Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

We have reported that norcantharidin (NCTD) induces human melanoma A375-S2cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in theapoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase(MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. We assessed theeffects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2 ,5-dipheyltetrazolium bromide ( MTT) assay, DNA fragmentation ( DNA agarose gel electrophoresis ) ,and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were alsocollected. The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKCinhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment withNCTD, and inhibitors of c-Jun NH2 - terminal kinase (JNK) and p38 ( SP600125 and SB203580,respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation ofphosphorylated JNK and phosphorylated p_(38), but had little effect on extracellularsignal-regulated kinase (ERK) expression. These results suggest that the activation of JNK andp_(38) MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays animportant regulation role in the activation of MAPKs.

Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis

Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.