Role And Mechanism Of Inhibition Of MTOR Signaling Pathway In ALA-PDT Killing A431 Cell

Background and Objective:Cutaneous squamous cell carcinoma is the second non-melanoma.It has the characteristics of invasive and destructiveness.The disease occurs mainly in the old people’s exposed parts.It is related to the ultraviolet radiation,exposure to chemical factors andvirus infection.The traditional treatments is surgery.but the photodynamic therapy is the best way to cure the people which the body resistance is poor or the location of skin lesion is special.Aminolevulinic acid photodynamic therapy(ALA-PDT)is a new way to cure cutaneous squamous cell carcinoma.It has the advantages of leaving smaller trauma,scar and letting people beautiful.But it also has disadvantages.The depth of its treatment is limited.It is low effect to cure the invasive squamous cell carcinoma.Mammalian target of rapamycin(m TOR)is a serine / threonine protein.It has a important effect to regulate cell growth and proliferation.The abnormal activation of m TOR is related with many malignant tumors.Rapamycin is a macrolide immunosuppressant which extract by streptomyces fermentation broth.Rapamycin is the inhibitor of m TOR.It can kill malignant tumors.m TOR is high expression in squamous cell carcinoma.Experiments were performed to study the A431 cell proliferation and apoptosis of combining ALA-PDT and rapamycin in skin squamous cell cancer cell.To explore the role and mechanism of m TOR in ALA-PDT damaging skin squamous cell carcinoma A431 cells,to provide new laboratory basis for ALA-PDT treated with patients with squamous cell carcinoma of the skin.Methods:A431 cells were divided into blank control group,laser group,ALA group,ALA-PDT group,inhibitor of m TOR(rapamycin)group and rapamycin combined with ALA-PDT group(combination group).CCK8 was used to detect the optical density value and calculating the cell survival rate of each group were obtained at 12 h,24h,48 h.Flow cytometry with annexin V-FITC/PI double staining was employed to detect apoptosis of eachgroup cell after intervening 12 h,24h and 48 h.Western blotting test was used to detect the expression of protein products m TOR and phosphorylation protein products of m TOR and 70 k Da ribosomal protein S6 kinase(p-p70S6K)after intervening 48 h in each of A431 cells.Apply the statistical software of SPSS20.0 for analysis.All data are presented as mean ± SD(?x ±s).Multiple comparisons were performed with one-way ANOVA,two planned primary comparisons were performed with LSD t.Data were analyzed using repeated measurements ANOVA.A value of P<0.05 was considered to denote statistical significance.Results:CCK8 detection results show that the cell survival rate had no obvious difference in blank control group,ALA group and laser group,but it had difference in ALA-PDT group that compared to blank control group,ALA group and laser group,it also had difference in rapamycin group that compared to blank control group.It was obviously lower in combintation group than the rest of the group.Flow cytometry instrument testing results showed that the rate of apoptosis had no difference in blank control group,ALA group and laser group,but it had difference in ALA-PDT group that compared to black control group,ALA group and laser group,it was significantly higher in combintation group than the other groups.By western-blot testing,the expression of m TOR,p-m TOR and p-p70S6 K had no obvious difference in blank control group,ALA group and light group,but the expression of m TOR,p-m TOR and p-p70S6 K was obviously lower in ALA-PDT group than the blank control group,ALA group and laser group,the expression of m TOR,p-m TOR and p-p70S6 K was obviously lower in rapamycin group than the blank control group.the expression of m TOR,p-m TOR and p-p70S6 K were evidently lower in combination group than the other group.Conclusion:1.ALA-PDT significantly inhibit the proliferation and promote the apoptosis of A431 cells in vitro experiments.2.The inhibitor of m TOR—rapamycin significantly inhibit the proliferation of A431 cells in vitro experiments.3.ALA-PDT combine with rapamycin has stronger lethality than ALA-PDT of A431 cells in vitro experiments.Blocking m TOR pathway can reduce the expression of m TOR,p-m TOR,p-p70S6 K and enhance the cell damage effect of ALA-PDT to A431 cells.