Targeted Intervention Of GSK-3βRegulating Proliferation, Invasion, Angiogenesis And Epithelial-mesenchymal Transition In Pancreatic Cancer And Its Molecular Mechanisms

ObjectiveDue to the rapid development in molecular biology and gene research, scientists have made great progress in mechanisms of carcinogenesis and cancer therapy. Many research outcomes have been used in clinical practice, like monoclonal antibody therapy and tumor vaccine. It has been widely accepted about the effectiveness of monoclonal antibody in cure of breast cancer and leukemia. But for malignant tumor of gastrointestinal system, especially for those of highly malignancy, like pancreatic cancer, surgery and chemotherapy are still the only effective treatment. Hence, it is of great importance to find new therapeutic targets to instruct clinical treatment and improve prognosis based on the research of the mechanism of carcinogenesis of pancreatic cancer.It has been discovered in recent years that GSK-3P widely participates in multiple cellular processes. It also involves in the regulation of carcinogenesis and metastasis of human cancer. But its role in these regulation are conflicting depending on the type of cancer. Molecular targeted therapy based on GSK-3βhas made progress in cancer treatment, some of them have been approved into clinical trials. Thus, we considered GSK-3βas a molecule target of great research value and potential of clinical translation. It is still unclear about the concrete role of GSK-3βin pancreatic cancer. In this research, we tried to explore the role of GSK-3βin proliferation, angiogenesis, invasion, metastasis and epithelial-mesenchymal transition of pancreatic cancer. Our research is funded by National Science Foundation of China (No.30801098 and 30972903).MethodsWe selected 60 cases of pancreatic adenocarcinoma of Union hospital from 2006 to 2010,35 of which have pared adjacent non-cancerous tissues. Immunohistochemistry stain of GSK-3β, Ser-9, Tyr-216, NF-(?)B, Sanil, E-cadherin and MMP-9 were performed. The correlation between GSK-3βand epithelial-mesenchymal transition, invasion of pancreatic cancer. The correlation between GSK-3βand clinic-pathologic features of pancreatic cancer.Lentivirus interference was used to inhibit the expression of GSK-3βin pancreatic cancer. CCK-8 and flow cytometry were applied to detect the effect of GSK-3βon proliferation and apotosis of pancreatic cancer cell lines. Western blot was used to investigate the expression of GSK-3βphosphorylation forms (Ser-9 and Tyr-216), angiogenesis, invasion and epithelial-mesenchymal transition related molacules. Wound healing and transwell test were ultilized to detect the effect of GSK-3βon migration and invasion of pancreatic cancer cell lines, respectively. Luciferase assay was used to investigate the effect of GSK-3βon the activity of NF-(?)B in pancreatic cancer cell lines.Xenograft tumor model in nude mice was established to explore the effect of GSK-3βknockdown on tumorigenesis of pancreatic cancer. Besides, intraperitonal injection of GSK-3βinhibitor(SB 216763) was also used to explore the effect of GSK-3βknockdown on tumorigenesis of pancreatic cancer. Immunohistochemistry stain of PCNA and CD31 were performed ot evaluate the the effect of GSK-3βknockdown on proliferation and angiogenesis of pancreatic cancer. TUNEL assay was ultilized to detect the effect of GSK-3βknockdown on apoptosis of pancreatic cancer. Finally, western blot was used to investigate the expression of VEGF, NF-(?)B, Snail, E-cadherin, mmp-9 in xenograft tumor tissues.ResultsGSK-3βexpression is significantly higher in primary pancreatic cancer than in adjacent non-cancerous tissues. The expression of GSK-3βhas positive correlation with clinical stage, TNM classification, tumor differentiation and lymph node metastasis, whereas it is irrelevant with patient gender and age. the expression of phosphor-GSK-3β(Ser-9) protein was lower in the cancerous tissues than in the noncancerous tissues. But the expression of phosphor-GSK-3β(Tyr-216), NF-(?)B,Snail and mmp-9 were higher in cancerous tissue than in noncancerous tissue The levels of total E-cadherin were low both in cancerous and noncancerous tissue. Pearson's test showed that GSK-3βcorrelates with all above-mentioned molecules.GSK-3βknockdown resulted in arrested proliferation and increased apoptosis in pancreatic cancer cell lines. It also inhibited migration, invasion, angiogenesis and. In a xenograft tumor model, GSK-3P knockdown inhibited tumor growth and angiogenesis and epithelial-mesenchymal transition in pancreatic cancer cell lines. Binding activity of NF-(?)B decreased significantly in GSK-3βknockdown group.GSK-3βknockdown and GSK-3βinhibitor could both inhibit tumorigensis in xenograft model. The expression of PCNA and CD31 decreased significantly in GSK-3βknockdown xenograft tissues. TUNEL assay showed apoptotic rate increased remarkably in GSK-3βknockdown group and SB 216763 intraperitonal injection group. Western blot showed decreased expression of phosphor-GSK-3β(Tyr-216), VEGF, NF-□B, Snail and mmp-9 in in GSK-3βknockdown group and SB 216763 intraperitonal injection group, while the expression of E-cadherin elevated.ConclusionsGSK-3P is closely related to the malignant biological behaviour of pancreatic cancer. GSK-3βwidely participates in the regulation of proliferation, apoptosis, invasion, migration, angiogenesis and epithelial-mesenchymal transition of pancreatic cancer. GSK-3P knockdown effectively inhibits proliferation, apoptosis, invasion, migration, angiogenesis and epithelial-mesenchymal transition of pancreatic cancer. The mechanism related to GSK-3βregualtion of malignant biological behaviour is probably through the NF-(?)B pathway. GSK-3βknockdown and GSK-3βinhibitor could both inhibit tumorigensis in xenograft model. Our results showed that GSK-3βis a promising therapeutic target in pancreatic cancer treatment, which might offer an new avenue for gene therapy.