Abstract
Objective
To investigate the inhibitory activities of norcantharidin (NCTD), a demethylated analogue of cantharidin, on Hep3B cells (a human hepatoma cell line) with deficiency of p53.
Methods
The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined.
Results
NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G2M phase arrest occurs at low concentration (⩽ 25 μmol/L) but G0G1 phase arrest at high concentration (50 μmol/L). The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation.
Conclusion
NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G2M or G0G1 phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Wang GS. Medical uses of mylabris in ancient China and recent studies. J Ethnopharmacol 1989;26:147–162.
Yang EB, Tang WY, Zhang K, Cheng LY, Mack PO. Norcantharidin inhibits growth of human HepG2 celltransplanted tumor in nude mice and prolongs host survival. Cancer Lett 1997;117:93–98.
Chen YN, Chen JC, Yin SC, Wang GS, Tsauer W, Hsu SF, et al. Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells. Int J Cancer 2002;100:158–165.
Tsauer W, Lin JG, Lin PY, Hsu FL, Chiang HC. The effects of cantharidin analogues on xanthine oxidase. Anticancer Res 1997;17:2095–2098.
Liu XH, Blazsek I, Comisso M, Legras S, Marion S, Quittet P, et al. Effects of norcantharidin, a protein phosphatase type-2A inhibitor, on the growth of normal and malignant haemopoietic cells. Eur J Cancer 1995;31(A):953–963.
Hong CY, Huang SC, Lin SK, Lee JJ, Chueh LL, Lee CH, et al. Norcatharidin-induced post-G(2)/M apoptosis is dependent on wild-type p53 gene. Biochem Biophys Res Commun 2000;276:278–285.
Sun ZX, Ma QW, Zhao TD, Wei YL, Wang GS, Li JS. Apoptosis induced by norcantharidin in human tumor cells. World J Gastroenterol 2000;6:263–265.
Yi SN, Wass J, Vincent P, Iland H. Inhibitory effect of norcantharidin on K562 human myeloid leukemia cells in vitro. Leuk Res 1991;15:883–886.
Lamboley C, Bringuier AF, Feldmann G. Apoptotic behaviour of hepatic and extra-hepatic tumor cell lines differs after Fas stimulation. Cell Mol Biol 2000;46:13–28.
Shin EC, Shin JS, Park JH, Kim JJ, Kim H, Kim SJ. Expression of Fas-related genes in human hepatocellular carcinomas. Cancer Letters 1998;134:155–162.
Lamboley C, Bringuier AF, Camus E, Lardeux B, Groyer A, Feldmann G. Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis. J Hepatol 2002;36:385–394.
Yamanaka T, Shiraki K, Sugimoto K, Ito T, Fujikawa K, Ito M, et al. Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology 2000;32:482–490.
Brosh R, Rotter V. When mutants gain new powers: news from the mutant p53 field. Nat Rev Cancer 2009;9:701–713.
Gerlier D, Thomasset N. Use of MTT colormetric assay to measure cell activation. J Immunol Methods 1986;94:57–63.
Peng F, Wei YQ, Tian L, Yang L, Zhao X, Lu Y, et al. Induction of apoptosis by norcantharidin in human colorectal carcinoma cell lines: involvement of the CD95 receptor/ligand. J Cancer Res Clin Oncol 2002;128:223–230.
Pop C, Salvesen GS. Human caspases: activation, specificity, and regulation. J Biol Chem 2009;284:21777–21781.
Salvesen GS. Caspases and apoptosis. Essays Biochem 2002;38:9–19.
Guicciardi ME, Gores GJ. Life and death by death receptors. FASEB J 2009;23:1625–1637.
Elrod HA, Sun SY. Modulation of death receptors by cancer therapeutic agents. Cancer Biol Ther 2008;7:163–173.
Keogh SA, Walczak H, Bouchier-Hayes L, Martin SJ. Failure of Bcl-2 to block cytochrome c redistribution during TRAIL-induced apoptosis. FEBS Lett 2000;471:93–98.
Zisman A, Ng CP, Pantuck AJ, Bonavida B, Belldegrun AS. Actinomycin D and gemcitabine synergistically sensitize androgen-independent prostate cancer cells to Apo2L/TRAIL-mediated apoptosis. J Immunother 2001;24:459–471.
Jiang S, Song MJ, Shin EC, Lee MO, Kim SJ, Park JH. Apoptosis in human hepatoma cell lines by chemotherapeutic drugs via Fas-dependent and Fas-independent pathways. Hepatology 1999;29:101–110.
Kim JS, Lee JM, Chwae YJ, Kim YH, Lee JH, Kim K, et al. Cisplatin-induced apoptosis in Hep3B cells: mitochondriadependent and -independent pathways. Biochem Pharmacol 2004;67:1459–1468.
Kuo KW, Hsu SH, Li YP, Lin WL, Liu LF, Chang LC, et al. Anticancer activity evaluation of the solanum glycoalkaloid solamargine. Triggering apoptosis in human hepatoma cells. Biochem Pharmacol 2000;60:1865–1873.
Kok SH, Cheng SJ, Hong CY, Lee JJ, Lin SK, Kuo YS, et al. Norcantharidin-induced apoptosis in oral cancer cells is associated with an increase of proapoptotic to antiapoptotic protein ratio. Cancer Lett 2005;217:43–52.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by Show Chwan Memorial Hospital
Rights and permissions
About this article
Cite this article
Yeh, Ch., Yang, Yy., Huang, Yf. et al. Induction of apoptosis in human Hep3B hepatoma cells by norcantharidin through a p53 independent pathway via TRAIL/DR5 signal transduction. Chin. J. Integr. Med. 18, 676–682 (2012). https://doi.org/10.1007/s11655-012-1206-8
Received:
Published:
Issue date:
DOI: https://doi.org/10.1007/s11655-012-1206-8