DOI QR코드

DOI QR Code

Cytotoxicity of Mifepristone via Calcium Modulation In Human Prostate Cancer Cells

인간 전립선암세포에 있어서 칼슘조절을 통한 mifepristone의 세포독성효과

  • Song, Hwi-June (Department of Biological Sciences, Pusan National University) ;
  • Kim, Byeong-Gee (Department of Biological Sciences, Pusan National University)
  • 송휘준 (부산대학과 자연과학대학 생명과학과) ;
  • 김병기 (부산대학과 자연과학대학 생명과학과)
  • Published : 2009.07.30

Abstract

MIF is a progesterone analogue and is known as a potent progesterone antagonist. Although MIF has been known to inhibit prostate cancer cell growth, its molecular mechanisms are not yet clear. In the present study, when the cells were treated for 2-4 days with 5-40 $\mu$M of MIF, the growth and viability of LNCaP cells were significantly decreased in a dose- and time-dependent manner. When the cells, cultivated in a normal 2 mM calcium concentration medium, were treated with 15 $\mu$M MIF for 1 day, the intracellular calcium level increased by 26% compared to the control. Similar results were also found in cells located in the calcium-free reaction buffer, indicating that MIF induced the increase of intracellular Ca$^{2+}$ levels, regardless of the presence of calcium in the surrounding medium. In the cells treated with various concentrations of MIF, the intracellular calcium levels increased in a dose dependent manner. Cells treated with MIF revealed typical early apoptotic signs, i.e., chromosome condensation and nuclei fragmentation. In cells treated with 40 11M MIF, Bcl-2 decreased to 19% of the control. The expression of Bax increased to almost 2 fold of the control. These results demonstrated very clearly that MIF treatment blocks the expression of Bcl-2 but stimulates the expression of Bax. According to the results of the present investigation, the apoptotic mechanism of MIF is triggered by intracellular modulation.

Mifepristone (MIF)은 프로게스테론 유사체이며, 강한 항프로게스테론 효과 때문에 전립선암 치료에 사용되고 있다. 본 연구에서는 MIF의 세포독성효과가 세포 내 칼슘농도 조절에 의한 것임을 밝힌다. 5-40 $\mu$M의 MIF를 처리 시 LNCaP 전립선암세포의 성장이 농도와 시간의존적으로 감소하였다. 반대로, 세포 내 칼슘의 레벨은 MIF의 처리시간과 농도도 의존적으로 증가하였다. MIF를 처리한 세포를 PI 혹은 Hoechst로 염색한 결과, 전형적인 세포자살의 징후인 응축된 염색질과 핵 조각단편들이 관찰되었다. 이들 세포자살징후 역시 MIF의 처리시간과 농도가 증가 할수록 심화되었다. 세포자살에 직접적으로 관여하는 중요한 단백질인 Bcl-2 그룹 단백질의 발현을 조사해 본 결과, 세포자살 억제단백질인 Bcl-2의 발현은 MIF처리시 치명적으로감소하였고, 대신에 세포자살 촉진단백질인 Bax의 발현은 2배로 증대되었다. 이상의 결과로 보아 MIF의 세포독성효과는 세포 내의 칼슘조절에 따른 세포자살에 의한 것으로 생각된다.

Keywords

References

  1. Arraudeau, S., M. Frieden, K. Nakamura, C. Castelbou, M. Michalak, and N. Demaurex. 2002. Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria. J. Biol. Chem. 277, 46696-46705 https://doi.org/10.1074/jbc.M202395200
  2. Cabeza, M., E. Bratoeff, I. Heuze, A. Guzman, G. Gomez, H. Berrios, and A. M. Rosales. 2007. Antiandrogenic and apoptotic effects of RU-486 on animal prostate. J. Steroid Biochem. Mol. Biol. 104, 321-325 https://doi.org/10.1016/j.jsbmb.2007.03.009
  3. Chami, M., A. Prandin, M. Campanella, P. Pinton, G. Szabadkai, J. C. Reed, and R. Rizzuto. 2004. Bcl-2 and bax exert opposing effects on $Ca^{2+}$ signaling, which do not depend on their putative pore-forming region. J. Biol. Chem. 279, 54581-54589 https://doi.org/10.1074/jbc.M409663200
  4. Chaudhary, K. S., P. D. Abel, and E. -N. Lalani. 1999. Role of the Bcl-2 gene family in prostate cancer progression and its implications for therapeutic intervention. Environmnt. Health Perspect. 107, 49-57 https://doi.org/10.2307/3434471
  5. El Etreby, M. F., Y. Liang, and R. W. Lewis. 2000. Induction of apoptosis by mifepristone and tamoxifen in human LNCaP prostate cancer cells in culture. The Prostate 43, 31-42. https://doi.org/10.1002/(SICI)1097-0045(20000401)43:1<31::AID-PROS5>3.0.CO;2-#
  6. Ferrari, D., P. Pinton, G. Szabadkai, M. Chami, M. Campanella, T. Pozzan, and R. Rizzuto. 2002. Endoplasmic reticulum, bcl-2 and $Ca^{2+}$ handling in apoptosis. Cell Calcium 32, 413-420 https://doi.org/10.1016/S0143416002002014
  7. Hajnoczly, G., E. Davies, and M. Muniswamy. 2003. Calcium signaling and apoptosis. Biochem. Biophys. Res. Commun. 304, 445-454 https://doi.org/10.1016/S0006-291X(03)00616-8
  8. Li, A., J. C. Felix, P. Minoo, C.A. Amezcua, and J. K. Jain. 2005. Effect of mifepristone on proliferation and apoptosis of ishikawa endometrial adenocarcinoma cells. Fertil. Steril. 84, 202-211 https://doi.org/10.1016/j.fertnstert.2005.01.126
  9. Madsen-Bouterse, S. A., G. M. Rosa, and J. L. Bruton. 2006. Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils. Endocrinol. 147, 8826-8834
  10. Moe, B. G., A. B. Vereide, A. Orbo, and G. Sager. 2009. High concentrations of progesterone and mifepristone mutually reinforce cell cycle retardation and induction of apoptosis. Anticancer Res. 29, 1053-1058
  11. Nutt, L .K., J. Cahndra, A. Pataer, B. Fang, J. A. Roth, S. G. Swisher, R. G. O’neil, and D. J. McConkey. 2002. Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis. J. Biol. Chem. 277, 20301-20308 https://doi.org/10.1074/jbc.M201604200
  12. Ozaki, M. A., H. Matsubara, T. Sato, K. Ikuta, Y. Nishizawa, and K. Suzumori. 2005. Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulose cells. Am. J. Reprod. Immunol. 53, 144-152 https://doi.org/10.1111/j.1600-0897.2005.00258.x
  13. Pinton, P., D. Ferrari, E. Rapizzi, F. Di Virgilio, T. Pozzan, and R. Rizzuto. 2002. A role for calcium in bcl-2 action? 195-201 84, 195-201 https://doi.org/10.1016/S0300-9084(02)01373-1
  14. Schneider, C. C., R.K. Gibb, D. D. Taylor, T. Wan, and C. Gercel-Taylor. 1998. Inhibition of endometrial cancer cell lines by mifepristone (RU486). J. Soc. Gynecol. Investig. 5, 334-338 https://doi.org/10.1016/S1071-5576(98)00037-9
  15. Scorrano, L., S. A. Oakes, J. T. Opferman, D. H. Cheng, M. D. Scorcinelli, T. Pozzan, and S. J. Korsmeyer. 2003. Bax and bak regulation of endoplasmic reticulum $Ca^{2+}$: A control point for apoptosis. Science 300, 135-139 https://doi.org/10.1126/science.1081208
  16. Sharma, A. K. and B. Rohrer. 2004. Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photpreceptor cell line. 35564-35572. 279, 35564-35572 https://doi.org/10.1074/jbc.M401037200
  17. Smaili, S. S., Y.-T. Hsu, A. C. P. Carvalho, T. R. Rosenstock J. C. Sharpe, and R. J. Youle. 2003. Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling. Braz. J. Med. Biol. Res. 36, 183-190
  18. Song, L.-N., M. Coghlan, and E. P. Gelmann. 2004. Antiandrogen effects of mifepristone on cocativator and corepressor interactions with the androgen receptor. Mol. Endocrinol. 18, 70-85 https://doi.org/10.1210/me.2003-0189
  19. Taplin, M.-E., J. Manola, W. K. Oh, P. W. Kantoff, G. L. Bubley, M. Smith, D. Barb, C. Mantzoros, D. P. Gelmann, and S. P. Balk. 2008. A phase II study of mifepristone (RU-486) in castration-resistnat prostate cancer, with a correlative assessment of androgen-related hormones. J. Compilation 101, 1084-1089
  20. Wertz, I. E. and V. M. Dixit. 2000. Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells. J. Biol. Chem. 275, 11470-11477 https://doi.org/10.1074/jbc.275.15.11470
  21. Zhang, H., J. J. Lu, Q. Z. Gao, and J. Zhang. 2006. Induction of apoptosis by mifepristone in androgen-independent prostate cancer cell lines in vitro. Zhonghua Wai Ke Za Zhi 44, 382-385
  22. Zhang W., L. Huang, Y. Zhuang, and W. Wang. 2008. The effect of mifepristone on apoptosis and caspase-3 activation in human ovarian luteinized granulose cells. Eur. J. Obstet. Gynecol. Reprod. Biol. 141, 131-136 https://doi.org/10.1016/j.ejogrb.2008.07.031