Warburg効果とTRAIL誘導がん細胞死
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言語: Japanese
出版者:
公開日: 2017-08-10
キーワード (Ja):
キーワード (En):作成者: 熊崎, 実南
メールアドレス:所属:

URL

http://hdl.handle.net/20.500.12099/56206

Warburg ຠᯝ࡜ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛ
Perturbation of the Warburg effect increases the sensitivity of cancer
cells to TRAIL-induced cell death2017⇃㷂 ᐇ༡┠ḟ

➨ 1 ❶ ⥴ゝ  1➨ 2 ❶ ◊✲ࡢ⫼ᬒ࡜┠ⓗ ➨㸯⠇ TRAIL ࢩࢢࢼࣝ ➨ 2 ⠇ TRAIL ࢩࢢࢼࣝࢆᶆⓗ࡜ࡋࡓ἞⒪ࡢኚ㑄࡜ၥ㢟Ⅼ ➨ 3 ⠇ TRAIL ⪏ᛶᶵᵓ

  4
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 ➨ 4 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰไᚚᶵᵓ㸸Warburg ຠᯝ➨ 5 ⠇ CCN1 ࡢⓎ⌧࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡬ࡢ㛵୚  1417➨ 3 ❶ Warburg ຠᯝ㛵㐃 PTBP1 ࡜ TRAIL ㄏᑟ࢔࣏ࢺ࣮ࢩࢫ
 ➨ 1 ⠇ ᗎ
 ➨ 2 ⠇ PTBP1 ࡢⓎ⌧࡜ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛ࡬ࡢ㛵୚
➨ 3 ⠇ TRAIL ⪏ᛶᶵᵓ࡟࠾ࡅࡿ PTBP1 ࡢᙺ๭ ➨ 4 ⠇ άᛶ㓟⣲㸦ROS㸧ࡢⓎ⏕࡜ TRAIL ⪏ᛶゎ㝖ᶵᵓ࡬ࡢ㛵୚ ➨ 5 ⠇ CCN1 ࡢⓎ⌧࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡬ࡢ㛵୚

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➨ 4 ❶ ⥲ᣓ

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37

ㅰ㎡

41

ᐇ㦂ࡢ㒊

42

ཧ⪃ᩥ⊩

51

◊✲ᴗ⦼┠㘓

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➨ 1 ❶ ⥴ゝ⭘⒆ቯṚᅉᏊ㸦TNF: tumor necrosis factor㸧ࡣࠊ1975 ᖺ࡟ Sloan-Kettering ⒴◊✲ᡤࡢ Carswell ࡽ࡟ࡼࡗ࡚࣐࢘ࢫ࡟⛣᳜ࡋࡓ Meth A ⫗⭘࡟ฟ⾑ᛶࡢቯṚࢆᘬࡁ㉳ࡇࡍᅉᏊ࡜ࡋ࡚Ⓨぢࡉࢀࡓࢧ࢖ࢺ࢝࢖࡛ࣥ࠶ࡿ 1ࠋࡑࢀ௨ᚋࠊTNF ࣇ࢓࣑࣮ࣜ࡟ᒓࡍࢧ࢖ࢺ࢝࢖ࣥ࡜ࡋ࡚࢔࣏ࢺ࣮ࢩࢫ࡟㛵୚ࡍࡿ TNF-DࠊFasLࠊTRAIL㸦Tumor necrosis-factor related apoptosis-inducing ligand㸧࡞࡝ 20 ✀㢮௨ୖࡀྠᐃࡉࢀ࡚࠸ࡿ 2, 3ࠋTRAIL ࡣࠊ⣽⬊⭷࡟Ꮡᅾࡍࡿ≉␗ⓗཷᐜయ࡬ࡢ⤖ྜࢆ௓ࡋ࡚࢔࣏ࢺ࣮ࢩࢫㄏᑟࢩࢢࢼࣝࢆ⣽⬊ෆ࡟ఏ㐩ࡍࡿᢠ⭘⒆ᛶࡢࢧ࢖ࢺ࢝࢖࡛ࣥ࠶ࡿࠋTRAIL ࡣ Death receptor㸦DR㸧4/5 ࡟⤖ྜࡋ࡚ࡀࢇ⣽⬊࡟ᑐࡋ࡚≉␗ⓗ࡟࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࡇ࡜࠿ࡽࠊࡀࢇ἞⒪ཬࡧࡀࢇண㜵࡟࠾࠸࡚ᴟࡵ࡚㔜せ࡞ാࡁࢆᢸ࠺ࡇ࡜ࡀᮇᚅࡉࢀ࡚࠾ࡾୡ⏺୰࡛◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿ 4ࠋ⌧ᅾࡲ࡛࡟ࣄࢺ⤌ࡳ᥮࠼ᆺ TRAIL ࡸ DR4/5 ࢆᶆⓗ࡜ࡋࡓᢠయ་⸆ࡀ㛤Ⓨࡉࢀᵝࠎ࡞ࡀࢇ࡟࠾࠸࡚⮫ᗋヨ㦂ࡀ⾜ࢃࢀ࡚࠸ࡿࡀ TRAIL ⪏ᛶࡢ⋓ᚓࠊ๪స⏝ࡢၥ㢟࠿ࡽ୰Ṇ࡜࡞ࡗࡓ 5, 6ࠋࡇࡢࡼ࠺࡞⫼ᬒ࠿ࡽ TRAIL ⪏ᛶᶵᵓࡢゎ᫂࡜ࠊ⪏ᛶゎ㝖࡟㛵ࢃࡿศᏊࢆྠᐃࡍࡿࡇ࡜ࡀᚲ㡲ㄢ㢟࡛࠶ࡿ࡜ゝ࠼ࡿࠋᮏ◊✲࡛ࡣࠊTRAIL ⪏ᛶゎ㝖࡟᭷ຠ࡞ᶆⓗศᏊ࡜ࡋ࡚ࠊࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝ ࢠ ࣮ ௦ ㅰ ไ ᚚ ᶵ ᵓ 㸦 Warburg ຠ ᯝ 㸧 ࡢ ᡂ ❧ ࡟ ᚲ 㡲 ࡞ 㑇 ఏ Ꮚ PTBP1
㸦Polypyrimidine tract-binding protein㸧࡟╔┠ࡋࡓࠋWarburg ຠᯝ࡜ࡣࠊࡀࢇ⣽1⬊ࡀ㓟⣲ࡢ᭷↓࡟㛵ࢃࡽࡎゎ⢾⣔࡟౫Ꮡࡋࡓ࢚ࢿࣝࢠ࣮௦ㅰࢆ⾜࠺⌧㇟࡛࠶ࡿ7

ࠋPTBP1 ࡣ PKM㸦Pyruvate kinase muscle㸧1/2 㑇ఏᏊࡢࢫࣉࣛ࢖ࢧ࣮࡜ࡋ࡚

ᶵ⬟ࡋࠊࡀࢇ⣽⬊࡟࠾࠸࡚ࡣ㑅ᢥⓗࢫࣉࣛ࢖ࢩࣥࢢ࡟ࡼࡾ PKM2 ࡢⓎ⌧ࡀඃ఩࡜࡞ࡗ࡚࠸ࡿ 8ࠋᡃࠎࡢ◊✲ࢢ࣮ࣝࣉࡣࡇࢀࡲ࡛࡟኱⭠ࡀࢇࡢ⮫ᗋ᳨యࡢ 90㸣
௨ୖ࡛ PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠾ࡾࠊPTBP1 ࡀࡀࢇ࡟࠾ࡅࡿ
Warburg ຠᯝࡢᡂ❧࡟ᚲ㡲࡞㑇ఏᏊ࡛࠶ࡿࡇ࡜ࢆ♧ࡋࡓ 9ࠋᮏ◊✲࡛ࡣࠊWarburg ຠᯝࡢᡂ❧࡟ᚲ㡲࡞㑇ఏᏊ PTBP1 ࡢⓎ⌧࡜ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛ࡬ࡢ㛵୚㸦➨ 3 ❶-2 ⠇㸧࡜ Warburg ຠᯝࡢ⬺ไᚚࢆ௓ࡋࡓάᛶ㓟⣲ࡢⓎ⏕ࡀ TRAIL ⪏ᛶゎ㝖ᶵᵓ࡟㛵୚ࡍࡿᶵᵓ㸦➨ 3 ❶-3ࠊ4 ⠇㸧ࢆ᪂ࡓ࡟᫂ࡽ
࠿࡟ࡋࡓࠋࡉࡽ࡟ࠊCCN1 ࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡬ࡢ㛵୚㸦➨ 3 ❶-5 ⠇㸧࡟ࡘ࠸࡚ሗ࿌ࡍࡿࠋ
ᮏ◊✲࡛ᚓࡽࢀࡓ▱ぢ࠿ࡽࠊWarburg ຠᯝࡢᡂ❧࡟ᚲ㡲࡞㑇ఏᏊ࡛࠶ࡿ PTBP1ࡀ TRAIL ⪏ᛶゎ㝖࡟᭷ຠ࡞ᶆⓗศᏊ࡜࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ௒ᚋࠊPTBP1
ࢆไᚚࡍࡿ໬ྜ≀ࡸ siRNA ࡀࡀࢇࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢ◚⥢ࡢࡳ࡞ࡽࡎ TRAIL ㄏᑟ⣽⬊Ṛࢆ฼⏝ࡋࡓࡀࢇࡢ་⸆ࢩ࣮ࢬ࡜ࡋ࡚ᮇᚅࡉࢀࡿࠋ

2ᮏᏛ఩ㄽᩥࡣࠊୗグࡢཎⴭㄽᩥࢆࡶ࡜࡟సᡂࡋᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉࡟ᥦฟࡋࡓࡶࡢ࡛࠶ࡿࠋ

(1) Perturbation of the Warburg effect increases the sensitivity of cancer cells to
TRAIL-induced cell death.
Minami Kumazaki, Haruka Shinohara, Kohei Taniguchi, Tomoaki Takai, Yuki
Kuranaga, Nobuhiko Sugito, Yukihiro Akao.
Experimental Cell Research; 347(1): 133-142 (2016)(2) Understanding of tolerance in TRAIL-induced apoptosis and cancelationof its machinery by D-mangostin, a xanthone derivative.
Minami Kumazaki, Haruka Shinohara, Kohei Taniguchi, Hiroshi Ueda, Mayuko Nishi, Akihide Ryo, Yukihiro Akao
Oncotarget; 6 (28): 25828-25842 (2015)3

➨ 2 ❶ ◊✲ࡢ⫼ᬒ࡜┠ⓗ➨ 1 ⠇ TRAIL ࢩࢢࢼࣝTRAIL ࡣࠊ1995 ᖺ࡟ Wiley ࡽࡢ◊✲ࢢ࣮ࣝࣉ࡟ࡼࡗ࡚༢㞳ࡉࢀ 10ࠊάᛶ໬ T⣽⬊ࡢ࡯࠿ࠊNK ⣽⬊ࠊ༢⌫ࠊᶞ≧⣽⬊ࠊዲ୰⌫࡞࡝ࡢච␿⣽⬊࡛ࡑࡢⓎ⌧ࡀㄆࡵࡽࢀ࡚࠾ࡾࠊ⣽⬊⭷࡟Ꮡᅾࡍࡿ≉␗ⓗཷᐜయ࡬ࡢ⤖ྜࢆ௓ࡋ࡚࢔࣏ࢺ࣮ࢩࢫ
ㄏᑟࢩࢢࢼࣝࢆ⣽⬊ෆ࡟ఏ㐩ࡍࡿ 11 ࠋTRAIL ཷᐜయ࡟ࡣ TRAIL-R1 㸦Death
receptor 4: DR4㸧ࠊTRAIL-R2 㸦Death receptor 5: DR5㸧ࠊTRAIL-R3 㸦Decoy
receptor 1: DcR1㸧ࠊTRAIL-R4 㸦Decoy receptor 2: DcR2㸧ࠊOsteoprotegerin
㸦OPG㸧ࡢ 5 ✀㢮ࡀᏑᅾࡍࡿ㸦Fig-1㸧12ࠋDR4/5 ࡣࠊࢩࢫࢸ࢖ࣥṧᇶ࡟ᐩࡴࢻ
࣓࢖ࣥࡢ⧞ࡾ㏉ࡋᵓ㐀ࢆ≉ᚩ࡜ࡋࡓ⣽⬊⭷ࣞࢭࣉࢱ࣮࡛࠶ࡾࠊ⣽⬊ෆ㡿ᇦ࡟ࡣDD 㸦Death domain㸧࡜࿧ࡤࢀࡿ࢔࣏ࢺ࣮ࢩࢫࡢㄏᑟ࡟ᚲ㡲ࡢᵓ㐀ࢆ᭷ࡋ࡚࠸
ࡿࠋ
ࡑࡢ௚ 3 ࡘࡢࣞࢭࣉࢱ࣮ࡣࠊTRAIL ㄏᑟ࢔࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡍࡿࠊ‛Decoy㸦࠾࡜ࡾ㸧‘࡜ࡋ࡚ᶵ⬟ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ 13ࠋDcR1/2 ࡣࠊDR4/5 ࡜⣽⬊እ㡿ᇦࡢ┦ྠᛶࡣ㧗࠸ࡀࠊ⣽⬊ෆ㡿ᇦࡢ DD ࡢḞኻཬࡧᶵ⬟୙඲࠿ࡽ TRAIL ࡀ⤖ྜࡋ࡚ࡶ࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡋ࡞࠸ 4, 13ࠋOPG ࡣ TRAIL ࡜ࡢ⤖ྜぶ࿴ᛶࡀ㠀ᖖ࡟ప࠸ࡓࡵ TRAIL ࣞࢭࣉࢱ࣮࡜ࡋ࡚ࡢ⏕≀Ꮫⓗព⩏࡟ࡘ࠸࡚ࡣ᫂ࡽ࠿࡟ࡉࢀ࡚࠸
࡞࠸ࠋ

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Figure-1 Characteristics of TRAIL-R in humans

