Stimulation of D-[14C]-Met uptake in cells
茨城県立医療大学紀要
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Original Article

Volume 23

Increased D-[S-methyl-14C]-methionine after Co-loading of
L-tyrosine ethyl ester in C6 Glioma Cells
Naoto Shikano1), Kentaro Kusanagi1), Masato Ogura1), Syuichi Nakajima1), Yuki Serizawa1),
Yuko Hamai1), Sanae Matsutani1), Masato Kobayashi2), Keiichi Kawai2)

1)

Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences

2)

Division of Health Science, Graduate School of Health Sciences, Kanazawa University

Abstract
Introduction: We investigated whether trans-stimulation by amino acid ethyl ester co-loading increases D-[S-methyl-14C]methionine (D-[14C]-Met) uptake into C6 glioma cells.
Methods: Effective amino acid ethyl esters for enhancing D/L-[14C]-Met transport were determined by incubating cells at 37°C
for 1 h in Na+-containing medium together with 18.5 kBq of non-carrier-added D/L-[14C]-Met and 1.0 mM of the ethyl ester
chloride salt of Gly, L-Arg, L-Ser, L-Leu, L-Met, L-Tyr, L-Val, L-Lys or L-Pro. Similarly, for the time course study of D/L-[14C]Met uptake with L-Tyr-OEt, the cells were incubated for 5, 10, 30, 60 and 90 min. In addition, the effects of MeAIB (a system
A-specific inhibitor) and BCH (a system L-specific inhibitor) at 1.0 mM were determined. Radioactivity was measured using aliquid scintillation counter.
Results: L-Tyr-OEt most effectively enhanced the uptake of both D- and L-[14C]-Met: 4.7-fold for D-[14C]-Met, and 2.5-fold forL-[14C]-Met. The highest uptake value of D-[14C]-Met with L-Tyr-OEt was observed 90 min or more after the start of incubation.
System L was primarily responsible for mediating D/L-[14C]-Met transport, and System A, which is not an amino acid exchanger,
contributed to D-[14C]-Met transport.
Conclusions: Trans-stimulation by L-Tyr-OEt co-loading increased D-[14C]-Met uptake into C6 glioma cells in vitro.
Key words: D-[S-methyl-14C]-methionine, L-tyrosine ethyl ester, trans-stimulation, system L, C6 glioma cell

1. Introduction

2-(methylamino)isobutyric acid (MeAIB), and system
ASC is Na+-dependent and intolerant of N-methylated

Neutral amino acids are transported by three main

substrates. Systems L and A have specific inhibitors,

systems, systems L, A, and ASC, that have overlappingwhereas system ASC does not.1,2) Kanai et al. previously

substrate specificities.1,2) System L is defined by being

identified a system L amino acid exchanger, LAT1 (L-type

Na+-independent and by its inhibition by 2-amino-amino acid transporter 1: system L transporter isoform), inbicyclo[2,2,1]heptane-2-carboxylic acid (BCH). System

C6 rat glioma cells and demonstrated that LAT1 requires

A is defined by its Na dependence and inhibition by

4F2 heavy chain (4F2hc) for its functional expression.3)+

Address: Naoto Shikano. Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences,
Ami4669-2, Ami, Inashiki, Ibaraki 300-0394, Japan
Phone: +81-29-840-2217, Fax: +81-29-840-2268, E-mail : sikano@ipu.ac.jp

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茨城県立医療大学紀要

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Expression of LAT1 in fetal rat liver and tumors suggests

whether trans-stimulation using L-tyrosine ethyl esterthat LAT1 plays a critical role in cell growth andaffects LAT1-mediated uptake of the D-isomer of [14C]-

proliferation. The degree of LAT1 and ATA1 (amino acidmethionine into tumor cells, such as C6 glioma cells.

transporter A1: system A transporter isoform) expression

There are no published data describing an in vitro method

reportedly indicates the extent of tumor malignancy.4,5)

for observing the amino acid exchange function for

Most transporters can only transport compounds of similar

especially D-isomer amino acids. For example, there

structure, whereas LAT1 can transport essential amino

is no current method for enhancing D-[S-methyl-14C]-

acids and many functionally designed non-natural amino

methionine (D-[14C]-Met) uptake or its trans-stimulation

acids.6-8) Four isoforms of system L transporter, LAT1

in C6 glioma cells in combination with an amino acid

to 4, are known. Of these, LAT1 and LAT2 are systemester.

