Regulation Of β2-AR/ERK1/2 Pathway On EMT,Migration And Invasion Of Gastric Cancer Cells

Gastric cancer（GC）is one of the malignant tumors that seriously threaten human life and health.According to the latest reports,its morbidity and mortality rank third in China.The research results in recent years showed that chronic stress can promote the occurrence and development of malignant tumors.When patients with malignant tumor are under chronic stress,excessive expression of catecholamine hormones will lead to increased invasiveness of tumor cells.Studies have pointed out that the key target that plays a role isβ2-adrenergic receptor（β2-AR）,and the high expression ofβ2-AR promotes the malignant progression of various tumors.Many studies have also pointed out thatβ2-AR can enhance the invasion ability of tumor cells by regulating epithelial-to-mesenchymal transition（EMT）of various malignant tumors.EMT is involved in many physiological and pathological processes of the body.The occurrence of EMT reduces the adhesion between cells,endows cells with strong interstitial cell characteristics,and then promotes the malignant progression of tumors.The signal pathway that regulates EMT is complicated,and extracellular signal-regulated kinase1/2（ERK1/2）changes into p-ERK1/2through phosphorylation,and plays a biological role by entering the nucleus from cytoplasm.Many studies have shown that ERK1/2 can mediate the EMT process of various malignant tumors,and then participate in various regulation including cell adhesion and migration.By blocking ERK1/2phosphorylation,it can effectively curb the malignant development of tumors,so ERK1/2 can mediate EMT and migration and invasion of gastric cancer cells.so far,most studies targetingβ2-AR cell levels have conducted experiments with catecholamines such as epinephrine,noradrenaline and synthetic isoproterenol,but they are all non-specific activationβ2-AR agonists.Therefore,this study will be explored with selectiveβ2-AR agonist salbutamol.The results of our previous experiments showed thatβ2-AR can accelerate the malignant progression of gastric cancer by promoting the angiogenesis of gastric cancer,but the molecular mechanism of its ability to mediate the EMT and migration of gastric cancer cells is unclear.Therefore,this study uses selectiveβ2-AR agonist salbutamol and selectiveβ2-AR antagonists to explore whetherβ2-AR regulate the migration and invasion of gastric cancer MGC-803 cells,using ERK1/2 phosphorylation inhibitors PD98059,explore whetherβ2-AR regulate the EMT,migration,and invasion ability of gastric cancer MGC-803 cells via ERK1/2.Part 1Effects of β2-AR on migration and invasion of gastric cancer cellsObjective:To investigate the effects of salbutamol,a selectiveβ2-AR agonist,and ICI,aβ2-AR specific blocker,on the migration and invasion of gastric cancer cells.Methods:1 The 50%inhibitory concentration(IC₅₀)of salbutamol on gastric cancer MGC-803 cells was screened by CCK8 method.2 CCK8 assay was used to detect the proliferation of gastric cancer MGC-803 cells in the control group,salbutamol group,ICI group and salbutamol+ICI group for 24h.3 Transwell method was used to detect the migration and invasion ability of gastric cancer MGC-803 cells in the control group,salbutamol group,ICI group and salbutamol+ICI group.Results:1 The IC50 values of salbutamol at 12h,24h,36h and 48h on gastric cancer MGC-803 cells were 133.73±18.89μmol/L,70.38±11.35μmol/L,44.00±2.20μmol/L and 31.77±2.19μmol/L,respectively.2 There was no statistical significance in the survival rate of gastric cancer MGC-803 cells among the control group,salbutamol group,ICI group and salbutamol+ICI group（P>0.05）.3 Compared with the control group,the migration and invasion ability of salbutamol group was significantly enhanced（P<0.05）,while that of ICI group was significantly decreased（P<0.05）.Compared with salbutamol group,the ability of migration and invasion in salbutamol+ICI group decreased significantly（P<0.05）.Compared with ICI group,the migration and invasion ability of salbutamol+ICI group was significantly enhanced（P<0.05）.Conclusion:The selectiveβ2-AR agonist albuterol can significantly enhance the migration and invasion of MGC-803 cells.β2-AR antagonist ICI118,551could significantly reduce the migration and invasion ability of MGC-803cells.In summary,β2-AR can regulate the migration and invasion of gastric cancer cells.Part 2β2-AR/ERK1/2 pathway regulates EMT,migration and invasion of gastric cancer cellsObjective:The ERK1/2 phosphorylation inhibitor PD98059 was combined with theβ2-AR agonist albuterol andβ2-AR antagonist ICI to explore the molecular mechanism ofβ2-AR in regulating EMT and migration and invasion ability of gastric cancer MGC-803 cells.Methods:1 Western Blot1.1Expression ofβ2-AR,ERK1/2,P-ERK1/2,P-ERK/ERK,E-Cadherin,N-Cadherin and snail in the control group,salbutamol group,ICI group and salbutamol+ICI group.1.2Expression ofβ2-AR,ERK1/2,P-ERK1/2,P-ERK/ERK,E-Cadherin,N-Cadherin and snail in gastric cancer MGC-803 cells in control group,salbutamol group,PD group and salbutamol+PD group.2 qRT-PCR2.1 Relative mRNA expression levels ofβ2-AR,ERK1/2,CDH1,CDH2 and snail in MGC-803 cells of control group,salbutamol group,ICI group and salbutamol+ICI group.2.2 Relative mRNA expression levels ofβ2-AR,ERK1/2,CDH1,CDH2 and snail in gastric cancer MGC-803 cells of control