the MRP family, MRP2 and MRP4, deserve particularattention with respect to renal physiology and pharmacology. One of the physiological roles of MRP2 is to transport
conjugated bilirubin from the liver into bile. In the kidney,
MRP2 plays a role in the transport of highly lipophilicorganic compounds across the apical membranes of tubularcells into urine. In contrast, the MRP4 isoform is involvedin the efflux of several small hydrophilic OAs, including
urate, prostaglandins, and cephem antibiotics into thetubular lumen.

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization

Analysis of the function of NPT4 (SLC17A3)
as OATv1
pOATv1 was first isolated as a voltage-driven OAT fromthe pig kidney cortex (Jutabha et al. 2003). It mediates
facilitated diffusion driven by membrane voltage. In
humans, NPT1 belongs to the SLC17 family and displays
the highest homology to pOATv1. Dehghan et al. havefound that NPT4, as well as URATv1, are causative genesof hyperuricemia (Dehghan et al. 2008). We have discovered that hNPT4, which belongs to the same family as
hNPT1, is a voltage-dependent OAT, as is porcine OATv1.
In 2010, Jutabha and Anzai et al. described an hNPT4protein localized to the apical membranes of renal proximal
tubular cells (Jutabha et al. 2010). The localization of thisprotein in the liver is still unknown and warrants further
investigation.
hNPT4 mediates the transport of PAH, estrone sulfate,estradiol glucuronide, bumetanide, and ochratoxin A (Anzai et al. 2010), and it interacts with various anioniccompounds, such as probenecid, diuretics, nonsteroidal
anti-inflammatory drugs, and steroid sulfates. Therefore, it
is a multispecific OAT that overlaps in substrate selectivity
with OAT family (SLC22) members (Anzai et al. 2006).
Many substrates for OATs, except for dicarboxylates andpenicillin G, interact with hNPT4. Thus, it would beinteresting to determine the common mechanisms responsible for the similar substrate selectivities of structurally
different OATs. hNPT4 interacts strongly with various
diuretics, such as thiazides and loop diuretics. Bumetanide
and furosemide inhibit PAH transport via hNPT4 in a dosedependent manner. It was reported that hNPT4 functions as
a luminal voltage-dependent excretion pathway for loop
diuretics in proximal tubules (Ellison and Wilcox 2008).
Considering that hNPT4 is a voltage-driven facilitatedtransporter which is localized on the apical membranes ofproximal tubules, it may represent a highly suitable exit
pathway for OAs via the apical membranes of renal
proximal tubules. Thus, the role of hNPT4 in PAH transport is similar to that of pOATv1, initially characterized byclassical expression cloning (Jutabha et al. 2003).
The transporter pOATv1 has an amino acid sequencethat is quite similar to that of hNPT1 (63 %) and lesssimilar to that of hNPT4 (45 %). Furthermore, pOATv1 isexpressed in the liver and in the kidney, the latter being
involved in the renal elimination of anionic drugs such as
PAH and diuretics and the secretion of endogenous substrates such as urate (Jutabha et al. 2010). PAH transport by
pOATv1, but not by hNPT4, has been shown to be chloride
sensitive. Since we could not determine the reason for thisdifference between the transporters, we were not able toconclude that hNPT4 is a human ortholog of pOATv1.

203

Two splice variants of NPT4
hNPT4 has two splice variants of NPT4 mRNA reported
in the GenBank database. One variant, hNPT4_L (variant
1: NM_001098486), contains an open reading frame
encodingaputative498-amino-acidprotein(NP_001091956), and the other, hNPT4_S (variant 2:
NM_006632), encodes a 420-amino-acid protein(NP_006623). The difference between these two isoforms
is the presence of a fourth exon in hNPT4_L. mRNAs forboth hNPT4 isoforms have been detected only in thekidneys and liver by reverse transcription polymerase
chain reaction (Jutabha et al. 2010; Ruddy et al. 1997).
Following injection of complementary RNAs (cRNAs)
into Xenopus oocytes, hNPT4_L, but not hNPT4_S, hasbeen found to be expressed on the plasma membrane(Jutabha et al. 2011a), corresponding to the results of aprevious study showing that NPT4 (U90545, short isoform) is localized on the endoplasmic reticulum following transfection of cDNA into COS (an abbreviation for‘‘CV-1 in origin with SV40 genes’’) cells (Melis et al.
2004).

