cytochrome-c-t and tanespimycin

We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

Topics: Adenosine Triphosphatases; Animals; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Benzoquinones; Blotting, Western; Caspase 3; Caspase 9; Cell Nucleus; Chromatin; Cytochromes c; Endodeoxyribonucleases; Enzyme Activation; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Intracellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Mice; Mitochondria; Mitochondrial Proteins; Myoblasts; Poly(ADP-ribose) Polymerases; Proteolysis; Proto-Oncogene Proteins c-akt; X-Linked Inhibitor of Apoptosis Protein

Recent studies have suggested that the proliferation of malignant gliomas may result from activation of protein kinase C (PKC)-mediated pathways. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of PKCbeta as well as other isoforms. The initial objective of this study was to assess the efficacy of enzastaurin in a series of malignant human glioma cell lines with diverse genomic alterations. Although enzastaurin independently produced a dose-dependent inhibition of cellular proliferation and decreased cell viability in each of the glioma cell lines examined, and partially down-regulated Akt and GSK3beta phosphorylation, median effective concentrations were at the upper limits of, or above, the clinically achievable range in all cell lines tested. We therefore examined whether the efficacy of enzastaurin could be enhanced by combination with the HSP90 antagonist, 17-AAG, which inhibits Akt and other signaling intermediates by a distinct mechanism. In comparison to the effect of enzastaurin alone, combination of enzastaurin with 17-AAG led to marked enhancement of antiproliferative and cytotoxic effects. Simultaneous exposure to both agents significantly increased the release of cytochrome c, as well as caspase 3 activation, Bax cleavage, and inhibition of Akt phosphorylation. Cells exposed to enzastaurin and 17-AAG also displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that the efficacy of enzastaurin can be potentiated by the addition of 17-AAG, and indicate that combining molecularly targeted therapies may provide a more effective strategy than single-agent therapy to treat patients with malignant gliomas.

Topics: Apoptosis; Benzoquinones; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Down-Regulation; Drug Synergism; Glioma; HSP90 Heat-Shock Proteins; Humans; Indoles; Lactams, Macrocyclic; Oncogene Protein v-akt; Phosphorylation

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.

Topics: Adenosine Triphosphate; Animals; Antennapedia Homeodomain Protein; Antimetabolites, Antineoplastic; Apoptosis; Benzoquinones; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Design; Enzyme Inhibitors; Female; Fibroblasts; Gene Products, tat; HeLa Cells; Homeodomain Proteins; HSP90 Heat-Shock Proteins; Humans; Inhibitor of Apoptosis Proteins; Lactams, Macrocyclic; Male; Mice; Mice, SCID; Microtubule-Associated Proteins; Models, Molecular; Molecular Mimicry; Neoplasm Proteins; Nuclear Proteins; Peptide Fragments; Prostatic Neoplasms; Protein Binding; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Rifabutin; Stem Cells; Survivin; Telomerase; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays