Abstract
IntroductionCholangiocarcinoma (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.Study methodsPost 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.ResultsCCLP-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 AKTSer473, while AKTThr308 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.ConclusionsThis 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.
Highlights
Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options
MK2206 treatment reduces Cholangiocarcinoma cellular proliferation in a dose and time dependent manner Cell viability results, as measured by MTT assay, indicated a dose and time correlate for growth reduction in both the CCLP-1 and SG231cell lines (Figure 1)
In both CCLP-1 and SG231 cell lines, AktSer473 was significantly reduced in MK2206 treated cells (0.1-2 μM; Figure 2)
Summary
Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options. Study 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. The majority of patients present with unresectable disease and even in those achieving a R0 resection, 5-year survival rates remain low (22-35% [3,4,6,7,8]) with greater than 50% recurrence at a median 31-month follow-up [8]. Because the Wilson et al Cancer Cell International (2015) 15:13 prognosis for CCA is dismal and no systemic therapy has demonstrated substantial efficacy [9], there exists an urgent need for identification of novel therapeutic targets
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