Redox regulation by andrographolide induced cell cycle arrest and cell death in HepG2 cells

The cytotoxicity of andrographolide to HelpG2 human hepatoma cells was investigated in the present study. Growth of HelpG2 cells was affected in the presence of andrographolide with IC50 of 40.2 µM after 48 h treatment. Flow cytometric analysis and DNA fragmentation assay revealed that andrographolide induced cell cycle arrest at G2/M phase and a late apoptosis of the cells. The occurrence of cell cycle arrest was accompanied by the collapse of mitochondrial membrane potential (MMP) and an intracellular increase of hydrogen peroxide (H2O2) but a decrease of superoxide radicals (O2-) and reduced glutathione. In the treated cells, expression of Bax as well as the transcriptional controller of this pro-apoptotic gene, p53, was upregulated but not other apoptotic proteins such as Bad, Bcl-2 and Bcl-XL. Although the activity of caspase-3. Which has direct effect on apoptosis, was also enhanced by the presence of andrographolide, cell death of HelpG2 could neither be prevented by a specific inhibitor of capsase-3 nor the pan-caspase inhibitor-zVAD (Val-Ala-Asp), indicating that it was a caspase-independent cell death. Since the overall percentage of apoptotic cells was relatively small throughout the experimental studies, we conclude that the sytotoxic effect of andrographolide on HelpG2 cells is primary attributed to the induction of cell cycle arrest via the alteration of cellular redox status.