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인체 방광암 및 백혈병세포에서 genistein에 의한 세포주기 G2/M arrest 유발에 관한 연구

Induction of G2/M Arrest of the Cell Cycle by Genistein in Human Bladder Carcinoma and Leukemic Cells

  • 김의겸 (한국과학영재학교) ;
  • 명유호 (한국과학영재학교) ;
  • 송관성 (한국과학영재학교) ;
  • 이기홍 (한국과학영재학교) ;
  • 류충호 (경상대학교 농과대학 식품공학과) ;
  • 최영현 (동의대학교 한의과대학 생화학교실 및 대학원 바이오물질제어학과)
  • Kim, Eu-Kyum (Korea Science Academy) ;
  • Myong, You-Ho (Korea Science Academy) ;
  • Song, Kwan-Sung (Korea Science Academy) ;
  • Lee, Ki-Hong (Korea Science Academy) ;
  • Rhu, Chung-Ho (Division of Applied Life Science, Gyeongsang National University) ;
  • Choi, Yung-Hyun (Department of Biochemistry, Dongeui University College of Oriental Medicine and Department of Biomaterial Control, Dongeui University Graduate School)
  • 발행 : 2006.07.31

초록

본 연구에서는 T24 인체방광암 및 U937 백혈병 세포의 증식에 미치는 genistein의 영향을 조사 하였다. Genistein이 처리된 T24 및 U937 세포는 처리 농도 의존적으로 세포의 증식이 현저히 감소되었으며 심한 형태적 변형이 동반되었으나, U937 세포에서 보다 높은 감수성을 보였다. 이러한 T24 및 U937 세포의 증식억제 및 형태 변형은 G2/M기의 세포주기 억제 및 apoptosis 유발과 연관성이 있음을 flow cytometry를 이용한 세포주기의 분석을 통하여 확인하였다. T24 세포에서 genistein에 의한 G2/M arrest는 cyclin A, cyclin B1 및 Cdc25C 등의 단백질 발현 감소와 연관성이 있었으나, 종양억제 유전자 p53 및 Cdk inhibitor p21의 발현에는 큰 변화가 없었다. U937 세포에서 genistein에 의한 G2/M arrest는 cyclin B1 및 p53 비의존적인 p21의 발현 증가와 연관성이 있었다. 이상의 결과들은 현재까지 거의 연구가 진행된 바 없는 인체방광암 및 백혈병 세포에서 genistein의 항암작용을 이해하는데 중요한 자료가 될 것이고 나아가 genistein을 포함한 그와 유사한 항암제 후보물질들의 연구에 있어서 기초 자료로서 사용될 수 있을 것으로 생각된다.

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinase and DNA topoisomerase activities. There are several studies documenting molecular alterations leading to cell cycle arrest and induction of apoptosis by genistein as a chemopreventive agent in a variety of cancer cell lines; however, its mechanism of action and its molecular targets on human bladder carcinoma and leukemic cells remain unclear. In the present study, we have addressed the mechanism of action by which genistein suppressed the proliferation of T24 bladder carcinoma and U937 leukemic cells. Genistein significantly inhibited the cell growth and induced morphological changes, and induced the G2/M arrest of the cell cycle in both T24 and U937 cells with a relatively stronger cytotoxicity in U937. The G2/M arrest in T24 cells was associated with the inhibition of cyclin A, cyclin B1 and Cdc25C protein expression without alteration of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1). However, the inhibitory effects of genistein on the cell growth of U937 cells were connected with a marked inhibition of cyclin B1 and an induction of Cdk inhibitor p21 proteins by p53-independent manner. These data suggest that genistein may exert a strong anticancer effect and additional studies will be needed to evaluate the different mechanisms between T24 and U937 cells.

