Efficient Killing Effect of Osteosarcoma Cells by Cinobufacini and Cisplatin in Combination

  • Huang, Tao (Department of Orthopedics, The First Affiliated Hospital of China Medical University) ;
  • Gong, Wei-Hua (Beth Israel Deaconess Medical Center, Harvard Medical School) ;
  • Li, Xiu-Cheng (Department of Orthopedics, The First Affiliated Hospital of China Medical University) ;
  • Zou, Chun-Ping (Department of Orthopedics, The First Affiliated Hospital of China Medical University) ;
  • Jiang, Guang-Jian (Beth Israel Deaconess Medical Center, Harvard Medical School) ;
  • Li, Xu-Hui (Beth Israel Deaconess Medical Center, Harvard Medical School) ;
  • Qian, Hao (Department of Orthopedics, The First Affiliated Hospital of China Medical University)
  • Published : 2012.06.30


Purpose: To study the killing effects on osteosarcoma cells of cinobufacini and cisplatin in combination and the related mechanisms so as to explore the chemotherapeutic method with integrated traditional Chinese and Western medicines. Methods: Cinobufacini and cisplatin were applied to OS732 cells singly or jointly and survival rates were measured by MTT assay. Changes in cellular shape were observed with inverted phase contrast and fluorescence microscopy and apoptosis rates were analyzed with flow cytometry (FCM). Immunocytochemistry were used to examine the Fas expression of OS732 cells. Results: The combination of cinobufacini and cisplatin had the effect of up-regulating Fas expression and inducing apoptosis. The survival rate of combined application of 100 ${\mu}g/ml$ cinobufacini and 1 ${\mu}g/ml$ cisplatin on OS-732 cells was significantly lower than with either of the agents alone (p<0.01). Changes in cellular shape and apoptotic rates also indicated the apoptosis-inducing effects of combined application were much enhanced. Conclusion: The combination of cinobufacini and cisplatin demonstrated strong killing effects on OS-732 cells which might be related to up-regulation of Fas expression.


