Study On Mechanism Of Hedgehog Signal Pathway In Development Of Cholangiocarcinoma

Cholangiocarcinoma accounts for 3% of the gastrointestinal cancer, and has an incidence of 2-4/100000/year, which is gradually increased in recent years. It is one of the lethal tumors with the highest malignancy. Because of a tendency to metastasize and lack of an effective method for early diagnosis, metastasis already occur in most of the cholangiocarcinoma patients at their diagnosis. Therefore, the prognosis of cholangiocarcinoma patients is really poor, and the median survival time is even less than 6 months after diagnosis. Despite the high mortality rate associated with cholangiocarcinoma,the causes of cholangiocarcinoma which hampered the treatment have not been identified。It is recognized that effective treatment of this fatal disease required detailed understanding of the molecular biology of cholangiocarcinoma. Accordingly, recognizing the molecular mechanisms of cholangiocarcinoma to make better diagnostic strategy and improving it’s prognosis is still a hard work to hepatobiliary surgery. Hedgehog signaling pathway is composed of Hh ligands(SHH,IHH,DHH),two transmembrane proteins PTCH (PTCH1, PTCH2) and SMO and three GLI transcriptional factors(GLI 1,GLI2,GLI3)。Recently,a series of studies have implicated that the over activation of Hedgehog signaling pathway is associated with many common human malignancies such as cholangiocarcinoma.Berman reported that 38 cell lines came from tumors from the esophagus, stomach, biliary tract, pancreas, and colon cancer had been detected the mRNA expression of Hh, and the maintenance of Hh on the proliferation of tumor cell growth and plays an important role。Artit Jinawath found that seven of cholangiocarcinoma cell lines (HuCCT1, HuH-28, MEC, TFK-1, RBE, TGBC24TKB andKKU-M156) and GLI1 also expressed in most cell lines (except HuCCT-1 andTFK-1 outside) in recent research, Hedgehog signaling pathway in cholangiocarcinoma cells was activated, which may be involved in the occurrence and development of cholangiocarcinoma.The close relationship between Hh pathway and cholangiocarcinoma suggests that it may be the ideal target for therapeutic strategy.By far,specific inhibitors of Hh pathway such as cyclopamine,a selective antagonist of SMO have been used in many studies of treatment of cancer. QBC939 cholangiocarcinoma cell line was established by Wang, et al. in 1993. This cell line exhibits a fast growth rate, hyperactivity, and high in vitro growth ability. It could respond to many hormones. Insulin and EGF could promote its proliferation, while dexamethasone could inhibit its growth. Cytogenetic analysis indicates that this cell line has obviously abnormal chromosome number and structure. The chromosomes display high frequency of fracture, displacement, loss and insertion. High percentage of polyploid cells was also found in this cell line. Since the establishment, this cell line has been widely used for tumor studies, especially in recent years. In our preliminary study, we found that signal molecules of HH signaling pathway were significantly higher in the QBC939 cholangiocarcinoma cells in comparison with those in the normal biliary epithelial cells (data not shown). This result suggest that as in the tumors of other organs, the Hh signaling pathway is only hyperactivated in the tumor tissue rather than the normal epithelium. Therefore, QBC939 cholangiocarcinoma cell line is an ideal cell line for HH signaling pathway study.This study use the nude mice xenograft model transplanted with QBC939 cholangiocarcinoma cells to investigate the impact of cyclopamine on the growth, proliferation and apoptosis of QBC939 cells, which may provide evidences for the clinical diagnosis, treatment, and prediction of prognosis of cholangiocarcinoma.Hedgehog signaling pathway proteins PTCH1, GLI1 expression in cholangiocarcinoma and its significance Objective:This study was to investigate the expression of PTCH1 and GLI1 of hedgehog signaling pathway in cholangiocarcinoma tissues,and explore their correlations to. the occurrence and development of cholangiocarcinoma.Methods:The expression of PTCHland GLI1 in 47 specimens of cholangiocarcinoma and 47 specimens of normal bile duct tissues adjacent to cancer was detected by immunohistochemistry,and their positive rate correlated with patients age、tumor size、differentiation grade neoplastic origin lymph node metastasis and TNM stage were investigated by statistical analysis.Result:The positive rates of PTCH1 and GLI1 were significantly higher in cholangiocarcinoma than in normal tissue adjacent to cancer (70.2%vs.14.9%, 89.4%vs.19.1%,P<0.05). The expression of PTCHland GLI1 in cholangiocarcinoma had no correlation to patients’age,tumor size and neoplastic origin (P>0.05),but were correlated to differentiation grade、lymph node metastasis and TNM stage(P<0.05). Conclusion:The elevated expressions of PTCH1 and GLI1 of Hh signaling pathway participated in the occurrence and development of cholangiocarcinoma. They may be ideal targets for therapy against cholangiocarcinoma.Blockage of Hedgehog Signaling Pathway Inhibits the Proliferation of QBC939 Cholangiocarcinoma CellObjective To study the impact of cyclopamine, a Hedgehog signaling pathway inhibitor, on the proliferation and apoptosis of QBC939 cholangiocarcinoma cells.Methods The proliferation of QBC939 cells were detected with MTT method. Cell apoptotic rate was analyzed with the flow-cytometry assay. The expressions of the tumor-related genes and proteins in QBC939 cells before and after cyclopamine treatment were detected with RT-PCR and Western blot. The inhibitory effect of cyclopamine on tumor growth was detected with the xenograft tumor model in nude mice. The expressions of tumor-related proteins in the xenograft were detected with the immunohistochemical method. The apoptosis of tumor cells upon cyclopamine treatment in the xenograft was analyzed by TUNEL assay.Results Cyclopamine inhibited the growth of QBC939 cells in time and dose dependent manners. After a 48h of 5,10, or 20μM cyclopamine treatment, QBC939 cells showed increased apoptotic rates, which were significantly higher than that in the control group (P＜0.01). Furthermore, cyclopamine downregulated the mRNA and protein levels of PTCH1、GLIland EGFR in QBC939 cells. Cyclopamine significantly inhibited tumor growth, promoted cell apoptosis, and downregulated PTCH1, GLI1, and EGFR expressions in the nude mice xenograft model. Conclusion Blockage of the Hedgehog signaling pathway with cyclopamine could suppress the proliferation and promote the apoptosis of QBC939 cholangiocarcinoma cells, and inhibit the tumorigenesis of QBC939 cells in nude mice.