A comparison of the signal pathways between the TNF alpha- and oridonin-induced murine L929 fibrosarcoma cell death.

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, we compared the signal transduction pathways between TNFalpha-and oridonin-induced L929 cell death. Oridonin and TNFalpha initiated apoptotic morphologic changes, but DNA fragmentation was found in TNFalpha-treated L929 cells but not in oridonin-treated ones. The pan-caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) augmented oridonin-and TNFalpha-induced cell death. However, the caspase-9 inhibitor (z-LEHD-fmk) only increased oridonin-induced L929 cell death. Moreover, poly (ADPribose) polymerase (PARP) was cleaved in oridonin-treated L929 cells but not in the TNFalpha-treated groups, and the caspase-3 inhibitor (z-DEVD-fmk) failed to inhibit PARP cleavage. These results showed that only oridonin-induced L929 cell death required PARP degradation in a caspase-3 independent manner. In addition, oridonin increased the ratio of Bax/Bcl-2 protein expression, but TNFalpha did not. TNFalpha induced p38 and ERK activation, whereas oridonin triggered only ERK activation. We also investigated the effect of oridonin on intracellular TNFalpha expression, and found that oridonin augmented endogenous pro-TNFalpha expression and its upstream protein IkB phosphorylation. These results indicated that although oridonin promoted endogenous pro-TNFalpha expression, a great difference existed between the signal pathways through which TNFalpha-and oridonin-induced cell death.</p

The augmentation of TNFalpha-induced cell death in murine L929 fibrosarcoma by the pan-caspase inhibitor Z-VAD-fmk through pre-mitochondrial and MAPK-dependent pathways.

We investigated the mechanism of the pan-caspase inhibitor z-VAD-fmk's augmentation of TNFalpha-induced L929 cell death and found this mechanism differs from that of TNFalpha-induced L929 cell death. In the presence of 20 ng/ml TNFalpha, z-VAD-fmk initiated apoptosis and necrosis in the majority of L929 cells as measured by an agarose gel electrophoresis and lactate dehydrogenase(LDH)activity based assay. Mitochondrial permeability transition (MPT) inhibitor (cyclosporine A) effectively inhibited z-VAD-fmk-augmented cell death. In addition, z-VAD-fmk plus TNFalpha increased Bax expression without affecting Bcl-2 and cytochrome expression. Western-blot analysis showed that z-VAD-fmk plus TNFalpha caused persistent JNK activation and ERK inactivation. Poly(ADP-ribose) polymerase (PARP) inhibitor (DPQ) effectively reversed the cell death which was augmented by z-VAD-fmk, and z-VAD-fmk plus TNFalpha also caused PARP cleavage to an 85 KDa fragment. These results indicate that in the presence of TNFalpha, z-VAD-fmk further augments cell death which requires the mitochondrial permeability transition and the JNK activation. However, we did not detect the changes in cytochrome c expression and the participation of caspase-9 in this process, suggesting that there might exist an unknown signal pathway(s) from the mitochondria to the downstream protein PARP, which is cleaved in a caspase-independent manner.</p

Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase-3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis

IL-1β Acts in Synergy with Endogenous IL-1β in A375-S2 Human Melanoma Cell Apoptosis Through Mitochondrial Pathway

Interleukin-1β (IL-1β) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1β-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1β did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1β (pro-IL-1β) and endogenous IL-1β production were involved in the apoptotic process. IL-1β enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1β induces endogenous IL-1β production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells