Nicotine Stimulates Biliary Proliferation and Fibrogenesis and Potentiates Cholangiocarcinoma Growth

Abstract

Nicotine is a major addictive substance in cigarette smoke, and has powerful biological properties throughout the body. Studies have demonstrated that nicotine promotes the progression of a number of disease processes, including cancer and fibrosis. Recent evidence suggests that nicotine plays a role in liver fibrosis. Cholangiocytes are the epithelial cells that line the intrahepatic and extrahepatic bile ducts. During biliary injury that occurs with diseases such as primary sclerosing cholangitis (PSC), cholangiocytes undergo a reactive process termed "ductular reaction," and display a neuroendocrine phenotype that may contribute to the fibrotic pathologies, and eventual development of cholangiocarcinoma. Epidemiological data indicate that smoking contributes to biliary fibrosis and cholangiocarcinoma. Due to this reason and the prevalence of nicotine usage, it was important to understand the role that nicotine plays in the progression of biliary fibrosis, and the regulators of this process. We first examined whether nicotine has pro-proliferative effects on the biliary system. We demonstrated in vitro that nicotine increases the proliferation of the normal cholangiocyte cell line (NRIC) by MTS assay and PCNA expression by western blot. We also demonstrated that nicotine works through a calcium/inositol triphosphate/phosphorylated extracellular regulated kinase (Ca2+/IP3/p-ERK) intracellular signaling pathway in NRIC. In vivo, rats were treated with nicotine via osmotic minipump infusion for two weeks. In the nicotine-treated group, we found an increase in the proliferation of cholangiocytes identified by cytokeratin (CK-19, a cholangiocyte specific marker) staining, and proliferating cell nuclear antigen (PCNA) expression. Increased expression of fibrotic genes/proteins was seen in liver tissue, and cholangiocytes isolated from rodent livers. Next, we sought to determine the effect of nicotine on cholangiocarcinoma proliferation. In vitro, nicotine stimulated the proliferation of cholangiocarcinoma growth, in the MzChA-1, CCLP1, and HuCC-Tl cell lines. In a nude mouse xenograft model, nicotine treatment caused an increase in tumor volume and PCNA expression. These data support the current literature suggesting that nicotine may induce fibrogenesis, and contributes to the liver pathology observed in smokers. They also support the notion that nicotine stimulates the growth of biliary cancer and call for further investigation of the subject.