Cisplatin-induced apoptosis in Hep3B cells: mitochondria-dependent and -independent pathways |
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Authors: | Kim Ji Su Lee Jae Myun Chwae Yong-Joon Kim Yeon Hyang Lee Jung Hwan Kim Kunhong Lee Tae Ho Kim Se Jong Park Jeon Han |
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Affiliation: | a Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Institute for Immunology and Immunological Diseases, Seoul, South Korea b Department of Microbiology, Pochon Cha University College of Medicine, Kyunggi-Do, South Korea c Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, South Korea d Department of Biology, College of Science, Yonsei University, Seoul, South Korea |
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Abstract: | Human hepatoma cell lines undergo apoptosis after treatment with cisplatin (CP), by mechanisms that are not fully understood, although our previous study demonstrated that Fas-dependent or -independent pathways are involved. To elucidate the mechanisms of CP-induced apoptosis in Hep3B cells, which are Fas- and p53-negative, we investigated mitochondria associated pathways, the involvement of NF-kappaB, and p73 activation. Results of Western blot and flow cytometry assay revealed that the translocation of Bax, resulted in the loss of mitochondrial membrane potential (Deltaphi(m)) and the efflux of cytochrome c and of second mitochondria-derived activator of caspase/DIABLO from mitochondria into the cytosol. Caspase-3, -8 and -9 were activated by CP treatment, however, CP-induced apoptosis was not completely blocked by pretreating with the pan-caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(O-methyl)-fluoromethylketone, indicating that caspase-independent apoptotic pathways might also be involved. RNase protection assay confirmed that NF-kappaB downregulation leading to the suppression of its target genes, such as XIAP and TRAF2, and p73 accumulation were also observed in Hep3B cells treated with CP. CP-induced apoptosis was inhibited to some extent by transiently overexpressed p73 dominant negative and XIAP, but not by p73DN or XIAP alone. In conclusion, this study demonstrates that CP-induced apoptosis in Hep3B cells is associated with mitochondrial dysregulation, NF-kappaB downregulation and p73 accumulation. |
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Keywords: | CP, cisplatin PT, permeability transition Smac, second mitochondria-derived activator of caspase Δ?m, mitochondrial membrane potential PI, propidium iodide z-VAD-fmk, benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(O-methyl)-fluoromethylketone RPA, RNase protection assay p73DN, p73 dominant negative IAP, inhibitor of apoptosis protein |
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