9)

L1 transporters and are amino acid exchangers. Trans-

In this study, we investigated the effectiveness ofstimulation by administration can occur in LAT1 andtrans-stimulation by co-loading of an amino acid ester

LAT2, because they are amino acid exchangers.9) Trans-

on D-isomer amino acid uptake, and we determined

stimulation by administration cannot occur in LAT3 andthe most effective amino acid ester for enhancing the

LAT4, which are system L2 transporters, because they are

accumulation of D-[14C]-Met and L-[14C]-Met in C6

not amino acid exchangers.9) The system L and ASC amino

glioma cells. Because D/L-[11C]-Met are clinically mostacid transporters LAT1 and ASCT-2 play roles in counter

popular labeled essential amino acids in positron emission

transport

and are very important for processes that use

computed tomography (PET) study today, and it is easylarge amounts of amino acids, such as tumor growth andto radiolabel. The results of this study could be usefulproliferation.10) In contrast, system A transporters do notfor developing molecular imaging and cancer treatment

play roles in counter transport, because they are not amino

agents based on D-amino acid.

10)acid exchangers.
To address the question “How can tumor uptake of
2. Materials and methods

amino acids be manipulated and improved?”, Jager et al.
commented that the main point is to find a tracer with highspecific uptake in tumor tissue (first approach) and to look2.1. Materials

for methods to improve tumor uptake or improve contrast

D-[14C]-Met (2.22 GBq/mmol) and L-[14C]-Met (2.22with the background (second approach).11) The second

GBq/mmol) were purchased from American Radiolabeledapproach comprises two tactics: manipulating the transport

Chemicals Co. (St. Louis, MO, USA). C6 (rat glial cellsystem (influx and especially efflux mechanisms), or

tumor) cells were purchased from JCRB (Osaka, Japan).

manipulating the kinetics of amino acid metabolism.

Plastic tissue culture dishes (60 mm diameter) and plastic

Lahoutte et al. found that tumors with pre-loaded natural

culture flasks (surface area 25 cm2) were purchased

amino acids increased

125/123

I-3-iodo-alpha-methyl-L-

from Nalge Nunc International (Rochester, NY, USA).

tyrosine (IMT) tumor uptake and increased contrast in an

2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH,in vivo animal imaging study.

These findings led us toa system-L specific inhibitor), 2-(methylamino)isobutyric

investigate methods for enhancing the accumulation ofacid (MeAIB, a system-A specific inhibitor), natural L/D-

drugs via LAT1 for improving diagnosis and treatment,amino acids, and reagent grade chemicals were purchased

and thus we attempted to enhance the function of LAT1

from Kanto Chemical Co., Inc. (Tokyo, Japan).

12)

for the trans-stimulation of neutral amino acid exchange
2.2. Cell cultureusing an amino acid ethyl ester as a prodrug.13,14)
We previously observed that L-tyrosine ethyl

C6 cells (105 cells/dish) were incubated in 60-mm

ester stimulated the uptake of IMT by CHO-K1 cells,13)

dishes with 5 ml of Dulbecco’s modified Eagle’s medium

which are not tumor cells. However, it remained unclear(DMEM: Cat. No. D5796; Sigma Chemical Co., St. Louis,

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Stimulation of D-[14C]-Met uptake in cells

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MO, USA) containing 10% fetal calf serum at 37°C in acells in good condition for an extended time but neutral

5% CO2 atmosphere (pH 7.4). The cells were sub-cultured

amino acids contained as nutrients in the uptake solution

every 5 days using 0.02% ethylenediamine tetraacetic

affect D/L-[14C]-Met uptake. Consequently, we used PBS

acid (EDTA) and 0.05% trypsin. Cells were used forcontaining sodium as the uptake solution in this study,

experiments on the 3rd or 4th day after sub-culturing. A cellconsistent with the sodium found in animals used in inproliferation curve was prepared to determine the durationvivo experiments.

of the logarithmic growth phase.
2.4. Time course of D/L-[14C]-Met uptake with L-Tyr-OEt
2.3. Radioactivity measurements for screening aminoacid esters

To determine the time course of D/L-[14C]-Met uptake
with L-Tyr-OEt, after removing the culture medium, cells

When the C6 cells reached approximately 80%

at approximately 80% confluence in 5 ml of PBS andconfluence (3–4 days after sub-culturing), the medium

incubated for 10 min were transferred to 2.0 ml of Na+

was replaced with 5 ml of phosphate-buffered saline

uptake solution containing 18.5 kBq of D/L-[14C]-Met(PBS) containing Na+ (37°C, pH 7.4) and the cells wereand 1.0 mM of L-Tyr ethyl ester chloride salt. The cells

incubated at 37°C for 10 min. Effective amino acid esters,

were then incubated for 5, 10, 30, 60 and 90 min using thetheir optimal concentration, and the duration of incubation

same conditions as used for screening effective amino acidrequired for enhanced D/L-[ C]-Met transport were

esters, and the radioactivity of each aliquot (200 μl) was

determined as follows. First, the cells were incubated atdetermined by liquid scintillation counting as described+