group,salbutamol group,PD group and salbutamol+PD group.Results:1 The reults of Western Blot1.1Expressions ofβ2-AR,ERK1/2,P-ERK1/2,p-ERK/ERK,E-Cadherin,N-Cadherin and snail proteins in MGC-803 cells of control group,salbutamol group,ICI group and salbutamol+ICI groupThere was no significant difference in ERK1/2 among each group（P>0.05）.Compared with the control group,the levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol group were significantly increased（P<0.05）,while the level of E-Cadherin was significantly reduced（P<0.05）.The levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in ICI group were significantly reduced（P<0.05）,while the level of E-Cadherin was significantly increased（P<0.05）.The levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol+ICI group were significantly reduced（P<0.05）,while the level of E-Cadherin was significantly increased（P<0.05）.Compared with salbutamol group,the levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol+ICI group were significantly decreased,while the level of E-Cadherin was significantly increased（P<0.05）.Compared with ICI group,the levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol+ICI group were significantly increased（P<0.05）,and the level of E-Cadherin was significantly reduced（P<0.05）.1.2Expressions ofβ2-AR,ERK1/2,P-ERK1/2,p-ERK/ERK,E-Cadherin,N-Cadherin and snail proteins in MGC-803 cells of control group,salbutamol group,PD group and salbutamol+PD groupThere was no significant difference in ERK1/2 among each group（P>0.05）.Compared with the control group,the levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol group were significantly increased（P<0.05）,and the level of E-Cadherin was significantly reduced（P<0.05）.The level ofβ2-AR in PD group was not significantly different（P>0.05）,the level of p-ERK1/2,p-ERK/ERK,N-Cadherin and snail were significantly reduced（P<0.05）,and the level of E-Cadherin was significantly increased（P<0.05）.The level ofβ2-AR in salbutamol+PD group was significantly increased（P<0.05）,the level of p-ERK1/2 and snail were significantly reduced（P<0.05）,and the level of E-Cadherin and N-Cadherin protein were not significantly different（P>0.05）.Compared with salbutamol group,the levels of p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol+PD group were significantly reduced（P<0.05）,the level ofβ2-AR had no significant difference（P>0.05）,and the level of E-Cadherin was significantly increased（P<0.05）.Compared with PD group,the levels ofβ2-AR,p-ERK1/2,p-ERK/ERK,N-Cadherin and snail in salbutamol+PD group were significantly increased（P<0.05）,while the level of E-Cadherin was significantly reduced（P<0.05）.2 The reults of qRT-PCR2.1 Expressions ofβ2-AR,ERK1/2,CDH1,CDH2 and snail mRNA in MGC-803 cells of control group,salbutamol group,ICI group and salbutamol+ICI groupCompared with the control group,the levels ofβ2-AR,ERK1/2,CDH2and snail in salbutamol group were significantly increased（P<0.05）,and the level of CDH1 was significantly reduced（P<0.05）.The levels ofβ2-AR,ERK1/2,CDH2 and snail in ICI group were significantly reduced（P<0.05）,and the level of CDH1 were significantly increased（P<0.05）.The levels of β2-AR,ERK1/2,CDH2 and snail in salbutamol+ICI group were significantly reduced（P<0.05）,and the level of CDH1 were significantly increased（P<0.05）.Compared with salbutamol group,the levels ofβ2-AR,ERK1/2,CDH2 and snail1 in salbutamol+ICI group were significantly reduced（P<0.05）,and the level of CDH1 was significantly increased（P<0.05）.Compared with ICI group,the levels ofβ2-AR,ERK1/2,CDH2and snail in salbutamol+ICI group were significantly increased（P<0.05）,and the level of CDH1 was significantly reduced（P<0.05）.2.2 Expression levels ofβ2-AR,ERK1/2,CDH1,CDH2 and snail mRNA in MGC-803 cells of control group,salbutamol group,salbutamol+PD group and PD groupCompared with the control group,the levels ofβ2-AR,ERK1/2,CDH2and snail in salbutamol group were significantly increased（P<0.05）,and the level of CDH1 was significantly reduced（P<0.05）.The levels ofβ2-AR and ERK1/2 in PD group were not significantly different（P>0.05）,the level of CDH1 was significantly increased（P<0.05）,and the levels of CDH2 and snail were significantly reduced（P<0.05）.The level of ERK1/2 in salbutamol+PD group was significantly reduced（P<0.05）,the levels ofβ2-AR and snail were significantly increased（P<0.05）,and the levels of CDH1 and CDH2 were not significantly different.Compared with salbutamol group,the level ofβ2-AR in salbutamol+PD group was not significantly different（P>0.05）,the levels of ERK1/2,CDH2 and snail were significantly reduced（P<0.05）,and the level of CDH1 was significantly increased（P<0.05）.Compared with PD group,the levels ofβ2-AR,ERK1/2,CDH2 and snail in salbutamol+PD group were significantly increased（P<0.05）,and the level of CDH1 was significantly reduced（P<0.05）.3 Transwell results:Compared with the control group,the migration and invasion ability of salbutamol group was significantly enhanced（P<0.05）,while that of PD group was significantly decreased（P<0.05）.Compared with salbutamol group,the ability of migration and invasion in salbutamol+PD group decreased significantly（P<0.05）.Compared with PD group,the migration and invasion ability of salbutamol+PD group was significantly enhanced（P<0.05）.Conclusion:β2-AR/ERK1/2 pathway can regulate EMT,migration and invasion of gastric cancer cells.