Modulation of STAT3 Signaling, Cell Redox Defenses and Cell Cycle Checkpoints by β-Caryophyllene in Cholangiocarcinoma Cells: Possible Mechanisms Accounting for Doxorubicin Chemosensitization and Chemoprevention

Cholangiocarcinoma (CCA) is an aggressive group of biliary tract cancers, characterized by late diagnosis, low effective chemotherapies, multidrug resistance, and poor outcomes. In the attempt to identify new therapeutic strategies for CCA, we studied the antiproliferative activity of a combination between doxorubicin and the natural sesquiterpene beta-caryophyllene in cholangiocarcinoma Mz-ChA-1 cells and nonmalignant H69 cholangiocytes, under both long-term and metronomic schedules. The modulation of STAT3 signaling, oxidative stress, DNA damage response, cell cycle progression and apoptosis was investigated as possible mechanisms of action. beta-caryophyllene was able to synergize the cytotoxicity of low dose doxorubicin in Mz-ChA-1 cells, while producing cytoprotective effects in H69 cholangiocytes, mainly after a long-term exposure of 24 h. The mechanistic analysis highlighted that the sesquiterpene induced a cell cycle arrest in G2/M phase along with the doxorubicin-induced accumulation in S phase, reduced the gamma H2AX and GSH levels without affecting GSSG. ROS amount was partly lowered by the combination in Mz-ChA-1 cells, while increased in H69 cells. A lowered expression of doxorubicin-induced STAT3 activation was found in the presence of beta-caryophyllene in both cancer and normal cholangiocytes. These networking effects resulted in an increased apoptosis rate in Mz-ChA-1 cells, despite a lowering in H69 cholangiocytes. This evidence highlighted a possible role of STAT3 as a final effector of a complex network regulated by beta-caryophyllene, which leads to an enhanced doxorubicin-sensitivity of cholangiocarcinoma cells and a lowered chemotherapy toxicity in nonmalignant cholangiocytes, thus strengthening the interest for this natural sesquiterpene as a dual-acting chemosensitizing and chemopreventive agent.