hNPT4 as a renal secretory pathway for urate
Urate is generated from purine metabolism, and sustained
hyperuricemia is a pathogenetic cause of gout, chronic
renal diseases, hypertension, and cardiovascular diseases.
The serum level of uric acid is largely determined by the
intrarenal reabsorption and secretion of urate. The renal
reabsorption of urate is controlled by two proximal tubularurate transporters: apical URAT1 (Enomoto et al. 2002)
and basolateral URATv1 (Vitart et al. 2008; Anzai et al.
2008; Caulfield et al. 2008). In contrast, hNPT4, an orphantransporter, is a multispecific OA efflux transporter that isexpressed by the kidneys and the liver. hNPT4 is localizedon the apical side of the renal tubule, and it functions as a
voltage-driven urate transporter (Jutabha et al. 2011b). In
addition, hNPT4 interacts significantly with loop diuretics
such as furosemide and bumetanide. Therefore, hNPT4
likely acts as a common secretion route for drugs, and may
play an important role in diuretic-induced hyperuricemia(Jutabha et al. 2010). After URATv1, which is a basolateralurate efflux transporter, and hNPT4, which is an apical OA
efflux transporter, were identified, a three-factor model was
proposed for the transepithelial transport of urate in renal
proximal tubules (Anzai and Endou 2012). However,
because the molecules involved in renal urate handlingplay physiological and pathophysiological roles that are
still somewhat unclear, it is important to continue toinvestigate those roles.

123

204

In 2008, a genome-wide association study found that the
NPT4-encoding gene SLC17A3 is, like two other genes, associated with the concentration of uric acid and the risk of gout
(Dehghan et al. 2008). Recently, NPT4 has been functionallycharacterized as a novel urate efflux transporter, and two lossof-function mutations (not polymorphisms) of SLC17A3 havebeen found in patients with gout and reduced excretion of renalurate (Jutabha et al. 2011b). Because it is a protein that is
apically expressed in renal tubules, NPT4 is believed to be anexit path for urate when it is transtubularly secreted.

Single nucleotide polymorphisms in SLC17A3/
NPT4There are currently 10 nonsynonymous SLC17A3 single
nucleotide polymorphisms (SNPs) reported in the public SNP
database (NCBI dbSNP). The functional properties of five of
the NPT4 variants (A100T, G239 V, V257F, G279R, andP378L, as reported at the time of determination) were characterized using the Xenopus oocyte expression system (Jutabha
et al. 2011a). The transport activities of urate, PAH, estrone
sulfate, and bumetanide were studied. Compared with a wildtype clone, P378L did not show any transport function for anyof the above four substrates tested. V257F and G279R displayed moderately reduced transport activities, whereas
A100T did not exhibit altered transport of any of the four
substrates. In terms of the structure–function relationship, thereduction of transport function in V257F but the lack of areduction in transport function in G239 V is interesting because
both residues are in the same (fifth) transmembrane domain.
The NPT4 protein has been detected at the plasma membranesof oocytes injected with cRNAs derived from all NPT4
mutants. It has been suggested that the reduced transport
activity caused by some SNPs is not due to an absence of
surface membrane expression. These functionally relevant
NPT4 variants may explain some of the interindividual differences in susceptibility to certain diseases, such as gout
(Pascual and Perdiguero 2006), that are observed in individuals
with altered NPT4 function. Diuretics are NPT4 substrates, so it
would be interesting to further investigate whether individuals
who possess SNPs in NPT4 are resistant to diuretic treatment.
Functional analysis of other hNPT isoforms that have sameamino acid replacement in the corresponding position tohNPT4 may provide further insight about the importance of
these residues on OA transport in SLC17 members.

