Cholangiocarcinoma and Pro-Inflammatory Cytokines
Cholangiocarcinoma and Pro-Inflammatory Cytokines
By Meeta Jaiswal, PhD Nicholas F. LaRusso, MD, and Gregory J. Gores, MD
The physiological importance of cholangiocytes, the epithelial cells lining bile ducts, is apparent from the fact that they contribute up to 40% of the daily output of bile and constitute only 3% of the liver cell population. Unfortunately, these cells are the targets of a wide variety of human inflammatory liver diseases. In these inflammatory cholangiopathies, there is an increased risk for the development of cholangiocarcinoma, usually a fatal malignant transformation of cholangiocytes.1-3 For example, the inflammatory cholangiopathies, primary sclerosing cholangitis, Caroli’s disease, and/or chronic infestations of the biliary tree all predispose to the development of cholangiocarcinoma.4,5 The precise cellular mechanism responsible for malignant transformation of cholangiocytes during chronic inflammation remains unexplored. Chronic inflammation is accompanied by the stimulation of phagocytes, cytokine generation, and production of reactive oxygen species. We have formulated the hypothesis that malignant transformation during chronic inflammation is induced by cytokine-mediated modulation of gene expression and generation of reactive oxygen species. These events promote mutagenic DNA alterations, cell proliferation, and other changes favoring carcinogenesis. (See Figure.)
Cytokines belong to a class of cell-derived proteins that actively protect the host against tissue injury, microbial infestations, and inflammatory mediators. However, over-production of certain cytokines during chronic infection and inflammation may cause injury to the host. There is a paucity of research regarding the contribution of cytokines in the initiation/progression or promotion of malignant transformation of the biliary epithelia.
Interleukin-6 (IL-6) is a critical mediator of the hepatic response to systemic inflammation and is elevated in patients with cholangitis.6 IL-6 can contribute to the autocrine and/or pancrine growth stimulation of cholangiocytes via activation of either p38 or p44/p42 mitogen activated protein Kinase 1 (MAPK) signaling pathways.7 The important role of IL-6 in cholangiocarcinoma is highlighted by the elevated circulating serum levels of IL-6 in patients with this cancer.8 Inhibition of IL-6 signaling has been found to inhibit anchorage independent growth of a malignant cholangiocyte cell line.9 Thus IL-6, via its mitogenic effects, appears to be important in cholangiocyte growth. These observations suggest that inhibition of IL-6 signaling is a potential therapeutic strategy for the treatment of this neoplasm.
Tumon Necrosis Factor-Alpha
Tumor necrosis factor-alpha (TNF-a), a pro-inflammatory cytokine, augments phagocyte accumulation at injured sites and stimulates hepatic cells to produce cytotoxic oxidants such as nitric oxide (NO), superoxide anion, and hydroxyl radicals. NO is generated in increased quantities when inducible nitric oxide synthase (iNOS) is expressed in response to cytokine stimulation. Cholangiocarcinoma cells in vitro express iNOS message and protein and generate high levels of NO when stimulated in vitro by a mixture of pro-inflammatory cytokines, including interleukin-1-beta (IL-1b), interferon gamma (IFN-g), and TNF-a.10 Moreover, liver tissue specimens from 18 cholangiocarcinoma patients and 30 primary sclerosing cholangitis uniformly showed intense staining for iNOS protein.10 Indeed, there is histological evidence of tumor-associated inflammatory cells, a potential source of inflammatory cytokines, in most cholangiocarcinoma patients. The near universal expression of NO by cholangiocarcinoma suggests it plays an important role in the biology of this cancer.10
Induction of iNOS with NO generation could explain, in part, the link between chronic inflammation and cholangiocarcinoma.12 For example, increased synthesis of NO and endogenous formation of nitrite and nitrosamines are risk factors for cholangiocarcinoma in subjects infested with liver flukes (Opisthorchis viverrini).13 The chronic infection of intrahepatic bile ducts renders the cholangiocytes with increased vulnerability to proliferation, inflammation, and carcinogens. It has been shown that NO and its derivatives can cause DNA strand breaks by deaminating purines and pyrimidines and oxidative damage to the bases. NO generated by induction of iNOS with inflammatory cytokines is sufficient to induce single-strand and double-strand breaks and oxidative DNA lesions in cholangiocarcinoma cells.10 The DNA damage was inhibited in the presence of iNOS substrate inhibitors, implicating NO as the mediator of DNA damage. Oxidative DNA lesions and damage caused by NO can be repaired by cellular repair processes. However, global repair in cholangiocarcinoma cells was reduced to 30% of its normal function in the presence of cytokine stimulated NO production.10 Inhibition of repair processes likely contributes to the accumulation of mutations in DNA. These observations suggest NO may play an important role in causing the oncogenic mutations important in the development of cholangiocarcinoma.
NO may affect proteins by nitrosylation of tyrosine and cysteine residues. Although tyrosine nitrosylation of proteins has been amply documented as a marker of protein oxidation, its effect on the catalytic functions of enzymes is obscure. In contrast, cysteine nitrosylation is known to directly inactivate enzymes.14 Inhibition of DNA repair enzymes by sulphydryl nitrosylation is the likely mechanism for the NO-dependent inhibition of global DNA repair. Indeed, exposure of O6-alkylguanine-DNA alkyltransferase to NO causes nitrosylation of its active thiol moiety inhibiting its activity.15 DNA repair proteins with zinc finger motifs, such as formamidopyrimidine DNA-glycosylase (Fpg), which repairs 8-oxodeoxyguanine residues,16 are also directly inhibited in the presence of aerobic NO.17 Our studies significantly extend these previous biochemical observations by demonstrating inhibition of DNA repair activity by a NO-dependent manner in intact cells during exposure to inflammatory cytokines. These data demonstrate that the magnitude of NO generated by iNOS in these cells is sufficient to inhibit DNA repair processes. The specific oxidative base excision DNA repair enzymes nitrosylated in these cells was not elucidated but currently is pursued in our laboratory.
The specific, potential oncogenic mutations induced by NO remain obscure. Recently, p53 mutations have been identified in many cholangiocarcinomas associated with primary sclerosing cholangitis.18 Whether NO can induce genetic alterations in p53 is unknown but would provide a mechanistic link between inflammation, NO formation, and the development of this malignancy. There are other potential mechanisms by which NO may be important in the initiation, promotion, and progression of this cancer. Data suggest NO could promote the accumulation of potential oncogenic mutations by inhibiting DNA repair enzymes. Inhibition of pro-apoptotic effector proteins by protein nitrosylation, such as caspase proteases, may also promote extended survival of malignant cells. Indeed, nitrosylation of caspases would disable apoptotic pathways promoting cell survival despite DNA damage. Finally, NO may confer a survival advantage for cancer by serving as an angiogenesis factor.19 Finally, NO may enhance expression of oncogenes.20
Conclusion
Pro-inflammatory cytokines appear to play a key role in the initiation, progression, and/or promotion of cholangiocarcinoma during inflammatory conditions of the bile ducts by inducing proliferation of cholangiocyts and stimulating the production of NO and reactive oxygen species. NO and associated reactive oxygen species, such as peroxynitrite, modify DNA bases and result in direct DNA damage. Concomitantly, nitrosylation of key repair proteins inhibit repair of the DNA alterations, promoting accumulation of potential oncogenic mutations important in the initiation and/or progression of this cancer. Based on this information, we speculate that iNOS inhibitors targeted to cholangiocytes could potentially have a chemo-preventive role in patients with chronic inflammatory cholangiopathies, such as patients with primary sclerosing cholangitis. Based on similar concepts, inhibition of IL-6 signaling could prove useful in the treatment of established cholangiocarcinoma. (Dr. Jaiswal is a Postdoctoral Fellow, Dr. LaRusso is Chairman of Internal Medicine, and Dr. Gores is a Professor of Medicine, Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic/Foundation/Medical School, Rochester, MN.)
Acknowledgement
This work was supported by grants from the National Institutes of Health (24031, NFL and DK41876, GJG), the American Liver Foundation (MJ), Cedar Grove, NJ, and by the Mayo Comprehensive Cancer Center, Rochester, MN.
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