Cotreatment with a novel phosphoinositide analogue inhibitor and carmustine enhances chemotherapeutic efficacy by attenuating AKT activity in gliomas

BACKGROUND. Heightened activity of the AKT signaling pathway is prominent in malignant gliomas and has been suggested to play a role in treatment resistance. Selective targeting of AKT, therefore, may increase chemosensitivity. Recently, a novel class of AKT-selective inhibitors has been described, including SH-6, a phosphatidylinositol analogue. METHODS. The effects of SH-6 on AKT signaling were tested in glioma cells, and the putative role of AKT signaling in chemoresistance was tested by attenuating AKT signaling pharmacologically and genetically. The initial characterization of SH-6 included treatment of glioma cells with increasing doses of SH-6 (0.30-30 mu M) and examining the effects on AKT signaling proteins by Western blot analyses and in kinase assays with immunoprecipitated AKT1. Dose-response studies with SH-6 administered to glioma cell lines were performed using a luminescent cell-viability assay (0.1-30 mu M). Studies examining the effect of carmustine, either alone or in combination with either the phosphatidylinositol 3-kinase inhibitor LY294002 or SH-6, were performed by cell viability assays and clonogenic survival assays. The effect of carmustine on AKT activity as a response to treatment also was examined. Caspase assays were used to examine the potential role of apoptosis in SH-6/ carmustine -elicited cell death. Finally, the induction of a dominant-negative AKT1 transgene was used in combination with carmustine to demonstrate the role of AKT1 in carmustine chemoresistance. RESULTS. Serum-stimulated phosphorylation of AKT1 was inhibited by SH-6 at doses >= 10 mu M (> 70% decrease in Threonine 308 and Serine 473 phosphorylation of AKT1). In adenosine triphosphate assays, 72 hours of treatment with SH-6 led to 50% lethal doses near 10 mu M for 2 cell lines tested. SH-6 enhancement of carmustine-mediated cell death led to synergistic increases in Caspase 3/Capsase 7 activity, implicating apoptosis as the cell death mechanism. In clonogenic assays, SH-6 cotreatment with carmustine significantly decreased the number of colonies at 10 mu M (P < .05) compared with carmustine alone. No decrease was observed in cells that were treated with SH-6 alone (10 mu M). LY294002 (10 mu M) was also able to enhance the effects of carmustine significantly in both cell lines. CONCLUSIONS. In the current study, the authors characterized the efficacy of a new class of adjuvant chemotherapeutics that show promise in enhancing the efficacy of standard chemotherapy regimens in gliomas.