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 microM) 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 microM). 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 > or =10 microM (>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 microM 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 microM (P < .05) compared with carmustine alone. No decrease was observed in cells that were treated with SH-6 alone (10 microM). LY294002 (10 microM) 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.
Van Meter
Timothy E
TE
Department of Neurosurgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, Virginia 23230, USA. tevanmet@hsc.vcu.edu
Broaddus
William C
WC
Cash
Dana
D
Fillmore
Helen
H
D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D002330:Carmustine / Q000008:administration & dosage / Q000627:therapeutic use*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D017024:Chemotherapy, Adjuvant; D002867:Chromones / Q000008:administration & dosage / Q000627:therapeutic use*; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000627:therapeutic use; D005910:Glioma / Q000188:drug therapy* / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000008:administration & dosage / Q000627:therapeutic use*; D050505:Mutant Proteins / Q000235:genetics / Q000494:pharmacology; D014411:Neoplastic Stem Cells / Q000187:drug effects; D010716:Phosphatidylinositols / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018906:Antineoplastic Agents, Alkylating; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D050505:Mutant Proteins; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; C516024:SH-6 compound; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases; D002330:Carmustine