cytochrome-c-t and geldanamycin

Geldanamycin and licochalcone A induce apoptosis in cancer cells. However, whether the combination of geldanamycin and licochalcone A-induced apoptosis in epithelial ovarian cancer cells is mediated by the formation of reactive oxygen species, leading to the activation of apoptotic caspase, has not been studied.. Using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3, we investigated the promoting effect of licochalcone A on geldanamycin-induced apoptosis.. Geldanamycin induced changes in apoptosis-related protein levels, loss of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases, cleavage of PARP-1, formation of reactive oxygen species and depletion of glutathione (GSH). Licochalcone A enhanced geldanamycin-induced apoptosis-related protein activation, formation of reactive oxygen species, caspase activation and cell death. The combined effect was inhibited by the addition of oxidant scavengers.. Licochalcone A may potentiate the apoptotic effect of geldanamycin on ovarian carcinoma cell lines by the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway. The apoptosis-promoting effect of licochalcone A may be mediated by its stimulatory action on the formation of reactive oxygen species and the depletion of GSH, which results in the activation of caspases.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzoquinones; Caspases; Cell Line, Tumor; Chalcones; Cytochromes c; Drug Synergism; Glutathione; Humans; Lactams, Macrocyclic; Membrane Potential, Mitochondrial; Protein Kinase Inhibitors; Reactive Oxygen Species

Hsp90 inhibitor geldanamycin and parthenolide have been shown to induce apoptosis in cancer cells. However, the combined effect of geldanamycin and parthenolide on epithelial ovarian cancer cells has not been studied. In respect of cell death process, we investigated the promoting effect of parthenolide on geldanamycin-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels; an increase in Bax and tumour suppressor p53 levels; loss of the mitochondrial transmembrane potential; cytochrome c release; activation of caspases (-8, -9 and -3); cleavage of PARP-1; and increase in the reactive oxygen species formation. Parthenolide enhanced geldanamycin-induced changes in the apoptosis-related protein levels, reactive oxygen species formation, nuclear damage and cell death. The combined effect was inhibited by the addition of oxidant scavengers. The results suggest that parthenolide may potentiate the apoptotic effect of geldanamycin on ovarian carcinoma cell lines by the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway. The apoptosis-promoting effect seems to be mediated by the stimulatory effect on the formation of reactive oxygen species.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Benzoquinones; Blotting, Western; Carcinoma, Ovarian Epithelial; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mitochondria; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Reactive Oxygen Species; Sesquiterpenes; Tumor Suppressor Protein p53

1. Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotective effect via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. 2. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (1 μg/mL). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis. 3. Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c. 4. In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3-K/Akt pathway. Key words: apoptosis, cardiomyocyte, heart failure, heat shock protein 90, hypoxia, phosphatidylinositol 3-kinase/Akt signalling pathway, serine/threonine protein kinase Akt.

Topics: Animals; Animals, Newborn; Apoptosis; bcl-Associated Death Protein; Benzoquinones; Blotting, Western; Caspase 3; Cell Hypoxia; Cells, Cultured; Cytochromes c; Cytoprotection; HSP90 Heat-Shock Proteins; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Lactams, Macrocyclic; Myocytes, Cardiac; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Time Factors

The Bcr-Abl fusion gene encodes for the p210(Bcr-Abl) or p185(Bcr-Abl) tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia, respectively. Because Bcr-Abl TK is chaperoned by Hsp90 (90 kDa heat-shock protein), we investigated the effects of novobiocin (NB), an Hsp90 C-terminal inhibitor, on the viability of the Bcr-Abl-positive human leukemia cells HL-60/Bcr-Abl and K562, the expression of Bcr-Abl protein and the interaction between Hsp90 and Bcr-Abl TK. Present studies demonstrate that NB is a potent inhibitor of the growth of Bcr-Abl-positive human leukemia cells. NB induces cytosolic accumulation of cytochrome c and activation of caspase-9 and caspase-3, triggering apoptosis of HL-60/Bcr-Abl and K562 cells. Treatment of cell lines with NB disrupts Bcr-Abl /Hsp90 and Bcr-Abl /Hsp70 interactions, resulting in a decreased amount of intracellular Bcr-Abl protein levels. Co-treatment with the proteasome inhibitor N-acetyl leucyl-leucyl norlucinal increases NB-mediated accumulation of Bcr-Abl in the detergent-insoluble cellular fraction, which demonstrates that NB promotes proteasomal degradation of Bcr-Abl. Moreover, both imatinib-resistant K562/G01 and primary CML CD34(+) cells are sensitive to NB.