Renal urate transporters as causes of diureticinduced hyperuricemia
Hyperuricemia is a well-known adverse effect of diuretic
treatment (Ravnan et al. 2002). Diuretics are thought tohave direct actions on urate transporters in renal proximal

123

N. Otani et al.

tubules by enhancing urate influx and causing an increasein the serum urate. We focused on whether hNPT4 is atransporter of urate.
Both membrane transporters and metabolic enzymes are
involved in the clearance of drugs from the kidney andliver; therefore, if these organs show altered drug transport
activity, the pharmacokinetics of the substrate drugs might
be affected (Anzai and Endou 2007). One substrate transported by hNPT4 is bumetanide, so polymorphic changes
in hNPT4 might affect the pharmacokinetics of this andother loop diuretics (Jutabha et al. 2010). For example, thephenomenon known as ‘‘diuretic resistance’’ is exhibitedby about one-third of patients with chronic heart failure(Ravnan et al. 2002). This phenomenon has been defined asa failure to decrease extracellular fluid volume despite theliberal administration of diuretics. A possible cause of thisphenomenon is decreased tubular delivery of loop diuretics, which is often attributed to decreased renal perfusion inpatients with heart failure. Furthermore, the differences infunction of the hNPT4 variants mentioned earlier probably
explain some of the interindividual differences in diuretic
response. Thus, it would be interesting to determine if
diuretic resistance is sometimes explained by alteredhNPT4 function (Jutabha et al. 2011b).
Common diuretics, such as loop diuretics (furosemide
and bumetanide) and thiazide, also reduce urinary uric acidexcretion and increase the serum uric acid level (Russel
et al. 2002). The protein hNPT4 likely serves as a common
secretion route for drugs and urate. The dose-dependent
inhibitory effects of furosemide and bumetanide on
hNPT4-mediated urate transport suggest that the inhibitionof urate efflux through this transporter by loop diuretics
may be responsible for diuretic-induced hyperuricemia(Jutabha et al. 2011b).

Conclusions
We have demonstrated that hNPT4 is a multispecific OA
efflux transporter expressed in the kidneys and liver. In thekidneys, it is localized on the apical sides of renal tubules
and functions as a voltage-driven urate transporter. The
kidneys play a dominant role in maintaining the serum
urate level. Renal urate excretion is a function of the balance between reabsorption and secretion. It has recently
been demonstrated that the uptake of luminal urate into
renal proximal tubular cells is mediated by the urate-anion
exchanger URAT1, and that the voltage-driven urate efflux
transporter URATv1/GLUT9 facilitates the exit of intracellular urate from the cells into the interstitium/blood
space. OAT1 and/or OAT3, located on the basolateral sidesof renal proximal tubular cells, have been proposed to act
as uptake transporters for urate from the interstitium/blood

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization

space into cells; however, the molecular identity of theapical urate transporter that excretes intracellular urate into
the luminal (urine) space is elusive. We suggest that NPT4/
OATv1 is the missing component of the three-factor model
because it functions in renal tubular efflux and the elimination of various anionic drugs, such as PAH and diuretics,
as well as in the secretion of endogenous substrates such asurate. In addition to a known effect on renal urate uptake
transporters, another mechanism for diuretic-induced
hyperuricemia may be an inhibition of NPT4-mediatedurate secretion. Therefore, the NPT4/OATv1-mediatedefflux of urate may represent a new drug target for
hyperuricemia.
Acknowledgments This study was supported in part by grants fromfrom the Japan Society for the Promotion of Science [JSPS
KAKENHI 24590328 (K.H.), 23590647 (P.J.), 26461258 (N.A.)], theStrategic Research Foundation Grant-aided Project for Private
Universities (S1412001), the Science Research Promotion Fund of theJapan Private School Promotion Foundation, the Gout Research
Foundation of Japan, the Shimabara Science Promotion Foundation(K.H.), a Dokkyo Medical University Investigator-Initiated Research
Grant (N.O., M.O.), and Research grant of Seki Minato Memorial
Awards of the Seki Minato Foundation (P.J., N.A.).
Compliance with ethical standards
Disclosure of potential conflicts of interest There are no conflicts ofinterest associated with the authors who contributed to thismanuscript.