TRAIL ࡀ DR4/5 ࡟⤖ྜࡍࡿ࡜ࠊ⭘⒆⣽⬊ࡸᙧ㉁㌿᥮ࡋࡓ⣽⬊࡟ᑐࡋ࡚≉␗ⓗ࡟࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࠋX ⥺⤖ᬗᵓ㐀ゎᯒࡢ⤖ᯝࠊTRAIL ࡀ⤖ྜࡍࡿ࡜ DRࡣ࣍ࣔ୕㔞య໬ࡋࠊ
⣽⬊ෆࡢ DD ࡀ࢔ࢲࣉࢱ࣮ศᏊ࡛࠶ࡿ FADD
㸦Fas-associateddeath domain㸧ࢆࣜࢡ࣮ࣝࢺࡉࡏࠊFADD ࢆ௓ࡋ࡚࢝ࢫࣃ࣮ࢮ 8 ࡀ⤖ྜࡍࡿࡇ࡜࡟ࡼࡾ DISC㸦Death-inducing signaling complex㸧࡜࿧ࡤࢀࡿ」ྜయࢆᙧᡂࡍ
ࡿ 14, 15ࠋࡑࡢᚋࠊ࢝ࢫࣃ࣮ࢮ 8 ࡣ⮬ᕫศゎ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࠊ2 ࡘࡢ⤒㊰ࢆ௓ࡋ࡚࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࠋ1 ࡘࡣᐇ⾜࢝ࢫࣃ࣮ࢮ࡛࠶ࡿ࢝ࢫࣃ࣮ࢮ 3 ࡢάᛶ໬࡬㐍ࡴእᅉᛶ⤒㊰࡛࠶ࡿ 15ࠋ௚࡟ࡣࠊBid ࡢษ᩿࠿ࡽ࣑ࢺࢥࣥࢻࣜ࢔⭷㟁఩

5ࡢᾘኻࠊࢩࢺࢡ࣒ࣟ c ࡢᨺฟࠊ࢝ࢫࣃ࣮ࢮ 9 ࡢάᛶ໬ࠊ࢝ࢫࣃ࣮ࢮ 3 ࡢάᛶ໬࡬࡜㐍ࡴෆᅉᛶ⤒㊰࡛࠶ࡿ㸦Fig-2㸧12, 14, 16ࠋ
TRAIL ࡢ⏕≀Ꮫⓗព⩏࡟ࡘ࠸࡚ࡣࠊච␿Ꮫⓗ┘どᶵᵓ࡜⭘⒆ᢚไ࡛࠶ࡿࡇ࡜
ࡀከᩘሗ࿌ࡉࢀ࡚࠸ࡿࠋTRAIL ࡣච␿⣽⬊ࡢࡳ࡟Ⓨ⌧ࡋ࡚࠾ࡾࠊ࣏ࣜከ⢾య㸦lipopolysacharides㸧ࡸ࢖ࣥࢱ࣮ࣇ࢙ࣟࣥ࡞࡝ࡢࢧ࢖ࢺ࢝࢖ࣥ࡟ࡼࡗ࡚ㄏᑟࡉ
ࢀࡿࡇ࡜࠿ࡽࡶච␿Ꮫⓗ┘どᶵᵓࢆᢸࡗ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ 17, 18ࠋࡲࡓࠊ
Zheng ࡽࡢ◊✲ࢢ࣮ࣝࣉ࡟ࡼࡾ TRAIL ࣀࢵࢡ࢔࢘ࢺ࣐࢘ࢫ࡟࠾࠸࡚ࠊࣜࣥࣃ⌫ᩘཬࡧ㦵㧊⣽⬊Ṛࡢῶᑡࠊࣜࢫࢸࣜ࢔⳦ࡢឤᰁ࡟ᑐࡋ࡚పឤཷᛶࢆ♧ࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ 19ࠋ୍᪉ࠊ⭘⒆ᢚไ࡟࠾ࡅࡿ TRAIL ࡢാࡁ࡟ࡘ࠸࡚ࡣࠊWalczackࡽࡢ◊✲ࢢ࣮ࣝࣉ࡟ࡼࡾࣄࢺࡢஙࡀࢇ⣽⬊ࢆ⛣᳜ࡋࡓ࣐࢘ࢫ࡟ recombinant
TRAIL ࢆᢞ୚ࡋࡓ⤖ᯝࠊ⭘⒆ࢧ࢖ࢬࡢ⦰ᑠຠᯝࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽึࡵ࡚᫂
ࡽ࠿࡜࡞ࡗࡓ 20ࠋTRAIL ࢩࢢࢼࣝࡢ⭘⒆ᢚไ࡟࠾ࡅࡿ⏕≀Ꮫⓗព⩏࡜ࡑࡢࡀࢇ
⣽⬊࡟ᑐࡍࡿ≉␗ᛶࡣ㠀ᖖ࡟⯆࿡῝࠸Ⅼ࡛࠶ࡿࠋTRAIL ࢩࢢࢼࣝࡣࠊࡀࢇ⣽⬊≉␗ⓗ࡟ㄏᑟࡉࢀࡿࡇ࡜࠿ࡽࠊࡀࢇ࡟ᑐࡍࡿ᭱ึࡢ㜵ᚚᶵᵓ࡛࠶ࡾࠊࡀࢇ⣽⬊࡜ṇᖖ⣽⬊࡟࠾ࡅࡿ DR4/5 ཬࡧ DcR1/2 ࡢⓎ⌧ࣞ࣋ࣝࡢᕪࡀࡑࡢ≉␗ᛶࢆࡶࡓ
ࡽࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀヲ⣽࡞ᶵᵓࡣᮍࡔ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ࠋ

6

Figure-2 TRAIL-induced apoptosis pathways7➨ 2 ⠇ TRAIL ࢩࢢࢼࣝࢆᶆⓗ࡜ࡋࡓ἞⒪ࡢኚ㑄࡜ࡑࡢၥ㢟Ⅼ
TRAIL ㄏᑟ⣽⬊Ṛࡣࡀࢇ⣽⬊࡟ᑐࡋ࡚㧗࠸≉␗ᛶࢆ᭷ࡍࡿࡇ࡜࠿ࡽࠊࡀࢇ἞⒪ཬࡧࡀࢇண㜵࡟࠾࠸࡚ᴟࡵ࡚㔜せ࡞ാࡁࢆᢸ࠺ࡇ࡜ࡀᮇᚅࡉࢀ࡚࠾ࡾୡ⏺୰࡛◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ1999 ᖺ࡟ࡣࣄࢺ⤌ࡳ᥮࠼ᆺ TRAIL ࢆ⏝࠸ࡓ๓⮫ᗋヨ㦂ࡀ⾜ࢃࢀࠊᙉຊ࡞⭘⒆㏥⦰ຠᯝࢆ♧ࡋࡓࡇ࡜࠿ࡽࠊTRAIL ⤒㊰ࢆ௓ࡍࡿ⣽⬊Ṛ࡟ὀ┠ࡀ㧗ࡲࡗࡓ 21ࠋDulanermin ࡣࠊDR4/5 ࢆⓎ⌧ࡋ࡚࠸ࡿࡀࢇ⣽⬊࡟ᑐࡋ࡚࢝ࢫࣃ࣮ࢮࡢάᛶ໬࡜ p53 ౫Ꮡᛶࡢ࢔࣏ࢺ࣮ࢩࢫࡢㄏᑟࢆᮇᚅࡋ࡚㛤Ⓨࡉࢀࡓࣄࢺ⤌ࡳ᥮࠼ᆺ TRAIL ࡛࠶ࡿࠋࣇࣛࣥࢫࡢ Soria ࡽࡣࠊ213 ྡࡢ㐍⾜ᛶ/෌Ⓨᛶ㠀ᑠ⣽⬊⫵ࡀࢇᝈ⪅ࢆᑐ㇟࡜ࡋ࡚ࠊDulanermin ࡢ paclitaxel࣭carboplatin㸦PC㸧/ paclitaxel࣭carboplatin࣭bevacizumab 㸦PCB㸧࡬ࡢే⏝ຠᯝཬࡧᏳ඲ᛶࢆホ
ࠋDulanermin ࡣࠊᝈ⪅࡟ᑐࡋ࡚ẘᛶ౯ࡍࡿ➨ II ┦ヨ㦂ࢆ⾜ࡗࡓ 5 㸦Table-1A㸧ࡸ๪స⏝ࡣ࡞ࡃⰋዲ࡞᭷⏝ᛶࢆ♧ࡋࡓࡀࠊPC/PCB ࡢ㏣ຍ࡟ࡼࡾ┦஌ⓗ࡞ᢠ⭘⒆ຠᯝࢆ♧ࡉ࡞࠿ࡗࡓࠋࡑࡢၥ㢟Ⅼ࡜ࡋ࡚ࠊࣄࢺ⤌ࡳ᥮࠼ᆺ TRAIL ࡣ⣽⬊Ṛࢆㄏᑟࡋ࡞࠸ DcR1/2 ࡬⤖ྜࡍࡿࡇ࡜ࠊ༙ῶᮇࡀ㠀ᖖ࡟▷࠸ࡇ࡜ࡀᣲࡆࡽࢀࡓ 22ࠋ
㏆ᖺ࡛ࡣࠊTRAIL ཷᐜయࢆάᛶ໬ࡍࡿ᏶඲ࣄࢺᆺࣔࣀࢡࣟࢼ࣮ࣝᢠయࡀ㛤Ⓨࡉࢀᵝࠎ࡞ࡀࢇ࡟ᑐࡋ࡚⮫ᗋヨ㦂ࡀ⾜ࢃࢀࠊᢠయ་⸆࡛ࡢ◊✲㛤Ⓨ౛ࡣᩘከࡃᏑ
ᅾࡍࡿࠋ኱⭠ࡀࢇཬࡧஙࡀࢇ⣽⬊ࡣ࢔࣏ࢺ࣮ࢩࢫࡢㄏᑟ࡟୺࡟ DR5 ࢆ౑⏝ࡋࠊᝏᛶࣜࣥࣃ⭘ࡸ⮅⮚ࡀࢇ⣽⬊ࡣ DR4 ࢆ౑⏝ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊࡀࢇ✀8࡟ࡼࡾᶆⓗ࡜࡞ࡿࣞࢭࣉࢱ࣮ࡀ␗࡞ࡿࡇ࡜࠿ࡽ DR4 㸦mapatumumab㸧ࡶࡋࡃࡣ DR5 㸦drozitumumab, conatumumab, lexatumumab, tigatuzumab, LY-135㸧ࢆࡑࢀࡒࢀᶆⓗ࡜ࡋࡓᢠయ་⸆ࡀ㛤Ⓨࡉࢀ࡚࠸ࡿ 6, 23-26ࠋᢠయ་⸆ࢆ⏝࠸ࡓ⮫ᗋヨ㦂ࡣࠊ࠸ࡃࡘ࠿ࡢ⑕౛࡛࣏ࢪࢸ࢕ࣈ࡞ഴྥࡀᚓࡽࢀࡓࡶࡢࡢᶆ‽ⓗ໬Ꮫ⒪ἲ࡜ࡢే⏝࡛㢧ⴭ࡞ຠᯝࡣᚓࡽࢀ࡞࠿ࡗࡓ㸦Table-1B㸧ࠋ

(A)

(B)

Table-1 Results of Dulanermin and Conatumumab in clinical trials(A and B) Abbreviations: BV, bevacizumab; Chemo, chemotherapy; CR,complete response; PR, partial response; n, number of patients; RCT,
randomized-controlled trials
Anticancer activity was considered when the addition of the TRAIL-receptor
aganist demonstrated statistically significant activity compared with the standard
therapy
9

TRAIL ཷᐜయࢆᶆⓗ࡜ࡋࡓᢠయ་⸆ࡢၥ㢟Ⅼ࡜ࡋ࡚ࠊTRAIL ⪏ᛶࡢ⋓ᚓࡀᣲࡆࡽࢀࡓ 27ࠋᐇ㝿࡟ࠊ㌿⛣ࡋࡓ⭘⒆ࡢ኱㒊ศࡣ TRAIL ࡟⪏ᛶࢆ♧ࡋࠊTRAIL ⪏ᛶࡣࡀࢇ἞⒪ࡢ㐣⛬࡛ᘬࡁ㉳ࡇࡉࢀ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ TRAIL ࢩࢢࢼࣝࢆᶆ
ⓗ࡜ࡋࡓ἞⒪ࡢ኱ࡁ࡞㞀ቨ࡜࡞ࡗ࡚࠸ࡿ 28ࠋTRAIL ⪏ᛶࢆ⋓ᚓࡋࡓ⣽⬊࡛ࡣࠊ࢔࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR4/5 ࡢⓎ⌧పୗࡸࠊ࢔࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ⌧␗ᖖࡢ㛵୚ࡀ♧၀ࡉࢀ࡚࠸ࡿ 29, 30ࠋ௨ୖࡢࡇ࡜࠿ࡽࡶ TRAIL ⪏ᛶᶵᵓࡢゎ᫂࡜ࠊ⪏ᛶゎ㝖࡟㛵ࢃࡿศᏊࢆྠᐃࡍࡿࡇ࡜ࡀᚲ㡲ㄢ㢟࡛࠶ࡿ࡜ゝ࠼ࡿࠋ

10

➨ 3 ⠇ TRAIL ⪏ᛶᶵᵓ௨๓ࡼࡾࠊTRAIL ⪏ᛶ࣓࢝ࢽࢬ࣒ࡣ࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡋ࡞࠸ DcR1/2 ࡟ࡼࡗ࡚ㄪ⠇ࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡓࠋᐇ㝿࡟ࠊDcR1/2 ࡢ㧗Ⓨ⌧ࡣึᮇࡢ⫶ࡀࢇࡸஙࡀࢇ࡛㧗㢖ᗘ࡟ㄆࡵࡽࢀ࡚࠾ࡾணᚋ୙Ⰻ࡜┦㛵ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚
࠸ࡿࡀࠊTRAIL ࡟ᑐࡍࡿឤཷᛶ࡜ࡢ┦㛵㛵ಀࡣᮍࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ㏆ᖺࠊ
TRAIL ⪏ᛶࢆ⋓ᚓࡋࡓ⣽⬊࡛ࡣࠊ࢔࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR4/5 ࡢⓎ⌧పୗࡸFLIP㸦FLICE-inhibitory protein㸧ࠊBCL-2㸦B-cell lymphoma 2㸧ࣇ࢓࣑࣮ࣜࠊIAP
㸦Inhibitor of apoptosis protein㸧ࣇ࢓࣑࣮ࣜ࡜࠸ࡗࡓ࢔࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ⌧␗ᖖࡢ㛵୚ࡀ♧၀ࡉࢀ࡚࠸ࡿ 29, 30ࠋ࢔࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR4/5 ࡢⓎ⌧పୗࡀࢇ࡟࠾ࡅࡿ㧗࣓ࢳࣝ໬ࡸⅬ✺↛ኚ␗࡞࡝ࡢ࢚ࣆࢪ࢙ࢿࢸࢵࢡ࡞ኚ໬࡟ࡼࡿ࢔
࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR4/5 ࡢⓎ⌧పୗࡀ TRAIL ⪏ᛶ࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌
ࡉࢀ࡚࠸ࡿ 30-32ࠋᚑࡗ࡚ࠊᑡ࡞ࡃ࡜ࡶᶵ⬟ⓗ࡞ DR4/5 ࡢⓎ⌧ࡣ TRAIL ㄏᑟ⣽⬊Ṛࡢάᛶ࡟ᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋᡃࠎࡣࡇࢀࡲ࡛࡟ࣄࢺ኱⭠⒴⣽⬊ᰴ
DLD-1 ࡟ᑐࡍࡿ TRAIL ⪏ᛶᰴ㸦DLD-1/TRAIL㸧ࢆస〇ࡍࡿࡇ࡜࡟ࡼࡾ TRAIL ⪏ᛶᶵᵓ࡜ࡋ࡚ DR5 ࡢⓎ⌧పୗ࡜ DR5 ࡢ⣽⬊⾲㠃ୖ࡬ࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ୙
Ⰻࢆ᫂ࡽ࠿࡟ࡋࡓ㸦Fig-3A and B㸧ࠋFLIP ࡢ㐣๫Ⓨ⌧