Topics: Apoptosis; Benzamides; Benzoquinones; Blast Crisis; Caspase 3; Caspase 9; Cell Proliferation; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Fusion Proteins, bcr-abl; HL-60 Cells; HSP90 Heat-Shock Proteins; Humans; Imatinib Mesylate; Immunoprecipitation; K562 Cells; Lactams, Macrocyclic; Leukemia; Novobiocin; Piperazines; Proteasome Endopeptidase Complex; Pyrimidines

We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.

Topics: alpha-Synuclein; Animals; Annexin A5; Apoptosis; Benzoquinones; Blotting, Western; Caspases; Cell Membrane; Cell Separation; Cysteine Proteinase Inhibitors; Cytochromes c; Flow Cytometry; Gene Deletion; Genotype; Glutathione; Green Fluorescent Proteins; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Mitochondria; Mutation; Nerve Tissue Proteins; Parkinson Disease; Plasmids; Quinones; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Synucleins; Time Factors

Two highly conserved mechanisms for maintaining cellular homeostasis are apoptosis and the cellular stress response. Hsp90 is one of the most abundant, highly conserved, and inducible Hsps in eukaryotes. Recently, Hsp90 has been shown to play important antiapoptotic roles through binding with Apaf-1, RIP and kinase domain of IKKalpha/beta. Our present studies demonstrate that Hsp90 can suppress tumor necrosis factor alpha (TNFalpha)-induced apoptosis in stable Hsp90-overexpressing NIH3T3 cells by preventing the cleavage of Bid. The prevention of the cleavage of Bid can be partially explained by the direct interaction between Hsp90 and Bid. Furthermore, disrupting the function of Hsp90 by the addition of its specific inhibitor, geldanamycin, blocked Hsp90's protection of Bid cleavage. These results show that Hsp90 can function at different levels within apoptotic signal transduction pathways.

Topics: Animals; Apoptosis; Benzoquinones; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspases; Cell Division; Cells, Cultured; Colorimetry; Cycloheximide; Cytochromes c; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mice; Mitochondria; NIH 3T3 Cells; Quinones; Signal Transduction; Tumor Necrosis Factor-alpha

Geldanamycin (GA) binds to heat shock protein 90 (Hsp90) and interferes with its function which is to protect various cellular proteins involved in signaling, growth control, and survival from ubiquitination and subsequent degradation by the proteasome. Recently, we demonstrated that GA inhibited migration of glioma cells in vitro associated with downregulation of hypoxia-inducible factor (HIF-1 alpha) and phosphorylation of focal adhesion kinase (FAK) (Zagzag et al., 2003, J Cell Physiol 196:394-402). Here, we have investigated the mechanisms through which GA treatment of the T98G glioma cell line induces apoptosis. We found that GA treatment induced cell death in a caspase-dependent manner through activation of caspase-3 and PARP cleavage together with release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria. Use of synchronized T98G cells showed that GA treatment of glioma cells during S-phase enhanced cytotoxicity followed by M-phase arrest, resulting in mitotic catastrophe. In addition, apoptosis was associated with the downregulation of the survival protein, phosphorylated Akt (pAkt), an important signaling protein in the PI3K pathway, that is overexpressed in many cancers including gliomas. Given that many glioma tumors show deregulation of the PI3K signaling pathway, either through loss of the tumor suppressor protein PTEN or overexpression of the growth factor EGFR, the ability to identify different subsets of patients using simple immunohistochemistry for the presence of absence of pAkt could enable selection of the appropriate kinase inhibitor, such as GA, for drug therapy. Based on our data presented here, GA or its analogs may have potential in the treatment of glioma.

Topics: Apoptosis; Apoptosis Inducing Factor; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Flavoproteins; Glioma; Humans; Lactams, Macrocyclic; Membrane Proteins; Mitochondria; Mitosis; Quinones