| Objective: Esophageal cancer is the eighth most common tumor in the world.Mortality ranks fourth among malignant tumor-related deaths in China and ranks sixth among all tumors in the world.Surgery is the preferred method for the treatment of resectable esophageal cancer.But the local recurrence rate is as high as 40% to60%.Nearly 50% of patients with esophageal cancer have distant metastases when they are diagnosed which lost the chance of surgical resection.These patients can only rely on chemotherapy and radiation therapy.Cisplatin is used as a first-line drugs for cancer chemotherapy which have a significant therapeutic effects on esophageal cancer.The mechanism by which cisplatin kills tumor cells as cytotoxic drugs is mainly to damage and inhibit DNA synthesis.During this process,multiple signaling pathways(mainly apoptotic pathways)are activated to avoid or promote cell death.In recent years,a large number of studies have shown that autophagy,as another form of cell death,can act alone or in combination with apoptosis to participate in the effects of chemotherapy drugs on tumor cells.The purpose of this experiment was to verify whether cisplatin induces autophagy in esophageal cancer EC109 cells and further explore the role of autophagy in cisplatin-induced esophageal cancer cell injury and the specific mechanism of its occurrence.Methods:(1)The EC109 cell line of human esophageal squamous cell carcinoma was cultured in vitro,and the different concentration of cisplatin was used to treat EC109 cells at different time points.The concentration gradient was 1uM,2.5uM,5uM,7.5uM,10 uM,each concentration was set up five time points of 0h,12 h,24h,48 h,72h.Cell viability was measured by using the CCK-8 method,the IC50 dose was calculated and the concentration of the next step of cisplatin intervention and the time of action were determined(2)Selection and grouping of cisplatin intervention concentration and time points: According to CCK-8 test results,5umol/L cisplatin was selected to establish a cisplatin treatment model at different time points Normal control group(NC),cisplatin treated group 24 h,cisplatin treated 48 h group,cisplatin treated 72 h group were set up.A 72-hour model with different concentrations of cisplatin was established.The normal control group(NC),cisplatin concentration1 uM group,cisplatin concentration 2.5uM group,and cisplatin concentration 5uM group were set up.Detection index: the expression level of autophagy marker protein LC3-II and Beclin1 in EC109 cells was detected by Western blotting.The expression level of autophagy substrate protein p62 and apoptosis related protein cleaved caspase-3 and XIAP in EC109 cells of each group were detected by Western blotting.Immunofluorescence was used to detect the expression of autophagic marker protein LC3 in EC109 cells.The apoptosis rate of EC109 cells in each group was detected by TUNEL.(3)Drug intervention and grouping: The autophagy inhibitor chloroquine was used to intervene EC109 cells and normal control group,chloroquine treatment group,cisplatin treatment group and cisplatin + chloroquine intervention group were set up.The most obvious treatment time and concentration of cisplatin in autophagy and apoptosis expression of EC109 cells were selected,namely 5uM cisplatin and 72 h.Chloroquine pretreatment method: chloroquine diluent was pretreated for 2 hours before cisplatin treatment.Detection index: Western blot technology was used to detect the expression of autophagy marker protein LC3-II,apoptosis related protein cleaved caspase-3 and XIAP protein in EC109 cells of each group.The apoptosis rate of EC109 cells in each group was detected by TUNEL.(4)SiRNA transfection and grouping: The expression of Atg5 was administrated by SiRNA,which was set as normal control group,SiRNA Atg5 intervention group,cisplatin treatment group,Cisplatin+ SiRNA Atg5 intervention group.The most obvious treatment time and concentration of cisplatin in autophagy and apoptosis expression of EC109 cells were selected,namely 5uM cisplatin and72 h.SiRNA Atg5 pretreatment method: SiRNA Atg5 was transfected for 6 h before cisplatin treatment.Detection index: The expression of Atg5,LC3-II,cleaved-caspase-3,and XIAP protein in each group of EC109 cells was detected by Western blotting.The apoptosis rate of EC109 cells in each group was detected by TUNEL.Results:(1)CCK-8 results showed that EC109 cells were sensitive to the effect of cisplatin,and the inhibitory effect of cisplatin on proliferation of EC109 cells was a dose-concentration effect.With the increase of time and concentration,the inhibitory effect of cell proliferation was significantly increased(P<0.05).In the 5uMcisplatin group,the cell proliferation inhibition rate was less than 50% at all 12 h,24h,48 h,and 72 h.