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9 号（9 月）2015〕

特集

PG・有機酸のトランスポーター

441

第 66 回大会シンポジウム「ビタミン・バイオファクタートランスポーター研究の最前線」
ミニレビュー

プロスタグランジンと有機アニオンのトランスポーター
獨協医科大学医学部薬理学講座

安西

尚彦，岡本

和久，花田

健治，阿部

＊

篤朗，大谷

直由，大内

基司

Vitamins (Japan), 89 (9), 441-445 (2015)

Transporters for prostaglandins and organic anions
Naohiko Anzai, Kazuhisa Okamoto, Kenji Hanada, Tokuro Abe, Naoyuki Otani, Motoshi Ouchi
Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi 321-0293, Japan

Organic anion transporters (OATs) play a fundamental role in the elimination of numerous endogenousand exogenous organic anions from the body. The secretion of numerous organic anions including
endogenous metabolites, drugs, and xenobiotics, is an important physiological function of excretoryorgans such as kidney and the process of secretion of organic anions through the proximal tubule cells is
DFKLHYHGYLDXQLGLUHFWLRQDOWUDQVFHOOXODUWUDQVSRUW7RGDWHVHYHUDOIDPLOLHVRIPXOWLVSHFL¿FRUJDQLFDQLRQtransporters, including organic anion transporter (OAT) family, organic anion-transporting polypeptide(OATP) family, sodium-phosphate transporter (NPT) family and ATP-dependent organic anion transporters
VXFKDVPXOWLGUXJUHVLVWDQFHDVVRFLDWHGSURWHLQ 053 DUHLGHQWL¿HGE\PROHFXODUFORQLQJ$PRQJWKHP
some members of OATP (SLC21/SLCO) family and OAT (SLC22) family are thought to contribute to themembrane permeation of prostglandins (PGs), for example, PGE2 and PGFĮ, that play various physiological
DQGSDWKRSK\VLRORJLFDOUROHV7KH¿UVW3*VSHFL¿FWUDQVSRUWHU3*7 SURVWDJODQGLQWUDQVSRUWHU DPHPEHU
RI6/&6/&2IDPLO\ZDVLGHQWL¿HGLQ7KLVWUDQVSRUWHUPD\SOD\DSULPDU\UROHLQWKHUHPRYDORIELRDFWLYH3*VIURPWKHFLUFXODWLRQ5HFHQWO\ZHLGHQWL¿HGDQRYHOPHPEHURIWKH6/&IDPLO\H[SUHVVHG
in mouse kidney and designated this member as OAT-PG (organic anion transporter for prostaglandins).
7KLV2$73*VSHFL¿FDOO\WUDQVSRUWV3*VZKLFKDUHORFDOL]HGLQSUR[LPDOWXEXOHVKey words: organic anion, prostaglandin, transporter, OAT, OATP
(Received May 11, 2015)
有機アニオンとは炭素骨格を有し，生理学的条件下に有機アニオンとはおいて陰性の荷電を帯びている物質の総称である（表ビタミン・バイオファクターには水溶性の高い物質

1）1）．臨床医療の現場において重要な有機アニオンとも含まれ，それらの一部は「有機イオン」に分類される．

いえば，抗生物質，利尿薬，非ステロイド性抗炎症薬

有機イオンは正電荷を持つ有機カチオン（有機塩基）と（NSAIDs），ACE 阻害薬などの薬物およびその代謝産負電荷を持つ有機アニオンに分けられる．すなわち，物がこれに該当する．有機アニオンの細胞膜輸送とい＊〒 321-0293 栃木県下都賀郡壬生町北小林 880

獨協医科大学医学部薬理学講座

E-mail：anzai@dokkyomed.ac.jp

442

安西

尚彦，岡本

和久，花田

健治，阿部

篤朗，大谷

直由，大内

基司

〔ビタミン 89 巻

うと生体異物の排出というイメージがあるが，実際にた発現クローニング法により OAT1（SLC22A6）が同定さは有機アニオンの中には，生体にとって（有用な）内因れ，その後ヒトでは OAT2，3，4，7，さらに URAT1 お

