Mechanistic insights of SRPK inhibitors in cholangiocarcinoma cell apoptosis

Abstract

Cholangiocarcinoma (CCA) is a cancer that arising from abnormal growth of bile duct epithelium. It is a common cancer among Thai population derived from unhygienic food consumption. Regarding no early-diagnosis and inefficient treatment, therefore, CCA represents very poor prognosis and low survival rate. The molecular mechanisms underlying CCA development are still unclear, however, dysregulation of mRNA splicing is suspected to play a major role as a number of oncogenic aberrant spliced-transcripts in CCA have been reported. Aberrant splicing is caused by the increased activity of Serine/Arginine rich-splicing factors (SRSFs) that translocate into the nucleus as phospho-SRSFs after activated by Serine-arginine protein kinases (SRPKs). In this study, the effects of two SRPK inhibitors (SRPIN340 and SPHINX31) in two CCA cell lines (KKU-213A and TFK-1) were investigated. SRPIN340 and SPHINX31 increased the number of dead cells once stained with calcein-AM/PI as dose-dependent manner. The type of dead cell induction was defined as apoptosis by increasing of apoptotic cell population, diffused cytoplasmic cytochrome c and upregulation of cleaved caspase-3 by Annexin-V/7AAD staining (flow cytometry), immunocytofluorescence and Western blot analysis, respectively. Moreover, Western blot analysis using anti-phospho-epitope of SRSF protein family members revealed lower phospho-SRSFs band intensities for representing the reduction of SRSFs phosphorylation. Particularly, inhibition of nuclear-cytoplasmic translocation of predominant SRSF1 was demonstrated by immunocytofluorescence and Western blotting of subcellular protein fractions. To link these phenotypes with aberrant gene splicing, Bridging Integrator 1 (BIN1) gene was selected. Both SRPIN340 and SPHINX31 can decrease BIN1+12A (oncogenic/anti-apoptotic isoform) once observed by RT-PCR. These results provide the strong evidences that suggests the targeting SRPKs could be an alternative strategy for developing the precise CCA treatment.

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