| Part Ⅰ Expression of β2-AR and AKR1B1 in Pancreatic Duct Adenocarcinoma Tissues and Its Clinical SignificanceObjective: To investigate the expressions of β2-adrenergic receptor(β2-AR,a mental factor related signaling molecule),aldo-keto reductase family 1 member B1(AKR1B1)in human pancreatic duct adenocarcinoma(PDAC),its relationship with clinicopathological parameters and prognosis assessmen in PDAC.Methods: The expressions of β2-AR and AKR1B1 were detected with immunohistochemistry in 138 cases of surgically resected specimens of human pancreatic ductal adenocarcinoma tissues,and expressions of β2-AR and AKR1B1 in 4 cases of fresh pancreatic cancer and paracancerous tissues were detected by Western blot(WB).Pearson correlation analysis was used to analyze the correlation between the total scores of β2-AR and AKR1B1.The correlations between β2-AR,AKR1B1 and clinicopathological parameters such as gender,age,tumor location,tumor diameter,lymph node metastasis etc were analyzed.The survival time of the PDAC patients was followed up after operation.Survival curves and hazard curves were calculated using the Kaplan-Meier method,and the prognosis of PDAC was analyzed using logrank univariate and multivariate Cox proportional hazards regression model.Results:(1)Immunohistochemical detection results showed that both β2-AR and AKR1B1 were mainly expressed in the cytoplasm,with expression of β2-AR on the cell membrane,expression of AKR1B1 in the nucleus,and lower expression of both of them in the corresponding paracancerous tissues,but the staining intensity in the paracancerous tissues was lower than that of the cancer tissues.Similar to the immunohistochemical results,WB results showed that both β2-AR and AKR1B1 were expressed in pancreatic cancer and paracancerous tissues,but the expression level in pancreatic cancer tissues was slightly higher than that in paracancerous tissues.(2)β2-AR was correlated with AKR1B1(r = 0.2061,P = 0.0153)in PDAC.(3)The expressions of β2-AR and AKR1B1 were not correlated with gender,age,tumor location,tumor diameter,distant metastasis,histological differentiation,TNM stage,levels of CEA and CA-199,but with lymph node metastasis.(4)Univariate analysis showed that the parameters of patient’s lymph node and distant metastasis,histological differentiation,TNM stage,β2-AR and AKR1B1 were correlated with the survival time of the PDAC patients after operation(P <0.05).The Cox proportional hazards regression model analysis showed that only lymph node and distant metastasis,β2-AR and AKR1B1 were independent prognostic indicators for pancreatic duct adenocarcinoma(P <0.05).Kaplan-meier analysis showed that expression levels of β2-AR and AKR1B1 in PDAC were negatively correlated with patient survival rate.Conclusions :(1)The expressions of β2-AR and AKR1B1 in pancreatic ductal adenocarcinoma were correlated and the levels of β2-AR or AKR1B1 were related to whether the patient has lymph node metastasis.(2)Lymph node and distant metastasis,expression levels of β2-AR and AKR1B1 are independent factors affecting the prognosis of pancreatic duct adenocarcinoma patients.The occurrance of lymph node metastasis and distant metastasis,high expressions of β2-AR and AKR1B1 can increase the survival risk of pancreatic duct adenocarcinoma patients.Part Ⅱ The Mechanism Research of Proliferation and Metastasis Promoted by β2-AR-induced AKR1B1 Activation in Pancreatic Cancer CellsObjective: To investigate the molecular mechanism of proliferation and metastasis promoted by β2-adrenergic receptor(β2-AR,a mental factor related signaling molecule)-induced aldo-keto reductase family 1 member B1(AKR1B1)activation in pancreatic cancer cells.Methods:(1)Western blot(WB)was used to confirm the protein expressions of β2-AR and AKR1B1 in multiple human pancreatic cancer cell lines BXPC-3,CFPAC-1,PANC-1 and human normal pancreatic duct epithelial cell line h TERT-HPNE.The interaction of β2-AR and AKR1B1 was identified by reciprocal immunoprecipitation.Immunofluorescence was conducted to detect the Co-localization of β2-AR and AKR1B1 in BXPC-3 pancreatic cancer cell lines.