Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Mechanism of Growth Inhibition by BCH in HEp2 Human Head and Neck Squamous Cell Carcinoma

사람 두경부 편평세포암종 HEp2 세포에서 BCH에 의한 세포성장 억제기전

  • Choi, Bong-Kyu (Dept. of Pharmacology, Wonkwang University School of Medicine) ;
  • Jung, Kyu-Yong (Dept. of Pharmacology, Wonkwang University School of Medicine) ;
  • Cho, Seon-Ho (Dept. of Oral Physiology and The Second Stage of BK21, Chosun University College of Dentistry) ;
  • Kim, Chun-Sung (Dept. of Oral Physiology and The Second Stage of BK21, Chosun University College of Dentistry) ;
  • Kim, Do-Kyung (Dept. of Oral Physiology and The Second Stage of BK21, Chosun University College of Dentistry)
  • 최봉규 (원광대학교 의과대학 약리학교실) ;
  • 정규용 (원광대학교 의과대학 약리학교실) ;
  • 조선호 (조선대학교 치과대학 구강생리학교실 및 2단계 BK21 프로젝트) ;
  • 김춘성 (조선대학교 치과대학 구강생리학교실 및 2단계 BK21 프로젝트) ;
  • 김도경 (조선대학교 치과대학 구강생리학교실 및 2단계 BK21 프로젝트)
  • Published : 2008.05.31
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Abstract

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the $Na^+$-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1), an isoform of system L amino acid transporter, is highly expressed in cancer cells to support their continuous growth and proliferation. 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is a model compound for the study of amino acid transporter as a system L selective inhibitor. We have examined the effect and mechanism of BCH on cell growth suppression in HEp2 human head and neck squamous cell carcinoma. The BCH inhibited the L-leucine transport in a concentration-dependent manner with a $IC_{50}$ value of $51.2{\pm}3.8{\mu}M$ in HEp2 cells. The growth of HEp2 cells was inhibited by BCH in the timeand concentration-dependent manners. The formation of DNA ladder was not observed with BCH treatment in the cells. Furthermore, the proteolytic processing of caspase-3 and caspase-7 in the cells were not detected by BCH treatment. These results suggest that the BCH inhibits the growth of HEp2 human head and neck squamous cell carcinoma through the intracellular depletion of neutral amino acids for cell growth without apoptotic processing.

사람 두경부 편평세포암종 HEp2 세포를 이용하여 아미노산 수송계 L 억제제인 BCH의 암세포 성장억제에 미치는 효과와 세포성장 억제기전을 밝히기 위해 HEp2 세포에서 uptake 실험, MTT 분석, DNA fragmentation 분석 및 immunoblotting 등을 시행하여 다음과 같은 결과를 얻었다. 아미노산 수송계 L 억제제인 BCH는 L-leucine uptake를 농도 의존적으로 억제하였으며, 그 $IC_{50}$은$ 51.2{\pm}3.8{\mu}M$로 산출되었다. BCH는 HEp2 세포의 성장을 시간과 농도에 의존적으로 억제하였다. BCH를 처리한 실험군에서 DNA fragmentation 현상은 볼 수 없었다. BCH를 처리한 실험군에서 procaspase-3과 procaspase-7의 proteolytic cleavage 현상은 볼 수 없었다. 본 연구의 결과로서 사람 두경부 편평세포 암종 HEp2 세포에서 아미노산 수송계 L 억제제 BCH는 LAT1 활성을 억제하여 세포성장에 필수적인 L-leucine 등 중성아미노산의 세포 내 고갈을 유도함으로써 HEp2 세포의 성장억제를 유도할 가능성이 있는 것으로 사료된다.

