Effect of Snake Venom Toxin from Vipera lebetina turanica on Breast Cancer Cells

Vipera lebetina turanica 사독이 인간 유방암 세포에 미치는 영향

  • Yang, Ka-Ram (Dept. of Acupuncture & Moxibustion, College of Oriental Medicine, Kyungwon University) ;
  • Song, Ho-Sueb (Dept. of Acupuncture & Moxibustion, College of Oriental Medicine, Kyungwon University)
  • 양가람 (경원대학교 한의과대학 침구학교실) ;
  • 송호섭 (경원대학교 한의과대학 침구학교실)
  • Published : 2009.06.20

Abstract

목적 : 이 연구는 Vipera lebetinat turanica의 사독약침액(蛇毒藥鍼液)(Snake venom toxin, SVT)이 인간 유방암 세포주인 MCF-7과 MDA-MB-231 세포에서 암세포성장의 억제 및 그 기전에 대하여 살펴보고자 하였다. 방법 : SVT를 처리한 후 MCF-7과 MDA-MB-231의 성장억제를 관찰하기 위해 CCK-8 assay를 시행하였고, apoptosis 평가에는 TUNEL assay를 시행하였다. 세포자멸사 관련 세포기전을 보기 위하여 세포내 활성산소량 및 미토콘드리아의 세포막전위 변화를 측정하였고, 세포자멸사 조절 단백인 Bax, Bcl-2 발현 변화 관찰에는 westem blot analysis를 시행하였다. 결과 : MCF-7과 MDA-MB-231 세포에 SVT를 처리한 후, 유방암 세포의 성장, Apoptosis의 유발 및 기전에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. MCF-7 세포와 MDA-MB-231 세포에서 SVT를 처리한 후 유방암 세포 성장이 억제되었다. 2. TUNEL assay를 통한 세포자멸사 평가에서 SVT를 처리한 MCF-7세포와 MDA-MB-231 세포 모두 세포자멸사 활성세포의 유의한 증가를 나타내었다. 3. 세포자멸사 관련 세포기전연구에서 SVT를 처리한 MCF-7 세포와 MDA-MB-231 세포에서 세포내 활성산소의 유의한 증가와 미토콘드리아 세포막 전위의 유의한 변동이 관찰되었다. 4. SVT를 처리한 MCF-7세포와 MDA-MB-231세포는 세포자멸사 관련 단백 발현에서 Bax의 유의한 증가와 Bcl-2의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 SVT가 세포내 활성산소를 증가시킴으로써 미토콘드리아의 세포막전위에 변화를 일으켜 유방암 세포주인 MCF-7과 MDA-MB-231 세포에 세포자멸사를 유발하여 증식억제 효과가 있음을 입증한 것이다.

