Abstract:Objective:To investigate the role of cytochrome C in the apoptosis of hepatoma cell induced by flurouracil , and to analyze the distribution of cytochrome C in cytosol and the relationship between the cytochrome C distribution and Caspase-9 activation.
Methods :The human hepatoma HepG2 cells were treated with flurouracil at 1×10-2mol/L and 2,4,8,16,24h respectively. The chang of cytochrome C in HepG2 apoptosis was detected by using Fluorescent Assay Kit; and proteolytic cleavage of caspase-9 and distribution of cytochrome C in cytosol or in mitochondria was analyzed by Western blot.
Results:Four hours after cells exposure to flurouracil, the caspase-9 activity in HepG2 cells increased gradually and reached the peak at 16h, compared with the control groups, the difference was significant (P<0.01). In Western blot analysis, caspase-9 was found to be cleaved into 10 kD fragment, and distribution of cytochrome C in cytosol became more and more obvious from 4h on and reached a peak at 16h，but the distribution of cytochrome C in mitochondria was opposite. The distribution change of cytochrome C and activity alteration of Caspase-9 were synchronous.
Conclusions:Caspase-9 was activated in the apoptosis of HepG2 cells induced by flurouracil, accompanied by release of cytochrome C from mitochondria to cytosol. This data implies that cytochrome C plays an important role in the activation of Caspase-9 and apoptosis of hepatoma cell induced by flurouracil.