Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins?

The present study is aimed to investigate the toxic effects of andrographolide (Andro) on    hepatoma cells and elucidate its preliminary mechanisms.  After cells were treated with different concentrations of Andro (0−50 μmol·L⁻¹) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-        diphenyltetrazolium bromide (MTT) assay.  Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0−30 μmol·L⁻¹) for 14 d, the number of colony formation was accounted under microscope.  Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining.  MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells.  Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner.  Moreover, Andro induced a decrease of Hep3B cells at the G₀-G₁ phase and a concomitant accumulation of cells at G₂-M phase.  At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2,     phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle  related proteins.  Taken together, the results demonstrated that Andro specifically inhibited the growth of  hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.