title: Inhibition of the AKT pathway in cholangiocarcinoma by MK2206 reduces cellular viability via induction of apoptosis.
abstract: BACKGROUND
  Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options. The PI3K/Akt/mTOR pathway represents an attractive therapeutic target due to its frequent dysregulation in CCA. MK2206, an allosteric Akt inhibitor, has been shown to reduce cellular proliferation in other cancers. We hypothesized that MK2206 mediated inhibition of Akt would impact CCA cellular viability.
  
  
  METHODS
  Post treatment with MK2206 (0-2 μM), cellular viability was assessed in two human CCA cell lines-CCLP-1 and SG231-using an MTT assay. Lysates from the MK2206 treated CCA cells were then examined for apoptotic marker expression levels using Western blot analysis. Additionally, the effect on cellular proliferation of MK2206 treatment on survivin depleted cells was determined.
  
  
  RESULTS
  CCLP-1 and SG231 viability was significantly reduced at MK2206 concentrations of 0.5, 1, and 2 μM by approximately 44%, 53%, and 64% (CCLP-1; p = 0.01) and 32%, 32%, and 42% (SG231; p < 0.00005) respectively. Western analysis revealed a decrease in AKT(Ser473), while AKT(Thr308) expression was unchanged. In addition, cleaved PARP as well as survivin expression increased while pro-caspase 3 and 9 levels decreased with treatment. Depletion of survivin in CCLP-1 cells resulted in apoptosis as evidenced by increased cleaved PARP. In addition, survivin siRNA further enhanced the antitumor activity of MK2206.
  
  
  CONCLUSIONS
  This study demonstrates that by blocking phosphorylation of Akt at serine473, CCA cellular growth is reduced. The growth suppression appears to be mediated via apoptosis. Importantly, combination of survivin siRNA transfection and MK2206 treatment significantly decreased cell viability.
authors: Medical College of Wisconsin, Translational and Biomedical Research Center, 8701 Watertown Plank Road, Milwaukee, WI 53226 USA.
authors: Jacob M
authors: JM
authors: Wilson
authors: Medical College of Wisconsin, Translational and Biomedical Research Center, 8701 Watertown Plank Road, Milwaukee, WI 53226 USA.
authors: Selvi
authors: S
authors: Kunnimalaiyaan
authors: Medical College of Wisconsin, Translational and Biomedical Research Center, 8701 Watertown Plank Road, Milwaukee, WI 53226 USA.
authors: Muthusamy
authors: M
authors: Kunnimalaiyaan
authors: Department of Surgery, Division of Surgical Oncology, Medical College of Wisconsin, 9200 W Wisconsin Ave, Milwaukee, WI 53226 USA.
authors: T Clark
authors: TC
authors: Gamblin
keywords: Apoptosis; Cholangiocarcinoma; MK2206; PI3K/mTOR/AKT