性物質，すなわち，プロスタグランジン（PG）やステ

よび OAT10 が 続 け て 同 定 さ れ た．OAT1 〜 4 およびロイド抱合体，その他に尿酸などが含まれており，有URAT1, OAT10 は腎臓に発現し，OAT7 は肝臓に存在し機アニオン輸送は必ずしも排泄のみを示すものではなている 3）．OATs の有機アニオン取り込みは，細胞内にい．有機アニオンはイオンという性質上，水溶性であ蓄積された有機酸との交換輸送により行われることがり，脂質で構成された細胞膜を自由に通過することは示唆されている．SLCO/SLC21 に分類される OATP ファ

できない．細胞膜透過のためには特定の膜タンパク質

ミリーは，肝機能検査試薬として知られる有機アニオであるトランスポーターが必要となり，いわゆる一群

ンの BSP（bromosulfophtalein）/ 胆汁酸トランスポーターの有機アニオントランスポーターがこれを担っていとしてクローニングされ，主に肝臓に発現し，大型の有機アニオンの輸送に関与する．近年，SLC17 に分類る．

される NPT ファミリーも有機アニオン排泄機構の一つ

有機アニオントランスポーターであると考えられている．MRPs は癌細胞に多剤耐性を有機アニオントランスポーターとして，これまでに付与する抗癌剤排出ポンプとして知られる MDR1 ととOAT（organic anion transporter）フ ァ ミ リ ー，OATP（or-もに，ABC（ATP Binding Cassette）トランスポータースー

ganic anion transporting polypeptide）フ ァ ミ リ ー，NPT

パーファミリーに属し，ATP の水解エネルギーを用い（sodium-phosphate transporter）ファミリー，そして ATPて細胞内から細胞外へ各種抗癌剤を排出する．肝臓で駆動型トランスポーターの MRP（multidrug resistance-as-の抱合型ビリルビン輸送に関与する MRP2（ABCC2）は，

sociated protein）ファミリーが分子同定されている（表腎臓では近位尿細管管腔側膜に存在して，アニオン性

2）2） ．SLC22 に分類される OAT ファミリーは，1997 年の高脂溶性化合物の尿中への分泌を担うと考えられてに PAH（パラアミノ馬尿酸）の取り込み活性を指標にしいる．また，同ファミリーの MRP4（ABCC4）も近位尿

表1

有機イオンの分類
輸送基質

性状

生体異物・薬物

内因性物質

有機カチオン 正電荷を持つ
利尿薬アミロライド，抗潰瘍薬シメチジ ヒスタミン，ドパミン，ノルアドレナリン，（有機塩基） 水溶性有機物質 ン，抗腫瘍薬シスプラチン
セロトニン，クレアチニン
抗生物質（PcG，セフェム），利尿薬（フ 胆汁酸，
クレアチニン，
ヌクレオチド（cAMP,，
有機アニオン 負電荷を持つ
ロセミド，チアジド），抗炎症薬 NSAIDs cGMP），
プロスタグランジン（PGE2, PGF2α）（有機酸）
水溶性有機物質 （サリチル酸，インドメタシン），抗腫瘍薬 尿酸，ケトン体，短鎖脂肪酸，TCA サイク
ル中間代謝体MTX，バルプロ酸
水溶性有機物質は荷電の状況により，正電荷を持つ有機カチオン（有機塩基）と陰電荷を持つ有機アニオン（有機酸）に分類される．

