benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Fibrosarcoma

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.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cell Line, Tumor; Fibrosarcoma; MAP Kinase Signaling System; Mice; Mitochondria; Necrosis; Poly(ADP-ribose) Polymerases; Signal Transduction; Tumor Necrosis Factor-alpha

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro. Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Dactinomycin; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Evodia; Fibrosarcoma; Fluorouracil; Furans; Gene Expression Regulation, Neoplastic; Genes, bcl-2; HeLa Cells; Hepatocytes; Heterocyclic Compounds, 4 or More Rings; Humans; Indole Alkaloids; Leukemia, Monocytic, Acute; Leukocytes, Mononuclear; Melanoma; Mice; Mitochondria; Molecular Structure; Neoplasm Proteins; Oligopeptides; Plant Extracts; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Rats; Rats, Inbred BUF; Sarcoma 180; Tumor Cells, Cultured

Previous studies have demonstrated the essential role of TNF-induced reactive oxygen intermediates (ROI) in the necrosis of L929 cells. We investigated the molecular basis for the interaction of hyperthermia and TNF in these cells. Hyperthermia, both febrile (40.0-40.5 degrees C) and acute (41.5-41.8 degrees C), strongly potentiated TNF killing, and sensistization was significantly quenched by the antioxidant, BHA. The broad-spectrum caspase inhibitor, Z-VAD, has been shown to markedly increase the TNF sensitivity of L929 cells at 37 degrees C; we observed that hyperthermia would also enhance the sensitivity of L929 cells to TNF + Z- VAD and that BHA could significantly quench the response, as well. The basis for hyperthermic potentiation was unlikely thermally-increased sensitivity to ROI, as treatment with hydrogen peroxide for 24 h killed L929 cells essentially equivalently, whether incubated continuously at 37 degrees C or at 40.0-40.5 degrees C, or for 2 h at 41.5-41.8 degrees C. However, febrile and acute hyperthermia markedly increased TNF-induced production of ROI, with or without Z-VAD. Hyperthermia dramatically accelerated the onset of this production, as well as the onset of necrotic death, as determined by oxidation of dihydro-rhodamine and propidium iodide staining, respectively, both of which were significantly quenchable with BHA. We conclude that hyperthermia potentiates TNF-mediated killing in this cell model primarily by increasing the afferent, and not the efferent, phase of TNF-induced necrosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Antioxidants; Butylated Hydroxyanisole; Caspases; Cell Survival; Cysteine Proteinase Inhibitors; Doxorubicin; Fever; Fibrosarcoma; Necrosis; Proteins; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

DNA topoisomerase inhibitors are effective chemotherapeutic agents on several solid tumor cells. They induce a specific signaling cascade that executes an active cell death process (apoptosis), including caspase activation, and the blockage of the signaling is associated with drug-resistance of tumor cells. However, little is known about the initial signal transduction induced by the agents. In the present study, we screened genes that are initially upregulated in caspase-independent manner. We found that the activating transcription factor 3 (ATF3) protein, a repressor of cyclic-AMP responsive element (CRE)-dependent transcription, was strongly induced among CRE-BP/ATF members and subsequently accumulated in nuclei following camptothecin or etoposide treatment. During induction of apoptosis, the accumulation and the nuclear translocation of ATF3 coincided with the activation of caspase protease and were not inhibited by the broad caspase inhibitor Z-VAD-fmk, indicating that ATF3 induction is not a downstream event of caspase activation. When stably or transiently overexpressed, ATF3 markedly accelerated the drug-induced apoptosis and enhanced caspase protease activation. ATF3 strongly downregulated CRE-dependent transcription, while ATF3 did not affect the expression levels of Bcl-2, Bcl-x, or Bax. Our present results indicate that ATF3 plays a critical role in accelerating caspase protease activation and apoptosis. Since CRE-dependent transcription functions as cell survival signaling, ATF3 could control the upstream signaling of apoptosis by repressing CRE-dependent gene expression of cell survival factors.

Topics: Activating Transcription Factor 3; Active Transport, Cell Nucleus; Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Camptothecin; Caspase Inhibitors; Caspases; Enzyme Activation; Enzyme Inhibitors; Etoposide; Fibrosarcoma; Gene Expression Regulation; HeLa Cells; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Topoisomerase I Inhibitors; Transcription Factors; Transfection; Tumor Cells, Cultured

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 7; Caspases; Cell Death; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; fas Receptor; Fibrosarcoma; Humans; Ligands; Mice; Necrosis; NF-kappa B; Oligopeptides; Receptors, Tumor Necrosis Factor; Signal Transduction; Transfection; Tumor Cells, Cultured