37°C for 1 h in PBS with Na uptake solution (NaCl; 137

above. Practical diagnostic imaging of a disease state with

mM, KCl; 3.7 mM, Na2HPO4; 8.0 mM, KH2PO4; 1.5 mM,

radiopharmaceuticals is based on net radiopharmaceutical

14

CaCl2･2H2O; 1.8 mM, MgCl･6H2O; 1 mM) containing

accumulation resulting from the influx and efflux of the18.5 kBq of non-carrier-added D/L-[14C]-Met and 1.0

radiopharmaceutical into and out of organs, tissues, andmM of the ethyl ester chloride salt of Gly, L-Arg, L-Ser,

cells. We did not measure the uptake rate (e.g.; mmol/

L-Leu, L-Met, L-Tyr, L-Val, L-Lys or L-Pro. Ethyl estersmin) of the labeled amino acid because this parameterof system L transport substrates and several additional

is not typically used in the diagnosis of cancer. In an inamino acid esters were chosen for screening effective

vitro cell study, the cell accumulation level of the labeled

compounds. Next, the medium was removed, the cells

amino acid following an adequate incubation time is more

were washed twice with 5.0 ml of cold PBS, and then theimportant than measurement of the uptake rate at the start

cells were solubilized using 2.0 ml of NaOH (2 N). The

of incubation. Although an effect of ester co-loading wasradioactivity of each aliquot (200μl) was measured using aobserved 5 to 15 min after the start of incubation, we

liquid scintillation counter (Perkin Elmer, Waltham, MA,

chose an incubation time of 60 min because this provided

USA, Tri-Carb 2910TR). Data are reported as the mean ofthe enough effect of ester co-loading.

the results from four dishes for each set of experimental
2.5. Inhibition of D/L-[14C]-Met uptake by MeAIB andconditions.
We used PBS uptake solution containing sodium

BCH

because it does not contain nutrients and other factors

Uptake inhibition studies were conducted using

found in culture medium. Consequently, if we incubate

MeAIB (a system A-specific inhibitor) and BCH (a systemcells in PBS for more than 90 min, the uptake data should

L-specific inhibitor) at a concentration of 1.0 mM. MeAIB

reflect the influence of cell starvation, possibly resulting in

was dissolved in Na+ uptake solution, pH 7.4, and BCH

autophagy. Furthermore, L-[14C]-Met has low metabolic

was dissolved in sodium-free uptake solution (containing

stability and thus the radioactivity values will include thecholine-Cl and K2HPO4 instead of the NaCl and Na2HPO4

effects of radioactive metabolites. Uptake experimentsin the Na+ uptake solution). After removing the culture

should be conducted in culture medium to maintain themedium, each dish was washed once with 5 ml of PBS for

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茨城県立医療大学紀要

10 min at 37°C. The cells were then incubated with 2 ml

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(2.73 ± 0.22 and 1.66 ± 0.15).

of incubation medium containing 18.5 kBq D/L-[ C]-Met
14

for 10 min at 37°C, after which the radioactivity of eachaliquot (200 μl) was determined as described above.
2.6. Statistical analysis
Data were collated as mean ± standard deviation
calculated from three to five measurements, and eachexperiment was performed in duplicate. The results wereanalyzed using Student’s t test. P-values of less than
0.0001 were taken to indicate statistical significance.

3. Results
3.1. Cell culture and proliferation curve
As shown in Fig. 1, the C6 cells showed continuous
logarithmic growth after seeding, and confluent growth
was seen on day 5. The cells were used in experiments on
day 3 or day 4 after seeding.

Fig. 2 Screening for effective amino acid esters
3.3. Time course of D/L-[14C]-Met uptake with L-Tyr-OEt
Figure 3A shows the time course of D-[14C]-Met
uptake with/without L-Tyr-OEt. The highest uptake valueof D-[14C]-Met without L-Tyr-OEt was observed 10 min
after starting the incubation, whereas the highest uptake
value of D-[14C]-Met with L-Tyr-OEt was observed after
90 min.

Fig. 1 Cell growth curve of C6 cells

Figure 3B shows the results of the L-[14C]-Met
uptake time course with/without L-Tyr-OEt. The highest

3.2. Screening effective amino acid esters

uptake points were 5 min for L-[14C]-Met without L-Tyr-

Figure 2A shows the uptake of D-[14C]-Met with

OEt and 90 min for L-[14C]-Met with L-Tyr-OEt.

various amino acid esters; the effective esters were L-Tyr-The use of L-Tyr-OEt to stimulate the uptake of

OEt, L-Lys-OEt and L-Pro-OEt (p < 0.001 vs. control).

