Acta Pharmacologica Sinica 2007 August; 28 (8): 1215-1223; doi: 10.1111/j.1745-7254.2007.00606.x

Aim: To investigate the antihepatocellular carcinoma effects of Chi-Shen extract (CSE) from the water-soluble compounds of Salvia miltiorrhiza and Paeoniae radix.

Methods: The effect of CSE on the growth of HepG2 cells (hepatocellular carcinoma cell line) was studied by 3-(4,5)-2,5-diphenyltetrazolium bromide assay. Apoptosis were detected through acridine orange (AO) and ethylene dibromide (EB) staining and DNA fragmentation assay. The effect of CSE on the cell cycle of HepG2 cells was studied by the propidium iodide staining method. The activation of caspases-3, -8 and -9 was examined by immunoassay kits. The transcription of the Bcl-2 family and p53 was detected by RT-PCR.

Results: Our data revealed that CSE strongly induced HepG2 cell death in a dose- and time-dependent manner. CSE-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by AO/EB staining and DNA fragmentation assay. The induction of HepG2 cell death was caused by an induction of apoptosis for the sub-G₁ proportion increase, the downregulation of Bcl-2, the upregulation of Bax and p53, and the activation of the caspases-3 and -9 pathways.

Conclusion: These results clearly demonstrated that CSE was able to inhibit the proliferation of HepG2 cells and cause apoptosis. Moreover, the anticancer effects of CSE were related to the Bcl-2 family pathway and the activation of caspases-3 and -9 in HepG2 cells.

Keywords: human hepatocellular carcinoma cells; apoptosis; Bcl-2; Bax; caspase-3; Salvia miltiorrhiza; Paeoniae radix