| BackgroundHepatolithiasis refers to the calculi occurred in the left/right hepatic biliary duct and its branches, which often associates with extrahepatic bile duct stones, bile duct stricture and liver fibrosis. The literature describing hepatolithiasis is found around the 16th centuries. Hepatolithiasis was frequently diagonozed in Asia, and the Chinese mainland reported the most number of cases. Hepatolithiasis with recurrent infection is the most common factors leading to cholangiocarcinoma.Cholangiocarcinoma is a malignant tumour disease with high rate of incidence, and the age of onset is often 50 to 70 years old. The cause of ICC is yet not clear,and studies have shown that patients with hepatolithiasis are high risk group of cholangiocarcinoma,about 2% to 10% of patients with hepatolithiasis will face the risk of cholangiocarcinoma,it is generally believed that it is the longterm mechanical stimulation of the hepatolithiasis to the bile duct and its chronic biliary tract infection which causes chronic bile duct hyperplasia inflammation/bile duct dilatation of the affected area/annular or segmental stenosis of bile duct; The affected bile duct wall will thicken, and its surrounding fibrous tissue hyperplasia with inflammatory cell infiltration; Lots of inflammatory cell infiltration in periportal and fibroblast proliferation, accompanied by liver damage, in severe case, it will result in liver segment or liver fibrosis atrophy and liver function damage.The inflammatory thickening of bile duct wall/fibrosis and stenosis will then cause epithelial dysplasia of bile duct. Pathology observed that epithelium dysplasia of bile duct can gradually migrate and change into adenocarcinoma. However, when hepatolithiasis with bile duct cancer occurs, since the symptom of most patients in the clinical manifestations is similar to hepatolithiasis with with bile duct cancer, without specificity, and in clinical examination, the presence of the tumor was easily missed diagnosed cause of the coexistence of stones and tumors coexist,which lead to the missing of best timing treatment.A lot of study on basic diease/clinical diease and epidemiological have proved that, inflammation is a distinct dangerous factor leading to tumor.The relationship between inflammation and tumor caused concern of researchers in early days cause infiltration of immune cells was found in tumor specimen.At that time it was explained as the response of immune system to tumour cell, but in later studies it is believed that "Tumour is originated from chronic inflammation". Besides, the type of immuse cell in tumor is the same to the inflammatory& nonneoplastic cell. After that more and more scientists further studies the relationship between tumour and inflammation, and it was found that chronic inflammation is a high risk factor of many malignant tumors, it joins in malignant transformation/tumor formation/development/affection/transfomation and several process, and it was listed as the ten biological characteristics of tumour cell.Inflammation, particularly chronic inflammation, is one of the most important factors leading to the occurrence and developing tendency of tumor, which is called "inflammation-related cancer" by some scholars. Pathogen and various inflammation-associated cancer is obviously irrelevant, but among them what they have in common is their key feature of sustainable inflammation:Initially, it was the infiltration by activated macrophage/lymphocyte, then it was generated by activated and formed by fibroblasts and vascular cells. At the same time it is believed that the ROS and NO which these infiltrating inflammatory cells produces may damage the normal cells,which in turn leads to the cell proliferation compensatory, and then accumulation of gene mutation and effectively brings endogenous and exogenous factor into normally growing cells. In addition, recently it is found that the inflammatory environment can accelerate epigenetic changes, which may lead to inflammation-associated tumorigenesis.c-Met is a protein product encoded by c-Met proto oncogene, it is the receptor of hepatocyte growth factor (HGF) with tyrosine kinase activity, and it is related to a variety of cancer genes product and regulatory proteins, the cells involved in signal transduction and regulation of cytoskeletal rearrangement, and it is an important facor for cell proliferation, differentiation and movement. HGF is the ligands of c-Met,and mainly produced