Department of Neurosurgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia.
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 muM) 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 muM). 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 muM (>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 muM 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 muM (P < .05) compared with carmustine alone. No decrease was observed in cells that were treated with SH-6 alone (10 muM). LY294002 (10 muM) 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. Cancer 2006. (c) 2006 American Cancer Society.
PMID: 17041888 [PubMed - as supplied by publisher]
