benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and rottlerin

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Topics: Acetamides; Acetophenones; Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Benzopyrans; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Histones; Humans; Indomethacin; Kinetics; Phenanthridines; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Pyrans; Spiro Compounds; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

We show here that JNK1 activity is rapidly up-regulated and prolonged by specific mechanisms during apoptosis induced by paclitaxel- or ginsenoside-Rh2 in SK-HEP-1 cells. The early phase of JNK1 activation is prevented in cells expressing the dominant negative SEK1 mutant, although this JNK1 perturbation does not prevent apoptotic cell death. The later phase of JNK1 activation, which is temporally coincided with caspase-dependent cleavage of JNK1-associated p21(WAF1/CIP1), is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21(WAF1/CIP1) and this perturbation of JNK1 activation results in prevention of apoptosis. The later JNK1 activation and apoptotic progression are also prevented by co-treatments of cells with rottlerin, a PKC-delta inhibitor or z-VAD-fmk, a pan caspase inhibitor. We also provide evidence that apoptotic cell death is significantly promoted in cells expressing JNK1, while this apoptotic cell death is effectively suppressed in cells expressing the dominant negative JNK1 mutant (DN-JNK1) or JBD, a JNK inhibitor protein. Thus, the later phase of JNK1 activation, which is linked to a caspase-dependent mechanism that requires PKC-delta activity, is associated with the induction of apoptosis, while the early JNK1 activation that is associated with a SEK1-mediated mechanism is not directly involved in apoptotic progression.

Topics: Acetophenones; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzopyrans; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; Cell Death; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Dominant; Ginsenosides; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Paclitaxel; Precipitin Tests; Protein Kinase C; Protein Kinase C-delta; Time Factors; Transfection; Up-Regulation