Keywords

References

  1. Christensen HN. 1990. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev 70: 43-77 https://doi.org/10.1152/physrev.1990.70.1.43
  2. Silbernagl S. 1979. Renal transport of amino acids. Klin Wochenschr 57: 1009-1019 https://doi.org/10.1007/BF01479986
  3. Kanai Y, Endou H. 2001. Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance. Curr Drug Metab 2: 339-354 https://doi.org/10.2174/1389200013338324
  4. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H. 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 273: 23629-23632 https://doi.org/10.1074/jbc.273.37.23629
  5. Yanagida O, Kanai Y, Chairoungdua A, Kim DK, Segawa H, Nii T, Cha SH, Matsuo H, Fukushima J, Fukasawa Y, Tani Y, Taketani Y, Uchino H, Kim JY, Inatomi J, Okayasu I, Miyamoto K, Takeda E, Goya T, Endou H. 2001. Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim Biophys Acta 1514: 291-302 https://doi.org/10.1016/S0005-2736(01)00384-4
  6. Uchino H, Kanai Y, Kim DK, Wempe MF, Chairoungdua A, Morimoto E, Anders MW, Endou H. 2002. Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition. Mol Pharmacol 61: 729-737 https://doi.org/10.1124/mol.61.4.729
  7. Mastroberardino L, Spindler B, Pfeiffer R, Skelly PJ, Loffing J, Shoemaker CB, Verrey F. 1998. Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family. Nature 395: 288-291 https://doi.org/10.1038/26246
  8. Pfeiffer R, Spindler B, Loffing J, Skelly PJ, Shoemaker CB, Verrey F. 1998. Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond. FEBS Lett 439: 157-162 https://doi.org/10.1016/S0014-5793(98)01359-3
  9. Sang J, Lim YP, Panzica M, Finch P, Thompson NL. 1995. TA1, a highly conserved oncofetal complementary DNA from rat hepatoma, encodes an integral membrane protein associated with liver development, carcinogenesis, and cell activation. Cancer Res 55: 1152-1159
  10. Wolf DA, Wang S, Panzica MA, Bassily NH, Thompson NL. 1996. Expression of a highly conserved oncofetal gene, TA1/E16, in human colon carcinoma and other primary cancers: homology to Schistosoma mansoni amino acid permease and Caenorhabditis elegans gene products. Cancer Res 56: 5012-5022
  11. Verrey F, Meier C, Rossier G, Kuhn LC. 2000. Glycoprotein- associated amino acid exchangers: broadening the range of transport specificity. Pflugers Arch 440: 503-512 https://doi.org/10.1007/s004240000274
  12. Pineda M, Fernandez E, Torrents D, Estevez R, Lopez C, Camps M, Lloberas J, Zorzano A, Palacin M. 1999. Identification of a membrane protein, LAT-2, that co-expressed with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small large zwitterionic amino acids. J Biol Chem 274: 19738-19744 https://doi.org/10.1074/jbc.274.28.19738
  13. Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, Kanai Y. 1999. Identification and functional characterization of a $Na^+$-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem 274: 19745- 19751 https://doi.org/10.1074/jbc.274.28.19745
  14. Rossier G, Meier C, Bauch C, Summa V, Sordat B, Verrey F, Kuhn LC. 1999. LAT2, a new basolateral 4F2hc/ CD98- associated amino acid transporter of kidney and intestine. J Biol Chem 274: 34948-34954 https://doi.org/10.1074/jbc.274.49.34948
  15. Kim DK, Kanai Y, Choi HW, Tangtrongsup S, Chairoungdua A, Babu E, Tachampa K, Anzai N, Iribe Y, Endou H. 2002. Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells. Biochim Biophys Acta 1565: 112-121 https://doi.org/10.1016/S0005-2736(02)00516-3
  16. Sloan JL, Mager S. 1999. Cloning and functional expression of a human $Na^+$ and $Cl^-$-dependent neutral and cationic amino acid transporter $B^{0+}$. J Biol Chem 274: 23740-23745 https://doi.org/10.1074/jbc.274.34.23740
  17. Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM, Lee SS, Yoon JH, Joo SY, Cha IH, Yook JI. 2002. Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett 177: 41-47 https://doi.org/10.1016/S0304-3835(01)00781-9
  18. Miller MC 3rd, Johnson KR, Willingham MC, Fan W. 1999. Apoptotic cell death induced by baccatin III, a precursor of paclitaxel, may occur without G(2)/M arrest. Cancer Chemother Pharmacol 44: 444-452 https://doi.org/10.1007/s002800051117
  19. Kok SH, Hong CY, Kuo MY, Lee CH, Lee JJ, Lou IU, Lee MS, Hsiao M, Lin SK. 2003. Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes. Oral Oncol 39: 19-26 https://doi.org/10.1016/S1368-8375(01)00129-4
  20. Datta R, Kojima H, Yoshida K, Kufe D. 1997. Caspase-3- mediated cleavage of protein kinase C theta in induction of apoptosis. J Biol Chem 272: 20317-20320 https://doi.org/10.1074/jbc.272.33.20317
  21. Liu X, Zou H, Slaughter C, Wang X. 1997. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89: 175- 184 https://doi.org/10.1016/S0092-8674(00)80197-X
  22. Cohen GM. 1997. Caspases: the executioners of apoptosis. Biochem J 326: 1-16 https://doi.org/10.1042/bj3260001