Effects And Mechanisms Of Simvastatin On Apoptosis Of Lung Adenocarcinoma A549 Cells

BackgroundLung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths.In recent years,the advent of targeted therapy and immunotherapy has greatly changed the prognosis of the patients with non-small cell lung cancer,but the 5-year survival rate is still low,and the treatment of lung cancer is still facing with great challenges.Finding new treatment strategies and drugs is essential for effective treatment of non-small cell lung cancer.Apoptosis is a type of programmed death,and dysregulation of this process can lead to uncontrolled cell proliferation,the occurrence and progression of cancer,and resistance to drug treatment.Studying the mechanism of apoptosis signal transduction,key proteins involved in apoptosis,and the mechanism by which cancer cells evade apoptosis can provide new ideas for the treatment of lung cancer.Statins have been shown to exert anti-tumor effects by inhibiting proliferation,inducing apoptosis and other mechanisms,and are promising drugs for the treatment of cancer.The JAK2/STAT3 pathway is one of the important signal pathways in the body.Studies have found that the JAK2/STAT3 pathway can be constitutively activated in various tumor cells and participate in physiological processes such as cell proliferation,invasion,metastasis,apoptosis,and angiogenesis.Therefore,inhibition of the JAK2/STAT3 signaling pathway is an attractive anti-cancer strategy.Previous studies have found that simvastatin can induce apoptosis of lung cancer cells,but the mechanism and related pathways are still unclear.ObjectiveTo explore the effect and mechanism of simvastatin on apoptosis of lung adenocarcinoma A549 cells.Methods1.After treating A549 cells with different concentrations of simvastatin for 24h and 48h,the effect of simvastatin on the survival rate of A549 cells was measured by CCK-8 method,and the concentration of simvastatin in the experiment was set according to the results;2.The effect of simvastatin on A549 cell cycle was detected by flow cytometry;3.The effect of simvastatin on mitochondrial membrane potential was detected by JC-1 fluorescent probe;4.The effect of simvastatin on apoptosis of A549 cells was detected by flow cytometry Annexin V-FITC/PI double staining method;5.The effects of different concentrations of simvastatin on Bax and Bcl-2 protein expression was detected by Western blot;6.The expression of Bax,Bcl-2 and Cytochrome C protein in mitochondria was detected by Western Blot method to further verify the role of mitochondrial pathway in the process of simvastatin-induced apoptosis;7.The CCK-8 method was used to detect the effect of caspase inhibitor on the survival rate of A549 cells;TUNEL method was used to detect the effect of caspase inhibitors on the apoptosis of A549 cells induced by simvastatin;8.After interfering with the expression of apoptosis inducing factor(AIF),Western Blot method was used to detect the expression level of AIF protein in mitochondria and cytoplasm;9.Immunofluorescence method was used to detect the nucleus transfer of AIF to explore the role of AIF in the process of simvastatin-induced apoptosis;10.The String database was used to analyze protein-protein interaction(PPI)of related proteins;11.In order to investigate the role of JAK2/STAT3 pathways in simvastatin-induced apoptosis,after treatment of A549 cells with different concentrations of simvastatin,Western Blot was used to detect the expression levels of JAK2/STAT3 pathway-related proteins such as p-JAK2,JAK2,p-STAT3,and STAT3;The cells were pretreated with JAK2/STAT3 pathway activator IL-6,and then treated with simvastatin.Western Blot was used to detect the expression levels of JAK2/STAT3.pathway related proteins to further explore the role of the JAK2/STAT3 pathway in simvastatin-induced apoptosis.Results1.Under the same action time,with the increase of simvastatin concentration,the cell survival rate gradually decreased,and under the same effect concentration,the cell survival rate of simvastatin after 48h was lower than that of the 24h group.It was shown that within a certain concentration and time range,the inhibitory effect of simvastatin on the growth of A549 cells was concentration-and time-dependent.The cell survival rate of the 20,40 and 80μg/ml simvastatin-treated group was lower than that of the control group(P<0.05);2.The results of cell cycle test showed that with the increase of simvastatin concentration,the proportion of cells in G0/G1 phase increased,and the proportion of cells in G2/M phase decreased.Compared with the control group,the simvastatin group at 20μg/ml and 40μg/ml had statistical differences(P<0.01);3.The results of mitochondrial membrane potential test showed that compared with the control group,with the increase of simvastatin concentration,mitochondrial membrane potential decreased(P<0.01);4.The results of flow cytometry showed that with the increase of the concentration of simvastatin,the apoptosis rate of the cells increased.Compared with the control group,the apoptosis rate of the 20μg/ml and 40μg/ml simvastatin-treated group had statistics difference(P<0.01);5.After treatment with simvastatin at different concentrations,as the concentration increased,the expression of apoptotic protein Bax increased,and the expression of apoptosis inhibitory protein Bcl-2 decreased.Compared with the control group,the expression of Bax protein was increased in the 10,20,and 40μg/ml simvastatin treatment group(P<0.05),and the expression of Bcl-2 protein was decreased in the 20,40 and 40μg/ml simvastatin group(P<0.01);6.Western Blot results showed that compared with the control group,the expression of apoptosis-inducing protein Bax in the mitochondria increased in the simvastatin-treated group(P<0.05),and the expression of Cytochrome C and the apoptosis-inhibiting protein Bcl-2 was decreased(P<0.05);7.CCK-8 results showed that,compared with the control group,the cell survival rate of the simvastatin-treated group,the simvastatin combined with the caspase inhibitor group decreased,but the difference between the two groups was not statistically significant(P>0.05);TUNEL results showed that compared with the control group,the number of fluorescently stained cells in the simvastatin-treated group was significantly increased;the co-treatment cells with caspase inhibitor and simvastatin,the number of fluorescently stained cells did not decrease;8.Western Blot results showed that compared with the control group,the expression of AIF protein in mitochondria of each group was reduced(P<0.01).In the cytoplasm,the expression of AIF protein was increased in the simvastatin-treated group,while the expression of AIF protein in the AIF inhibitor or AIF-siRNA combined with simvastatin groups was lower than that in the simvastatin-treated group(P<0.01);9.The immunofluorescence method was used to further explore the situation of AIF entering the nucleus.The results showed that the AIF positive fluorescence of control group A549 cells mainly concentrated in the cytoplasm and did not overlap with DAPI significantly.While in the simvastatin-treated group,the nuclear AIF fluorescence and DAPI overlap,and there is a tendency to enter the nucleus;10.The PPI network diagram suggests the interaction between AIF,Bcl-2,Bax,STAT3,and JAK2 proteins;11.After treating A549 cells with simvastatin,the levels of phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)in(20,40μg/ml)simvastatin-treated group were decreased,compared with the control group,the difference was statistically significant(P<0.05).After pretreatment of cells with JAK2/STAT3 pathway activator IL-6,p-JAK2 and p-STAT3 protein levels were higher than those of the control group.As the concentration of simvastatin increases,the levels of JAK2 and STAT3 phosphorylation induced by IL-6 in the cells were inhibited,and the protein expression level was lower than that in the IL-6 treatment group(P<0.05).Conclusions1.Simvastatin can inhibit the survival of A549 cells,induce cell cycle arrest in G0/G1 phase,and can induce apoptosis of A549 cells.2.Simvastatin can activate caspase-independent,AIF-mediated mitochondrial apoptosis pathway in A549 cells,and the mechanism of its function is related to the inhibition of JAK2/STAT3 signaling pathway.