Keywords

References

  1. E Marshall. Epidemiology. Search for a killer: focus shifts from fat to hormones. Science. 1993 ; 259 : 618-21. https://doi.org/10.1126/science.8430308
  2. NM Perera, GP Gui. Multi-ethnic differences in breast cancer : current concepts and future directions. Int J Cancer. 2003 ; 106 : 463-7. https://doi.org/10.1002/ijc.11237
  3. Bange J, Zwick E, Ullrich A. Molecular targets for breast cancer therapy and prevention. Nat Med. 2001 ; 7 : 548-52. https://doi.org/10.1038/87872
  4. Roy AM, Baliga MS, Katiyar SK. Epigallocatechin-3-gallate induces apoptosis in estrogen receptor-negative human breast carcinoma cells via modulation in protein expression of p53 and Bax and caspase- 3 activation. Mol. Cancer Ther. 2005 ; 4 : 81-90.
  5. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics. CA Cancer J Clin. 2007 ; 57 : 43-66. https://doi.org/10.3322/canjclin.57.1.43
  6. Feig DI, Reid TM, Loeb LA. Reactive oxygen species in tumorigenesis. Cancer Res. 1994 ; 54 : 1890-4.
  7. Fujino G, Noguchi T, Takeda K, Ichijo H. Thioredoxin and protein kinase in redox signaling. Semin Cancer BioI. 2006 ; 16 : 427-35. https://doi.org/10.1016/j.semcancer.2006.09.003
  8. Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer Cell. 2006 ; 10 : 175-6. https://doi.org/10.1016/j.ccr.2006.08.015
  9. Kondo N, Nakamura H, Masutani H, Yodoi J. Redox regulation of human thioredoxin network. Antioxid. Redox Signal. 2006 ; 8 : 1881-90. https://doi.org/10.1089/ars.2006.8.1881
  10. Kim BC, Kim HG, Lee SA, Lim S, Park EH, Kim, S.J, Lim CJ. Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondria pathway. Biochem Pharmacol. 2005 ; 70 : 1398-407. https://doi.org/10.1016/j.bcp.2005.07.025
  11. Zhang Y, Chen F. Reactive oxygen species (ROS), troublemaker between nuclear factorkappaB(NF -kappaB) and c-Jun NH(2)-terminal kinase(JNK). Cancer Res. 2004 ; 64 : 1902-5. https://doi.org/10.1158/0008-5472.CAN-03-3361
  12. Kuo PL, Chen CY, Hsu YL. Iso-obtusilactone A induces cell cycle arrest and apoptosis through reactive oxygen species/apoptosis signal- regulating kinase 1 signaling pathway in human breast cancer cells. Cancer Res. 2007 ; 67 : 7400-20.
  13. Siigur E, Aaspollu A, Siigur J. Sequence diversity of Vipera lebetina snake venom gland serine proteinase hornologs-result of alternative-splicing or genome alteration. Gene. 2001 ; 263 : 199-203. https://doi.org/10.1016/S0378-1119(00)00571-0
  14. Shim JY, Song HS. Effect of Snake venom toxin from Vipera Iebetina tursnica on Neuroblastoma Cells. Journal of Korean Acupuncture and Moxibustion Society. 2008 ; 25(3) : 53-69.
  15. Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinase. J Biol Chem. 1999 ; 274 : 20049-52. https://doi.org/10.1074/jbc.274.29.20049
  16. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000 ; 21 : 485-95. https://doi.org/10.1093/carcin/21.3.485
  17. Guseva NV, Taghiyev AF, Rokhlin OW, Cohen MB. Death receptor-induced cell death in prostate cancer. J Cell Biochem. 2004 ; 91 : 70-99. https://doi.org/10.1002/jcb.10707
  18. Keith W Crawford and Wayne D Bowen. Sigma-2 Receptor Agonist Activate a Novel Apoptotic Pathway and Potentiate Antineoplastic Drugs in Breast Tumor Cell Lines. Cancer Res. 2002 ; 62 : 313-22.
  19. Manisha Nigam, Vishal Ranjan, Swasti Srivastava, Ramesh Sharma, Anil K Balapure. Centchroman induces G0/G1 arrest and Caspase dependent Apoptosis involving Mitochondria Membrane Depolarization in MCF-7 and MDA MB-231 Human Breast Cancer Cells. Life Sciences. 2008 ; 82 : 577-90. https://doi.org/10.1016/j.lfs.2007.11.028
  20. LA Chodosh, CMD Cruz, HP Gardner, SI Ha, ST Marquis, JV Rajan, DB Stairs, JY Wang, M Wang. Mammary gland development, reproductive history, and breast cancer risk. Cancer Res. 1999 ; 59 : 1765-71.