11

FLIP ࡣࠊ࢝ࢫࣃ࣮ࢮ 8 ࡜ᵓ㐀ⓗ࡟㢮ఝࡋ࡚࠾ࡾ FLIPS ࡜ FLIPRࠊFLIPL ࡢ 3 ࡘࡢ࢔࢖ࢯࣇ࢛࣮࣒ࡀᏑᅾࡍࡿ 33ࠋFLIPS ࡜ FLIPR ࡣ࢝ࢫࣃ࣮ࢮ 8 ࡜➇ྜࡋ DISC
ᙧᡂࢆ㜼ᐖࡍࡿ 34, 35ࠋ୍᪉ࠊFLIPL ࡢ࢔࣏ࢺ࣮ࢩࢫ࡟࠾ࡅࡿാࡁࡣ」㞧࡛ࡑࡢⓎ⌧ࣞ࣋ࣝ࡟౫Ꮡࡋ࡚࠸ࡿࠋ㐣๫Ⓨ⌧ࡋࡓ FLIPL ࡣ࢝ࢫࣃ࣮ࢮ 8 ࡜࣊ࢸࣟ஧㔞యࢆᙧᡂࡍࡿࡇ࡜࡛࢝ࢫࣃ࣮ࢮࡢάᛶ໬ࢆ㜼ᐖࡍࡿ 36ࠋFLIP ࡢ㐣๫Ⓨ⌧ࡣከࡃࡢࡀࢇ࡛ㄆࡵࡽࢀ࡚࠾ࡾࠊTRAIL ࢩࢢࢼࣝࢆ฼⏝ࡋࡓࡀࢇ἞⒪ࡢᶆⓗ࡜ࡋ࡚ᮇᚅ
ࡉࢀ࡚࠸ࡿࠋ
BCL-2 ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢⓎ⌧␗ᖖ
BCL-2 ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣࠊ࣑ࢺࢥࣥࢻࣜ࢔ࡢ⭷㏱㐣ᛶࢆไᚚࡋ࣑ࢺࢥࣥ
ࢻࣜ࢔ࢆ௓ࡋࡓ࢔࣏ࢺ࣮ࢩࢫࡢಁ㐍ࡸ㜼ᐖ࡟㛵୚ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛ࡢ◊✲࠿ࡽ࢔࣏ࢺ࣮ࢩࢫಁ㐍ᛶࡢ BAX/BAK ࡢⓎ⌧పୗ࡜ྠᵝ࡟ࠊ࢔࣏ࢺ࣮ࢩࢫᢚไᛶࡢ
BCL-2ࠊ BCL-XLࠊMCL-1 ࡢ㐣๫Ⓨ⌧ࡣࠊࡀࢇ࡟࠾ࡅࡿ໬Ꮫ⒪ἲࡸᨺᑕ⥺⒪ἲ
࡟᢬ᢠᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿ 37-39ࠋTRAIL ࢩࢢࢼࣝ࡟㛵ࡋ࡚ࡶ
BCL-2 ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢⓎ⌧␗ᖖࡀ TRAIL ⪏ᛶ࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ
IAP ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢ㐣๫Ⓨ⌧
IAP ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣ࢝ࢫࣃ࣮ࢮࢆ┤᥋ⓗ࡟㜼ᐖࡍࡿࡇ࡜࡟ࡼࡗ࡚ෆᅉ
ᛶࠊእᅉᛶ࠸ࡎࢀࡶࡢ࢔࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡍࡿࠋࡀࢇ࡟࠾࠸࡚ IAP ࡸ XIAP ࡢ㐣

12๫Ⓨ⌧ࡀㄆࡵࡽࢀ࡚࠸ࡿࠋXu ࡽࡢ◊✲ࢢ࣮ࣝࣉࡣ IAP ࡢⓎ⌧ᢚไ࡟㛵୚ࡍࡿࢩࢫࣉࣛࢳࣥࢆᢞ୚ࡍࡿࡇ࡜࡟ࡼࡾ TRAIL ࡢឤཷᛶࡀᅇ᚟ࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠾
ࡾ IAP ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢ㐣๫Ⓨ⌧࡜ TRAIL ⪏ᛶࡢ㛵୚ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ
௒ᚋ TRAIL ࣞࢭࣉࢱ࣮࡛࠶ࡿ DR4/DR5 ࡢⓎ⌧ࢆࡉࡽ࡟ቑᙉࡍࡿపศᏊ໬ྜ≀࡜ TRAIL ࡢే⏝ࡶࡋࡃࡣࠊ࢔࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ⌧ᢚไ࡜ TRAIL ࡜ࡢే⏝࡟ࡼࡾእᅉᛶ⤒㊰ࡢ࢔࣏ࢺ࣮ࢩࢫࢆቑᙉࡉࡏࡿ࡞࡝ࠊTRAIL ⪏ᛶᶵᵓ࡟ྜࢃࡏ
ࡓඞ᭹ἲࡀᮇᚅࡉࢀࡿࠋ
(A)

(B)

㸦Kumazaki et al., 2015 ࡼࡾᢤ⢋)Figure-3(A) Western blot analysis was performed to determine steady-state expression of DR5,
DR4, and adaptor molecule FADD. E-actin was used as an internal control. Also shown
are the steady-state expression levels of DR5 mRNA as relative ratios with respect to
the GAPDH expression level. The expression level of mRNA was calculated by the''Ct method. Means S.D. indicated by error bars are shown. (B)The photomicrographshows the results of immunofluorescence staining for DR5 (anti-DR5) on the cellsurface and in the cytosol of DLD-1 and DLD-1/TRAIL cells. Nuclei were counterstainedin blue with Hoechst33342.
13

➨ 4 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰไᚚᶵᵓ㸸Warburg ຠᯝ㏆ᖺࠊࡀࢇ࡟ᑐࡍࡿ᪂ࡓ࡞๰⸆ࡢࢱ࣮ࢤࢵࢺ࡜ࡋ࡚࢚ࢿࣝࢠ࣮௦ㅰࡀὀ┠ࡉࢀ࡚࠸ࡿࠋࡀࢇ⣽⬊ࡣప㓟⣲࣭పᰤ㣴≧ែ࡟࠾࠸࡚㝈ࡽࢀࡓ࢚ࢿࣝࢠ࣮※ࢆ᭷ຠ฼⏝ࡍࡿࡓࡵ࡟௦ㅰᶵᵓࡢࣜࣉࣟࢢ࣑ࣛࣥࢢࢆ⾜ࡗ࡚࠸ࡿࠋࡑࡢ୍ࡘࡀ
ࠕWarburg ຠᯝࠖ࡜࠸࠺⌧㇟࡛࠶ࡿࠋWarburg ຠᯝࡣࠊࠕࡀࢇ⣽⬊ࡣዲẼⓗ᮲௳ୗ࡟࠾࠸࡚ࡶ TCA ࢧ࢖ࢡࣝࢆά⏝ࡏࡎࠊᖖ࡟᎘Ẽⓗゎ⢾ࢆ฼⏝ࡋ࡚ࢢࣝࢥ࣮ࢫ
ࢆ௦ㅰࡋࠊ኱㔞ࡢங㓟ࢆศἪࡍࡿࠖ࡜࠸࠺⌧㇟ࢆ Otto Warburg ࡀᥦၐࡋࡓࡶࡢ
࡛࠶ࡿ 7, 40ࠋࡀࢇ⣽⬊ࡀ Warburg ຠᯝࢆ⋓ᚓࡍࡿࡢ࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠸
ࡿࡢࡀࠊPKM ࡛࠶ࡿࠋWarburg ຠᯝࡢ⋓ᚓᶵᵓࢆ Figure-4 ࡟♧ࡋࡓࠋPKM ࡣゎ⢾⣔ࡢ᭱⤊ࢫࢸࢵࣉ࡛࠶ࡿ࣍ࢫ࢚࣍ࣀ࣮ࣝࣆࣝࣅࣥ㓟㸦PEP㸧࠿ࡽࣆࣝࣅࣥ
㓟࡬ࡢ཯ᛂࢆゐ፹ࡍࡿ㓝⣲࡛࠶ࡾࠊゎ⢾⣔ࡢᚊ㏿㓝⣲࡜ࡋ࡚ാࡃ 41ࠋPKM ࡟ࡣ
PKM1 ࡜ PKM2 ࡢ 2 ࡘࡢ࢔࢖ࢯࣇ࢛࣮࣒ࡀᏑᅾࡋࠊࢫࣉࣛ࢖ࢩࣥࢢ㐣⛬࡟࠾࠸࡚࢚࢟ࢯࣥ 8ࠊ9ࠊ11 ࢆྲྀࡾ㎸ࡴ࡜ PKM1ࠊ࢚࢟ࢯࣥ 8ࠊ10ࠊ11 ࢆྲྀࡾ㎸ࡴ࡜
PKM2 ࡀసࡽࢀࡿ 42 ࠋࡀࢇ⣽⬊࡟࠾࠸࡚ࡣࢫࣉࣛ࢖ࢧ࣮࡛࠶ࡿ hnRNPA1ࠊ
hnRNPA2ࠊPTBP1 ࡀ㧗Ⓨ⌧ࡍࡿࡇ࡜࡛࢚࢟ࢯࣥ 9 ࡢྲྀࡾ㎸ࡳࡀ㜼ᐖࡉࢀࠊ࢚࢟
ࢯࣥ 10 ࢆྲྀࡾ㎸ࢇࡔ PKM2 ࡀ㧗Ⓨ⌧ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ 43-45ࠋⴭ⪅ࡽࡣࠊ኱⭠⒴ࡢ⮫ᗋ᳨యࡢ 90%௨ୖ࡛ PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠾ࡾࠊ࡯ࡰࡍ࡭࡚ࡢࡀࢇ✀࡟࠾࠸࡚ PKM2 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇ࡜ࢆ☜ㄆࡋ࡚࠸ࡿ 46ࠋ

14

TCA ࢧ࢖ࢡࣝࢆ௓ࡋࡓࢢࣝࢥ࣮ࢫ௦ㅰ࡛ࡣࢢࣝࢥ࣮ࢫ 1 ࣔࣝᙜࡓࡾ 36ATP ࡀ⏘⏕ࡉࢀࡿࡢ࡟ᑐࡋࠊゎ⢾⣔࡛ࡣ 2ATP ࡋ࠿⏘⏕ࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠸ࡓࡵ ATP ࡢ⏘⏕ຠ⋡ࡀప࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊゎ⢾⣔ࡣ TCA ࢧ࢖ࢡࣝ࡜ẚ㍑ࡋ࡚཯ᛂࢫࢸࢵ
ࣉࡀ༢⣧࡛࠶ࡿࡓࡵ ATP ࡢ⏘⏕㏿ᗘࡀ㏿ࡃࠊࡀࢇ⣽⬊ࡣࢢࣝࢥ࣮ࢫࡢྲྀࡾ㎸ࡳࢆஹ㐍ࡉࡏࡿࡇ࡜࡛኱㔞ࡢ ATP ⏘⏕ࢆྍ⬟࡟ࡋ࡚࠸ࡿࠋࡲࡓࠊゎ⢾⣔ࡢ౑⏝࡟
ࡣ 2 ࡘࡢ฼Ⅼࡀᣲࡆࡽࢀࠊࡘࡲࡾ TCA ࢧ࢖ࢡࣝࡢ㐣⛬࡛Ⓨ⏕ࡍࡿάᛶ㓟⣲㸦reactive oxygen species; ROS㸧ࡢⓎ⏕ࢆᢚไ࡛ࡁࡿࡇ࡜࡛࠶ࡿࠋ⣽⬊ෆ࡛㐣๫⏘⏕ࡉࢀࡓ ROS ࡣ࢔࣏ࢺ࣮ࢩࢫࡸ࣮࢜ࢺࣇ࢓ࢪ࣮➼ࡢࣉࣟࢢ࣒ࣛ⣽⬊Ṛࡢཎᅉ࡜࡞ࡿࡓࡵࠊゎ⢾⣔ࢆ౑⏝ࡍࡿࡇ࡜࡛ ROS ࡢⓎ⏕ࢆᢚไࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡶ࠺୍ࡘࡢ฼Ⅼࡣࠊゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࢆ฼⏝ࡋࡓ࣌ࣥࢺ࣮ࢫࣜࣥ
㓟ᅇ㊰࡟ࡼࡿ᰾㓟ྜᡂࡢಁ㐍࡛࠶ࡿࠋPKM1 ࡀᖖ࡟άᛶᆺࡢᅄ㔞య࡛Ꮡᅾࡍࡿ
ࡢ࡟ᑐࡋ࡚ࠊPKM2 ࡣẚ㍑ⓗάᛶࡢప࠸༢㔞యࡶࡋࡃࡣ஧㔞య࡛Ꮡᅾࡍࡿࠋࡇ
ࡢከ㔞య໬ࡢไᚚᶵᵓ࡟ࡣ PKM2 ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࡀ㛵୚ࡋ࡚࠾ࡾࠊࣜࣥ㓟໬ࢆཷࡅࡓ PKM2 ࡣᅄ㔞యࡢᙧᡂࡀᅔ㞴࡟࡞ࡿࠋPKM2 ࡢ࢟ࢼ࣮ࢮάᛶࡀప࠸
≧ែ࡟ಖࡓࢀࡿࡇ࡜࡛ࠊ⣽⬊ෆ࡛ࡣゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࡀ⵳✚ࡋࡸࡍࡃ࡞ࡿࠋ
⣽⬊ࡣࡇࡢ୰㛫௦ㅰ⏘≀ࢆ࣌ࣥࢺ࣮ࢫࣜࣥ㓟⤒㊰㸦PPP㸧࡟ືဨࡋࠊ⣽⬊ศ⿣࡟ᚲせ࡞᰾㓟ྜᡂ࡟౑⏝ࡋ࡚࠸ࡿࠋ
௨ୖࡢ㏻ࡾࠊWarburg ຠᯝࡣࡀࢇ⣽⬊ࡢቑṪࠊ⏕Ꮡࠊ࢚ࢿࣝࢠ࣮ࡢ⋓ᚓ࡟ᴟࡵ15࡚㔜せ࡞ᶵᵓ࡛࠶ࡿࠋ

