Radiation Oncology Journal

  • 제22권2호
  • /
  • Pages.145-154
  • /
  • 2004
  • /
  • 2234-1900(pISSN)
  • /
  • 2234-3156(eISSN)
대한방사선종양학회 (The Korean Society for Radiation Oncology)
권호 표지

담즙산 합성유도체(HS-1200)가 인체 유방암 세포주(MCF-7)에서 유도하는 방사선 감작 효과

A Novel Chenodeoxycholic Derivative HS-1200 Enhances Radiation-induced Apoptosis in Human MCF-7 Breast Cancer Cells

  • 이형식 (동아대학교 의과대학 방사선종양학교실) ;
  • 최영민 (동아대학교 의과대학 방사선종양학교실) ;
  • 권혁찬 (동아대학교 의과대학 내과학교실) ;
  • 송연숙 (동아대학교 의과대학 해부학교실)
  • Lee Hyung Sik (Department of Radiation Oncology, College of Medicine, Ding A University) ;
  • Choi Young Min (Department of Radiation Oncology, College of Medicine, Ding A University) ;
  • Kwon Hyuk Chan (Department of Hemato-oncology College of Medicine, Ding A University) ;
  • Song Yeon Suk (Department of Anatomy and cell Biology College of Medicine, Ding A University)
  • 발행 : 2004.06.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

목적 : 인체 유방암 세포주인 MCF극에 새로운 CDCA합성유도체인 HS-1200을 방사선과 함께 처치하여 아포토시스 유도 활성 및 방사선 감작 효과를 관찰하고자 하였다. 대상 및 방법 : MCF-7 세포에 2$\~$8 Gy의 X-ray와 16$\mu$M 농도의 HS-1200을 처리한 세포들의 세포 생존 곡선을 clonogenic assay를 통하여 구하였다. 아포토시스 유도 확인은 8 Gy의 X-ray와 40$\mu$M 농도의 HS-1200을 전 처치하여 구한 agarose gel 전기영동 및 Hoechst staining을 이용하였다. 면역형광법을 이용한 cytochrome c, Bax 및 AIF들의 관찰과 미토콘드리아 막전위 측정을 시행하였다 Western blotting을 통한 PARP (poly (ADP-ribose) poly-merase) cleavage, Bax, Bcl-2, Bak 및 AIF 들의 발현을 관찰하였다. 결과 : 2$\~$8 Gy의 X-ray를 조사한 군(R)과 HS-1200 처리 후 2$\~$8 Gy의 X-ray를 조사 한 군(HR)의 세포 생존 곡선을 비교하니 HR군에서 세포 감작 효과를 관찰할 수 있었다. DNA ladder는 R군에서는 72시간재 관찰되는 반면에 HR 군에서는 24시간째 관찰되어 DNA 분절이 빠르게 진행됨을 알 수 있었고, PARP cleavage의 관찰에서도 R 군에 비해 24시간 빠르게 진행되었다. 면역 형광법을 이용한 실험에서도HR군이 R 근에 비하여 미토콘드리아 막전위($\Delta$$\psi$$_{m}$)의 급격한 감소, cytochrome의 다량 방출, Bax의 증가된 점상 변화 등이 관찰되었고, AIF의 변화는 뚜렷하지 않았다. Western blotting을 이용한 Bax, Bcl-2, Bak 및 AIF들의 발현을 관찰하였을 때 Bax 만 HR 군에서 시간대별로 증가되는 추세를 보인 반면 Bcl-2, Bak 및 AIF들의 발현은 특이한 차이를 발견할 수 없었다. 결론 : 인체 유방암 세포주(MCF-7)에서 새로운 담즙산 합성 유도체인 HS-1200은 방사선 조사에 의한 아포토시스의 유도를 감작시키는 사실을 관찰하였다. 아포토시스 유도감작 증가는 Bax/Bcl-2 분율의 상대적 증가로 기인한다고 생각한다. 상기 결과를 토대로 HS-1200의 항암 치료제로서의 역할에 관한 기초 자료로서의 유용성을 제시할 수 있었다.