by mesenchymal cell, and effected in ways of autocrine and paracrine, it was a typical growth factor for interstitial cell; HGF can stimulates the growth of many cells, and takes part in the regulation of angiogenesis and immunological activity,and it can promotes the proliferation/metastasis and invasion of cancer cells. When c-Met and its ligand combined with HGF, autophosphorylation of Tyr1234/Tyr1235 will happen,which activate tyrosine kinase in c-Met cytoplasm, the activated PTK will lead to the autophosphorylation of tyrosine. Afterwards, lots of effectors in cytoplasm raise carboxyl terminal and fast phosphorylation occurs, which finally activate many singal path with the cells.It is also found in the study that c-Met pathway can induce tumorigenesis and metastasis; C-Met is related to the resistance of tumour to many kinase; C-Met can interact with a variety of membrane receptors which promotes the tumorigenesis and metastasis, inducing resistance. Many molecular mechanisms can activate c-Met in tumour cells, the most common way is the combination of HGF and c-Met, which leads to the autophosphorylation and enhances the activity of c-Met tyrosine kinase, which leads to the tyrosine phosphorylation of a variety of substrate protein. The risistence of tyrosine kinases which fuctioned at c-Met is gradually into the step of clinical trial,while PF-2341066 was accepted by US FDA that enters into the preferential assessment procedure in 2011 as a c-Met inhibitor.The COX-2 gene of human is located in the 1q25.2-q25.3 of number one chromosome, the length of it is 8.3 kb, and it is composed of 10 exon exons and 9 introns, coding polypeptides which is made up of 604 amino acid residues. COX-2 and COX-1 have homology of 62%, but they showed distinct function difference. COX-2 is mainly located on cell membrane, its expression and regulation was focused on transcription level.The factors which inducing its expression including cancer gene/nterleukin-1/hypoxia/ultraviolet rays/transforming growth factor and tumor necrosis factor-a and etc, while dexamethasone/antioxidant/tumor suppressor protein P53 can inhibite its expreession. And about the carcinogenic mechanism of COX-2, it’s generally accepted that COX-2 can join in formation and development of tumour by means of promoting cell proliferation/inhibiting cell apoptosis/boosting the formation of blood vessel and restraining immuse system, which promoting cell proliferation and inhibiting cell apoptosis as well as boosting the formation of blood vessel.Celecoxib is the first listed selective inhibitor of COX-2, Xu and other researchers in the National Cancer Institute in Frederick.US found that the inhibitor of COX-2 celecoxib having an anti-angiogenic effect. A research made by Emory University researchers showed that celecoxib, possibly by activating the extrinsic death pathway of receptor, can induces apoptosis of non-small cell lung cancer cells. The researchers used a mouse model, by inhibiting the expression of COX-2, it was found that the inhibition of VEGF pathway is greatly enhanced, and in the end it effectively inhibit tumorigenesis and metastasis, which prolongs survival period.With the further study of biological behavior of tumour, it is found that angiogenesis plays an important role for the growth and development of tumour. The angiogenesis is adjusted by positive and negative ways, while VEGF is an important promoting factor for angiogenesis.The developing procedures of tumour can be divided into two steps:no blood vessel and with blood vessel.In the steps of no blood vessel, the tumour cell moves by dispersion, and it requires oxygen and nutrients and moves away metabolic product, at this point the qpoptosis rate of tumour rate is high, and the size of the volumn is rarely larger than l-2mm3, the tumour lies dormant, and this period may last several weeks to several years.But once the tumour cell mutate with anti-apoptosis characteristic, it enters into the with blood vessel step. When entering into the with blood step, the size of the volumn increases and it may transfer. The growth factor of vascular endothelium affected the endothelial cells of blood vessel, and joins the rebirth of tumour vessel, and offer stroma for the transfer of endothelial cells of vessel and tumour cess. When the diamereur of tumour tissue grows to the size of larger than 1cm, the activated tumour tissue synthesizes and secrete VEGF by autocrine or paracrine mechanism, ptomotes