Figure-4 Warburg effect16

➨ 5 ⠇ CCN1 ࡢⓎ⌧࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡬ࡢ㛵୚
 CCN1㸦CYR61㸧ࡣ CCN ࣇ࢓࣑࣮ࣜ࡟ᒓࡋࠊቑṪᅉᏊࠊࢧ࢖ࢺ࢝࢖ࣥࠊప㓟⣲≧ែ࡟ࡼࡿ⣽⬊ࢫࢺࣞࢫ࡞࡝ᵝࠎ࡞่⃭࡟ࡼࡗ࡚ㄏᑟࡉࢀࡿ⣽⬊እ࣐ࢺࣜࢵ
ࢡࢫࢱࣥࣃࢡ㉁࡛࠶ࡿ 47ࠋCCN1 ࡣ 4 ࡘࡢࢻ࣓࢖ࣥ࠿ࡽᵓᡂࡉࢀ࡚࠾ࡾࡑࢀࡒ
ࢀࡀ⤖ྜࡍࡿ࢖ࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮ࡀ␗࡞ࡿࡇ࡜࠿ࡽ⣽⬊ࡢ᥋╔ࠊ㐟㉮ࠊቑṪࠊ⏕Ꮡ࡞࡝ࡢᵝࠎ࡞⣽⬊άᛶࡢไᚚ࡟㛵୚ࡋ࡚࠸ࡿ 48ࠋ࢖ࣥࢸࢢࣜࣥࡣ 2 㔞యࢱࣥࣃࢡ㉁࡛࠶ࡾࠊȘ࡜șࡢࢧࣈࣘࢽࢵࢺࡀ㠀ඹ᭷⤖ྜ࡛఍ྜࡋάᛶࡢ࠶ࡿ 2㔞యࢆᙧᡂࡋ࡚࠸ࡿ 49, 50ࠋȘ࡜șࡢࢧࣈࣘࢽࢵࢺࡣᩘከࡃᏑᅾࡋࠊ20 ✀㢮௨ୖࡢȘșࡢ࢖ࣥࢸࢢࣜࣥࡀྠᐃࡉࢀ࡚࠾ࡾࠊࡑࡢ⤌ࡳྜࢃࡏ࡟ࡼࡾ⤖ྜࡍࡿศᏊ
ࡀ␗࡞ࡿ 50, 51ࠋCCN1 ࡣ⣽⬊ࡢቑṪࡸ⏕Ꮡ࡟㛵୚ࡍࡿ୍᪉ࠊ⥺⥔ⱆ⣽⬊࡟࠾࠸࡚ CCN ࢱࣥࣃࢡ㉁ࡣࠊTNF-Dࡶࡋࡃࡣ FasL Ꮡᅾୗ࡛࢔࣏ࢺ࣮ࢩࢫࢆቑᙉࡍࡿ
ࡇ࡜ࡀ in vitro ཬࡧ in vivo ࡢ⣔࡛᫂ࡽ࠿࡟࡞ࡗ࡚࠸ࡿ 52ࠋࡑࡢ࣓࢝ࢽࢬ࣒ࢆ
Figure-5 ࡟♧ࡍࠋCCN1 ࡣ࢖ࣥࢸࢢࣜࣥD6E1 ࡜ࢩࣥࢹ࢝ࣥ 4㸦Syndecan-4㸧࡬ࡢ⤖ྜࢆ௓ࡋ࡚ 5-࣏ࣜ࢟ࢩࢤࢼ࣮ࢮ㸦5-Lox㸧ࡢάᛶ໬ࢆྵࡴᵝࠎ࡞࣓࢝ࢽࢬ࣒
࡟ࡼࡾ኱㔞ࡢ ROS ࢆⓎ⏕ࡉࡏࡿࠋ㏻ᖖ TNF-D༢⊂࡛ࡣࠊNF-NEࡀ࢝ࢫࣃ࣮ࢮࡢ
άᛶࢆ㜼ᐖࡍࡿ c-FLIP ࡢㄏᑟࡸ ROS ࢆᢚไࡍࡿࡇ࡜࡛ JNK㸦c-Jun N-terminal
kinase㸧ࡢάᛶ໬ࢆ㜼ᐖࡍࡿࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮࢆㄏᑟࡍࡿࡇ࡜࡟ࡼࡾ TNF-Dㄏᑟᛶ࢔࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡋ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊCCN1 ࡟ࡼࡗ࡚⏘⏕ࡉࢀࡓ17

ROS ࡣ JNK ࡢάᛶ໬ࢆಁ㐍⥔ᣢࡋࠊࡑࡢ⤖ᯝ FLIP ࡢാࡁࢆ㜼ᐖࡍࡿࡇ࡜࡛
TNF-Dㄏᑟᛶࡢ࢔࣏ࢺ࣮ࢩࢫࢆಁ㐍ࡍࡿࠋࡉࡽ࡟ࠊROS ࡢⓎ⏕ࡣ p38/MAPK ࢆάᛶ໬ࡉࡏࠊBAX ࡢ࣑ࢺࢥࣥࢻࣜ࢔࡬ࡢᒁᅾཬࡧࢩࢺࢡ࣒ࣟ c ࡢᨺฟࢆㄏᑟࡋࠊ
FasL ࢆ௓ࡋࡓ࢔࣏ࢺ࣮ࢩࢫࢆಁ㐍ࡍࡿࠋࡀࢇ⣽⬊࡟࠾ࡅࡿ CCN1 ࡢാࡁ࡟ࡘ࠸
࡚ࡶ⥺⥔ⱆ⣽⬊࡜㢮ఝࡋ࡚࠸ࡿࠋࡀࢇࡢ㛫㉁࡛ࡣ⥺⥔ⱆ⣽⬊ࡀάᛶ໬ࡉࢀ࡚࠾
ࡾࠊࡀࢇ࡟࠾ࡅࡿ CCN1 ࡢ㐣๫Ⓨ⌧ࡣࠊAKT ࡸ ERK ࢩࢢࢼࣝࢆάᛶ໬ࡉࡏࡿࡇ࡜࡛⭘⒆ቑṪࡸ⾑⟶ᐦᗘࢆୖ᪼ࡉࡏࡿࡇ࡜ࡀච␿୙඲࣐࢘ࢫࢆ⏝࠸ࡓᐇ㦂ࡼࡾ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿ 51ࠋ㏆ᖺࠊ๓❧⭢ࡀࢇ࡟࠾࠸࡚ࡶ TRAIL Ꮡᅾୗ࡛ࡣ࢖ࣥ
ࢸࢢࣜࣥࣞࢭࣉࢱ࣮DvE3ࠊD6E4 ࡬ࡢ⤖ྜࢆ௓ࡋ࡚ TRAIL ㄏᑟ⣽⬊Ṛࢆቑᙉࡍࡿ
ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ 52ࠋ๓❧⭢ࡀࢇ⣽⬊ PC3 ࡟࠾ࡅࡿ TRAIL ࡜ࡢ┦஌ຠᯝ࡟ࡣ ROS ࡢⓎ⏕ࡣ㛵୚ࡋ࡚࠾ࡽࡎ PKC 㸦Protein kinase C㸧Dࡢάᛶ໬౫Ꮡⓗ࡟ㄏᑟࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀヲ⣽࡞ᶵᵓࡣ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ 52ࠋ
CCN1 ࡢ࢔࣏ࢺ࣮ࢩࢫㄏᑟᶵᵓࡣ CCN1 ࡢ࢖ࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮௨እ࡬ࡢ⤖ྜࡶࡋࡃࡣ TRAIL ㄏᑟ⣽⬊Ṛ࡟㛵୚ࡍࡿࣞࢭࣉࢱ࣮㸦DR4/5㸧ࡢ఍ྜ࡟㛵୚ࡍࡿࡇ࡜࠿ࡽ TRAIL ㄏᑟ⣽⬊Ṛࡀಁ㐍ࡉࢀࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼ࡽࢀࡿࠋ

18

Figure-5 Signaling crosstalk between CCN1 and TNF-D
D or FasL in fibroblasts19➨ 3 ❶ Warburg ຠᯝ㛵㐃 PTBP1 ࡜ TRAIL ㄏᑟ࢔࣏ࢺ࣮ࢩࢫ
➨ 1 ⠇ ᗎ
TRAIL ࢆ௓ࡋࡓࡀࢇ⣽⬊≉␗ⓗ࡞⣽⬊Ṛࡣᢠ⭘⒆⸆ࡢᶆⓗ࡜ࡋ࡚ᮇᚅࡉࢀ࡚
࠸ࡿࠋࣄࢺ⤌ࡳ᥮࠼ᆺ TRAIL ࡣࣄࢺࡢ⏕యෆ࡟࠾ࡅࡿ༙ῶᮇࡀ㠀ᖖ࡟▷ࡃࠊ㔜⠜࡞⫢ᶵ⬟㞀ᐖࡢᠱᛕࡀ࠶ࡿࡇ࡜࠿ࡽࠊ⌧ᅾࡣ DR4/5 ࢆᶆⓗ࡜ࡋࡓᢠయ་⸆ࡢ
㛤Ⓨࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊTRAIL ⪏ᛶࡢ⋓ᚓࡀ኱ࡁ࡞㞀ቨ࡜࡞ࡗ࡚
࠾ࡾᮍࡔ᭷ຠ࡞἞⒪ἲࡣ☜❧ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊTRAIL ㄏᑟ⣽⬊Ṛࢆ฼⏝ࡋࡓࡀࢇࡢ἞⒪⸆ࡢ㛤Ⓨ࡟ྥࡅ TRAIL ⪏ᛶゎ㝖࡟㛵ࢃࡿศᏊࡢྠᐃཬ
ࡧࡑࡢ⪏ᛶゎ㝖ᶵᵓࡢゎ᫂ࢆヨࡳࡓࠋ20

➨ 2 ⠇ PTBP1 ࡢⓎ⌧࡜ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛ࡬ࡢ㛵୚ࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰᶵᵓ࡛࠶ࡿ Warburg ຠᯝࡣ PTBP1/PKM ࢝ࢫࢣ࣮ࢻ࡟ࡼࡗ࡚ᡂ❧ࡋ࡚࠾ࡾࠊࡇࢀࡲ࡛࡟኱⭠ࡀࢇࡢ⮫ᗋ᳨యࡢ 90%௨ୖ࡛
PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋ2 ✀⣽⬊ᰴࢆ⏝࠸࢔ࣞ࢖ゎᯒ㸦㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝ㸧ࢆ⾜ࡗࡓ࡜ࡇࢁࠊDLD-1 ࡜ẚ㍑ࡋ࡚ DLD-1/TRAIL ࡟࠾
࠸࡚ PTBP1 ࡢ㧗Ⓨ⌧ࡀㄆࡵࡽࢀࡓࠋᐇ㝿࡟ᐃᖖ≧ែ࡟࠾ࡅࡿ PTBP1 ࡢࢱࣥࣃࢡ㉁࡛ࣞ࣋ࣝࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲ࡟ࡼࡾ᳨ドࡋࡓ࡜ࡇࢁࠊ࢔ࣞ࢖ゎᯒ ࡢ ⤖ ᯝ࡜ྠᵝ࡟ DLD-1/TRAIL ࡟࠾࠸࡚ PTBP1 ࡢ㧗Ⓨ⌧ࡀ ☜ㄆࡉࢀࡓ㸦Fig-6A㸧ࠋ(A)

Figure-6(A) Western blot analysis was performed to determine the steady-state expression of
PTBP1 in TRAIL-sensitive DLD-1 and –resistant DLD-1 cells. E-actin was used as aninternal control.

21ࡑࡇ࡛ࠊsiRNA ࢆ⏝࠸࡚ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ᫬ࡢ TRAIL ࡟ᑐࡍࡿឤཷᛶ࡬ࡢᙳ㡪ࢆホ౯ࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࡟࠾࠸࡚ࡶ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥ ࡋ ࡓ ⣽ ⬊ ࡛ ࡣ ࠊ ⣙ 20 㸣 ࡢ ⏕ ⣽ ⬊ ᩘ ࡢ ῶ ᑡ ࡀ ㄆ ࡵ ࡽ ࢀ ࡓ 㸦 Fig-6B 㸧ࠋ
DLD-1/TRAIL ࡟࠾࠸࡚ቑṪᢚไຠᯝࢆ♧ࡉ࡞࠸ TRAIL 5 ng/ml ࡟࠾࠸࡚ࡶ
PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡛ TRAIL ࡟ࡼࡿ⣽⬊Ṛࡀ┦஌ⓗ࡟ㄏᑟࡉࢀࡿࡇ
࡜ࡀศ࠿ࡗࡓ㸦Fig-6B㸧ࠋFigure-6C ࡛♧ࡍࡼ࠺࡟ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡛ࡣ TRAIL ㄏᑟ࢔࣏ࢺ࣮ࢩࢫࡢᣦᶆ࡛࠶ࡿ࢝ࢫࣃ࣮ࢮ 8 ࡢ㢧ⴭ࡞άᛶ໬ࡀㄆ
ࡵࡽࢀࡓࠋ
(B)

22

(C)

Figure-6(B) TRAIL-sensitive and -resistant DLD-1 cells were transfected with siR-PTBP1 (2 nM)
for 48 h and then treated with rTRAIL (5, 10 ng/ml) for 24 h. The cell viability wasestimated at 72 h after the transfection. The cell viability of the control (0; PBS alone) is
indicated as 100 %. The growth inhibition effect by TRAIL with and without thetransfection with siR-PTBP1 was assessed by the average value of the growth inhibition
ratios (GI) at each TRAIL concentrations (5, 10 ng/ml). We defined the synergistic effect(Syn) by introduction of siR-PTBP1 as the ratios of siR-PTBP1 to control siRNA GI value.
(C) Western blot analysis was performed to determine the level of the active form of
caspase-8. E-actin was used as an internal control.

ࡉࡽ࡟ࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡟࠾ࡅࡿ࢚ࢿࣝࢠ࣮௦ㅰ࡬ࡢᙳ㡪ࢆ᳨ドࡍࡿ࡜ PKM2 ࠿ࡽ PKM1 ࡬ࡢࢫ࢖ࢵࢳࡀほᐹࡉࢀࡓ(Fig-6D)ࠋPKM1 ࡢⓎ⌧ࡀ᭷ព࡜࡞ࡿࡇ࡜࡛ゎ⢾⣔࠿ࡽ㓟໬ⓗࣜࣥ㓟໬࡬ࡢࢩࣇࢺࡀண
ࡢ ATP ࣞ࣋ࣝ࡜ゎ⢾⣔ࡢ᭱⤊⏘≀࡛࠶ࡿங㓟ࣞ࣋ࣝࡢࡉࢀࠊ⣽⬊ෆ

ᐃࢆ⾜ࡗࡓࠋPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡛ࡣࠊ⣽⬊ෆࡢ ATP ࣞ࣋ࣝࡢቑຍ࡜ங㓟ࣞ࣋ࣝࡢῶᑡࡀ
ㄆࡵࡽࢀࡓ㸦Fig-6E㸧ࠋࡇࡢ⤖ᯝ࠿ࡽࠊPTBP1 ࡢⓎ⌧పୗ࡟ࡼࡾ⣽⬊ෆࡢ࢚ࢿࣝ
ࢠ࣮௦ㅰࡀ୍㒊ゎ⢾⣔࠿ࡽ㓟໬ⓗࣜࣥ㓟໬࡬ࢩࣇࢺࡋ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ

23

(D)

(E)

Figure-6(D) TRAIL-sensitive and -resistant DLD-1 cells were transfected with control or PTBP1siRNA (siR-PTBP1; 2 nM) for 48 h. Western blot analysis was performed to determine
the expression of Warburg effect-related genes. E-actin was used as an internal control.
(E) TRAIL-sensitive and -resistant DLD-1 cells was transfected with control or
siR-PTBP1 (2, 5 nM) for 72 h. The ATP and lactate production were normalized to cellnumbers, and that of the Control (0) is indicated as “1”.