表2

有機アニオントランスポーターの分類

1．Organic Anion Transporter（OAT）family：SLC22（有機アニオントランスポーターファミリー）OAT1, OAT2, OAT3, OAT4, OAT7, URAT1
2．Sodium-phosphate transporter family：SLC17（Na＋依存性リン酸トランスポーターファミリー）NPT1, NPT4, OATv1
3．
Organic Anion Transporting Polypeptide（OATP）family：SLCO（肝特異的有機酸トランスポーターファミリー）OATP1A2（OATP-A），OATP1B1（OATP-C），OATP1B3（OATP8），OATP2A1（PGT），OATP2B1（OATP-B），
OATP4C1（OATP-R）
4．MRP（multidrug resistance-associated protein）family：ABCC（多剤耐性関連タンパク質ファミリー）MRP1, MRP2, MRP3, MRP4, MRP5, MRP6
5．Organic Solute Transporter（OST）（有機溶質トランスポーター）
OSTα-OSTβ
ヒトの体内での有機アニオン輸送を担う分子群として5つのトランスポーターファミリーが知られている．

9 号（9 月）2015〕

PG・有機酸のトランスポーター

443

細管管腔側膜に存在し，プロスタグランジンや尿酸に作用するためには，①細胞内での産生，②細胞外での加え，セフェム系抗生物質などのアニオン性薬物の分膜受容体への作用，③細胞内での不活性化，という 3泌を担うと考えられている 4）．

つの段階を経る必要がある（図 1）．極めて膜透過性の低い PG が①から②，そして②から③という細胞膜のプロスタグランジンの細胞膜透過

内外での移動を迅速に行うためにはトランスポーター

有機アニオンの一つであるプロスタグランジン（PG）は，アラキドン酸を基質として PGH 合成酵素であるシクロオキシゲナーゼ（COX）により生成されるが必要となる．

PG トランスポーター

生理活性物質である．細胞内で合成された PG は，オー

PG トランスポーターの分子実体としては，1995 年

トクラインないしパラクラインにより細胞外に放出さSchuster らの研究グループが世界に先駆けて PG に特異れ，細胞膜表面にある受容体に結合し，多彩な生理作性の高い分子として PGT（SLCO/SLC21 ファミリー）の用を発現する．細胞外に放出されて作用発現に寄与しクローニングに成功している 5）．PGT に続き著者らのた PG は，細胞内に取込まれて 15-ヒドロキシ PG 脱水

研究グループが，薬物等の体外排泄を担う有機酸トラ素酵素（15-PGDH）による代謝を受けて 15-ケト-PG とンスポーター OAT（SLC22 ファミリー）が PG を輸送すなり不活性化される．PG は高い疎水性を持つため，ることを明らかにした．また，ABC トランスポーター

脂質二重層を容易に透過するものと考えられてきたの MRP4 や，新規有機溶質トランスポーターの OSTα-が，実際にはその膜透過性は非常に低いため，合成さOSTβによる PG 輸送も報告されている（表 3）．

れた PG の細胞外放出および作用した PG の細胞内への取込みには，トランスポーターと呼ばれる膜輸送タ
4）ンパク質が必要となる ．PG がオータコイドとして

A）PGT（Prostaglandin transporter）
ステロイドホルモン抱合体を輸送することが知られ㻔㻞㻕⭷ཷᐜయ䜈䛾స⏝䠄⣽⬊እ䜈䛾ᨺฟ䠅䠄⣽⬊ෆ䜈䛾ྲྀ㎸䜏䠅㻼㻳䜰䝷䜻䝗䞁㓟
䝥䝻䝇䝍䜾䝷䞁䝆䞁
ཷᐜయ

㻯㻻㼄

㻼㻳

㻔㻝㻕⣽⬊ෆ䛷䛾⏘⏕
䠄PG⏘⏕⣽⬊䠅図1

㻼㻳㻔㻟㻕⣽⬊ෆ䛷䛾୙άᛶ໬
䠄PG䛾ᶆⓗ⣽⬊䠅

プロスタグランジン（PG）の経細胞膜輸送（文献 4）改変）

PG がオータコイドとして作用するためには，①細胞内での産生，②細胞外での膜受容体への作用，③細胞内での不活性化，という 3 つの段階を経る必要がある．①と②，および②と③の段階をそれぞれつなぐものがトランスポーターによる（1）細胞外への放出と（2）細胞内への取込みである．

