Ceramide Induces The Apoptosis Of Non-small Cell Lung Cancer Cells Through The Txnip/Trx1 Complex

Background:Ceramide is a biologically active sphingolipid substance that can participate in the composition of biological cell membranes,especially in endothelial cells.It can respond to a variety of internal and external stresses such as ultraviolet rays,radiation,bacteria and various drugs,and plays an important role in the process of cell growth,proliferation,differentiation,aging and apoptosis.Ceramide is still a frontier topic in cancer research.In previous studies,we confirmed that LPS promotes lung cancer cell apoptosis by inducing the acid sphingomyelase/ceramide pathway,but the specific pathway of ceramide-induced cancer apoptosis is not clear.The TXNIP plays an important role in the redox reaction signal transmission inside and outside the cell.Therefore,we studied the role of Txnip/Trx1 complex in ceramide-induced apoptosis of human lung adenocarcinoma cells A549 and PC9,and provided new clinical ideas for future lung cancer treatment options.Methods:1.Treat A549 and PC9 cells with different concentrations of C2-ceramide(0,20,50,100 and 200 μmol/L)for 12,24 and 36 hours,and select the appropriate time and concentration to induce apoptosis through CCK-8 kits to detect cell viability.2.Use different concentrations of verapamil(VER,a thioredoxin-interacting protein inhibitor)to treat the cells,the CCK-8 kit was used to detect cell viability to screen the appropriate VER concentration.3.Group the cells,treat A549 and PC9 cells with 0,50 μmol/L C2-ceramide,100μmol/L VER + 50 μmol/L C2-ceramide,100 μmol/L VER for 24 hours,and then use Hoechst Apoptosis Staining Kit detect cell apoptosis.4.Treat the two cells seeded in a 6-well plate under the conditions of 0,50 μmol/L C2-ceramide,100 μmol/L VER + 50 μmol/L C2-ceramide,and 100 μmol/L VER for 24 hours,using fluorescence after incubation for 15 minutes in the dark FITC-labeled Annexin V-FITC,it was stained with propidium iodide(PI)for 5 minutes under dark conditions,and then the apoptosis rate was detected by flow cytometry.5.After exposing two cells to 0,50 μmol/L C2-ceramide,100 μmol/L VER + 50μmol/L C2-ceramide,100 μmol/L VER for 24 hours.Reverse Transcription Polymerase Chain Reaction(RT-PCR)technology was used to detect thioredoxin interacting protein,thioredoxin-1(TRX1)and cysteine aspartate protein(Caspase-3)gene expression,using Western blotting(WB)to detect TXNIP,Caspase-3,TRX1 and cl-Caspase-3 protein expression.6.After treating the cells with 0,50 μmol/L C2-ceramide,100 μmol/L VER + 50μmol/L C2-ceramide,and 100 μmol/L VER for 24 hours,the expression levels of TXNIP and TRX1 were analyzed by immunofluorescence technology.7.Two kinds of cells were treated with 0,50 μmol/L C2-ceramide,100 μmol/L VER + 50 μmol/L C2-ceramide and 100 μmol/L VER,and Caspase-3 activity detection kit was used to detect Caspase-3 activity.Results:1.With the gradual increase of the ceramide concentration and duration,the survival rate in the two kinds of cells is gradually reduced.Compared with the control group,it can be seen that the ceramide concentration with a cell viability of about 70%is 50 μmol/L,so we choose 50 μmol/L ceramide concentration treatment for 24 hours is the best experimental condition;for the treatment concentration of verapamil,we choose a concentration of 100 μmol/L for subsequent experiments.2.In terms of molecular and protein levels,compared with the control group,the expression levels of TXNIP,P38,Caspase-3 and cl-Caspase-3 in the ceramide treatment group increased(P<0.05).Compared with the ceramide group,the expression level of the ceramide and verapamil co-incubation group was significantly suppressed(P<0.05);while the protein TRX1 showed the opposite result.3.Compared with the control group,the results of Hoechst 33258 staining showed that in the two types of cells,the apoptosis rate of the ceramide group was significantly increased,and the verapamil group had no significant change,while the co-incubation with verapamil could significantly save the cells(P<0.05).Compared with the control group,the academic results of flow cytometry also appeared similar phenomena(P<0.05).4.Immunofluorescence staining showed that the fluorescence intensity of Txnip expression in the ceramide group was significantly higher than that in the control group(P<0.05);and the expression of Txnip was inhibited in the VER inhibitor and ceramide co-incubation group compared with the ceramide group(P<0.05),Trx1 has the opposite result,and it can be seen that Txnip and Trx1 may change position.5.From the results of the Caspase-3 activity test,compared with the control group,the ceramide group Caspase-3 activity increased(P<0.05),but there was no significant change in the VER group;while the Cer + VER group compared with the ceramide group,Caspase-3 activity is reduced(P<0.05).Conclusions:After ceramide induces apoptosis of lung adenocarcinoma cells A549 and PC9,it must involve the participation of Txnip and Trx1.These results indicated that ceramide induced apoptosis of lung adenocarcinoma A549 and PC9 by Txnip/Trx1 complex.