ᐇ㝿࡟ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿ Warburg ຠᯝࡢ⬺ไᚚ㸦ゎ⢾⣔࠿ࡽ㓟໬ⓗࣜࣥ㓟໬࡬ࡢࢫ࢖ࢵࢳ㸧ࡀ TRAIL ࡟ᑐࡍࡿឤཷᛶࡢᅇ᚟࡟㛵୚ࡋ࡚࠸ࡿࡢ࠿
ࢆゎ⢾⣔ࡢ㜼ᐖ๣࡛࠶ࡿ 2-ࢹ࢜࢟ࢩࢢࣝࢥ࣮ࢫ㸦2-DG㸧ࢆ⏝࠸᳨࡚ドࡋࡓࠋ
DLD-1/TRAIL ࡟ 2-DG ࢆ 48 ᫬㛫స⏝ࡉࡏࡓᚋ TRAIL ࢆᢞ୚ࡋࡓ⣽⬊࡛ࡣ⏕⣽⬊ᩘࡢ㢧ⴭ࡞ῶᑡ࡜ࠊ࢝ࢫࣃ࣮ࢮ 8 ࡢάᛶ໬ࡀㄆࡵࡽࢀࡓ㸦Fig-6F㸧ࠋࡇࢀࡽࡢ

24⤖ᯝ࠿ࡽࠊWarburg ຠᯝࡢᡂ❧࡟ᚲ㡲࡞㑇ఏᏊ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜
࡟ࡼࡿ Warburg ຠᯝࡢ⬺ไᚚࢆ௓ࡋ࡚ TRAIL ࡟ᑐࡍࡿឤཷᛶࡀᅇ᚟ࡍࡿࡇ࡜ࡀ
᫂ࡽ࠿࡜࡞ࡗࡓࠋ

(F)

Figure-6(F) TRAIL-resistant DLD-1 cells were pretreated with 2-DG (5 mM) for 24 h and thentreated with rTRAIL (5, 10, 25 ng/ml) for 24 h. The cell viability was estimated at 48 h
after the start of treatment. The cell viability of the control (0; DMSO alone) is indicatedas 100 %. Western blot analysis was performed to determine the level of the active formof caspase-8. E-actin was used as an internal control.

25ࡉࡽ࡟ࠊPTBP1 ࡢⓎ⌧࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡜ࡢ㛵ಀࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࠊࢺ
ࣜࣃࣥࣈ࣮ࣝⰍ⣲᤼㝖ヨ㦂ἲ࡟ࡼࡾ TRAIL ࡢ IC50 ್ࡀ 20 ng/ml ௨ୖ࡛࠶ࡗࡓࣄ
ࢺ኱⭠⒴⣽⬊ᰴ SW480 ࡜ࠊṇᖖࣄࢺங⭢ୖ⓶⣽⬊ᰴ MCF10A ࢆ⏝࠸࡚ྠᵝࡢᐇ㦂ࢆ⾜ࡗࡓࠋ
ࡑࡢ⤖ᯝ 2 ✀⣽⬊ᰴࡢ TRAIL ࡢ IC50 ್ࡣࠊࡑࢀࡒࢀ 20.2 ng/mlࠊ
102.1 ng/ml ࡜࡞ࡗࡓࠋึࡵ࡟ࠊᐃᖖ≧ែ࡟࠾ࡅࡿ PTBP1 ࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥ
ࣈࣟࢵࢺἲ࡟ࡼࡾ᳨ドࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࡟࠾࠸࡚ࡶ DLD-1/TRAIL ࡜ྠᵝ࡟ࠊPTBP1 ࡢ㧗Ⓨ⌧ࡀㄆࡵࡽࢀࠊMCF10A ࡛ࡑࡢⓎ⌧ࣞ࣋ࣝࡀ᭱ࡶ㧗࠸ࡇ࡜ࡀศ࠿
ࡗࡓ㸦Fig-6G㸧ࠋḟ࡟ࠊPTBP1 ࡢⓎ⌧ࢆ siRNA ࢆ⏝࠸࡚ࣀࢵࢡࢲ࢘ࣥࡋࡓ᫬ࡢTRAIL ࡟ᑐࡍࡿឤཷᛶ࡬ࡢᙳ㡪ࢆ᳨ドࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࡟࠾࠸࡚ࡶ TRAIL 5
ng/ml ࡟࠾࠸࡚ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡛ TRAIL ࡟ࡼࡿ⣽⬊Ṛࡀ┦஌ⓗ࡟ㄏᑟࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦Fig-6H㸧ࠋ୰࡛ࡶ TRAIL ࡢ IC50 ್ࡀ᭱ࡶ㧗࠸
MCF10A ࡛ TRAIL ࡢឤཷᛶࡢᅇ᚟ຠᯝࡀᙉࡃㄆࡵࡽࢀࡓ
㸦Syn:2.94㸧ࠋFigure-6G࡟♧ࡍࡼ࠺࡟ࠊDLD-1/TRAIL ࡜ྠᵝ࡟ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡟
ࡼࡾ PKM2 ࠿ࡽ PKM1 ࡬ࡢࢫ࢖ࢵࢳࡀㄆࡵࡽࢀࡓ㸦Fig-6G㸧ࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ
PTBP1 ࡢ㧗Ⓨ⌧ࡣ TRAIL ⪏ᛶ࡜㛵㐃ࡋ࡚࠾ࡾࠊTRAIL ⪏ᛶࡀᙉ࠸⣽⬊ᰴ࡯࡝
PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡟ࡼࡾ TRAIL ࡟ᑐࡍࡿឤཷᛶࡢᅇ᚟ຠᯝ
ࡀᙉ࠸ࡇ࡜ࡀศ࠿ࡗࡓࠋ

26

(G)

(H)

Figure-6(G) TRAIL-sensitive DLD-1, TRAIL-resistant SW480 and MCF10A cells weretransfected with control or siR-PTBP1 (2 nM) for 48 h. Western blot analysis was27performed to determine the expression of Warburg effect-related genes. E-actin wasused as an internal control. (H) TRAIL-resistant SW480 and MCF10A cells weretransfected with siR-PTBP1 (2 nM) for 48 h and then treated with rTRAIL (5, 10 ng/ml)
for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viabilityof control (0; PBS alone) is indicated as 100 %. The growth inhibition effect by TRAIL
with and without the transfection with siR-PTBP1 was assessed by the average value ofthe growth inhibition ratios (GI) at each TRAIL concentrations (5, 10 ng/ml). We defined
the synergistic effect (Syn) by introduction of siR-PTBP1 as the ratios of siR-PTBP1 tocontrol siRNA GI value. Data are expressed as means r SD of 3 different experiments.
*p<0.05, as indicated by the brackets (Student’s t test).

28➨ 3 ⠇ TRAIL ⪏ᛶᶵᵓ࡟࠾ࡅࡿ PTBP1 ࡢᙺ๭ࡇࢀࡲ࡛ࡢ◊✲࠿ࡽ DR5 ࡢⓎ⌧పୗ࡜⣽⬊⾲㠃ୖ࡬ࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ୙
Ⰻࡀ TRAIL ⪏ᛶࡢせᅉ࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ 53ࠋᮏ⠇࡛ࡣࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ㝿࡟ 2 ࡘࡢ TRAIL ⪏ᛶᶵᵓࡀゎ㝖ࡉࢀࡿ࠿࡝࠺࠿ࢆ᳨ドࡋࡓࠋึࡵ࡟ࠊsiR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 48 ᫬㛫࡛ࡢ DR5 ࡢⓎ⌧ࣞ࣋ࣝࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲ࡟ࡼࡾ᳨ドࡋࡓࠋࡑࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ
⣽⬊࡟࠾࠸࡚ DR5 ࡢⓎ⌧ࡀ㢧ⴭ࡟ቑຍࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦Fig-7A㸧ࠋࡑࡇ࡛ࠊPTBP1 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆస〇ࡋࠊPTBP1 ࡀ DR5 ࡢ㌿෗࡟୚࠼ࡿᙳ㡪ࢆ᳨ドࡋࡓࠋDLD-1 ࡟ PTBP1 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋ 24 ᫬㛫ᚋ࡟࠾ࡅࡿ DR5 ࡢⓎ⌧ࣞ࣋ࣝࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲ࡟ࡼࡾホ౯ࡋࡓ࡜ࡇࢁࠊ
DR5 ࡢⓎ⌧పୗࡀㄆࡵࡽࢀࡓࡀ㸦Fig-7B㸧ࠊࢡ࣐ࣟࢳࣥච␿ỿ㝆ἲ࡛ࡣ PTBP1
࡜ DR5 ࡢ┤᥋⤖ྜࡀㄆࡵࡽࢀࡎ PTBP1 ࡀ DR5 ࡢ㌿෗࡟㛵୚ࡋ࡚࠸࡞࠸ࡇ࡜ࡀ
᫂ࡽ࠿࡜࡞ࡗࡓࠋḟ࡟ DR5 ࡢ⣽⬊⾲㠃ୖ࡬ࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ୙Ⰻ࡬ࡢᙳ㡪ࢆ᳨ドࡋࡓࠋ2 ✀⣽⬊ᰴ࡟ siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 48 ᫬㛫࡛ࡢDR5 ࡢ⣽⬊ෆᒁᅾࢆච␿ᰁⰍἲ࡟ࡼࡾホ౯ࡋࡓ⤖ᯝࠊ࠸ࡎࢀࡢ⣽⬊ᰴ࡟࠾࠸࡚
ࡶ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡟ࡼࡾ⣽⬊⾲㠃ୖ࡬ࡢ DR5 ࡢⓎ⌧ቑຍࡀほᐹࡉࢀࡓ㸦Fig-7C㸧ࠋ௨ୖࡢ⤖ᯝ࠿ࡽ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ
ࡇ࡜࡛ DR5 ࡢⓎ⌧࡜⣽⬊⾲㠃ୖ࡬ࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡀㄏᑟࡉࢀ TRAIL ⪏ᛶࡀ29ゎ㝖ࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ

(A)

(B)

(C)

Figure-7(A) TRAIL-sensitive and -resistant DLD-1 cells were transfected with control or
siR-PTBP1 (2, 5 nM) for 48 h. Western blot analysis was performed to determine theexpression levels of PTBP1 and DR5. (B) TRAIL-resistant DLD-1 cells were transfectedwith control and PTBP1 expression plasmid vectors (0.2 Pg/ml) for 24 h. Western blot
analysis was performed to determine the levels of PTBP1 and DR5. (C) TRAIL-sensitiveand -resistant DLD-1 cells were transfected with siR-PTBP1 (2, 5 nM) for 48 h. The
results of immunofluorescence staining for anti-DR5 antibody binding on the cell surface
and in the cytosol of untreated (0: Control siRNA) or siR-PTBP1 transfected cells areshown. Nuclei were counterstained in blue with Hoechst33342. Anti-DR5 antibody
bound to the cell surface, which is indicated by the white arrows.
30

➨ 4 ⠇ άᛶ㓟⣲㸦ROS㸧ࡢⓎ⏕࡜ TRAIL ⪏ᛶゎ㝖ᶵᵓ࡬ࡢ㛵୚๓⠇࡟࠾࠸࡚ࠊPTBP1 ࡣ DR5 ࡢ㌿෗࡟┤᥋㛵୚ࡋ࡚࠸࡞࠸ࡇ࡜࠿ࡽࠊ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢኚ໬㸦Warburg ຠᯝࡢ⬺ไᚚ㸧ࡀ DR5 ࡢⓎ⌧ቑຍ࡟㛵୚ࡋ࡚࠸ࡿ࠿ࢆ᳨ウࡋࡓࠋ➨ 2 ⠇࡟࠾࠸࡚ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡟
ࡼࡾ PKM2 ࠿ࡽ PKM1 ࡬ࡢࢫ࢖ࢵࢳࡀㄏᑟࡉࢀࠊ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮௦ㅰࡀ୍
㒊ゎ⢾⣔࠿ࡽ࣑ࢺࢥࣥࢻࣜ࢔࡟࠾ࡅࡿ TCA ࢧ࢖ࢡࣝ࡟ࡼࡿ㓟໬ⓗࣜࣥ㓟໬࡟ࢩࣇࢺࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࠊ⣽⬊ෆ࡛ࡣάᛶ㓟⣲㸦ROS㸧ࡀⓎ⏕ࡋ࡚࠸ࡿྍ⬟ᛶࡀணࡉࢀࡓࠋᡃࠎࡢ◊✲ࢢ࣮ࣝࣉࡣࡍ࡛࡟ࠊPTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥ

ࡍࡿࡇ࡜࡟ࡼࡾ⣽⬊ෆ࡛ ROS ࡢⓎ⏕ࡀቑຍࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ 9ࠋࡑࡇ࡛ࠊ⣽⬊ෆ࡛Ⓨ⏕ࡋࡓ ROS ࡜ TRAIL ⪏ᛶゎ㝖ᶵᵓ࡬ࡢ㛵୚ࢆ᳨ドࡍࡿࡓࡵ࡟ࠊᢠ
㓟໬๣࡛࠶ࡿ N-࢔ࢭࢳࣝࢩࢫࢸ࢖ࣥ㸦NAC㸧ࢆ⏝࠸࡚ᐇ㦂ࢆ⾜ࡗࡓࠋNAC ࢆ 6᫬㛫స⏝ࡋࡓᚋ TRAIL ࡟ᑐࡍࡿឤཷᛶ࡬ࡢᙳ㡪ࢆࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲᤼㝖ヨ㦂ἲ࡟ࡼࡾᐃࡋࡓࠋࡑࡢ⤖ᯝࠊNAC ࢆస⏝ࡉࡏࡓ⣽⬊࡟࠾࠸࡚ࠊPTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿ TRAIL ㄏᑟ⣽⬊Ṛࡀ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓ
㸦Fig-8A㸧ࠋ㠀ᖖ࡟⯆࿡῝࠸ࡇ࡜࡟ࠊNAC ࢆస⏝ࡉࡏࡓ⣽⬊࡛ࡣ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡾゎ㝖ࡉࢀ࡚࠸ࡓ DR5 ࡢⓎ⌧࡜ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢㄏᑟࡶ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦Fig-8B and C㸧ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ
PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿ DR5 ࡢ Ⓨ⌧࡜ࣜࢡࣝ ࣮ࢺ࣓ ࣥࢺࡢㄏᑟࡣ