表3

プロスタグランジントランスポーターの分類

1．Organic Anion Transporting Polypeptide（OATP）family：SLCO（肝特異的有機酸トランスポーターファミリー）OATP2A1（PGT）
2．Organic Anion Transporter（OAT）family：SLC22（有機アニオントランスポーターファミリー）OAT1, OAT2, OAT3, OAT43．MRP（multidrug resistance-associated protein）family：ABCC（多剤耐性関連タンパク質ファミリー）MRP4
4．Organic Solute Transporter（OST） （有機溶質トランスポーター）
OSTα-OSTβ
ヒトの体内でのプロスタグランジン輸送を担う分子群として有機アニオントランスポーターの中でも4つのファミリーが担うことが報告されている．

安西

444

尚彦，岡本

和久，花田

健治，阿部

篤朗，大谷

直由，大内

基司

〔ビタミン 89 巻ている OATP（SLCO/SLC21）ファミリーの中から同定

作用，代謝が原則的に腎臓内部で行われ，PG は局所されたものが PGT である 5）．OATP ファミリーには輸調節に関与していると考えられている．

送基質の一つとして PG を輸送するクローンがいくつかあることが知られているが，PGT は OATP ファミリーB）OAT-PG（prostaglandin-specific organic anionの中では，構造的にも機能的にも特異な存在であり，

transporter）

輸送基質は PG に限られている．PGT による各種プロ

OATs による PG 輸送の特性は 2002 年に Kimura らにス タ グ ラ ン ジ ン の 細 胞 内 取 り 込 み 速 度 は，PGE1 〜

より報告されている 8）．ヒト OAT1（hOAT1），hOAT2，

PGE2 〜 PGF2α＞TXB2≫6- ケ ト -PGF1α 〜 iloprost（PGI2

hOAT3，hOAT4 それぞれの安定発現細胞を用いて，RI

アナログ）の順である．PGT は PGH2 も輸送すること

標識 PGE2 と PGF2α の細胞内取込みを調べたところ，

6）が報告されている（Km＝376±34 nM） ．最近，ラット PGT mRNA は 肺， 肝， 腎， 胃， 十 二 指 腸， 空 腸，

それぞれの細胞で時間および濃度依存性の取込みが認められ，その Km は PGE2 に対しては 154 〜 970 nM で

回腸，結腸，睾丸，子宮，脳，眼などに存在するが，あり，PGF2αに対しては 575 〜 1092 nM であった．最近，

心 筋， 骨 格 筋 な ど に は 認 め ら れ な い． ラ ッ ト PGTShiraya らは SLC22 ファミリーのオーファントランス

mRNA は，PG 代謝が最も盛んな肺に非常に多く存在

ポーターの中から，PG 特異的な輸送を行うクローンする．また，ラット PGT mRNA は腎では乳頭部＞髄を見出し，prostaglandin-speci¿c organic anion transporter

質＞皮質の順に発現しているとされており，糸球体，（OAT-PG）と名付けた 9）．OAT-PG は，近位尿細管の基

集合管主細胞，髄質の間質細胞と乳頭部細胞などに発底側膜に限局して発現し，先述の 15-PGDH の局在と7）現するとされる ．ヒト PGT mRNA は，さらに心筋一致することから，PG シグナルの不活化に重要な役と骨格筋にも認められる．これまでに述べたように，割を持つことを示唆している（図 3）．OAT-PG は，エ

強力な作用を有する PG は受容体に作用した後，速やイ コ サ ノ イ ド で は PGE2 や PGH2 を 対 向 基 質 と し てかに局所で代謝されなければならない．しかし，PG