D-[14C]-Met and L-[14C]-Met resulted in up to 4.7-

The results for L-[14C]-Met are shown in Fig. 2B and thefold higher uptake than that obtained with D-[14C]-Met

effective esters were L-Arg-OEt and L-Tyr-OEt (p < 0.001

alone, and 2.5-fold higher uptake than that observed

vs. control). The uptake of L-[ C]-Met was inhibited bywith L-[14C]-Met alone in a time course study (Fig. 3).

L-Val-OEt, L-Met-OEt and L-Leu-OEt (p < 0.001 vs.

These trans-stimulation effects were observed 5 to 15

control). L-Tyr-OEt was the most effective amino acidmin after the start of incubation with L-Tyr-OEt, and theester for enhancing the uptake of both D- and L-[ C]-Met

effects increased with time. Furthermore, some amino

14

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Stimulation of D-[14C]-Met uptake in cells

13acid esters stimulated the transport of D-[14C]-Met (Fig.

The present results verify that the uptake of both D-[14C]-

2). Incubation for 90 min or more may result in increased

Met and L-[14C]-Met by C6 cells is mediated primarily by

D/L-[14C]-Met uptake, but uptake saturated within several

the system L transporter. Furthermore, we identified forhours and then decreased. Observation with an opticalthe first time that D-[14C]-Met uptake may be mediatedmicroscope showed no morphological changes in the cellsby system A, as shown in Figure 4A.

during the 90 min experiment.

Fig. 3 Time course of D-[14C]-Met (A) and L-[14C]-Met(B) uptake

Fig. 4 Uptake inhibition study of D-[14C]-Met (A) andL-[14C]-Met (B)
4. Discussion

14

3.4. Inhibition of D/L-[ C]-Met uptake by MeAIB andBCH

It is noteworthy that neutral amino acid transport in

Figures 4A and B show the results of the analysis

cultured glioma cells is dominated by LAT1,15) and C6of the transport mechanism of D/L-[14C]-Met. D-[14C]-

glioma cells express LAT1 but not LAT2 in the presence

Met uptake was inhibited by the addition of MeAIB orof 4F2hc.16) Four isoformes of system L transporter are

BCH. L-[14C]-Met uptake was inhibited by the addition

known, LAT1 to 4.9) LAT1 and LAT3 are tumor-cell-of BCH. These data suggest that system L played a major

type transporters. LAT3 is expressed in specific minor

role in mediating the transport of both D-[14C]-Met (bytumor cells in, for example, hepatocarcinoma andabout 59.6%) and L-[ C]-Met (by about 71.4%), andprostate cancer.9) LAT2 and LAT4 are normal-cell-type

that sodium-dependent transport played a minor role intransporters9) and there is no report suggesting LAT4

D-[14C]-Met transport. MeAIB enhanced L-[14C]-Met

expression in C6 glioma cells. Trans-stimulation of

uptake by about 19.6% in Na uptake solution, but thisLAT1 and LAT2 can occur following the administration

increase was not statistically significant.

of L-Tyr-OEt because LAT1 and LAT2 are amino acid14+

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茨城県立医療大学紀要

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exchangers. In contrast, trans-stimulation of LAT3 andcompetitive inhibition of influx. Competitive inhibition

LAT4 by L-Tyr-OEt cannot occur because those isoforms

could be caused by preloading the amino acid which could

cannot exchange amino acids. System A transporters are

not be removed before uptake of D- and L-[14C]-Met, andnot trans-stimulation sensitive, because they are not amino

would minimize the enhancement of labeled amino acidacid exchangers. Therefore, LAT1 is likely the main trans-

influx.

stimulation sensitive transporter mediating D/L-[14C]-

In this study, we selected L-Tyr combined with ethyl

Met transport in C6 cells. We are currently planning toalcohol, but ethyl alcohol may not be the best alcohol fortest the effect of knockout of LAT1 RNA using siRNAin vivo use because it does not specifically distribute toon trans-stimulation or inhibition using a LAT1-specifictarget tumors in vivo. The expression levels of the amino

compound.

acid exchangers ASCT2 and LAT1 are coordinately

Figure 2 shows that L-Tyr-OEt was the most effective

elevated in a wide spectrum of primary human cancers.17)

amino acid ester for enhancing the uptake of both D- andTherefore, our next study will investigate the effect of otherL-[14C]-Met in C6 cells, although the factors underlying

