S. T. Koprowski1, K. M. Sokolowski1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Surgical Oncology/Department Of Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA
Introduction: Cholangiocarcinoma (CCA) is highly malignant and characterized by poor prognosis with chemotherapeutic resistance. Therefore, continued development of novel, effective approaches are needed. Notch1 is highly expressed in CCA, but the utility of Notch1 inhibition is not defined. Based on recent findings, we hypothesized that curcumin, a polyphenolic phytochemical, suppresses CCA growth in vitro via inhibition of Notch1 signaling.
Methods: Established CCA cell lines CCLP-1 and SG-231 were treated with varying concentrations of curcumin (0-20µM). Viability was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colonogenic assays. Cell lysates were analyzed via Western blotting for Notch1/HES1/Survivin pathway expression, apoptosis, and cell cycle progression. Following curcumin treatment, quantitative real-time polymerase chain reaction (qPCR) was utilized to determine mRNA expression of Notch signaling components.
Results: Curcumin-treated CCA cells exhibited reduced viability compared to control treatment. Statistically significant reductions in cell viability were observed with curcumin treatment at concentrations of 7.5, 10, and 15µM by approximately 10%, 48%, and 56% for CCLP-1 and 13%, 25%, and 50% for SG-231 respectively. Upon Western analysis, concentrations of 10µM and above showed reductions in Notch1, HES1, and Survivin. Apoptosis was evidenced by an increase in expression of cleaved PARP (Poly [ADP] ribose polymerase). Cyclin D1 (cell cycle progression) expression levels were also reduced with treatment. Data collected from qPCR similarly indicated significant reductions in Notch1, HES-1, and Survivin mRNA expression in CCA treatment groups.
Conclusion: Curcumin effectively induces CCA (CCLP-1 and SG-231) growth suppression and apoptosis at relatively low treatment concentrations when compared to previous research. A concomitant reduction of Notch1, HES1, and Survivin expression in CCA cell lines provides novel evidence for a potential anti-tumorigenic mechanism-of-action. To our knowledge, this is the first report showing reduction in HES-1 expression via both mRNA and protein analysis following treatment with curcumin. Such findings merit further investigation of curcumin-mediated inhibition of Notch signaling in CCA either alone or in combination with chemotherapeutic agents.