| Cholangiocarcinoma(CCA)is a kind of aggressive tumor of the liver derived from the epithelial cells of the bile duct.Because of the late discovery,high incidence of multiple diseases and old age,nearly half of the patients with cholangiocarcinoma can only use palliative treatment.Radiotherapy plays an important role in palliative treatment of cholangiocarcinoma.Postoperative adjuvant radiation therapy is also of great significance to the patients undergoing surgery.However,regardless of the treatment regimen,cholangiocarcinoma is still prone to invasion and metastasis of blood vessels and distant lymph nodes,leading to cancer-related death.It has been reported that radiation therapy may be at risk of promoting tumor metastasis.Therefore,the mechanism of tumor metastasis related to radiation treatment may be involved in the invasion and metastasis of cholangiocarcinoma,and the potential role of platelet and granulocyte-associated protein 8(S100A8)in the metastasis of cholangiocarcinoma has attracted our attention.On one hand,a large number of studies have shown that the interaction between tumor cells and platelets in the blood flow microenvironment is an inevitable factor for early distant metastasis.Studies of lung cancer,pancreatic cancer and breast cancer have shown that the interactions between platelets and tumor cells activate platelets,many kinds of cytokines can influence the adhesion,angiogenesis and remodeling of tumor cells.In the bloodstream,platelets participate in the formation of blood clots around tumor cells,preventing them from being cleared by natural killer cells(NK).Platelets form a coating around the tumor cells,which allows the tumor cells to adhere to the vessel wall and enhance the tumor cells’ extravasation from the vessel wall.The interaction between platelets and tumor cells enables platelet-specific Histocompatibility I to adhere to tumor cells and prevent the recognition of NK cells and epithelial-mesenchymal transition(EMT),thus promoting tumor invasion and metastasis.These studies confirm that platelets play an important role in promoting tumor metastasis,and studies of radiation injury have shown that radiation has a significant destructive effect on local endothelium,that can cause platelets to clump together and form clots.In radiotherapy for cholangiocarcinoma,this process may increase the interaction between tumor cells and platelets,thus increasing the risk of metastasis.Therefore,previous studies suggest that platelets may be an important microenvironmental factor in promoting the invasion and metastasis of cholangiocarcinoma cells,and its specific effects and related molecular mechanisms are worth exploring.On the other hand,studies on various tumor cells themselves have shown that irradiation can induce an increase in the expression of granulocyte-associated protein A8(S100 calcium-binding protein A8,S100A8).The expression of S100A8 in lung cancer,colon cancer,stomach cancer,pancreatic cancer and primary liver cancer was significantly increased.Recent studies have reported that over-expression of S100A8 can promote the invasion and distant metastasis of breast cancer and is significantly associated with poor clinical prognosis.S100A8 is a well-known endogenous ligand of toll-like receptor-4(TLR4).The TLR4 pathway is known to be involved in a wide range of malignancies.However,the role of S100A8 in the metastasis of cholangiocarcinoma remains unknown,based on the uncertainty of clinical efficacy of radiotherapy in the treatment of cholangiocarcinoma metastasis and the discovery that irradiation can induce the expression of S100A8 in some tumor cells,it is suggested that S100A8 may be involved in the invasion and metastasis of cholangiocarcinoma cells.Therefore,this topic takes the bile duct cancer cell line as the main research object,unifies the nude mouse bile duct cancer cell liver metastasis model as well as the clinical pathology specimen correlation molecular target detection,to study the effect of platelet and S100A8 gene on the invasion and metastasis of cholangiocarcinoma,the p38MAPK-MMP2/MMP9 pathway regulated by platelet-derived growth factor(PDGF)and TLR4/NF-κB-VEGF pathway regulated by S100A8 were investigated,to investigate the molecular mechanism of invasion and metastasis of cholangiocarcinoma cells induced by platelet and S100A8.The study covered the following two aspects:Firstly,the co-culture system of RBE and HCCC-9810 with human peripheral blood platelets was established.The invasion and migration of transwell cells were tested,western blot was used to detect the expression of p38MAPK-MMP2/MMP9 signal pathway and EMT related molecules in bile duct carcinoma cells.Furthermore,platelet-derived growth factor receptor(PDGFR)inhibitor CP-673451 and p38 MAPK inhibitor SB203580 were added to the cell co-culture model to detect the changes of EMT invasion and migration,signal pathway molecules and related molecules expression in cholangiocarcinoma cells,observe the change of cell morphology.In addition,western blot and ELISA were used to detect the expression of MMP2/MMP9 protein in cholangiocarcinoma tissues,and the correlation between the expression of MMP2/MMP9 protein and clinicopathological parameters was analyzed.Finally,the hepatic metastasis model of BALB/c-nu cholangiocarcinoma cells was established,and the hepatic metastasis was detected;hematoxylin and eosin staining was observed.Secondly,S100A8 overexpressed RBE cells and S100A8 knock-out HCCC-9810 cells were constructed by lentiviral transfection using western blot to detect the effect of radiation on the expression of S100A8 cells,the invasion and migration ability of tumor cells were detected by transwell cell invasion test and cell migration test,and the secretion level of vascular endothelial growth factor(VEGF)in cell culture medium was detected by ELISA,the effect of tumor cell culture medium on the microvascular lumen formation of human umbilical vein endothelial cells(HUVEC)was observed under microscope.Furthermore,TAk242 of TLR4 and PDTC of NF-κB were added into the transfected cell model to detect the invasion and migration ability of cholangiocarcinoma cells and the secretion level of VEGF,western blot was used to detect the expression of VEGF,P65 and other