D-amino acids and the substrates for ASCT2 (D-[14C]-Ala

this observation are currently unclear. If the pool of L-Tyrand D-[14C]-Ser) and LAT1 (D-[14C]-Met, D-[14C]-Pheamino acid is smaller than the amount of generated L-Tyrand D-[14C]-Tyr),18-20) together with the amino acid esters

inside C6 glioma cells from L-Tyr-OEt, L-Tyr efflux

studied in the current investigation and other amino acidshould increase due to the exchanger and D/L-[ C]-Met

esters. Our aim will be to identify promising amino acidinflux should also increase due to the exchanger. The

esters for targeting tumor tissue in mouse or rat and tonormal concentration range for each amino acid pool andaddress concentration dependence. Investigations on thethe Km value for the efflux of each substrate amino acid via

effects of other D-amino acids with other amino acidthe exchanger may be very important factors underlying

esters are also anticipated in the near future.

14

why L-Tyr-OEt was the most effective amino acid ester
for enhancing the uptake of both D- and L-[14C]-Met in
Conflict of interest

C6 cells.
As shown in Fig. 3, effects of ester co-loading wereobserved 5 to 15 min after the start of incubation with
L-Tyr-OEt, suggesting that 5 to 15 min may be required

The authors declare that they have no conflict ofinterest.

to synthesize the amino acid inside the cell following
passage of the co-loaded ester across the cell membrane
Acknowledgement

and enzymatic hydrolysis inside the cell. Use of the
opposite enantiomer, D-Tyr-OEt, which is not a substrate
for hydrolysis enzymes, resulted in no enhanced labeled
tyrosine uptake in CHO-K1 cells.

We wish to thank Naoto Muramatsu, Sayaka Imai,

Consequently, we

Kei Satoh, Yumi Suzuki, Yuko Hirotsu and Naomi Tojyo

believe there is also a large increase caused by enzymatic(Ibaraki Prefectural University of Health Sciences)hydrolysis in the amount of L-Tyr inside the C6 cells thatfor their excellent technical assistance. This work was

becomes available due to an amino acid exchanger, most

supported by Grants-in-Aid for Scientific Research

likely LAT1.

(#26461801) from the Ministry of Education, Science,

13)

Trans-stimulation using L-tyrosine ethyl ester

Sports and Culture of Japan and the Japan Society forenhanced the uptake of the D-isomer of [ C]-methionine

the Promotion of Science. Financial support was also

by C6 glioma cells, possibly via LAT1. In this paper

provided by a Japan Atherosclerosis Research Foundation

we propose an in vitro amino acid ester (prodrug of an

Grant in 2016.

14

amino acid that is a substrate for efflux by an aminoacid exchanger) co-loading method that might prevent

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Stimulation of D-[14C]-Met uptake in cells

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93.

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和文抄録
アミノ酸エチルエステル負荷によるトランス刺激が D-[S-methyl-14C]-methionine (D-[14C]-Met) の C6 神経膠腫細胞への取り込みを増加させるかどうかを検討した。どのアミノ酸エチルエステルが D/L-[14C]-Met 輸送を増強するかの検討では，細胞を 1.0 mM の塩化エチルエステル塩 Gly，L-Arg，L-Ser，L-Leu，L-Met，
L-Tyr，L-Val，L-Lys，L-Pro または L-Arg を添加した 18.5kBq の無担体 D/L-[14C]-Met とともに，Na+ 含有
培地中，37℃で 1 時間インキュベートし集積を計測した。また，L-Tyr-OEt を添加し D/L-[14C]-Met の取り込みの経時的測定を行った実験では，細胞を最大 90 分間までインキュベートして集積の計測を行った。さらに，MeAIB（A 系特異的阻害剤）および BCH（L 系特異的阻害剤）の 1.0mM における効果を測定した。
L-Tyr-OEt は D- および L-[14C]-Met の取り込みを最も効果的に増強したが，D-[14C]-Met では 4.7 倍，L-[14C]Met では 2.5 倍であった。L-Tyr-OEt を用いた D-[14C]-Met の最も高い取り込み値は，インキュベーション
開始後 90 分かそれ以上の時間で観測された。システム L は主に D/L-[14C]-Met 輸送の媒介に関与し，シス
テム A は D-[14C]-Met 輸送に寄与した。L-Tyr-OEt 同時負荷によるトランス刺激は，インビトロで C6 グリオー
マ細胞への D-[14C]-Met 取り込みを増加させた。
キーワード : D-[S-methyl-14C]-methionine, L- チロシンエチルエステル , トランス刺激 , L 系 ,C6 グリオーマ細胞

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