Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells

✍ Scribed by Yan-Nian Chen; Jung-Chou Chen; Sui-Chu Yin; Guang-Sheng Wang; Wei Tsauer; Sheng-Feng Hsu; Shih-Lan Hsu

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: French
• Weight: 796 KB
• Volume: 100
• Category: Article
• ISSN: 0020-7136
• DOI: 10.1002/ijc.10479

✦ Synopsis

Abstract

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh‐7. NCTD treatment (5 μg/ml) enhanced the expression of Cdc25C and p21^Cip1/Waf1^, increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1–associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27^Kip1^. Moreover, NCTD treatment also increased the phosphorylation of Bcl‐2 and Bcl‐X~L~ but did not affect the expression of Bax or Bad. Bcl‐2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl‐2 in NCTD‐treated HepG2 cells. In addition, NCTD treatment caused activation of caspase‐9 and caspase‐3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad‐spectrum caspase inhibitor z‐VAD‐fmk markedly inhibited NCTD‐induced caspase‐3 activity and cell death. These results suggest that phosphorylation of p21^Cip1/Waf1^ and Cdc25C and biphasic regulation of cyclin B1–associated kinase activity may contribute to NCTD‐induced M‐phase cell‐cycle arrest. Furthermore, the increase of p21^Cip1/Waf1^, phosphorylation of Bcl‐2 and Bcl‐X~L~, activation of caspase‐9 and caspase‐3 may be the molecular mechanism through which NCTD induces apoptosis. © 2002 Wiley‐Liss, Inc.