calpain and rottlerin

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Benzopyrans; Biphenyl Compounds; Buthionine Sulfoximine; Calpain; Caspases; Catalase; Cell Line, Tumor; DNA Damage; Glutathione; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Onium Compounds; Protein Kinase C; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1

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