D. Walden1, K. Sokolowski1, S. Kunnimalaiyaan1, C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Department Of Surgical Oncology,Milwaukee, WI, USA
Introduction: Cholangiocarcinoma (CCA) remains the second most prevalent hepatic neoplasm in the United States. Novel therapies are imperative as aggressive metastasis, chemo-resistance, and limited treatment options precipitate a 5-year survival to less than 10%. We have shown that Notch1 alteration reduces CCA cell growth in vitro; therefore, suggesting the importance of Notch1 in carcinogenesis. More recently, we have identified xanthohumol (XN), a prenylated chalcone from hop plants, inhibits both hepatocellular and pancreatic carcinoma through Notch1 reduction. Despite this investigation, the role of XN in CCA remains unclear. We hypothesize that XN inhibits CCA proliferation through reducing Notch1 signaling.
Methods: Notch isoform expression in CCA cell lines (CCLP-1 and SG-231) was determined via Western analysis. The anti-proliferative effect of XN on CCA cell lines was assessed through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), real-time imaging and colony forming assays. To determine mechanism of action, cell lysates were analyzed via Western blot for Notch1 signaling pathway members and apoptotic markers.
Results: Western analysis revealed that basal levels of the Notch1 isoform were highly expressed indicating an integral relationship in CCA carcinogenesis. Statistically significant (p-value<0.01) reductions in cell viability, when compared with control, were observed with XN treatment at concentrations of 5, 10, and 15µM by approximately 23%, 46%, and 56% for CCLP-1 and 9%, 25%, and 38% for SG-231, respectively. The colony formation assay showed apparent growth reductions formation starting at 5 µM XN. Both cell lines demonstrated a dose dependent decrease in Notch1 expression. Reduced viability was mediated through apoptosis evidenced by decreases in cyclin D1 and survivin proteins.
Conclusion: Xanthohumol effectively inhibits CCA growth through the inhibition of Notch. Based on these findings along with reports in other organ-specific cancers, this provides further evidence of the importance of Notch signaling in carcinogenesis as well as XN as a novel anti-carcinogenic treatment.
