The In Vitro Study Of Autophagy Of Prostate Cancer Cell Line PC-3 Under The Induction Of Salinomycin

Objective:This thesis is to study the autophagy and possible mechanism of Prostate cancer cell line PC-3 under the induction of salinomycin,providing experimental basis for the treatment of prostate cancer in the clinical application of salinomycin.Materials and methods:(1)The Salinomycin of different concentrations were treated with Prostate cancer cells line PC-3 which are in the logarithmic phase.The autophagosome and other ultrastructure were observedbyemployingtransmissionelectron microscope.Immunofluorescence was used to detected the expression of microtubule-associated protein 1 light chain 3 II.The expressions of microtubule-associated protein 1 light chain 3 II,sequestosome1/SQSTM1,Beclin-1,autophagyrelated gene 3 and autophagyrelated gene 9 were be detected by employing Western Blot method.(2)The Salinomycin were treated with Prostate cancer cells line PC-3 for different time.The expressions of microtubule-associated protein 1 light chain 3II,sequestosome 1/SQSTM1,Beclin-1,autophagyrelated gene 3and autophagyrelated gene 9 were be detected by employing Western Blotmethod.(3)ThepcDNA3.1green fluorescent protein-microtubule-associated protein 1 light chain 3 plasmid were introduced into Prostate cancer cells line PC-3 by applying transfection technology.After that,he Salinomycin of different concentrations were treated with it.In the end,the expression of GFP-LC3fusion protein were observed by fluorescence microscopy.(4)Statistical method:The experimental data are expressed with x~—±s and use graphPad prism and SPSS19.0 statistical analysis software to analyze.The comparison of the experimental group and control group will employ T test,and more than two groups of data comparison use analysis of variance.Result:(1)The study observed autophagosome by employing transmission electron microscope.The blank control group did not appear autophagosome and autophagie vacuole,1,2?mol/L salinomycin treatment groups appeared a large number of autophagosome and autophagie vacuole,and 2?mol/L salinomycin treatment group appeared more autophagosome than 1?mol/L group.(2)The study observed the expression of microtubule-associated protein 1 light chain 3 II by employing immunofluorescence.Microtubule-associated protein 1 light chain 3 II,the marker protein of autophagy,which is marked with red fluorescent expressed relatively weakly in blank control group.Compared with blank control group,the expression of microtubule-associated protein 1 light chain 3 II is gradually stronger in 2,5?mol/L treatment group,and fluorescence intensity is strongest in 5?mol/L treatment group.(3)The study detected the expression of cell autophagy related protein by employing Western Blot method.Concentration group:After Prostate cancer cells line PC-3 are respectively dealt with by 0.25,0.5,1,2 and5?mol/Lsalinomycinfor24hours,theexpressionof microtubule-associatedprotein1lightchain3II,the marker protein of autophagy,significantly enhanced,the expression of sequestosome 1/SQSTM1 weakened,and the expressions of Beclin-1 and autophagyrelated gene 3 also enhanced.Compared with control group,concentration group existed statistical differences(p<0.05)and the comparative difference among various treatment groups possess statistical significance(p<0.05).However,the expression of autophagyrelated gene 9 in various treatment groups enhanced when the concentration of salinomycin increased.Compared with blank control group,those groups shown that the statistical differences existed(p<0.05),but those expressions change irregularly.Time group:After Prostate cancer cell line PC-3 are respectively dealt with by 2?mol/L salinomycin for 4,6,8,12 and 24 hours,the expression of microtubule-associated protein 1 light chain 3?,the marker protein of autophagy,significantly enhanced,the expression of sequestosome 1/SQSTM1 weakened,and the expressions of Beclin-1 and autophagyrelated gene 3 also enhanced.Compared with control group,this group existed statistical differences(p<0.05)and the comparative difference among various treatment groups possess statistical significance(p<0.05).However,compared with blank control group,the expression of autophagyrelated gene 9 in various treatment groups enhanced when the time of exposure to salinomycin increased,which has statistical differences but the expressions in various treatment groups change irregularly.(4)The study observed the green fluorescent protein-microtubule-associated protein 1 light chain 3fusion protein's expression by employing fluorescence microscope.The green fluorescent protein-microtubule-associated protein 1 light chain 3fusion protein of blank control group dispersed in the cytoplasm and scarcely formed fluorescence spots which marks autophagosome.In this group,the average spot of individual cells in this horizon had six spots.The result shows that autophagosome is extremely rare.Fluorescence spots could be observed in 1?mol/L,2?mol/L and 5?mol/L treatment groups,and the average spot of individual cells in their horizons in those groups respectively has 28,54 and 86 spots.The experimental result manifested the statistical differences(p<0.05)existed between the experimental group and control group,also in experimental group and experimental group.The results indicates that the fluorescence spots under inverted fluorescence microscope gradually increased when the drug concentration of salinomycin becomes stronger within a certain range.Among those experimental groups,the number of fluorescence spots in 5?mol/L treatment group is the most.Conclusion:(1)The Salinomycin could induce autophagy of prostate cancer cell line PC-3 and the action intensity of autophagy is positively correlated to dosage and time of salinomycin.(2)The autophagy mechanism of prostate cancer cell line PC-3 induced by salinomycin probably is related with the expressions of up-regulation autophage gene Beclin-1and autophagyrelated gene 3.