  21. RR Singh, R Kumar. Steroid hormone receptor signaling in tumorigenesis. J Cell Biochem. 2005.
  22. G Jasienska, I Thune, PT Ellison. Energetic factors, ovarian steroids and the risk of breast cancer. Eur J Cancer Prev. 2000 ; 9 : 231-9. https://doi.org/10.1097/00008469-200008000-00003
  23. J Bergh, PE Jonsson, B Glimelius, P Nygren. A systematic overview of chemotherapy effects in breast cancer. Acta Oncol. 2001 ; 40 : 253-81.
  24. E Rock, A DeMichele. Nutritional approaches to late toxicities of adjuvant chemotherapy in breast cancer survivors. J Nutr. 2003 ; 133 : 3785-93.
  25. Brown RL, Haley TL, West KA, Crabb JW. Pseudechetoxin : a peptide blocker of cyclic nucleotide-gated ion channels. Proc Natl Acad Sci USA. 1999 ; 96 : 754-9. https://doi.org/10.1073/pnas.96.2.754
  26. Sher E, Giovannini F, Boot J, Lang B. Peptide neurotoxins, small-celllung carcinoma andneurological paraneoplastic syndromes. Biochimie. 2000 ; 82 : 927-36. https://doi.org/10.1016/S0300-9084(00)01165-2
  27. van Lunteren E, Moyer M. Peptide toxin blockers of voltage-sensitive K+ channels: inotropic effects on diaphragm. J Appl Physiol. 1999 ; 3 : 1009-16.
  28. Sattler R and Tymianski M. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Mol Neurobiol. 2001 ; 24 : 107-29. https://doi.org/10.1385/MN:24:1-3:107
  29. Duchen MR. Mitochondria and calcium: from cell signaling to cell death. J Physiol. 2000 ; 529 : 57-68.
  30. Halliwell B and Gutteridge JMC. Free Radicals in Biology and Medicine(3rd ed), Oxford : Oxford Univ Press. 1999.
  31. Nicholls DG, Budd SL, Ward MW and Castilho RF. Excitotoxicity and mitochondria. Biochem Soc Symp. 1999 ; 66 : 55-67.
  32. Stout AK, Raphael HM, Kanterewicz BI, Klann E and Reynolds IJ. Glutamate-induced neuron death requires mitochondria calcium uptake. Nat Neurosci 1998 ; 1 : 366-73. https://doi.org/10.1038/1577
  33. Balaban RS. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol 2002 ; 34 : 1259-71. https://doi.org/10.1006/jmcc.2002.2082
  34. Hansford RG and Zorov D. Role of mitochondria calcium transport in the control of substrate oxidation. Mol Cell Biochem 1998 ; 184 : 359-69. https://doi.org/10.1023/A:1006893903113
  35. McCormack JG and Denton RM. Mitochondria Ca2transport and the role of intra mitochondria Ca2in the regulation of energy metabolism. Dev Neurosci. 1993 ; 15 : 165-73. https://doi.org/10.1159/000111332
  36. Pieper AA, Verma A, Zhang J and Snyder SH. Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci 1999 ; 20 : 171-81. https://doi.org/10.1016/S0165-6147(99)01292-4
  37. Szabo C and Dawson VL. Role of poly(ADP-ribose) synthetase in inflammation and ischaemiareperfusion. Trends Pharmacol Sci. 1998 ; 19 : 287-98. https://doi.org/10.1016/S0165-6147(98)01193-6
  38. Virag L, Salzman AL, and Szabo C. Poly (ADP-ribose) synthetase activation mediates mitochondria injury during oxidant-induced cell death. J Immunol 1998 ; 161 : 3753-9.
  39. Saksela M, Lapatto R and Raivio KO. Irrevers-ible conversion of xanthine dehydrogenase into xanthine oxidase by a mitochondria protease. FEBS Lett. 1999 ; 443 : 117-20. https://doi.org/10.1016/S0014-5793(98)01686-X
  40. Iverson SL and Orrenius S. The cardiolipincytochrome c interaction and the mitochondria regulation of apoptosis. Arch Biochem Biophys: 2004 ; 423 : 37-46. https://doi.org/10.1016/j.abb.2003.12.002
  41. Brookes PS and Darley-Usmar VM. Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition. Am J Physiol Heart Circ Physiol 2004 ; 286 : 39-46. https://doi.org/10.1152/ajpheart.00742.2003
  42. Packer MA and Murphy MP. Peroxynitrite causes calcium efflux from mitochondria which is prevented by cyclosporine A. FEBS Lett. 1994 ; 345 : 237-40. https://doi.org/10.1016/0014-5793(94)00461-7