31

Warburg ຠᯝࡢ⬺ไᚚ࡟ࡼࡿ ROS ࡢⓎ⏕ࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ
(A)

(B)

(C)

Figure-8(A)TRAIL-sensitive DLD-1 cells were pretreated with NAC (1 mM) for 6 h and thenincubated with siR-PTBP1 (5 nM) and/or rTRAIL (5, 10 ng/ml) for 24 h. The cell viability
was estimated at 72 h after the treatment. Data were obtained from 3 independent
experiments. The cell viability of the control (Control; Control-siRNA alone) is indicatedas 100 %. (B) TRAIL-sensitive DLD-1 cells were pre-treated with NAC (1 mM) before
the transfection with siR-PTBP1. Western blot analysis was performed to determine theexpression level of DR5 protein. E-actin was used as an internal control. (C)TRAIL-sensitive DLD-1 cells treated with NAC (1 mM) and/or siR-PTBP1 (5 nM). The
results of immunofluorescence staining for anti-DR5 binding on the cell surface and in
the cytosol of untreated (0: Control-siRNA) or siR-PTBP1 transfected cells are shown.
Nuclei were counterstained in blue with Hoechst33342. Anti-DR5 antibody bound to thecell surface, which is indicated by the white arrows. *p<0.05, as indicated by thebrackets (Student’s t test).
32➨ 5 ⠇ CCN1 ࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡬ࡢ㛵୚ ᮏ⠇࡛ࡣࠊDLD-1/TRAIL ࡟࠾࠸࡚ Control ⣽⬊࡜ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ
⣽⬊ࢆ⏝࠸࡚࢔ࣞ࢖ゎᯒࢆ⾜࠸ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿࡑࡢ௚ࡢ TRAIL⪏ᛶゎ㝖࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡟࠾࠸࡚ CCN1 㑇ఏᏊࡢ㢧ⴭ࡞Ⓨ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓࠋCCN1 ࡣࠊ࢖ࣥࢸࢢ
ࣜࣥ⤖ྜࢱࣥࣃࢡ㉁࡛࠶ࡾ࢖ࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮࡟⤖ྜࡍࡿࡇ࡜࡛⣽⬊᥋╔ࠊ㐟㉮ࠊ⏕Ꮡ࡞࡝ᵝࠎ࡞⣽⬊άᛶไᚚ࡟㛵୚ࡋ࡚࠸ࡿ 48ࠋ୍᪉ࠊCCN1 ࡣ๓❧⭢ࡀࢇ⣽⬊࡟࠾࠸࡚ TRAIL ㄏᑟ⣽⬊Ṛࢆቑᙉࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࠾ࡾࠊ࢔࣏ࢺ࣮
ࢩࢫࡢㄏᑟ࡟㛵୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿ 52ࠋ
DLD-1/TRAIL ࡬ siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ㝿ࡢ CCN1 ࡢ mRNAࠊ
ࢱࣥࣃࢡ㉁Ⓨ⌧ࣞ࣋ࣝࢆ᳨ドࡋࡓࠋᐃᖖ≧ែ࡟࠾࠸࡚ࡑࡢⓎ⌧ࡀㄆࡵࡽࢀ࡞࠸
ࡢ࡟ᑐࡋ࡚ࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡛ࡣ㢧ⴭ࡟ mRNAࠊࢱࣥࣃࢡ㉁ࣞ
࡛࣋ࣝⓎ⌧ࡀቑຍࡋ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦Fig-9A㸧ࠋࡑࡇ࡛ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡾㄏᑟࡉࢀࡓ CCN1 ࡢⓎ⌧ࡀ TRAIL ⣽⬊Ṛࡢㄏᑟಁ㐍࡟࡝ࡢࡼ࠺࡟㛵 ୚ ࡋ ࡚ ࠸ ࡿ ࡢ ࠿ ᳨ ド ࡋ ࡓ ࠋ ึ ࡵ ࡟ ࠊ CCN1 ࡢ Ⓨ ⌧ ࣋ ࢡ ࢱ ࣮ ࢆ స 〇 ࡋ
DLD-1/TRAIL ࡟ CCN1 ࢆ㐣๫Ⓨ⌧ࡋࡓ᫬ࡢ TRAIL ࡟ᑐࡍࡿឤཷᛶࢆホ౯ࡋࡓࠋCCN1 ࣋ࢡࢱ࣮ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 24 ᫬㛫࡟࠾࠸࡚ࡑࡢⓎ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓࡓࡵᮏᐇ㦂࡛ࡣࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 24 ᫬㛫ࡢ⣽⬊ࢆ CCN1 㐣๫Ⓨ⌧

33⣽⬊࡜ࡋ࡚౑⏝ࡋࡓ㸦Fig-9B㸧ࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊCCN1 ࢆ㐣๫Ⓨ⌧ࡋࡓ⣽⬊࡛ࡣࠊࢥࣥࢺ࣮ࣟࣝ⣽⬊࡜ẚ㍑ࡋ࡚࢝ࢫࣃ࣮ࢮ 8 ࡢάᛶ໬ࢆకࡗࡓ TRAIL ㄏᑟ⣽⬊Ṛࡀቑᙉࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦Fig-9C㸧ࠋࡲࡓࠊPTBP1 ࡢ CCN1 ࡟ᑐࡍࡿ㌿෗࡬ࡢᙳ㡪ࢆࢡ࣐ࣟࢳࣥච␿ỿ㝆ἲ࡟ࡼࡾホ౯ࡋࡓ⤖ᯝࠊPTBP1 ࢱࣥࣃࢡࡀ
CCN1 ࡢ mRNA ࡜┤᥋⤖ྜࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓ㸦Fig-9D㸧ࠋࡉࡽ࡟ࠊPTBP1
ࡢⓎ⌧࣋ࢡࢱ࣮ࢆ⏝࠸ PTBP1 ࢆ㐣๫Ⓨ⌧ࡋࡓ⣽⬊࡟࠾࠸࡚ CCN1 ࡢⓎ⌧ቑຍࡀ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡓ㸦Fig-9E㸧ࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊPTBP1 ࡣ TRAIL ㄏᑟ⣽⬊Ṛ࡟㛵୚ࡍࡿ CCN1 ࡢࣜࣉࣞࢵࢧ࣮࡜ࡋ࡚ᶵ⬟ࡋ࡚࠾ࡾࠊPTBP1 ࢆࣀࢵࢡࢲ࢘
ࣥࡍࡿࡇ࡜࡟ࡼࡿ CCN1 ࡢⓎ⌧ቑຍࢆ௓ࡋ࡚ TRAIL ㄏᑟ⣽⬊Ṛࡀቑᙉࡉࢀࡿྍ
⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
(A)

34

(B)

(C)

(D)

(E)

Figure-9(A) DLD-1/TRAIL cells were transfected with siR-PTBP1 (2, 5 nM) for 48 h. The
expression levels of CCN1 mRNA as a relative ratio with respect to the GAPDH
expression level was evaluated by RT-qPCR. Also shown is the expression level of
CCN1 protein determined by performing Western blot analysis. Means (S.D) indicatedby error bars are shown. (B) Control and CCN1-expression plasmid vectors (0.2 Pg/ml)
were used to transfect DLD-1/TRAIL cells for 24 or 48 h. Western blot analysis was
35performed to determine the expression of CCN1 with E-actin used as the internal control.
(C) Control and CCN1-expression plasmid vectors (0.2 Pg/ml) were used to transfect
DLD-1/TRAIL cells for 24 h, and the cells were then exposed to rTRAIL (10, 25, 50
ng/ml) for 24 h. The cell viability was estimated at 48 h after the treatment. The cell
viability of the control (0; PBS alone) is indicated as 100 %. Western blot analysis was
performed to determine the expression of activation of caspase-8 with E-actin used as
the internal control. (D) MCF10A cells transfected with control or siR-PTBP1 (2 nM) for
48 h. The expression levels of CCN1 mRNA as relative ratios with respect to the
GAPDH expression level was evaluated by RT-qPCR. The expression level of input
DNA is indicated as “1”. (E) DLD-1/TRAIL cells were transfected with control or
PTBP1-expression plasmid vectors (0.2 Pg/ml) for 24 h. Western blot analysis was
performed to determine the expression of PTBP1 and CCN1 proteins, with E-actin usedas the internal control. *p<0.05, as indicated by the brackets (Student’s t test).

36➨ 4 ❶ ⥲ᣓࡀࢇ࡟ᑐࡍࡿ TRAIL ㄏᑟ࢔࣏ࢺ࣮ࢩࢫࢆᶆⓗ࡜ࡋࡓከࡃࡢ⮫ᗋヨ㦂ࡢ⤖ᯝࡣࠊ
TRAIL ⪏ᛶࡢ⋓ᚓࠊࡉࡽ࡟ࡣ㔜⠜࡞⫢ᶵ⬟㞀ᐖ➼ࡢ๪స⏝ࡢࡓࡵኻᩋ࡟⤊ࢃࡗࡓࠋࡇࡢࡼ࠺࡞⫼ᬒ࠿ࡽ TRAIL ⪏ᛶᶵᵓࡢゎ᫂࡜ࠊ⪏ᛶゎ㝖࡟᭷ຠ࡞ᶆⓗศᏊࢆྠᐃࡍࡿࡇ࡜ࡀࡀࢇ⣽⬊≉␗ⓗ࡞ TRAIL ㄏᑟ⣽⬊Ṛࢆᶆⓗ࡟ࡍࡿ᪂つ἞⒪⸆ࡢ㛤Ⓨ࡟ồࡵࡽࢀ࡚࠸ࡿࠋ
ᮏ◊✲࡛ࡣࠊWarburg ຠᯝ࡜ࡋ࡚▱ࡽࢀࡿࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰ
ᶵᵓࡢᡂ❧࡟ᚲ㡲࡞㑇ఏᏊ PTBP1 ࡟╔┠ࡋࡓࠋPyruvate kinase 㸦PK㸧ࡣࠊ4ࡘࡢ࢔࢖ࢯࣇ࢛࣮࣒ࡀᏑᅾࡍࡿゎ⢾⣔ࡢᚊ㏿㓝⣲࡛࠶ࡿࠋᐇ㝿࡟ PKM1/2 ࡢⓎ⌧ࣞ࣋ࣝࡣࠊࢫࣉࣛ࢖ࢧ࣮ࢱࣥࣃࢡ㉁࡛࠶ࡿ PTBP1 ࡟ࡼࡗ࡚ไᚚࡉࢀ࡚࠸ࡿࠋ
ࡀࢇ⣽⬊࡟࠾࠸࡚ࡣࠊPTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠾ࡾ㑅ᢥⓗࢫࣉࣛ࢖ࢩࣥࢢ࡟ࡼࡾ
PKM2 ࡢⓎ⌧ࡀ᭷ព࡜࡞ࡿࡇ࡜࡛ Warburg ຠᯝࢆᡂ❧ࡉࡏ࡚࠸ࡿࠋᮏ◊✲࡛ࡣ
TRAIL ⪏ᛶᰴ࡟࠾࠸࡚ PTBP1 ࡢ㧗Ⓨ⌧ࢆㄆࡵࠊ
PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ࡜ࠊTRAIL ࡢឤཷᛶࡀᅇ᚟ࡍࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦➨ 3 ❶-2 ⠇㸧ࠋࡑࡢຠᯝࡣ TRAIL࡟ᑐࡋ࡚᭱ࡶᙉ࠸⪏ᛶࢆ♧ࡋࡓṇᖖࣄࢺங⭢ୖ⓶⣽⬊ᰴ MCF10A ࡟࠾࠸࡚㢧ⴭ࡟ㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊPTBP1 ࡢⓎ⌧࡜ TRAIL ⪏ᛶ࡟ࡣṇࡢ┦㛵ࡀ࠶ࡾࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ࡜ TRAIL ឤཷᛶࡀᅇ᚟ࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ
ᡃࠎࡣࡇࢀࡲ࡛࡟ࠊ࢔࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR5 ࡢⓎ⌧పୗ࡟ຍ࠼࡚ DR5 ࡢ⣽