the establishment of abundant capillary network around the tumour tissue, and under the effect of growth factor of vessel, endothelial cells of veseel secrete collagenase and Plasminogen so as to degrade the basement membrane of blood vessel, meanwhile, the basement membrane of microblood vessel which was produced by the inside of tumour tissue is not perfect which enable the tumour cell to enters into the blood vessel without compilicated process of invasion, which promotes the tumour cell to reborn and proliferate in new part.Studies have shown that, c-Met may act on COX-2. Jones studies on the gastric cells of stomach, and it is believed that by c-Met phosphorylation and ERK-2, signal transduction pathway can activate the gene expression of COX-2. The mean micro vessel density of c-Met gene positive expression in gastric carcinoma tissue was significantly higher than the negative group, indicating that gastric cancer cells with positive c-Met gene expression is more likely to generate tumor micro vessels, it can possiblely upregulated COX-2 expression and increase prostaglandin synthesis, thereby affecting the growth of gastric cancer and metastasis.ObjectiveStudy on the combination of COX-2 and C-Met in hepatolithiasis with cholangiocarcinoma and their respective tissue expression; Observe the combined fuction of PF-2341066 and celecoxib on cholangiocarcinoma cell line QBC939 and the experi,emtal effect of tumor bearing model of nude mice of cholangiocarcinoma; Discuss whether the HGF-c-Met-COX-2-PGE2-VEGF is a potential signal pathway of angiogenesis of bile duct cancer; Investigate whether the combination use of inhibitor of c-Met PF-2341066 and cox-2 inhibitor celecoxib can offer a new target treatment strategy for the future treatment strategy for this kind of cancer.Method1.Western blot and RT-PCR to test the expression of c-Met/COX-2 in cholangiocarcinoma tissue and analyze its relationship with clinical pathology:2.The analysis method of the relationship between the expression leve of c-Met & COX-2 and clinicopathological parameters and prognosis:Conduct Mann-Whitney U and Kruskal-Wallis test to analyze the relationship between c-Met & COX-2 expression level and clinicopathological parameters of hepatolithiasis and cholangiocarcinoma, using the Cox proportional hazards model to study the relation between the expression leve of c-Met & COX-2 and prognosis.3.MTT assay of cell proliferation:QBC939 cholangiocarcinoma cells in vitro and then treat with a combination of c-Met inhibitor PF-2341066 and celecoxib to detect cell proliferation.4.Test apoptosis by flow cytometry:QBC939 cholangiocarcinoma cells in vitro, and then treated by c-Met inhibitor PF-2341066 and celecoxib to intervene, using Annexin V-EGFP and Propidium Iodide staining, and after staining, ustilize flow cytometry to detect cell apoptosis.5.Build the transplanted tumors in nude mice.Using PF-2341066 combined with celecoxibon in mice, then assay the cell proliferation/apoptosis by MTT/flow cytometry.Result1.The protein expression of c-Met & COX-2 in the tissue of hepatolithiasis and cholangiocarcinoma is the highest, and their expression in carcinoma adjacent tissue is higher than in normal tissues, and there is a statistically significant difference (P <0.05) among the groups.2.Expression level of c-Met/COX-2 was closely associated with pathological grade of cholangiocarcinoma, and expression level of c-Met/COX-2 of histological grade Ⅲ & Ⅳ is significantly higher than of grade Ⅰ and Ⅱ(P<0.05); Expression level of c-Met/COX-2 also have significant correlation with the transfer of lymph node,the expression level of c-Met/COX-2 within the cholangiocarcinoma tissue of metastasis-positive patients is significantly higher than that of those without lymph node metastasis of cholangiocarcinoma(P<0.05); In addition, the expression level of c-Met/COX-2 within the cholangiocarcinoma tissue of patients who have distant metastasis is also significantly higher than those who have no distant metastasis of cholangiocarcinoma (P<0.05); Thus, c-Met/COX-2 may play an important role in clinical progression and metastasis of cholangiocarcinoma3.Utilize COX multivariate regression method to analyze the relative prognosis factors of patients.lt is found that prognosis of the patients who have higher expression level of c-Met/COX-2 is worse than those with lower expression of c-Met/COX-2, it indicates that the expression level of c-Met/COX-2 have negative relation