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

Cancer. 2006 Nov 15;107(10):2446-54. doi: 10.1002/cncr.22248.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Alkylating / therapeutic use
  • Brain Neoplasms / drug therapy*
  • Brain Neoplasms / metabolism
  • Carmustine / administration & dosage
  • Carmustine / therapeutic use*
  • Caspases / metabolism
  • Cell Survival / drug effects
  • Chemotherapy, Adjuvant
  • Chromones / administration & dosage
  • Chromones / therapeutic use*
  • Enzyme Inhibitors / administration & dosage
  • Enzyme Inhibitors / therapeutic use
  • Glioma / drug therapy*
  • Glioma / metabolism
  • Humans
  • Morpholines / administration & dosage
  • Morpholines / therapeutic use*
  • Mutant Proteins / genetics
  • Mutant Proteins / pharmacology
  • Neoplastic Stem Cells / drug effects
  • Phosphatidylinositols / pharmacology
  • Phosphoinositide-3 Kinase Inhibitors*
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents, Alkylating
  • Chromones
  • Enzyme Inhibitors
  • Morpholines
  • Mutant Proteins
  • Phosphatidylinositols
  • Phosphoinositide-3 Kinase Inhibitors
  • SH-6 compound
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Proto-Oncogene Proteins c-akt
  • Caspases
  • Carmustine