37⬊⾲㠃ୖ࡬ࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ୙Ⰻࡀ TRAIL ⪏ᛶࡢせᅉ࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿
࡟ࡋࡓ 53ࠋPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡜࡟ࡼࡾࠊDR5 ࡢⓎ⌧పୗ࡜ࣜࢡ࣮ࣝ
ࢺ࣓ࣥࢺࡢ୙Ⰻࡀゎ㝖ࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦➨ 3 ❶-3 ⠇㸧ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ
PTBP1 ࡣ DR5 ࡢ㌿෗࡟┤᥋ⓗ࡟㛵୚ࡋ࡚࠸࡞࠸ࡇ࡜࠿ࡽ⣽⬊ෆ࡛ࡢ࢚ࢿࣝࢠ
࣮௦ㅰࡢኚ໬࡟╔┠ࡋ TRAIL ⪏ᛶゎ㝖ᶵᵓࡢゎ᫂ࢆヨࡳࡓࠋPTBP1 ࢆࣀࢵࢡࢲ
࢘ࣥࡋࡓ⣽⬊࡛ࡣࠊPKM2/PKM1 ࡢẚࡀᐃᖖ≧ែ࡜ẚ㍑ࡋ࡚㢧ⴭ࡟ῶᑡࡋ࡚࠾
ࡾࠊࡉࡽ࡟ࡣ⣽⬊ෆࡢ ATP ࣞ࣋ࣝࡢቑຍ࡜ࠊゎ⢾⣔ࡢ᭱⤊⏘≀࡛࠶ࡿங㓟㔞ࡢῶᑡࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮௦ㅰࡀ୍㒊ゎ⢾⣔࠿ࡽ࣑ࢺࢥࣥ
ࢻࣜ࢔࡟࠾ࡅࡿ㓟໬ⓗࣜࣥ㓟໬࡬ࢩࣇࢺࡋ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ2006 ᖺ࡟ࠊ࢘ࢥࣥࡢ୺せ࡞ᵓᡂᡂศ࡛࠶ࡿࢡࣝࢡ࣑ࣥࡀ ROS ࡢⓎ⏕ࢆ௓ࡋ࡚ DR5 ࡢⓎ⌧ࡀቑຍࡍࡿࡇ࡜࡟ࡼࡾ TRAIL ࡢឤཷᛶࢆᅇ᚟ࡉࡏࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ 54ࠋ
ᡃࠎࡣࠊPTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ࡜ PKM2 ࠿ࡽ PKM1 ࡬ࡢࢫ࢖ࢵࢳ࡟
ࡼࡾ ROS ࡢ⏘⏕㔞ࡀୖ᪼ࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠸ࡿ 46ࠋᐇ㝿࡟ࠊᢠ㓟໬๣࡛࠶ࡿ NAC ࢆస⏝ࡉࡏࡿࡇ࡜࡛ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿ TRAIL ㄏᑟ⣽⬊Ṛࡀ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊDR5 ࡢⓎ⌧࡜ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢㄏᑟࡶ࢟ࣕࣥࢭࣝࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ
Warburg ຠᯝࡢ⬺ไᚚ࡟ࡼࡿ ROS ࡢⓎ⏕ࡀ DR5 ࡢⓎ⌧࡜ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ
ㄏᑟ࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸦➨ 3 ❶-4 ⠇㸧ࠋࡋ࠿ࡋࠊROS ࡢⓎ⏕38ࡀ DR5 ࡢⓎ⌧ࢆቑຍࡉࡏࡿ࣓࢝ࢽࢬ࣒࡟ࡘ࠸࡚ࡣ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ࠋࡉࡽ
࡟ᮏ◊✲࡛ࡣࠊPTBP1 ࡢࣀࢵࢡࢲ࢘ࣥ࡟ࡼࡿࡑࡢ௚ࡢ TRAIL ⪏ᛶゎ㝖࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ⾜ࡗࡓࠋ࢔ࣞ࢖ゎᯒࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡟࠾࠸࡚Ⓨ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓ CCN1 㑇ఏᏊ࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡜ࡢ㛵㐃࡟ࡘ࠸࡚ࡶ
᳨ドࢆ⾜ࡗࡓࠋChen ࡽࡣࠊCCN1 ࡀ࢖ࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮ཬࡧࢩࣥࢹ࢝ࣥ 4࡟⤖ྜࡍࡿࡇ࡜࡛኱㔞ࡢ ROS ࢆⓎ⏕ࡉࡏ JNK ࡢάᛶ໬ࢆ௓ࡋ࡚ TRAIL ㄏᑟ⣽⬊Ṛࢆಁ㐍ࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ 52ࠋᐇ㝿࡟ࠊCCN1 ࢆ㐣๫Ⓨ⌧ࡉࡏࡓ⣽⬊࡟࠾࠸࡚ TRAIL ㄏᑟ࡟ࡼࡿ⣽⬊Ṛࡀቑᙉࡉࢀࡿࡇ࡜ࡀศ࠿ࡗࡓࠋPTBP1 ࡣࠊ
Pre-miRNA ࡸᵝࠎ࡞㑇ఏᏊࡢࢫࣉࣛ࢖ࢩࣥࢢࡢไᚚ࡟㛵୚ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ
࡚࠸ࡿ 55ࠋᡃࠎࡣࠊࢡ࣐ࣟࢳࣥච␿ỿ㝆ἲ࡟ࡼࡾ PTBP1 ࡀ CCN1 ࡢ㌿෗࡟࠾࠸࡚ࣜࣉࣞࢵࢧ࣮࡜ࡋ࡚ᶵ⬟ࡋ࡚࠸ࡿࡇ࡜ࢆ᪂ࡓ࡟᫂ࡽ࠿࡟ࡋࡓ㸦➨ 3 ❶-5 ⠇㸧ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊDR5 ࡢⓎ⌧ቑຍ࡟ࡣ Warburg ຠᯝࡢ⬺ไᚚࡢࡳ࡞ࡽࡎࠊ
CCN1 ࡢⓎ⌧ቑຍ࡟ࡼࡿ ROS ࡢ㛵୚ࡀ♧ࡉࢀࡓࠋᮏ◊✲ࡼࡾࠊ
Warburg ຠᯝ࡜ TRAIL ㄏᑟ⣽⬊Ṛ࡜ࡢ㛵㐃ࡀ᫂ࡽ࠿࡟࡞ࡾࠊPTBP1ࡣ TRAIL ⪏ᛶゎ㝖࡟᭷ຠ࡞ᶆⓗศᏊ࡜࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ௒ᚋࠊPTBP1
ࢆไᚚࡍࡿ໬ྜ≀ࡸ siRNA ࡀࡀࢇࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢ◚⥢ࡢࡳ࡞ࡽࡎ TRAIL ㄏᑟ⣽⬊Ṛࢆ฼⏝ࡋࡓࡀࢇࡢ་⸆ࢩ࣮ࢬ࡜ࡋ࡚ᮇᚅࡉࢀࡿࠋ

39

Figure-10 Schematic diagram of the mechanism and machinery involved in the
TRAIL-induced apoptosis by silencing PTBP1

40

ㅰ㎡

ᮏ◊✲ࡢ㐙⾜࠾ࡼࡧㄽᩥࡢసᡂ࡟࠶ࡓࡾࠊ⤊ጞࡈ᠓⠜࡞ࡿࡈᣦᑟࠊࡈ㠴᧡ࢆ㈷ࡾࡲࡋࡓᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉ ㉥ᑿ ᖾ༤ ᩍᤵ࡟ᚰࡼࡾ
ឤㅰࡢពࢆ⾲ࡋࡲࡍࠋࡲࡓࠊᮏㄽᩥࡢసᡂ࡟㝿ࡋࠊࡈຓゝ࠾ࡼࡧࡈᰯ㜀ࢆ㈷ࡾࡲࡋࡓᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉ Ᏹ㔝 ᩥ஧ ᩍᤵࠊ ୖ⏣ ᾈ ᩍᤵࠊ⏣୰ 㤶࠾㔛 ᩍᤵ࡟῝ࡃឤㅰ࠸ࡓࡋࡲࡍࠋ

ᮏ◊✲ࡢ㐙⾜࡟࠶ࡓࡾࠊ◊✲άືࢆඹ࡟ࡋ࡚ࡁࡓᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉㉥ᑿ◊✲ᐊࡢⓙᵝ࡟ᚰࡼࡾ࠾♩⏦ࡋୖࡆࡲࡍࠋ

41

ᐇ㦂ࡢ㒊
1. ヨ⸆
Recombinant Human TRAIL-Apo2L Ligand (#4354-10; BioVision, CA, USA)ࠊ2ࢹ࢜࢟ࢩ-D-ࢢࣝࢥ࣮ࢫ(Sigma-Aldrich, St. Louis, MO,USA)ࠊN-࢔ࢭࢳࣝ-L-ࢩࢫ
ࢸ࢖ࣥ(Sigma-Aldrich, St. Louis, MO, USA)ࢆ౑⏝ࡋࡓࠋ

2. ⣽⬊ᇵ㣴ࣄ ࢺ ኱ ⭠ ⒴ ⣽ ⬊ ᰴ DLD-1 ࠊ SW480 ࡣ ࠊ Japanese Collection Research
Bioresources Cell Bank (Osaka, Japan)ࡼࡾ㉎ධࡋࡓࠋMCF10A ࡣࠊAmerican
Type Culture Collection (ATCC Manassas, VA, USA)ࡼࡾ㉎ධࡋࡓࠋ⣽⬊ࡣ㉎ධ
ᚋ 6 ࠿᭶௨ෆࡶࡋࡃࡣ MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza,
Rockland, ME, USA)࡛⟶⌮ࡋࡓࡶࡢࢆ౑⏝ࡋࡓࠋDLD-1ࠊDLD-1/TRAIL ཬࡧ
SW480 ࡣ 10 %FBS ྵ᭷ RPMI-1640(189-02025; Invitrogen, Carlsbad, CA, USA)୰࡛ 5 % CO2ࠊ37Υࡢ᮲௳ୗ࡛ᇵ㣴ࡋࡓࠋMCF10A ࡣ MEBM(CC3150; Lonza,
Tokyo, Japan)୰࡛ 5 % CO2ࠊ37Υࡢ᮲௳ୗ࡛ᇵ㣴ࡋࡓࠋ⏕⣽⬊ᩘࡣࠊࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲᤼㝖ヨ㦂ἲ࡟ࡼࡾホ౯ࡋࡓࠋᇵ㣴ᚋࡢ⣽⬊⁐ᾮࢆࢺࣜࣃࣥࣈ࣮ࣝ࡜➼㔞ΰྜࡋࠊ⾑⌫ィ⟬┙࡟࡚⏕⣽⬊ᩘࢆィᩘࡋࡓࠋࢥࣥ

42ࢺ࣮ࣟࣝ⣽⬊࡟ᑐࡍࡿ⏕⣽⬊ᩘ(%)ࢆ⣽⬊⏕Ꮡ⋡࡜ࡋࡓࠋ3. ࢚࢘ࢫࢱࣥࣈࣟࢵࢺゎᯒ
3-1 ࢱࣥࣃࢡ㉁ᢳฟ
ࢱࣥࣃࢡ㉁ᢳฟᾮ࡟ࡣࠊProtein lysis buffer (10 nM Tris-HCLࠊ0.1% SDSࠊ1%
NP-40ࠊ0.1% ࢹ࢜࢟ࢩࢥ࣮ࣝ㓟ࢼࢺ࣒ࣜ࢘ࠊ150 mM NaClࠊ1 mM EDTA)࡟ 1%
Protease inhibitor cocktailࠊPhosphatase inhibitor cocktail II ཬࡧ III ࢆΰྜࡋ࡚
⏝࠸ࡓࠋࢱࣥࣃࢡ㉁ᢳฟᾮ࡟ᅇ཰ࡋࡓ⣽⬊ࢆᠱ⃮ࡉࡏࠊ20 ศ㛫ị୰࡟㟼⨨ࡉࡏࡓࠋࡑࡢᚋࠊ13,000rpmࠊ4Υࠊ20 ศ㛫㐲ᚰศ㞳ࡋࡓࠋ㐲ᚰศ㞳ࡋࡓୖΎࢆᅇ཰
ࡋࠊࢱࣥࣃࢡ㉁ࢧࣥࣉࣝ࡜ࡋࡓࠋࢱࣥࣃࢡ㉁ᐃ㔞ࡣࠊDC Protein assay kit (Biorad,
Hercules, CA, USA)ࢆ⏝࠸࡚⾜ࡗࡓࠋᐃ㔞ࡋࡓࢱࣥࣃࢡ㉁ࢆ SDS sample buffer(62.5 mM Tris-HCLࠊ2% SDSࠊ10% ࢢࣜࢭ࣮ࣟࣝࠊ50 mM DTTࠊ0.01%ࣈࣟࣔ
ࣇ࢙ࣀ࣮ࣝࣈ࣮ࣝ)࡜ΰ࿴ࡋ࡚ 50 Pg/μL ࡟ㄪᩚࡋࠊ98Υ࡛ 5 ศ㛫Ἓ㦐ฎ⌮ࡋࡓᚋࠊịୖ࡛ 5 ศ㛫㟼⨨ࡋࡓࠋ

3-2 㟁ẼὋື࠾ࡼࡧ㌿෗㟁ẼὋື࡟ࡣࠊ࢖࣮ࢪ࣮ࢭࣃ࣮ࣞࢱ࣮(Wako)ཬࡧ Super Sep Ace (Wako)ࢆ⏝࠸

43ࡓࠋὋືᚋࠊࢤࣝࢆ blotting buffer (25 mM Trisࠊ0.2 M ࢢࣜࢩࣥࠊ20%࣓ࢱࣀ࣮ࣝ)࡟ 5 ศ㛫ᾐࡋࡓࠋPVDF ࣓ࣥࣈࣞࣥ(PerkinElmer Life Sciences, Boston, MA,USA)ࡣ࣓ࢱࣀ࣮ࣝ࡟ 3 ศ㛫ᾐࡋࠊ㉸⣧Ỉ࡟ 5 ศ㛫ᾐࡋࡓࠋࡑࡢᚋࠊblotting buffer࡟ 5 ศ㛫ᾐࡋࡓࠋ㝧ᴟഃ࠿ࡽࠊblotting buffer ࡟ᾐࡋࡓࢁ⣬ࠊPVDF ࣓ࣥࣈࣞࣥࠊ
ࢤࣝࠊࢁ⣬ࡢ㡰࡟㔜ࡡࠊ15 Vࠊ370 mA ࡛ 40 ศ㛫㌿෗ࡋࡓࠋ

3-3 ࢚࢘ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢ
㌿෗ᚋࠊ0.1% Tween 20 ྵ᭷ 50 mM Tris-HCL buffer (TBST)࡛Ὑίࡋࠊ5%ࢫ࢟
࣒࣑ࣝࢡ⁐ᾮ࡟ᾐࡋ࡚ 1 ᫬㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋTBST ࡛Ὑίࡋࠊᢠయᕼ㔘ᾮ
(2%BSAࠊ0.01%࢔ࢪ໬ࢼࢺ࣒ࣜ࢘ࠊTBST)࡛ᕼ㔘ࡋࡓ୍᫬ᢠయ࡟ᾐࡋ࡚ 4Υ୍࡛ᬌ཯ᛂࡉࡏࡓࠋTBST ࡛Ὑίࡋࡓᚋࠊ5%ࢫ࣒࣑࢟ࣝࢡ⁐ᾮ࡛ᕼ㔘ࡋࠊLuminate
Forte Western HRP Substrate (WBLUF0500; Millipore)࡛Ⓨගࡉࡏࡓᚋ࡟ࠊ
Luminescent image analyzer LAS-4000 UV mini (Fujifilm, Tokyo, Japan)ࢆ⏝࠸᳨࡚ฟࡋࡓࠋ୍ḟᢠయࡣ PTBP1 (#8776)ࠊDR5 (#8074)ࠊ Caspase-8 (#9496)ࠊ
CCN1 (#14479)ࡣ Santa Cruz Biotechnology (Santa Cruz, CA, USA)ࡼࡾࠊFADD(M035-3)ࡣ MBL (MEDICAL & BIOLOGICAL LABORATORIES CO, LTD, Nagoya,Japan) ࡼ ࡾ ࠊ PKM1 (NBP2-14833SS) ࠊ PKM2 (NBP1-48308SS) ࡣ Novus
Biologicals (Littleton, CO, USA)ࢆ౑⏝ࡋࡓࠋࢥࣥࢺ࣮ࣟࣝ࡟ࡣ anti-E-actin

44

antibody (A5316; Sigma-Aldrich)ࢆ౑⏝ࡋࡓࠋ

4. Real-time PCR
4-1 RNA ᢳฟ⣽⬊ࡢ RNA ࡣ NucleaseSpin miRNA kit (TaKaRa, Osaka, Japan)ࢆ౑⏝ࡋᢳฟࡋࡓࠋRNA 㔞ࡣ UV spectrophotometry ࡟࡚ᐃ㔞ࡋࡓࠋ