with prognosis, and c-Met/COX-2 may be dangerous factor for the prognosis of cholangiocarcinom(P<0.05). Further statistical analysis also showed that the tumor histological grade (P<0.05)/lymph node metastasis (P<0.05) and distant metastases (P<0.05) are also risk factors for the prognosis of patients with cholangiocarcinoma.4. Conduct experiment in vitro by using cell line of cholangiocarcinom QBC939, after utilizing c-Met inhibitor with different concentrations on QBC939 cell, test the variation of its proliferation and apoptosis, it turns out that inhibition rate of QBC939 cell increases with the concentration rate of c-Met inhibitor, and it is also found that the inhibition rate in the combination of PF-2341066& Celecoxib is the highest with statistically significance compared with the control group(P <0.05).After the same method was used on the cell, test the apoptosis of QBC939 cell by means of flow cytometry, the result shows that the apoptosis rate of QBC939 cells increases with the concentration rate of c-Met inhibitor, and apoptosis rate is the highest in combined group of PF-2341066 and Celecoxib, there is significant difference (P<0.05) as well.5. Utilize c-Met inhibitor of different concentrations rate on QBC939 cells, test the expression level of c-Met, COX-2, VEGF protein and mRNA, it is found the expression level of c-Met, COX-2, VEGF protein and mRNA decreases with the increasing concentrations rate of c-Met inhibitor, and there are statistically significant differences (P<0.05) comparing with the control group.6.Build tumor-bearing nude mice, intervene the nude mice with combination of c-Met inhibitor with celecoxib, and compared its volumn of tumor, it is found that the volumn of tumor decreases with the increasing comcentration rate of c-Met inhibitor, and the average volumn of tumor of combination of PF-2341066 and celecoxib is the smallest, and there is significant statistical difference (.P<0.05) compared with the control group.7. Using c-Met inhibitor with different concentrations rate on nude mice, test the variation of proliferation and apoptosis.Ttransplanted tumors in nude mice, detect changes in proliferation and apoptosis.The results show that the rate of cell proliferation inhibition increases with the concentration rate of c-Met inhibitor, and it is the highest in the group of combined PF-2341066 and celecoxib, and there is statistically significance compared with the control group(P<0.05). After the same method was used,test the apoptosis of cell by means of flow cytometry, the result shows that the apoptosis rate of cells increases with the concentration rate of c-Met inhibitor, and the apoptosis rate is the highest in the combined group of PF-2341066 and celecoxib, and there is significant difference (P<0.05) as well.8.Using c-Met inhibitor with different concentrations rate on nude mice, test the expression level of c-Met, COX-2, VEGF protein and mRNA,it is found that the expression level of c-Met, COX-2, VEGF protein and mRNA decreases with the increasing concentrations rate of c-Met inhibitor, and the lowest level appears in the combined group of PF-2341066 and celecoxib, and there is a significant statistical difference (P<0.05).Conclusion1. The expression level of c-Met and COX-2 in the tissue of hepatolithiasis and cholangiocarcinoma is higher compared to para-tumorous tissues and normalbile duct tissue, suggesting that c-Met and COX-2 play an important role in development of cholangiocarcinoma.2. The expression level of c-Met/COX-2 in hepatolithiasis with cholangiocarcin-oma is closely related with histological grade and distant metastasis, suggesting that c-Met and COX-2 are closely related with the malignant degree of hepatolithiasis with cholangiocarcinoma and invasive transfer.3. Combination of c-Met inhibitor and Celecoxib can reduce the size of tumor volumn and promote cell apoptosis, suggesting that c-Met and COX-2 may be important factors affecting the development of cholangiocarcinoma.4. c-Met and COX-2 inhibitor may be future potential drug for hepatolithiasis with cholangiocarcinoma, it may bring new prospects for the adjuvant treatment of cholangiocarcinoma.5.c-Met inhibitor PF-2341066 and COX-2 Inhibitor celecoxib have synergistic inhibitory effect on the growth of cholangiocarcinoma cell.The exist of the potential signal path on carcinoma angiogenesis between the c-Met and COX-2 need further research. |
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