Connexin 43 plays an important role in the transformation of human cholangiocytes upon stimulation with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine

Abstract

BackgroundClonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear.\n\nMethodologyUsing the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell cycle protein E2F1 and the cell proliferation-related proteins Ki-67 and cytokeratin 19 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 also increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Cox-2 was also upregulated. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression.\n\nConclusionsThese results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease.\n\nAuthor summaryClonorchis sinensis, a human fluke, resides in the liver of humans and is commonly found in the common bile duct and gall bladder. This parasite is the main cause of cholangiocarcinoma, also called bile duct cancer, in humans. Of note, the excretory-secretory products (ESP) of C. sinensis are known to cause inflammation in the biliary epithelium, which may ultimately result in neoplasms via production of reactive oxygen species and subsequent DNA damage. Together with N-nitrosodimethylamine (NDMA), a potent hepatotoxin that can cause fibrosis and tumors in the liver, ESP led to an increase in the growth and proliferation of cholangiocytes. Our results showed that the ESPs of C. sinensis induced pro-inflammatory responses by increasing the levels of proinflammatory cytokines and nuclear factor kappa B (NF{kappa}B), which in turn, enhanced the production of connexin 43 (Cx43), a gap-junction protein. Therefore, Cx 43 can serve as a potential target for developing a therapeutic strategy for the treatment of cholangiocarcinoma in humans.