  1. Akimzhanov AM, Wang X, Sun J and Boehning D (2010). T-cell receptor complex is essential for Fas signal transduction. Proc Natl Acad Sci USA, 107, 15105-10.
  2. Bhurgri Y, Usman A, Bhurgri H, et al (2009). Primary malignancies of bone and cartilage in Karachi. Asian Pac J Cancer Prev, 10, 891-4.
  3. Broadhead ML, Dass CR, Choong PF (2011). Systemically administered PEDF against primary and secondary tumours in a clinically relevant osteosarcoma model. Br J Cancer, 105, 1503-11.
  4. Chen Z, Chen HY, Lang QB et al (2012). Preventive effects of jiedu granules combined with cinobufacini injection versus transcatheter arterial chemoembolization in post-surgical patients with hepatocellular carcinoma: a case-control trial. Chin J Integr Med, 18, 339-44.
  5. Chen Z, Zhai XF, Su YH, et al (2003). Clinical observation of cinobufacini injection used to treat moderate and advanced primary liver cancer. Zhong Xi Yi Jie He Xue Bao, 1, 184-6 (in Chinese).
  6. Cui X, Inagaki Y, Xu H, et al (2010). Anti-hepatitis B virus activities of cinobufacini and its active components bufalin and cinobufagin in HepG2.2.15 cells. Biol Pharm Bull, 33, 1728-32.
  7. Dai ZJ, Gao J, Ji ZZ, et al (2009). Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3. J Ethnopharmacol, 123, 91-6.
  8. Dong HJ, Zhang LJ, Zhai XF, et al (2007). Effects of different nursing methods in treatment of phlebitis caused by cinobufacini injection. Zhong Xi Yi Jie He Xue Bao, 5, 585-7 (in Chinese).
  9. Dotan A, Dadia S, Bickels J, et al (2010). Expandable endoprosthesis for limb-sparing surgery in children: long-term results. J Child Orthop, 4, 391-400.
  10. Inoue S, Harper N, Walewska R, Dyer MJ, Cohen GM (2009). Enhanced Fas-associated death domain recruitment by histone deacetylase inhibitors is critical for the sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis. Mol Cancer Ther, 8, 3088-97.
  11. Kim JS, Lee YC, Nam HT, et al (2007). Apicularen A induces cell death through Fas ligand up-regulation and microtubule disruption by tubulin down-regulation in HM7 human colon cancer cells. Clin Cancer Res, 13, 6509-17.
  12. Konstantakou EG, Voutsinas GE, Karkoulis PK, et al (2009). Human bladder cancer cells undergo cisplatin-induced apoptosis that is associated with p53-dependent and p53-independent responses. Int J Oncol, 35, 401-16.
  13. LaPensee EW, LaPensee CR, Fox S, et al (2010). Bisphenol A and estradiol are equipotent in antagonizing cisplatin-induced cytotoxicity in breast cancer cells. Cancer Lett, 290, 167-73.
  14. Liedtke C, Trautwein C (2012). The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer. Eur J Cell Biol, 91, 582-9.
  15. Limmahakhun S, Pothacharoen P, Theera-Umpon N, et al (2011). Relationships between serum biomarker levels and clinical presentation of human osteosarcomas. Asian Pac J Cancer Prev, 12, 1717-22.
  16. Li XY, Lu Y, Shan QX and Xia Q (2006). Effect of cinobufacini on vascular contractile of rat thoracic aorta. Zhejiang Da Xue Xue Bao Yi Xue Ban, 35, 178-81 (in Chinese).
  17. Ma L, Song B, Jin H, et al (2012). Cinobufacini induced MDA-MB-231 cell apoptosis-associated cell cycle arrest and cytoskeleton function. Bioorg Med Chem Lett, 22, 1459-63.
  18. Maroto P, Huddart R, Garcia del Muro X, et al (2011). Brief report: phase II multicenter study of temozolomide in patients with cisplatin-resistant germ cell tumors. Oncology, 80, 219-22.
  19. Matsuda N, Yamamoto S, Takano K, et al (2009).Silencing of fas-associated death domain protects mice from septic lung inflammation and apoptosis. Am J Respir Crit Care Med, 179, 806-15.
  20. Moulian N and Berrih-Aknin S (1998). Fas/APO-1/CD95 in health and autoimmune disease: thymic and peripheral aspects. Semin Immunol, 10, 449-56.
  21. O'Callaghan G, Kelly J, Shanahan F, Houston A (2008). Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells. Br J Cancer, 99, 502-12.
  22. Qi FH, Li AY, Lv H, et al (2008). Apoptosis-inducing effect of cinobufacini, Bufo bufo gargarizans Cantor skin extract, on human hepatoma cell line BEL-7402. Drug Discov Ther, 2, 339-43.
  23. Qi FH, Li AY, Zhao L, et al (2010b). Apoptosis-inducing effect of cinobufacini on human hepatoma cell line HepG2 and its mechanism of action. Yao Xue Xue Bao, 45, 318-23 (in Chinese).
  24. Qi F, Li A, Zhao L, et al (2010a). Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells. J Ethnopharmacol, 128, 654-61.
  25. Okamoto K, Fujisawa J, Reth M, Yonehara S (2006). Human T-cell leukemia virus type-I oncoprotein Tax inhibits Fas-mediated apoptosis by inducing cellular FLIP through activation of NF-kappaB. Genes Cells, 11, 177-91.
  26. Rotte A, Garmann D, Buss I, Jaehde U (2010). Effect of extracellular ATP on cisplatin-induced cytotoxicity in human ovarian carcinoma cells. Chemotherapy, 56, 1-8.
  27. Shiu LY, Chang LC, Liang CH, et al (2007). Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin. Food Chem Toxicol, 45, 2155-64.
  28. Sprowl JA, Gregorc V, Lazzari C, et al (2012). Associations Between ABCC2 Polymorphisms and Cisplatin Disposition and Efficacy. Clin Pharmacol Ther.
  29. Wang DL, Qi FH, Xu HL, et al (2010). Apoptosis-inducing activity of compounds screened and characterized from cinobufacini by bioassay-guided isolation. Mol Med Report, 3, 717-22.
  30. Wang SS, Zhai XF, Li B (2009). Effects of cinobufacini injection on contents of serum thyroid-stimulating hormone and adrenaline in rats. Zhong Xi Yi Jie He Xue Bao, 7, 228-31 (in Chinese).
  31. Wang SS, Zhai XF, Li B (2011). Effect of cinobufacini injection on the tumor growth of tumor bearing rats of different constitutions]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 31, 1101-3 (in Chinese).
  32. Wei X, Li CG, Nong S, Zhu XY, Huang XM (2006). The influence of Solanum lyratum Thunb extract on apoptosis and the expression of fas/fasL genes in Hela cells. Zhong Yao Cai, 29, 1203-6 (in Chinese).

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