cytochrome-c-t and rottlerin

Activation of protein kinase C (PKC) has been implicated in the protection of ischemic preconditioning (IPC), but the exact role of PKC in early and late hepatic IPC is still unclear. The present study was conducted in order to investigate the differential role of PKC during early and late hepatic IPC. Rats were subjected to 90 min of partial hepatic ischemia followed by 3 (early IPC) and 24 h (late IPC) of reperfusion. IPC was induced by 10 min of ischemia following 10 min of reperfusion prior to sustained ischemia, and chelerythrine, a PKC inhibitor, was injected 10 min before IPC (5 mg/kg, i.v.). Chelerythrine abrogated the protection of early IPC, as indicated by increased serum aminotransferase activities and decreased hepatic glutathione content. While the IPC-treated group showed a few apoptotic cell deaths during both phases, chelerythrine attenuated these changes only at late IPC and limited IPC-induced inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) overexpression. Membrane translocation of PKC-δ and -ε during IPC was blocked by chelerythrine. Our results suggest that PKC might play a differential role in early and late IPC; activation of PKC-δ and -ε prevents necrosis in early IPC through preservation of redox state and prevents apoptosis in late IPC with iNOS and HO-1 induction. Therefore, PKC represents a promising target for hepatocyte tolerance to ischemic injury, and understanding the differential role of PKC in early and late IPC is important for clinical application of IPC.

Topics: Acetophenones; Animals; Apoptosis; Benzophenanthridines; Benzopyrans; Cytochromes c; Heme Oxygenase-1; Ischemic Preconditioning; Liver Diseases; Male; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Rats; Reperfusion Injury

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment.

Topics: Acetophenones; Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-Associated Death Protein; bcl-X Protein; Benzamides; Benzopyrans; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Humans; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasm Transplantation; Pancreatic Neoplasms; Protein Kinase C-delta; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Proteinuria may contribute to progressive renal damage by inducing tubulointerstitial inflammation, fibrosis, and tubular cell injury and death, but the mechanisms underlying these pathologic changes remain largely unknown. Here, in a rat kidney proximal tubular cell line (RPTC), albumin induced apoptosis in a time- and dose-dependent manner. Caspase activation accompanied albumin-induced apoptosis, and general caspase inhibitors could suppress this activation. In addition, Bcl-2 transfection inhibited apoptosis and attenuated albumin-induced Bax translocation to mitochondria and cytochrome c release from the organelles, further confirming a role for the intrinsic pathway of apoptosis in albuminuria-associated tubular apoptosis. We observed phosphorylation and activation of PKC-delta early during treatment of RPTC cells with albumin. Rottlerin, a pharmacologic inhibitor of PKC-delta, suppressed albumin-induced Bax translocation, cytochrome c release, and apoptosis. Moreover, a dominant-negative mutant of PKC-delta blocked albumin-induced apoptosis in RPTC cells. In vivo, we observed activated PKC-delta in proteinuric kidneys of streptozotocin-induced diabetic mice and in kidneys after direct albumin overload. Notably, albumin overload induced apoptosis in renal tubules, which was less severe in PKC-delta-knockout mice. Taken together, these results suggest that activation of PKC-delta promotes tubular cell injury and death during albuminuria, broadening our understanding of the pathogenesis of progressive proteinuric kidney diseases.

Topics: Acetophenones; Albumins; Animals; Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Caspases; Cell Line; Cytochromes c; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kidney Tubules, Proximal; Male; Mice; Mice, Knockout; Protein Kinase C-delta; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Rats; Streptozocin

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin. In elucidating its mechansim, rottlerin prevented the release of cytochrome c from mitochondria into cytosol, and also prevented the cleavage of caspase-3. Yet, rottlerin by itself markedly decreased mitochondrial membrane potential and increased mitochondrial superoxide production, but this did not result in cytochrome c release or in caspase-3 cleavage. At a higher test concentration, two other phenolic phytochemicals, namely, quercetin and resveratrol, were each able to largely prevent the occurrence of apoptosis in cells exposed to DOC. In contrast, epigallocatechin gallate, curcumin, and genistein were ineffective.

Topics: Acetophenones; Apoptosis; Benzopyrans; Biphenyl Compounds; Caspase 3; Colon; Cytochromes c; Cytoprotection; Deoxycholic Acid; DNA Damage; DNA, Neoplasm; Epithelial Cells; HCT116 Cells; Humans; Hydrazines; Mitochondria; Phenols; Picrates; Quercetin; Resveratrol; Stilbenes; Superoxides

Ginsenoside-Rh2 (G-Rh2) has been shown to induce apoptosis in a variety of cell types. In this study, we show that G-Rh2-induced apoptosis is accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in the human hepatoma cell line, SK-HEP-1. Furthermore, protein kinase C delta (PKCdelta) activity was markedly up-regulated in a lipid activator-independent manner with kinetics similar to those of PKCdelta and PARP cleavages during the apoptotic progression. Pre-treatment of cells with the caspase-3 specific inhibitor (z-DEVD-fmk) effectively prevented the G-Rh2-induced proteolytic activation of PKCdelta. Moreover, rottlerin, a specific PKCdelta inhibitor blocked G-Rh2-induced proapoptotic effects on the cells including the release of cytochrome c, activation of caspase-3 activity, and proteolytic cleavage and activation of PKCdelta. These results suggest that G-Rh2-induced apoptosis is functionally linked to mitochondrial dysfunction and caspase-3 activity is regulated by positive feedback with PKCdelta via the mitochondrial pathway.

Topics: Acetophenones; Apoptosis; Benzopyrans; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Inhibitors; Ginsenosides; Humans; Protein Kinase C-delta

Se-methylselenocysteine (Se-MSC) has been shown to possess potent chemopreventive and anti-tumor properties. However, its exact mechanism of action is still not well understood. The present study investigated the mechanism of Se-MSC on the induction of apoptosis using U937 human leukemia cells. Se-MSC induced dose- and time-dependent apoptosis of U937 cells as assessed by flow cytometry analysis, DNA fragmentation, and proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Se-MSC increased time- and dose-dependent cytochrome c accumulation in the cytosol, which was greatly inhibited by overexpression of Bcl-2, suggesting that the apoptotic effect by Se-MSC in U937 cells is mitochondrial-dependent. Se-MSC also induced activation of caspases, followed by proteolytic cleavage of PKC-delta. The Se-MSC-induced apoptosis required activities of caspases since pretreatment of a pan-caspase inhibitor z-VAD-fmk greatly suppressed the Se-MSC-induced apoptosis as well as proteolytic cleavage of PKC-delta, suggesting activation of caspases is critical for the Se-MSC-induced apoptosis, and caspases lie upstream of PKC-delta. The Se-MSC-induced apoptosis of U937 cells also required activity of PKC-delta because pretreatment of rottlerin, a specific PKC-delta inhibitor greatly blocked the Se-MSC-induced apoptosis as well as processing and activities of caspases, suggesting activation of PKC-delta is also important for the Se-MSC-induced apoptosis of U937 cells, and PKC-delta lies upstream of caspases. Together, our data suggest the apoptotic mechanism by Se-MSC in U937 cells may be related to cytochrome c release from the mitochondria, and mutual activation between caspases and PKC-delta via a positive feedback mechanism, which may potentiate the apoptotic action by Se-MSC in U937 cells.

Topics: Acetophenones; Apoptosis; Benzopyrans; Caspases; Cysteine; Cytochromes c; Enzyme Activation; Feedback, Physiological; Humans; Organoselenium Compounds; Poly(ADP-ribose) Polymerases; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Selenocysteine; U937 Cells