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-κB, 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 (Δφm) 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-κB 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-κB downregulation and p73 accumulation.
Bibliographical noteFunding Information:
We would like to thank Dr. Jean Y.J. Wang for p73-α and -β constructs, Dr. William G. Kaelin Jr. for p73 DN construct, and Dr. Xiaodong Wang for anti-Smac antibody. This research was supported by a grant from the Korea Science and Engineering Foundation (2000-2-20900-011-3).
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