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.
Huang
Jian
J
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang, PR China.
Wu
Lijun
L
Tashiro
Shin-Ichi
S
Onodera
Satoshi
S
Ikejima
Takashi
T
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D005354:Fibrosarcoma / Q000473:pathology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008928:Mitochondria / Q000502:physiology*; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases