cytochrome-c-t and enzastaurin

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.

Topics: Adult; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Brain Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Mitochondria; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand

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