Purpose : To examine whether a synthetic bile acid derivatives (HS-1200) sensitizes the radiation-induced apoptosis in human breast cancer cells (MCF-7) and to investigate the underlying mechanism. Materials and Methods : Human breast cancer cells (MCF-7) in exponential growth phase were treated with HS-1200 for 24 hours at 37$^{\circ}C$ with 5$\%$ CO$_{2}$ in air atmosphere. After removal of HS-1200, cells were irradiated with 2$\~$8 Gy X-ray, and then cultured Ii drug-free media for 24-96 hours. The effect of radiation on the clonogenicity of MCF-7 cells was determined with clonogenic cell survival assay with 16$\mu$M of HS-1200. The induction of apoptosis was determined using agarose gel electrophoresis and Hoechst staining. The expression level of apoptosis-related molecules, such as PARP, Bax, Bcl-2, Bak and AIF, were assayed by Western blotting analysis with 40$\mu$M of HS-1200 combined with 8 Gy irradiation. To examine the cellular location of cytochrome c, bax and AIF immunofluorescent stainings were undertaken. Results : Treatment of MCF-7 cells with 40$\mu$M of HS-1200 combined with 8 Gy irradiation showed several changes associated with enhanced apoptosis by agarose gel electrophoresis and Hoechst staining. HS-1200 combined with 8 Gy irradiation treatment also enhanced production of PARP cleavage products and increased Bax/Bcl-2 ratio by Western blotting. Loss of mitochondrial membrane potential ($\Delta$$\psi$$_{m}$) and increased cytochrome c staining indicated that cytochrome c had been released from the mitochondria in HS-1200 treated cells. Conclusion : We demonstrated that combination treatment with a synthetic chenodeoxycholic acid derivative HS-1200 and irradiation enhanced radiation-induced apoptosis of human breast cancer cells (MCF-7). We suggest that the increased Bax/Bcl-2 ratio In HS-1200 co-treatment group underlies the increased radio sensitivity of MCF-7 cells. Further futures studies are remained elusive.