proteins.In addition,western blot,immunohistochemistry and ELISA were used to detect the expression and localization of S100A8 and VEGF in clinical cholangiocarcinoma tissues,and the correlation between the expression of S100A8 and VEGF and clinicopathological parameters was analyzed.Finally,in the liver metastasis model of mouse cholangiocarcinoma cells,the metastatic foci formed by S100A8 overexpression or knock-out tumor cells were detected,and the changes of histopathology were observed by H&E staining,the metastasis of cholangiocarcinoma cells was observed by cell fluorescence labeling combined with liver biopsy.The main results and conclusions of this study are as follows:1.The experiments of cell invasion and migration showed that RBE and HCCC-9810,which were co-incubated with platelets respectively,could enhance the ability of invasion and migration of tumor cells.In addition,the cell adhesion test showed that there was obvious adhesion between platelets and tumor cells.The results showed that platelets could promote the invasion and metastasis of cholangiocarcinoma cells.2.In the co-culture system of bile duct cancer cells and platelets,PDGFR inhibitor can significantly inhibit the invasion and metastasis of bile duct cancer cells induced by platelets,and affect the morphological changes of tumor cells induced by platelets,at the same time,the changes of platelet-induced EMT related molecules in tumor cells were eliminated.The expression of MMP2 / MMP9 was significantly higher in clinical cholangiocarcinoma than that in adjacent normal tissues.The expression of MMP2 / MMP9 was significantly correlated with lymph node metastasis and poor prognosis,kaplan-meier survival analysis showed that high expression of MMP2/ MMP9 was associated with decreased overall survival in patients with cholangiocarcinoma.Platelet co-culture can increase the expression of MMP2/ MMP9 in cholangiocarcinoma cells,while PDGF inhibitor can significantly decrease the expression of MMP2/MMP9,suggesting that platelets are involved in the regulation of MMP2/MMP9 in cholangiocarcinoma cells.3.p38 MAPK inhibitor treatment can block the invasion and metastasis of bile duct carcinoma cells induced by platelets in co-culture system.At the same time,selective inhibition of p38 MAPK could significantly down-regulate the expression of MMP2/ MMP9 induced by platelets,and eliminate the expression of platelet-induced EMT related molecules.Platelet-induced phosphorylation of p38 MAPK was also found to be elevated.These results suggest that PDGF may up-regulate MMP2/MMP9 by activating p38 MAPK pathway,and participate in inducing EMT changes and invasion and metastasis of cholangiocarcinoma cells.4.In the model of liver metastasis in nude mice,RBE cells pretreated with platelets,RBE cells pretreated with platelet and PDGFR inhibitor were found to form metastatic foci in the liver of the control group,however,the number of transformed foci of platelet-pretreated cells was significantly higher than that of control cells,and inhibition of PDGF could decrease the number of liver transformed foci of platelet-induced RBE cells.The results showed that PDGF could promote the invasion and metastasis of cholangiocarcinoma cells in vivo.5.The expression of S100A8 protein in cholangiocarcinoma cells was significantly increased by ionizing radiation.In S100A8 overexpressing RBE cells and S100A8 interfering HCCC-9810 cells,the invasiveness of S100A8 overexpressing cells was increased,but the invasiveness of S100A8 interfering cells was decreased.The expression level of S100A8 in clinical bile duct carcinoma was significantly higher than that in adjacent normal tissues,and the expression of S100A8 protein was mainly in cytoplasm and membrane of bile duct carcinoma,the expression level of S100A8 protein was significantly correlated with tumor differentiation and lymph node metastasis in patients with cholangiocarcinoma.Kaplan-meier survival analysis showed that high expression of S100A8 was significantly correlated with lower overall survival in patients with cholangiocarcinoma.Finally,we found that S100A8 could induce the invasion and metastasis of cholangiocarcinoma cells in vivo,and inhibition of S100A8 could decrease the invasion and metastasis of cholangiocarcinoma cells.These results confirm that S100A8 is involved in promoting the metastasis of cholangiocarcinoma cells.6.The expression level of VEGF protein in clinical bile duct carcinoma tissues was significantly higher than that in adjacent tissues.immunohistochemical detection showed that VEGF protein was mainly expressed in cytoplasm and cell membrane,kaplan-meier survival analysis showed that the high expression of VEGF was significantly associated with lower overall survival in patients with cholangiocarcinoma.Furthermore,the high expression of VEGF was associated with lower overall survival in patients with cholangiocarcinoma,the high expression of S100A8 and VEGF was associated with poor prognosis in patients with cholangiocarcinoma.The results showed that S100A8 could regulate the secretion of VEGF in HCCC-9810 cells,and the supernatant of HCCC-9810 cells overexpressing S100A8 could promote the formation of microvascular lumen in HUVEC cells,anti-VEGF neutralizing antibody can inhibit the invasion and migration of cholangiocarcinoma cells induced by S100A8.After blocking TLR4 and NF-κB with TAk242 and PDTC respectively,we found that the invasion and metastasis of cholangiocarcinoma cells were decreased,and the secretion of VEGF,the expression of VEGF protein and phosphorylated P65 induced by S100A8 were significantly decreased.These results confirm that S100A8 promotes the metastasis of cholangiocarcinoma cells by regulating TLR4/NF-κB-VEGF pathway.To sum up,in this study,we firstly explain the possible effects of radiotherapy on the invasion of cholangiocarcinoma in terms of platelet microenvironment factors and tumor cells,then the p38MAPK-MMP2/MMP9 pathway regulated by PDGF and TLR4/NF-κB-VEGF pathway regulated by S100A8 were analyzed.These results deepen the understanding of the mechanism of invasion and metastasis of cholangiocarcinoma,and provide a new direction and therapeutic strategy for the potential target of cholangiocarcinoma therapy. |
PDF Full Text Request