Induction of apoptosis in human Hep3B hepatoma cells by norcantharidin through a p53 independent pathway via TRAIL/DR5 signal transduction

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.