Apaf-1 overexpression partially overcomes apoptotic resistance in a cisplatin-selected HeLa cell line.
Inhibition of caspase-3-mediated apoptosis has been hypothesized to be associated with chemoresistance. Investigations of apoptosis revealed that cytosolic cytochrome c is associated with a complex of apoptotic protease activating factor-1 (Apaf-1), an adapter molecule, and caspase-9 to activate caspase-3. However, whether these apoptotic molecules are involved in acquired cisplatin resistance is not understood. The present work shows reduced activation of caspase-3 and apoptosis in a cisplatin-selected HeLa cell line. Ac-DEVD-CHO, a caspase-3 inhibitor, inhibited cisplatin-induced apoptosis about 60-70% in both cell lines. Ac-LEHD-CHO, a caspase-9 inhibitor or Ac-IETD-CHO, a caspase-8 inhibitor, inhibited cisplatin-induced caspase-3 activation and apoptosis similarly in both cell lines. In addition, cisplatin induced the activation of caspase-9, the upstream activator of caspase-3, in a dose-dependent manner, and the activation of caspase-9 was less induced in resistant cells. The accumulation of cytosolic cytochrome c, an activator of caspase-9, and the induction of the mitochondrial membrane-associated voltage-dependent anion channel were also reduced in cisplatin-resistant cells. However, the concentration of Bcl-2 family proteins in cisplatin-resistant cells was normal. The concentration of Apaf-1 was unaltered in both cell lines. Increasing the cellular concentration of Apaf-1 through the transient expression of the gene increased the induction of apoptosis in resistant cells, associated with enhanced activation of caspase-9, caspase-3 and DNA fragmentation factor. Regression analysis reveals that the modification factor, the ratio of the slope in the linear range of the dose-response curve with Apaf-1 to the slope without Apaf-1, is 1.5 and 4.75 in the HeLa and cisplatin-resistant HeLa cells, respectively. These results indicate that apoptosis and caspases are less induced in cisplatin-selected HeLa cells. They also suggest that ectopic overexpression of Apaf-1 may partially reverse the acquired cisplatin resistance.
Kamarajan
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Tumor Biology Laboratory, Department of Biochemistry, Chang Gung University, Taoyuan 33332, Taiwan, ROC.
Sun
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Sun
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Chao
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D000256:Adenoviridae / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D002945:Cisplatin / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012044:Regression Analysis; D014407:Tumor Cells, Cultured
C506592:APAF1 protein, human; D000970:Antineoplastic Agents; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D002945:Cisplatin