키워드

참고문헌

  1. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya and Y. Fukami. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinase. J. Biol. Chem. 262, 5592-5595
  2. Bektic, J., R. Guggenberger, I.E. Eder, A.E. Pelzer, A.P. Berger, G. Bartsch and H. Klocker. 2005. Molecular effects of the isoflavonoid genistein in prostate cancer. Clin. Prostate Cancer 4, 124-129 https://doi.org/10.3816/CGC.2005.n.021
  3. Belenguer, P., L. Pelloquin, V. Baldin, M.L. Oustrin and B. Ducommun. 1995. The fission yeast Nim1/Cdr1 kinase: a link between nutritional state and cell cycle control. Prog. Cell Cycle Res. 1, 207-214
  4. Biggs, J.R., J.E. Kudlow and A.S. Kraft. 1995. The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells. J. Biol. Chem. 271, 901-906 https://doi.org/10.1074/jbc.271.2.901
  5. Choi, Y.H., W.H. Lee, K.Y. Park and L. Zhang. 2000. p53-independent induction of p21 (WAF1/CIP1), reduction of cyclin B1 and G2/M arrest by the isoflavone genistein in human prostate carcinoma cells. Jpn. J. Cancer Res. 91, 164-173 https://doi.org/10.1111/j.1349-7006.2000.tb00928.x
  6. Choi, Y.H., L. Zhang, W.H. Lee and K.Y. Park. 1998. Genistein-induced G2/M arrest is associated with the inhibition of cyclin B1 and the induction of p21 in human breast carcinoma cells. Int. J. Oncol. 13, 391-396
  7. Constantinou, A., K. Kiguchi and E. Huberman. 1990. Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res. 50, 2618-2624
  8. Cooper, M.J., J.J. Haluschak, D. Johnson, S. Schwartz, L.J. Morrison, M. Lippa, G. Hatzivassiliou and J. Tan. 1994. p53 mutations in bladder carcinoma cell lines. Oncol. Res. 6, 569-579
  9. Datto, M.B., Y. Yu and X.-F. Wang. 1995. Functional analysis of the transforming growth factor ${\beta}$ responsive elements in the WAF/Cip1/p21 promoter. J. Biol. Chem. 270, 28623-28628 https://doi.org/10.1074/jbc.270.48.28623
  10. Dorai, T. and B.B. Aggarwal. 2004. Role of chemopreventive agents in cancer therapy. Cancer Lett. 215, 129-140 https://doi.org/10.1016/j.canlet.2004.07.013
  11. Dulic, V., G.H. Stein, D.F. Far and S.I. Reed. 1998. Nuclear accumulation of $p21^{Cip1}$ at the onset of mitosis: a role at the G2/M-phase transition. Mol. Cell. Biol. 18, 546-557 https://doi.org/10.1128/MCB.18.1.546
  12. Elledge, S.J. and J.W. Harper. 1994. Cdk inhibitors: on the threshold of checkpoints and development. Curr. Opin. Cell Biol. 6, 847-852 https://doi.org/10.1016/0955-0674(94)90055-8
  13. Fattaey, A. and R.N. Booher. 1997. Myt1: a Wee1-type kinase that phosphorylates Cdc2 on residue Thr14. Prog. Cell Cycle Res. 3, 233-240
  14. Fotsis, T., M. Pepper, H. Adlercreutz, G. Fleishmann, T. Hase, R. Montesano and L. Schweigerer. 1993. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc. Natl. Acad. Sci. USA 90, 2690-2694
  15. Girard, F., U. Strausfeld, A. Fernandez and N.J.C. Lamb. 1991. Cyclin A is required for the onset of DNA replication in mammalian fibloblasts. Cell 67, 1169-1179 https://doi.org/10.1016/0092-8674(91)90293-8
  16. Guadagno, T.M., M. Ohtsubo, J.M. Roberts and R.K. Assoian. 1993. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science 262, 1572-1575 https://doi.org/10.1126/science.8248807
  17. Jiang, H., J. Lin, Z.Z. Su, F.R. Collart, E. Huberman and P.B. Fisher. 1994. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF/CIP1, expression in the absence of p53. Oncogene 9, 3397-3406
  18. Koff, A., A. Giordano, D. Desai, K. Yamashita, J.W. Harper, S. Elledge, T. Nishimoto, D.O. Morgan, B.R. Franza and J.M. Roberts. 1992. Formation and activation of a cyclin E-Cdk2 complex during the G1 phase of the human cell cycle. Science 257, 1689-1694 https://doi.org/10.1126/science.1388288
  19. Krek, W. and E.A. Nigg. 1991. Differential phosphorylation of vertebrate $p34^{Cdc2}$ kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. EMBO J. 10, 305-316
  20. Kumagai, A. and W.G. Dunphy. 1991. The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system. Cell 64, 903-914 https://doi.org/10.1016/0092-8674(91)90315-P
  21. Li, Y., C.W. Jenkins, M.A. Nichols and Y. Xiong. 1994. Cell cycle expression and p53 regulation of the cyclin-dependent kinase p21. Oncogene 9, 2261-2268
  22. Markovits, J., C. Linassier, P.H. Fosse, J. Couprie, A. Jacquemin-Cablon, J.M. Saucier, J.B. Le Pecq and A. Larsen. 1989. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase. Cancer Res. 49, 5111-5117
  23. Matsushime, H., D.E. Quelle, S.A. Shurtleff, M. Shibuya, C.J. Sherr and J.Y. Kato. 1994. D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol. 14, 2066-2076 https://doi.org/10.1128/MCB.14.3.2066
