| Objective Acid sphingomyelinase(ASMase)is a key enzyme in the sphingomyelin/ceramide(Cer)signaling channel,which plays an important role in the process of Cer production by cells.Ceramide,a bioactive sphingolipid,is now at the forefront of cancer research,is highly enriched in endothelial cells,and can respond to various cellular stresses.Classically,ceramide plays a vital role in many physiological and pathological processes,which is thought to induce death,growth inhibition,and senescence in cancer cells.Lipopolysaccharide(the endotoxin of Gram-negative bacteria,composed of phospholipid-polysaccharide-protein complex,LPS)can induce apoptosis in lung cancer cells when given a certain stimulation of concentration and dose,but the molecular mechanisms involved are not clear.Herein,we studied the role of ASMase and the role of ASMase/Cer pathway in LPS-induced apoptosis of human lung adenocarcinoma cells(pc9,A549),providing new clinical treatment strategies for the treatment of lung cancer and even various tumors.Methods 1.the pc9 and A549 cells were treated with different concentrations of LPS(200,150,100,50,10 and 0?g/ml)for 24,36 and 48 hours.Appropriate concentrations and times of induction of apoptosis were selected through the CCK-8 kit.2.Cells were treated with IM at different concentrations.Cell viability were measured by the CCK-8 method.Selecting the largest concentration of IM concentration but meaningless to harm the cell itself.3.Cells were grouped and treated with 0,150 ?g/m L LPS,20 ?mol/L IM + 150 ?g/m L LPS,and 20 ?mol/L IM for 36 hours.Hoechst apoptosis staining was performed to observe cell shrinkage and nuclear Shattere.4.After exposing two cells to 0,150 ?g/m L LPS,20 ?mol/L IM + 150 ?g/m L LPS for 36 hours,RT-PCR and WB technology were used Detection of ASK1,Caspase3,ASMase and cl-Caspase3.5.the expression levels of Cer were analyzed using immunofluorescence technology after treatment of cells with 0,150 ?g/m L LPS,20 ?mol/L IM + 150 ?g/m L LPS for 36 hours.6.the exposure different time and concentrations of Cer(80,60,40,10 and 0 ?mol/L)for 12,24 and 36 hours for two cells.The CCK-8 kit observed the change of OD.Hoechst staining observed the change of cell apoptosis with different concentrations.7.the cells were treated with Cer concentrations of 0 and 60 ?mol/L,respectively,and the changes of ASK1,Caspase3,and cl-Caspase3 m RNA and proteins were detected by RT-PCR and WB.Results 1.With the increase of LPS concentration and exposure time,the pc9 and A549 survival rate gradually decreased,compared with the control group,the cells viability was significantly decreased.The survival rate was about 70%,so the treatment conditions of 150 ?g/m L and 36 hours were selected;the results showed that the IM concentration ranged from 0 to 20 ?mol/L had no obvious toxicity to the cells at 36 hours,so the treatment dose of 20 ?mol/L was selected.2.Compared with the control group,the expressions of the m RNA and proteins ASK1,ASMase,Caspase3 and cl-Caspase3 in the LPS-treated group were significantly increased(P <0.01),and there were no significant change in the IM group(P > 0.05);Compared with the LPS-treated group,the expression level of inhibitor IM pretreatment group was significantly inhibited(P <0.01);the results of apoptosis staining showed the same change.3.It can be seen from the immunofluorescence staining that the expression of Cer in the LPS group was significantly increased compared with the control group(P <0.01),but there was no significant change in the IM group;while the expression of Cer was significantly inhibited in the inhibitor with LPS group compared the LPS group alone(P <0.01).4.From the results of CCK-8 and fluorescence microscope,with the increase of time and concentration in the Cer group,the cells survival rate decreased significantly,and the number of apoptotic cells increased significantly.5.Compared with the control group,the expression levels of ASK1,Caspase3 and cl-Caspase3 in the Cer group were evidently agumented(P <0.05).Conclusions From the above results,we found out that LPS induces apoptosis of human lung adenocarcinoma cells pc9 and A549,and the expression of apoptosis-related proteins,ASMase and Cer are increase,which indicates that the participation of ASMase and Cer must be involved.The underlying mechanism needs further investigation,but,Overall,these results at least indicate that the ASMase/Cer pathway plays an important role in the apoptosis of human lung adenocarcinoma cells pc9 and A549 induced by LPS. |
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