K. M. Sokolowski1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Surgical Oncology/Surgery,Milwaukee, WI, USA
Introduction:
Cholangiocarcinoma (CCA) is characterized by poor prognosis and therapeutic inefficacy. The current standard of practice for advanced CCA is systemic chemotherapy with gemcitabine and cisplatin. However, a limited survival benefit along with increasing resistance has ushered in a renaissance in alternative approaches. Increased understanding and application of new technologies has led to the identification of more pathway-focused treatment strategies. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target as it is overexpressed in various cancer types including CCA. However, the role of targeting GSK-3 in CCA progression remains elusive. We hypothesize that GSK-3 stabilizes cellular growth thereby promoting proliferation and with subsequent inhibition, leading to a reduction in cellular growth in vitro. For this, we have used a clinically well-characterized GSK-3 inhibitor, LY2090314.
Methods:
The effects of LY2090314 on CCA cell lines (CCLP-1 and SG-231) were assessed by MTT and colonogenic assay. Cell lysates were analyzed via Western blotting for pro-apoptotic, anti-apoptotic and cell cycle proteins. Apoptotic induction was further evaluated through caspase-glo 3/7 assay. Mechanism of LY2090314 administration on CCA cellular proliferation was also examined.
Results:
Increasing LY2090314 treatment (0μM – 20μM) had a significant dose and time-dependent growth reduction (p<0.01) compared to control. The concentration at which 50%growth inhibition (IC50) was observed in vitro following 96hr treatment for CCLP-1 and SG-231 is 13.7µM and 9µM respectively. Similar concentrations inhibited colony formation in both CCA cell lines. Western analysis indicated that growth suppression was due to apoptosis as well as cell cycle arrest as evidenced by increased expression of pro-apoptotic (cleaved PARP and cleaved caspase-3), reduced anti-apoptotic (survivin) proteins and mitigation of cell cycle arrest proteins (p21 and cyclin D1). Functionally, this was confirmed by an increase in caspase activity. Importantly, LY2090314 treatment reduced the expression levels of only GSK-3β in CCA cell lines.
Conclusion:
LY2090314, a GSK-3 inhibitor, effectively reduces CCA growth in vitro and appears through reduction of GSK-3β. To our knowledge, this is the first study on the effect of LY2090314 in cholangiocarcinoma cell lines in vitro and provides rationale for further preclinical analysis.