(2)Detection of autophagy in EC109 cells treated with cisplatin at different time points.Western blotting results showed that the expression of autophagy-labeling protein LC3-II and Beclin1 increased significantly in cisplatin-treated group,and it increased with the increase of drug concentration and the prolongation of action time.The expression was highest at 5uM cisplatin treatment for 72 hours.the difference was statistically significant(P<0.05);The expression of autophagic substrate protein p62 was significantly reduced and it increased with the increase of drug concentration and the prolongation of action time.The expression was highest at 5uM cisplatin treatment for 72 hours.the difference was statistically significant(P<0.05).The results of immunofluorescence showed that with the increase of cisplatin concentration and the prolongation of action time,the LC3-II green fluorescence point of EC109 cells increased,and the expression of 5uM cisplatin treated group 72 h was the most,the difference was statistically significant(P<0.05).(3)Detection of EC109 cell apoptosis and XIAP expression at different time points after cisplatin treatment.Western blotting results showed that the expression of cleaved caspase-3 in EC109 cells increased gradually with the increase of cisplatin concentration and the prolongation of action time.The expression of cleaved caspase-3 in EC109 cells increased gradually,and the expression of cleaved caspase-3in 5uM cisplatin treatment for 72 hours was the highest.the difference was statistically significant(P<0.05);The results of Western blot showed that the expression of XIAP in EC109 cells decreased gradually with the increase of cisplatin concentration and the prolongation of action time.The expression of XIAP in EC109 cells was gradually decreased after 5uM cisplatin treatment for 72 hours,and the difference was statistically significant compared with the normal control group(P<0.05).TUNEL results showed that with the increase of cisplatin concentration and the prolongation of action time,the expression of EC109 cell apoptosis positive points increased gradually and the expression of 5uM cisplatin treated group 72 h was the most,compared with the normal control group,the difference was statistically significant(P<0.05).(4)Effects of chloroquine on autophagy and apoptosis in EC109 cells.Western blot results showed that compared with the NC group,the expressionof LC3-II in the cisplatin group was increased(P<0.05),LC3-II expression in the cisplatin+chloroquine group was significantly higher than that in the cisplatin group(P<0.05);Compared with NC group,cleaved caspase-3 expression was increased in cisplatin group,and cleaved caspase-3 expression in cisplatin + chloroquine group was significantly lower than that in cisplatin group(P<0.05).TUNEL results showed that compared with the NC group,the expression of apoptosis positive cells in the cisplatin group EC109 cells increased(P<0.05),and the apoptosis positive cells in the cisplatin + chloroquine group decreased significantly compared with the cisplatin group(P<0.05).TUNEL results showed that compared with the NC group,the expression of apoptosis positive cells in the cisplatin group EC109 cells increased(P<0.05),and the apoptosis positive cells in the cisplatin + chloroquine group decreased significantly compared with the cisplatin group(P<0.05).(5)The effect of SiRNA on autophagy and apoptosis of EC109 cells.Western blot results showed that compared with the NC group,the expression of LC3-II and cleaved caspase-3 in the cisplatin group increased(P<0.05).The expression of LC3-II and cleaved caspase-3in the cisplatin+SiRNA group was significantly decreased compared with the cisplatin group.(P<0.05).TUNEL results showed that compared with the NC group,the positive expression of apoptosis in EC109 cells of cisplatin group was increased(P<0.05),and the positive expression of apoptosis in cisplatin+SiRNA group was significantly lower than that of cisplatin group(P<0.05).(6)The effect of autophagy on the expression of XIAP.Western blotting results showed that compared with the NC group,the expression of XIAP in the cisplatin group EC109 cells was decreased(P<0.05);Compared with the cisplatin group,the XIAP expression was increased in the cisplatin+chloroquine group and the cisplatin+SiRNA group(P<0.05).Conclusion: This study confirms that(1)After treated with cisplatin,the apoptosis of EC109 cells increased,accompanied with the activation of autophagy and decreased expression of XIAP.(2)After inhibited autophagy by Chloroquine and SiRNA Atg5,Cisplatin treatment reduced the expression of apoptosis related protein cleaved Caspase3 and decreased the apoptosis rate.(3)The expression of XIAP increased after chloroquine inhibited autophagy and SiRNA Atg5 intervened autophagy.In summary,we conclude(1)Cisplatin induces autophagy activation in esophageal cancer cells.(2)Autophagy activation aggravates cisplatin-induced damage to esophageal cancer cells.(3)Autophagy may aggravate apoptosis of esophageal cancer cells by over-removing XIAP. |