키워드

참고문헌

  1. Im EO, Lee S, Suh H, Kim KW, Bae YT, Kim ND. A novel ursodeoxycholic acid derivative induces apoptosis in human MCF-7 breast cancer cells. Pharm Pharmacol Commun 1999;5:293-298 https://doi.org/10.1211/146080899128734875
  2. Park YH, Kim J, Baek J, et al. Induction of apoptosis in HepG2 human hepatocellular carcinoma cells by a novel derivative of ursodeoxycholic acid (UDCA). Arch Pharm Res 1997;20:29-33 https://doi.org/10.1007/BF02974038
  3. Baek JH, Kim J, Kang C, Lee YS and Kim KW. Induction of apoptosis by bile acids in HepG2 human hepatocellular carcinoma cells. Korean J Physiol Pharmacol 1997;1:107-115
  4. Kim DK, Lee JR, Kim A, et al. Inhibition of initiation of simian virus 40 DNA replication in vitro by the ursodeoxycholic acid and its derivatives. Cancer Lett 1999;146:147-153 https://doi.org/10.1016/S0304-3835(99)00251-7
  5. Im EO, Choi YH, Paik KJ, et al. Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells. Cancer Lett 2001;163:83-93 https://doi.org/10.1016/S0304-3835(00)00671-6
  6. Choi, YH, Im EO Suh H, et al. Apoptotic activity of novel bile acid derivatives in human leukemic T cells through the activation of caspases. Int J Oncol 2001;18:979-984
  7. Morgan WA, Sharma P, Kaler B, Bach PH. The modulation of protein kinase C by bile salts. Biochem Soc Trans 1997;25:75S
  8. Ward NE, OBrian CA. The bile acid analog fusidic acid can replace phosphatidylserine in the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate in vitro. Carcinogenesis 1988;9:1451-1454 https://doi.org/10.1093/carcin/9.8.1451
  9. Oh SG, Yang KM, Hur WJ, Yoo YH, Suh HS, Lee HS. A novel chenodeoxycholic derivative HS-1200 induces apoptosis in human HT-29 colon cancer cells. J Korean Soc Ther Radiol Oncol 2002;20(4):367-374
  10. Faubion WA, Guicciardi ME, Miyoshi H, et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 1999;103:137-145 https://doi.org/10.1172/JCI4765
  11. Di Cristofano A, Kotsi P, Peng YF, Cordon-Cardo C, Elkon KB, Pandolfi PP. Impaired Fas response and autoimmunity in Pten+/-mice. Science 1999;285:2122-2125 https://doi.org/10.1126/science.285.5436.2122
  12. Rust C, Karnitz LM, Pays CV, Moscat J, Simari RD, Gores GJ. The bile acid taurochenodeoxycholate activates a phoshatidylinositol 3-kinase-dependent survival signaling cascade. J Biol Chem 2000;275:20210-20216 https://doi.org/10.1074/jbc.M909992199
  13. Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 1990;3:543-553
  14. Qiao D, Chen W, Stratagoules, ED, Martinez JD. Bile acid-induced activation of activator protein-l requires both extracellular signal-regulated kinase and protein kinase C signaling. J Biol Chem 2000;275:15090-15098 https://doi.org/10.1074/jbc.M908890199
  15. Craven PA, Pfanstiel J, Derubertis FR. Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids. J Clin Invest 1987;79:532-541 https://doi.org/10.1172/JCI112844
  16. Song CS, Echchgadda I, Baek BS, et al. Dehydroepiandrosterone sulfotransferase gene induction by bile acid activated farnesoid x receptor. J Biol Chem 2001;276:42549-42556 https://doi.org/10.1074/jbc.M107557200
  17. Park DJ, Blanchard SG, Bledsoe RK, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365-1368 https://doi.org/10.1126/science.284.5418.1365
  18. Hallahan DE, Sukhatme VP, Sherman ML, et al. Protein kinase C mediates X-ray inducibility of nuclear signal transducers EGR1 and JUN. Proc Natl Acad Sci USA 1991;88:2156-2160 https://doi.org/10.1073/pnas.88.6.2156
  19. Uckun FM, Tuel-Ahlgren L, Song CW, et al. Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death. Proc Natl Acad Sci USA 1992;89:9005-9009 https://doi.org/10.1073/pnas.89.19.9005
  20. Findik D, Song Q, Hidaka H, Lavin M. Protein kinase A inhibitors enhance radiation-induced apoptosis. J Cell Biochem 1995;57:12-21 https://doi.org/10.1002/jcb.240570103
  21. Wang CY, Mayo MW, Baldwin AS, Jr. TNF-and cancer therapy-induced apoptosis; potentiation by inhibition of NFkappaB. Science 1996;274:784-787 https://doi.org/10.1126/science.274.5288.784
  22. Kim SH, Seong JS. The Fas/FasL in radiation-induced apoptosis in vivo. J Korean Soc Ther Radiol Oncol 2003;21(3):222-226
  23. Reap EA, Roof K, Maynor K, et al. Radiation and stress induced apoptosis; A role for Fas/Fas ligand interactions. Proc Natl Acad Sci USA 1997;94:5750-5755 https://doi.org/10.1073/pnas.94.11.5750
  24. Sheard MA, Vojtesek B, Janakova L, Kovarik J, Zaloudik J. Up-regulation of Fas(CD95) in human p53 wild-type cancer cells treated with ionizing radiation. Int J Cancer 1997;73: 757-762 https://doi.org/10.1002/(SICI)1097-0215(19971127)73:5<757::AID-IJC24>3.0.CO;2-1
  25. Savitsky K, Bar-Shira A, Gilad S, et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995;268:1749-1753 https://doi.org/10.1126/science.7792600
  26. Weichselbaum RR, Hallahan D, Fuks Z, Kufe D. Radiation induction of immediate early genes; effectors of the radiation stress response. Int J Radiat Oncol Biol Phys 1994;30:229-234 https://doi.org/10.1016/0360-3016(94)90539-8
  27. Hockenbery D, Nunez G, Miliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990;348:334-336 https://doi.org/10.1038/348334a0
  28. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 1997;3:614-620 https://doi.org/10.1038/nm0697-614
  29. Cherbonnel-Lasserre C, Gauny S and Kronenberg A. Suppression of apoptosis by Bcl-2 or Bcl-xL promotes susceptibility to mutagenesis. Oncogene 1996;13:1489-1497
  30. Cherbonnel-Lasserre C and Dosanjh MK. Suppression of apoptosis by overexpression of Bcl-2 or Bcl-xL promotes survival and mutagenesis after oxidative damage. Biochimie 1997;79:613-617 https://doi.org/10.1016/S0300-9084(97)82011-1
  31. Pauwels B, Korst AEC, Pattyn GGO et al. Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro. Int J Radiat Oncol Biol Phys 2003;57:1075-1083 https://doi.org/10.1016/S0360-3016(03)01443-3
  32. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997;275:1126-1132 https://doi.org/10.1126/science.275.5303.1126
  33. Rodrigues CM, Fan G, Ma X, Kren BT, Steer CJ. A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane pertubation. J Clin Invest 1998; 101:2790-2799 https://doi.org/10.1172/JCI1325
  34. Zamzami N, Susin SA, Masse B, et al. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995;182:367-377 https://doi.org/10.1084/jem.182.2.367
  35. Zamzami N, Marchetti P, Castedo M, et al. Inhibitors of permeability transition interfere with the disruption of the mitochondrial membrane potential during apoptosis. FEBS Lett 1996;384:153-157
  36. Yershalmi B, Dahl R, Devereaux MW, Gumpricht E, Sokol RJ. Bile acid induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology 2001;33:616-626 https://doi.org/10.1053/jhep.2001.22702
  37. Yoon HS, Rho JH, Yoo KW, et al. Synthetic bile acid derivatives induce nonapoptotic death of human retinal pigment epithelial cells. Curr Eye Res 2001;22:367-374 https://doi.org/10.1076/ceyr.22.5.367.5499