Study On Apoptotic Signaling Pathway Of Hepatoma Cells Induced By Fluorouracil

PART I Study on intrinsic signal pathway in apoptosis of hepatoma cell induced by fluorouracilObjective To determine whether intrinsic signal pathway is involved in apoptosis of hepatoma cell induced by fluorouracil.Methods The human hepatoma HepG2 cells were treated with flurourail at 1 × 10-2mol/L for 2,4,8,16,24h respectively. The caspase-9 activity was detected using caspase-9 Fluorescent Assay Kit, and the small subunit (p10), which is a 10KDa fragment generated by proteolytic cleavage of procaspase-9 was examined by Westeron blot. Distribution of cytochrome C and Smac/DIABLO in cytosol or in mitochondria was analyzed by Westeron blot and indirect immuofluorescence assay. The apoptotic rates of HepG2 cells induced by fluorouracil with or without the caspase-9 inhibitor were measured by flow cytometry.Results 4h after exposure to fluorouracil, the caspase-9 activity in HepG2 cells increased gradually and reached the peak at 16h, compared with the void control groups, the difference was significant (p<0.01). The p10 protein band which represented small subunit was observed by Western blot 4h after treatment with drug, and became clearest at 16h. In Westeron blot analysis, distribution of cytochrome C and Smac/DIABLO in cytosol increased gradually from 4h on and reached the peak at 16h, characterized by the protein band indicative of the two proteins in cytosol becoming clearer and clearer, but the distribution of cytochrome C in mitochondria was opposite. Consistent with the results of Westeron blot, indirect immunofluorescence assay found that fluorescence of cytochrome C and Smac/DIABLO in the non-treated hepatoma cells had a punctate or plotted staining pattern which indicated cytochrome C andSmac/DIABLO were located in mitochondria, but since 2 hours after treatment with drug, the fluorescence of cytochrome C and Smac/DIABLO was found in a diffuse distribution pattern implied release of cytochrome C and Smac/DIABLO from mitochondria to cytosol. In the presence of caspase-9 inhibitor, the apoptotic rates of HepG2 cells induced by fluorouracil were remarkablely decreased (p<0.05). But comparing to the correspondent negative control group, the apoptotic rate of inhibitor group at 16h was still significant higher.Conclusion Caspase-9 was activated in the apoptosis of HepG2 cells induced by fluorouracil, and its activivity was markedly increased. During this process, cytochrome C and Smac/DIABLO were involved in activation of procaspase-9 after release from mitochondrial intermembrane space to cytosol; the inhibitor of caspase-9 was able to partial blocked the apoptosis of HepG2 cells induced by fluorouracil. In summary, this data suggested that apoptosis of hepatoma cell induced through by fluorouracil is via intrinsic signal pathway.PART II Role of intrinsic and extrinsic signalingpathway inapoptosis of hepatoma cells induced by fluorouracilObjective To investigate the roles of intrinsic and extrinsic signaling pathway in apoptosis of HepG2 cells induced by fluorouracil (5-Fu), as well as the relationship between the two signaling pathway.Methods The human hepatoma HepG2 cells were treated with fluorouracil at 1 × 10-2mol/L for 2, 4, 8, 16, 24h respectively. After administration of special inhibitor of caspase-8 or caspase-9, the activity of caspase-3 was detected using caspases Fluorescent Assay Kit. The apoptotic rates of HepG2 cells induced by 5-Fu were measured by flowcytometry.Results During the apoptosis of HepG2 cells induced by fluorouracil, after caspase-8 special inhibitor was put in, the caspase-3 activity in the inhibitor groups decreased markedly compared with the drug goups (p<0.05), the apoptotic rate also reduced significantly (p<0.05), but the apoptotic rate of inhibitor group at 16h was still remarkably higher than that of corresponding void control group (p<0.05). Similarly, after administration of caspase-9 special inhibitor, the caspase-3 activity and apoptotic rate in the inhibitor groups fell down significantly compared with drug group...