  24. Messina, M.J., V. Persky, K.D. Setchell and S. Barnes. 1994. Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr. Cancer 21, 113-131 https://doi.org/10.1080/01635589409514310
  25. Meyerson, M. and E. Harlow. 1994. Identification of G1 kinase activity for Cdk6, a novel cyclin D partner. Mol. Cell. Biol. 14, 2077-2086 https://doi.org/10.1128/MCB.14.3.2077
  26. Mezencev, R., J. Mojzis, M. Pilatova and P. Kutschy. 2003. Antiproliferative and cancer chemopreventive activity of phytoalexins: focus on indole phytoalexins from crucifers. Neoplasma 50, 239-245
  27. Morgan, D.O. 1995. Principles of CDK regulation. Nature 374, 131-134 https://doi.org/10.1038/374131a0
  28. Mueller, S.C., Y. Yeh and W.T. Chen. 1992. Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells. J. Cell Biol. 119, 1309-1325 https://doi.org/10.1083/jcb.119.5.1309
  29. Ohsumi, K., C. Katagiri and T. Kishimoto. 1993. Chromosome condensation in Xenopus mitotic extracts without histone H1. Science 262, 2033-2035 https://doi.org/10.1126/science.8266099
  30. Ohtsubo, M. and J.M. Roberts. 1993. Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science 259,1908-1912 https://doi.org/10.1126/science.8384376
  31. Rauth, S., J. Kichina and A. Green. 1997. Inhibition of growth and induction of differentiation of metastatic melanoma cells in vitro by genistein: chemosensitivity is regulated by cellular p53. Bri. J. Cancer 75, 1559-1566 https://doi.org/10.1038/bjc.1997.268
  32. Ravindranath, M.H., S. Muthugounder, N. Presser and S. Viswanathan. 2004. Anticancer therapeutic potential of soy isoflavone, genistein. Adv Exp Med Biol 546, 121-165 https://doi.org/10.1007/978-1-4757-4820-8_11
  33. Sanchez, V., A.K. McElroy and D.H. Spector. 2003. Mechanisms governing maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human cytomegalovirus. J. Virol. 77, 13214-13224 https://doi.org/10.1128/JVI.77.24.13214-13224.2003
  34. Santibanez, J.F., A. Navapro and J. Martinez. 1997. Genistein inhibits proliferation and in vitro invasive potential of human prostate cancer cell lines. Anticancer Res. 17,1199-1204
  35. Sarkar, F.H. and Y. Li. 2002. Mechanisms of cancer chemoprevention by soy isoflavone genistein. Cancer Metastasis Rev 21, 265-280 https://doi.org/10.1023/A:1021210910821
  36. Shao, Z.-M., M.L. Alpaugh, J.A. Fontana and S.H. Barsky. 1998. Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by $p21^{WAF1/CIP1}$ induction, G2/M arrest, and apoptosis. J. Cell. Biochem. 69, 44-54 https://doi.org/10.1002/(SICI)1097-4644(19980401)69:1<44::AID-JCB5>3.0.CO;2-V
  37. Spinozzi, F., C. Pagliacci, M. Graziella, M. Rasalba, F. Grignani, C. Riccardi and I. Nicoletti. 1994. The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in jurkat T-leukemia cells. Leuk. Res. 18, 431-439 https://doi.org/10.1016/0145-2126(94)90079-5
  38. Steele, V.E., M.A. Pereira, C.C. Sigman and G.J. Kelloff. 1995. Cancer chemoprevention agent development strategies for genistein. J. Nutri. 125, 713S-716S
  39. Tchou, W.-W., W.N. Rom and K.-M. Tchou-Wong. 1996. Novel form of $p21^{WAF1/CIP1/SDI1}$ protein in phorbol ester-induced G2/M arrest. J. Biol. Chem. 271, 29556-29560 https://doi.org/10.1074/jbc.271.47.29556
  40. Traganos, F., B. Ardelt, N. Halko, S. Bruno and Z. Darzynkiewicz. 1992. Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells. Cancer Res. 52, 6200-6208
  41. Waga, S., G.J. Hannon, D. Beach and B. Stillman. 1994. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369, 574-578 https://doi.org/10.1038/369574a0
  42. Xiong, Y., G. Hannon, H. Zhang, D. Casso, R. Kobayashi and D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366,701-704 https://doi.org/10.1038/366701a0
  43. Zhang, W., L. Grasso, C.D. McClain, A.M. Gambel, Y. Cha, S. Travali, A.B. Deisseroth and W.E. Mercer. 1995. p53-independent induction of WAF1/CIP1 in human leukemia cells is correlated with growth arrest accompanying monocyte/marcrophage differentiation. Cancer Res. 55, 668-674
  44. Zi, X., D.K. Feyes and R. Agarwal. 1998. Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast canecr cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinase and associated cyclins. Clinical Cancer Res. 4, 1055-1064

피인용 문헌

  1. The Anticancer Effect of Combination of Genistein and Photofrin PDT in Human AMC-HN3 Head and Neck Cancer Cell Lines vol.18, pp.9, 2008, https://doi.org/10.5352/JLS.2008.18.9.1257