Studies On The Mechanisms Of Norcantharidin-induced Tumor Cell Apoptosis

This dissertation reports that norcantharidin (NCTD) induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies demonstrate that NCTD inhibited the proliferation of human melanoma A375-S2, mouse fibrosarcoma L929 and human cervical HeLa cells in a dose- and time-dependent manner. MTT assay, photomicroscopical observation and DNA agarose gel electrophoresis showed that NCTD induced cell apoptosis in A375-S2, L929 and HeLa cells.In NCTD-treated A375-S2 cells, cysteine aspartic specific proteases: caspase-3 and -9 were activated, and the expression of ICAD and PARP was decreased in a time-dependent manner. NCTD increased the expression of the apoptosis inducer, Bax, decreased the expression of the anti-apoptotic protein, Bcl-xL and Bcl-2, promoted the release of cytochrome c, and activated down-stream caspase-9 in mitochondrial apoptotic pathway. The change of Bcl-2/Bax or Bcl-xL/Bax expression was reversed by caspase-3 inhibitor, and caspase-3 inhibitor had no significant effect on the release of cytochrom c. These observations indicated that appropriate dose of NCTD activated the mitochondrial apoptotic pathway that involved the Bcl-2 family including Bcl-2, Bcl-xL and Bax. At the same time, the participation of caspase-9 and caspase-3 was required in this apoptotic pathway. The activity of caspase-8 increased at 28 h and suggested that caspase-8 was involved in the later stage of apoptosis. The inhibitory effect of NCTD on A375-S2 cells was partially reversed by the inhibitors of MAPK inhibitors and PKC inhibitors. The expression of JNK phosphorylation and p38 phosphorylation was increased after treatment with NCTD and the inhibitor of JNK and p38 had significant inhibitory effects on the protein up-regulation of phosphorylated JNK and p38 expression. Simultaneously, PKC family-inhibitor, staurosporine, blocked the up-regulation of phosphorylated JNK and phosphorylated p38 and had little effect on the ERK expression. Taken together, NCTD induced A375-S2 cell apoptosis by activating JNK and p38 pathways, increasing the ratio of Bax/Bcl-xL or Bax/Bcl-2 protein expression, and promoting the release of cytochrome c...