(2)p CDNA3.1-β2AR was transfected into relatively β2-AR lower expression cell line,CFPAC-1 cells for 48 h.Cell counting kit-8(CCK-8)was used to detect the cell proliferation,and the protein levels of β2-AR,AKR1B1,P-ERK1/2 and ERK1/2 were tested using western blot analyses,as well as the apoptosis and cell cycle distribution were detected by Flow Cytometry analysis.The p CMV-AKR1B1 was subsequently transfected into PANC-1 cells(relatively AKR1B1 lower expression),analyzing the protein levels of β2-AR,AKR1B1,P-ERK1/2 and ERK1/2,and counting the pancreatic cancer cells proliferation rate,cell cycle distribution and apoptosis rate.(3)Western blot analysis was performed to detect the protein expression levels of β2-AR,AKR1B1,P-ERK1/2 and ERK1/2 in pancreatic cancer cells BXPC-3 following β2-AR antagonist ICI 118,551(100 μmol/L)treatment for 24 h.As well as the cell proliferation was examined by CCK-8,the apoptosis and cell cycle distribution were detected by Flow Cytometry analysis.(4)The migration ability of pancreatic cancer cell BXPC-3 was observed following β2-AR agonist ISO(10 μmol/L)and ISO+ICI 118,551 treatment for 24 h,48 h.The competitive ELISA(Enzyme-Linked Immunosorbent Assay)kit was used to detect prostaglandin F2α(PGF2α)and the expressions of matrix matalloproteinases 2(MMP-2),matrix matalloproteinases 9(MMP-9)were detected by WB.Results:(1)β2-AR expressed in all human pancreatic cancer cell lines BXPC-3,CFPAC-1,PANC-1,especially strong expression in PANC-1,but mild expression in human normal pancreatic duct epithelial cell line h TERT-HPNE;In all cell lines(BXPC-3,CFPAC-1,PANC-1 and h TERT-HPNE),there was a stongest expression in CFPAC-1 cells and a weakest expression in PANC-1 cells.Distinct interaction of β2-AR and AKR1B1 was found in BXPC-3 cells.What’s more,we also discovered the co-localization of the two proteins in Bxpc-3 pancreatic cancer cells.(2)Compared with the control group,β2-AR and AKR1B1 overexpression observably promoted CFPAC-1 cells and PANC-1 cells proliferation and decreased the percentage of early apoptotic cells,respectively.Flow cytometric analysis showed that the proportion of CFPAC-1 and PANC-1 cells in the S phase was distinctly elevated and cell percentage in the G0/G1 phase reduced in response to β2-AR and AKR1B1 overexpression(all P <0.05).At the same time,overexpression of β2-AR led to a significant increase in AKR1B1 protein expression(P <0.05).Overexpression of AKR1B1 could feedback lightly inhibited β2-AR expression.Compared with the control group,the protein level of P-ERK1/2 was significantly increased after β2-AR overexpression and AKR1B1 overexpression,and there was no significant change in total ERK1/2 protein level.(3)Compared with the control group,there was a higher expression of β2-AR,AKR1B1,P-ERK1/2,but not ERK1/2 in pancreatic cancer cells BXPC-3 following β2-AR antagonist ICI 118,551(100 μmol/L)treatment for 24 h,also there were a lower cell proliferation rate,higer apoptosis rate and more G0/G1 phase cells in group of ICI 118,551.(4)An enhancing migration ability of pancreatic cancer cell BXPC-3,higer level of PGF2α and overexpression of MMP-2 and MMP-9 was observed after pancreatic cancer BXPC-3 cells were stimulated by β2-AR agonist ISO.Conclusions:(1)AKR1B1 is a potential interacting ligand of β2-adrenergic receptor(β2-AR),which can interact and co-localize.β2-AR can up-regulate the expression of P-ERK1/2 by activating the AKR1B1 to promote proliferation of pancreatic cancer cells.(2)The activation of AKR1B1 mediated by β2-AR can increase the cell migration ability,and increase the synthesis of the tumor-associated multi-pathway activator PGF2 of AKR1B1 product and the expression of the pro-metastasis related molecules MMP-2 and MMP-9,thus causing the metastasis of pancreatic cancer.(3)The β2-AR/AKR1B1 axis may be associated with chronic stress-associated pancreatic cancer and can serve as a potential target for pancreatic cancer intervention. |
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