PGE2 排出が促進される（トランス亢進）だけでなく，の代謝酵素は細胞内にあるので，PG を不活性化する

他 の エ イ コ サ ノ イ ド で あ る 12-HPETE（12-hydroper-

ためにはまず細胞内へ取り込まれる必要がある（図2）．生体全体としては肺循環が重要で，各臓器で作用㻼㻳㻱㻞

を終え代謝し切れなかった PG は肺で代謝され，体循環に回らないようにしている．腎臓では PG の産生，

㻼㻳㻱㻞

OAT
-PG

㻝㻟㻘㻝㻠㻙㼐㼕㼔㼥㼐㼞㼛㻙㻝㻡㻙㼗㼑㼠㼛㻙㻼㻳㻱㻞㻼㻳㻱㻞䠄䝬䜴䝇㏆఩ᒀ⣽⟶⣽⬊䠅㻝㻟㻘㻝㻠㻙㼐㼕㼔㼥㼐㼞㼛㻙㻝㻡㻙㼗㼑㼠㼛㻙㻼㻳㻱㻞図3
㻝㻡㻙㻼㻳㻰㻴

㻼㻳䕧㻝㻟㑏ඖ㓝⣲㻝㻡㻙㼗㼑㼠㼛㻙㻼㻳㻱㻞
䠄PGTⓎ⌧⣽⬊䠅

図2

㻝㻡㻙㼗㼑㼠㼛㻙㻼㻳㻱㻞㻝㻡㻙㻼㻳㻰㻴

PGT㻼㻳㻱㻞㻝㻡㻙㼗㼑㼠㼛㻙㻼㻳㻱㻞

プロスタグランジントランスポーター PGT と PG
不活性化の連関PG は受容体に作用した後，PGT により取込まれ，細胞内の PG 代謝酵素（15-PGDH）により代謝される．さらにPG △ 13 還 元 酵 素 な ど に よ り 分 解 さ れ た 代 謝 産 物（13,14-dihydro-15-keto-PGE2）は PG 取込みの交換基質となって効率的に細胞外に放出される．

OAT-PG と PG 不活性化酵素（15-PGDH）との連関

マウスの近位尿細管の基底側膜に限局して発現し PG 特
異 的 な 輸 送 を 行 う prostaglandin-speci¿c organic aniontransporter（OAT-PG）は，先述の PG 代謝酵素 15-PGDH の局在と一致し，受容体に作用した後の PG を細胞内に取込み続けて酵素 15-PGDH による不活化が行われると考えられている．OAT-PG は，エイコサノイドでは PGE2 やPGH2 を対向基質として PGE2 排出が促進される（トランス 亢 進 ）だ け で な く， 他 の エ イ コ サ ノ イ ド で あ る
12-HPETE（12-hydroperoxyeicosatetraenoic acid），
15-HPETE，15-HETE（15-hydroxyeicosatetraenoic acid） などもトランス亢進を示す．15-ケト-PGE2 などの PGE2 不活性化代謝産物によるトランス亢進が存在するのかどうかについては今後の検討が待たれる．

9 号（9 月）2015〕

PG・有機酸のトランスポーター

445

oxyeicosatetraenoic acid），15-HPETE，15-HETE（15-hy-

4）安西尚彦（2011）生理活性脂質のトランスポーター．栄養・droxyeicosatetraenoic acid）などもトランス亢進を示すこ

食品機能とトランスポーター（日本栄養・食糧学会監修）．

とが示され，興味深いがその生理学的意味は不明であり，PGT に み ら れ る よ う な 13,14-ジ ヒ ド ロ-PGE2 や
13,14-ジヒドロ-15-ケト-PGE2 などの PGE2 不活性化代
謝産物によるトランス亢進が存在するのかどうかについては解析がなされておらず，不活性化酵素との機能的な連関についての今後の検討が待たれる．
（平成 27.5.11 受付）

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5）Kanai N, Lu R, Satriano JA, Bao Y, Wolkoff AW, Schuster VL(1995) Identi¿cation and characterization of a prostaglandin transporter. Science 268, 866-869
6）Chi Y, Schuster VL (2010) The prostaglandin transporter PGTtransports PGH2. Biochem Biophys Res Commun 395, 168-172
7）Bao Y, Pucci ML, Chan BS, Lu R, Ito S, Schuster VL (2002)
Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids. Am J Physiol Renal Physiol 282,
F1103-F1110
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文

献H (2002) Human organic anion transporters and human organic
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