4-2 mRNA ࡢᐃ㔞
PrimeScript RT reagent kit (TaKaRa)ࢆ౑⏝ࡋࠊ37Υ15 ศࠊ85Υ5 ⛊ࠊ4Υ࡛ RNAࢧࣥࣉࣝࡢ㏫㌿෗཯ᛂࢆ⾜࠸ࠊ㗪ᆺ cDNA ࢆྜᡂࡋࡓࠋQuantitative reverse
transcription-PCR (qRT-PCR) ཯ ᛂ ࡟ ࡣ Universal SYBR select Master Mix(Applied Biosystems, Forester City, CA)ཬࡧ Tabele-2 ࡢࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ౑
⏝ࡋࡓࠋGAPDH ࡢ mRNA 㔞ࢆෆ㒊ࢥࣥࢺ࣮ࣟࣝ࡜ࡋࡓࠋ95Υ30 ⛊࡛ึᮇኚᛶ
ࢆ⾜ࡗࡓᚋࠊ95Υ5 ⛊ࡢኚᛶ཯ᛂཬࡧ 60Υ60 ⛊ࡢ࢔ࢽ࣮ࣜࣥࢢ࣭ఙ㛗཯ᛂࢆ40 ࢧ࢖ࢡࣝ⾜࠸ࠊ95Υ15 ⛊ࠊ60Υ30 ⛊ࠊ95Υ15 ⛊ࡢࢫࢸࢵࣉ࡛⼥ゎ᭤⥺ࢆศᯒࡋࡓࠋྛࢧࣥࣉࣝࡢ཯ᛂࡣ 3 ᅇࡎࡘ⾜࠸ࠊ''Ct ἲ࡟࡚ mRNA 㔞ࢆィ⟬ࡋࡓࠋ

45

Primers

Sequences

DR5 forward

5’-GAGAGACTATAAGAGCGT-3’

DR5 reverse

5’-CTTCCTGAAGAGAACCACAC-3’

GAPDH forward

5’-TCTAGACGGCAGGTCAGGTCCACC-3’

GAPDH reverse

5’-CCACCCATGGCAAATTCCATGGCA-3’

Table-2 Sequences of primers using in this study

4-3 miRNA ࡢᐃ㔞miRNA ࡢᐃ㔞࡟ࡣ TaqMan MicroRNA Assay (Applied Biosystems)ࢆ౑⏝ࡋࡓࠋࡲ ࡎ TawMan MicroRNA Reverse Transcription Kit (Applied Biosystems) ࠊstem-loop RT primer (Applied Biosystems)ཬࡧ RNA ࢧࣥࣉࣝࢆΰྜࡋࠊ16Υ࡛
30 ศࠊ42Υ࡛ 30 ศࠊ
85Υ࡛ 5 ศࠊ4Υ࡛ 10 ศࡢ㡰࡛ RT ཯ᛂࢆ⾜ࡗࡓࠋqRT-PCR
࡟ࡣ TapMan MicroRNA Assay ෆࡢ PCR primer (Applied Biosystems)ཬࡧ
THUNDERBIRD Probe qPCR Mix (TOYOBO, Osaka, Japan)ࢆ౑⏝ࡋࡓࠋ95Υ
30 ⛊࡛ኚᛶࡉࡏࡓᚋࠊ95Υ5 ⛊ࠊ60Υ60 ⛊ࡢ཯ᛂࢆ 40 ࢧ࢖ࢡࣝ⾜࠸''Ct ἲ࡟࡚ miRNA 㔞ࢆィ⟬ࡋࡓࠋRNU6B ࢆෆ㒊ࢥࣥࢺ࣮ࣟࣝ࡜ࡋࠊྛࢧࣥࣉࣝࡢ཯ᛂ
ࡣ 3 ᅇࡎࡘ⾜ࡗࡓࠋ46

5. 㑇ఏᏊᑟධᐇ㦂ྛ⣽⬊ࡣ 0.5×105 cells/mL ࡢ⣽⬊ᐦᗘ࡛ 6 ࢙࢘ࣝࣉ࣮ࣞࢺ࡟᧛✀ࡋࡓࠋࢺࣛࣥ
ࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜࠺ 24 ᫬㛫๓࡟᧛✀ࡋࠊࣉ࣮ࣞࢺ࡟᥋╔ࡉࡏࡓࠋᮏ◊✲࡟౑⏝ࡋࡓ siRNA (Invitrogen)ࡢ㓄ิࡣ Tabel-3 ࡟♧ࡋࡓࠋControl ࡜࡞ࡿ㠀≉␗ⓗRNA ࡣ Hokkaido System Sciences (Sapporo, Japan)ࡼࡾ㉎ධࡋࡓࠋsiRNA ࡣ
Lipofectamine RNAiMAX (Invitrogen)ࢆ౑⏝ࡋ࡚࢝ࢳ࢜ࣥᛶ࣏ࣜࢯ࣮࣒ࢆᙧᡂࡉ
ࡏࠊ⣽⬊ෆ࡟ᑟධࡋࡓࠋ㑇ఏᏊᑟධࢆ⾜ࡗࡓ 48 ᫬㛫ᚋ࡟ᅇ཰ࡋࠊྛ࢔ࢵࢭ࢖࡟
౑⏝ࡋࡓࠋ

Gene

Sequences

PTBP1

5’-AUCUCUGGUCUGCUAAGGUCACUUC-3’ (siR-PTBP1)

Control

5’-GGCCUUUCACUACUCCUCA-3’

Table-3 Sequences of siRNAs using in this study

6. ChIP ࢔ࢵࢭ࢖DLD-1/TRAIL ࡟ siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ5%CO2ࠊ37Υ࡛ 48 ᫬㛫ᇵ㣴ࡋࡓࠋ
ChIP ࢔ࢵࢭ࢖࡟ࡣ ChIP-IT Express Enzymatic (53009; Active Motif,

47

Carlsbad, CA, USA)ཬࡧ DR5 antibody (Cell Signaling Technology)ࢆ⏝࠸ࡓࠋࡲࡓࠊ࣏ࢪࢸ࢕ࣈࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚ Antibody against Suz12 (39357; Active Motif)ࠊ
ࢿ࢞ࢸ࢕ࣈࢥࣥࢺ࣮ࣝ࡜ࡋ࡚ rabbit IgG (53025; Active Motif)ࢆ౑⏝ࡋࡓࠋච␿
ỿ㝆཯ᛂ࡛ᅇ཰ࡋࡓ DNA ࡣ Real-time PCR ࡟࡚ቑᖜࡋࠊInput DNA ࡢ඲య㔞࡟ᑐࡍࡿቑᖜ⋡ࢆồࡵࡓࠋ

7. L-lactate ࢔ࢵࢭ࢖DLD-1 ཬࡧ DLD-1/TRAIL ࡟ᑐࡋࠊsiR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ
5%CO2ࠊ37Υ࡛ 48 ᫬㛫ᇵ㣴ࡋࡓࠋ⣽⬊ࢆᅇ཰ࡋࠊL-Lactate Assay Kit (700510;
Cayman Chemical Company, Ann Arbor, MI, USA)ࢆ⏝࠸࡚⣽⬊ෆࡢ L-lactate ࢆᢳฟཬࡧᐃࢆ⾜ࡗࡓࠋ

ᐃ್ࡣྛࢧࣥࣉࣝࡢ⏕⣽⬊ᩘ࡛⿵ṇࡋࠊ⣽⬊ෆங㓟⏘⏕㔞࡜ࡋࡓࠋ

8. ATP ࢔ࢵࢭ࢖DLD-1 ཬࡧ DLD-1/TRAIL ࡟ siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ5%CO2ࠊ
37Υ࡛ᇵ㣴ࡋࡓࠋ48 ᫬㛫ᚋ࡟⣽⬊ࢆᅇ཰ࡋࠊATP Determination Kit (A22066;

48

Invitrogen)࡟࡚ ATP ࣞ࣋ࣝࢆᐃࡋࡓࠋᐃ್ࡣྛࢧࣥࣉࣝࡢ⏕⣽⬊ᩘ࡛⿵ṇࡋࡓࠋ

9. ච␿⺯ගᰁⰍච␿⺯ගᰁⰍࡣ Cell Signaling Technology ࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗࡓࠋDLD-1 ཬࡧ DLD-1/TRAIL ࡟ siR-PRBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ48 ᫬㛫ᚋࡢ⣽⬊ࢆᅇ཰ࡋࡓࠋSmear Gell (SG-01; GenoStaff, Tokyo, Japan)ࢆ⏝࠸࡚ࢫࣛ࢖ࢻ࢞ࣛ
ࢫୖ࡟⣽⬊ࢆ௜╔ࡉࡏࠊ4%࣒࣍ࣝ࢔ࣝࢹࣄࢻ࡟ᾐࡋ࡚ᐊ

࡛ 15 ศ㛫⣽⬊ࢆᅛ

ᐃࡋࡓࠋPBS ࡟࡚⣽⬊ࢆὙίࡋࣈࣟࢵ࢟ࣥࢢࣂࢵࣇ࢓࣮(1×PBSࠊ5%ṇᖖࣖࢠ⾑Ύࠊ0.3% TritonTMX100)࡛ 60 ศ㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋࡑࡢᚋࠊࣈࣟࢵ࢟ࣥࢢ
ࣂࢵࣇ࢓࣮ࢆ྾ᘬ㝖ཤࡋࠊ୍᫬ᢠయࢆ࢔ࣉࣛ࢖ࡋ࡚ 4Υ୍࡛ᬌ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋPBS ࡛Ὑίࡋࡓᚋࠊ⺯ගᶆ㆑஧ḟᢠయࢆຍ࠼࡚ᬯᡤࠊᐊ

࡛ 2 ᫬㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ஧ḟᢠయ࡟ࡣࠊAnti-Rabbit IgG (H+L), F (ab’)2 Fragment Alexa
Fluor 488 (#4412; Cell Signaling Technology)ࢆ౑⏝ࡋࡓࠋࡲࡓࠊ⣽⬊᰾ᰁⰍ࡟
Hoechst33342ࠊ⣽⬊㦵᱁ᰁⰍ࡟ fluorescent F-actin probe Rhodamine Phalloidin(Cytoskeleton, Denver, Co, USA)ࢆ౑⏝ࡋࠊ஧ḟᢠయ࡜ྠ᫬࡟࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓ ࠋ ෌ ᗘ Ὑ ί ࢆ ⾜ ࠸ ࠊ ᑒ ධ ๣ ࢆ ῧ ຍ ࡋ ࡓ ࡶ ࡢ ࢆ BIOREVO fluorescence49

microscope (Keyence, Osaka, Japan)࡟࡚ほᐹࡋࡓࠋ10. ⤫ィᏛⓗゎᯒྛᐇ㦂ࡣ 3 ᅇࡎࡘ⾜ࡗࡓࠋᐇ㦂ᡂ⦼ࡣᖹᆒ್±ᶆ‽೫ᕪ࡛♧ࡋࠊ⤫ィᏛⓗ࡞ẚ㍑
ࡣ Student’s t test ࡟ࡼࡾ⾜ࡗࡓࠋ༴㝤⋡ 5%ᮍ‶ࢆ᭷ពᕪ࠶ࡾ࡜ࡋࡓࠋྛࢢࣛࣇ
ࡢ᭷ពᕪࡣ p0.05,

p0.01,p0.001 ࡛♧ࡋ࡚࠸ࡿࠋ

50

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56◊✲ᴗ⦼┠㘓➨୍ⴭ⪅࡛ሗ࿌ࡋࡓㄽᩥ
1. Perturbation of the Warburg effect increases the sensitivity to TRAIL-induce
cell death.
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1. A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human
Bladder Cancer T24 Cells.
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2. Colorectal cancer cell-derived extracellular vesicles induced phenotypicalteration of T cells into tumor-growth supporting cells with transforming growth
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3. PKM1 is involved in resistance to anti-cancer drugs.
Taniguchi K, Sakai M, Sugito N, Kuranaga Y, Kumazaki M, Shinohara H, Ueda
H, Futamura M, Yoshida K, Uchiyama K, Akao Y.
Biochemical and Biophysical Research Communications. 2016 Apr 22.

57

4. PTBP1-associated microRNA-1 and -133b suppress the Warburg effect incolorectal tumors.
Taniguchi K, Sakai M, Sugito N, Kumazaki M, Shinohara H, Yamada N,
Nakayama T, Ueda H, Nakagawa Y, Ito Y, Futamura M, Uno B, Otsuki Y,
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Shinohara H, Sugito N, Taniguchi K, Ito Y, Nakayama T, Uno B, Itoh A, Akao Y.
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6. Perturbation of energy metabolism by fatty-acid derivative AIC-47 and imatinibin BCR-ABL-harboring leukemic cells.
Shinohara H, Kumazaki M, Minami Y, Ito Y, Sugito N, Kuranaga Y, Taniguchi K,
Yamada N, Otsuki Y, Naoe T, Akao Y.
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7. MicroRNA-145 repairs infarcted myocardium by accelerating cardiomyocyte
autophagy.
Higashi K, Yamada Y, Minatoguchi S, Baba S, Iwasa M, Kanamori H, Kawasaki
M, Nishigaki K, Takemura G, Kumazaki M, Akao Y, Minatoguchi S.
American Journal of Physiology: Heart and Circulatory Physiology. 2015 Dec 1.
8. Positive feedback of DDX6/c-Myc/PTBP1 regulated by miR-124 contributes tomaintenance of the Warburg effect in colon cancer cells.
Taniguchi K, Sugito N, Kumazaki M, Shinohara H, Yamada N, Matsuhashi N,
Futamura M, Ito Y, Otsuki Y, Yoshida K, Uchiyama K, Akao Y.
Biochimica et Biophysica Acta. 2015 Sep 1.
9. MicroRNA-124 inhibits cancer cell growth through PTBP1/PKM1/PKM2
feedback cascade in colorectal cancer.
Taniguchi K, Sugito N, Kumazaki M, Shinohara H, Yamada N, Nakagawa Y, Ito
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58

10. Organ-specific PTB1-associated micoRNAs determine expression of
pyrucate kinase isoforms.
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11. Anti-cancer fatty-acid derivative induced autophagic cell death throughmodulation of PKM isoform expression profile mediated by bcr-abl in chonicmyeloid leukemia.
Shinohara H, Taniguchi K, Kumazaki M, Yamada N, Ito Y, Otsuki Y, Uno B,
Hayakawa F, Minami Y, Naoe T, Akao Y.
Cancer Letters. 2015 Apr 28.
12. Colorectal cancer cell-derived microvesicles containing microRNA-1246
promotes angiogenesis by activating Smad 1/5/8 signaling elicited by PML
down-regulation in endothelial cells.
Yamada N, Tsujimura N, Kumazaki M, Shinohara H, Taniguchi K, Nakagawa Y,
Naoe T, Akao Y.
Biochimica et Biophysica Acta. 2014 Nov.

59

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