| Objective: To explore the possibility of enhancing the therapeutic effect of cisplatin(DDP),a conventional chemotherapeutic drug,in the treatment of neuroblastoma by inhibiting autophagy,and explore possible molecular mechanisms.Method:1 Human neuroblastoma cell SH-SY5 Y cultured in vitro with different concentrations(0,1.25,2.5,5,10,20,40ug/ml)of the cisplatin treatment in logarithmic growth phase SH-SY5 Y cells in 24 h,48h,72 h,and different concentrations(0,1.25,2.5,5,10mM)were treated with 3-MA in neuroblastoma SH-SY5 Y cells in logarithmic growth phase after 24 h,colorimetric determination of cell viability using MTS;Human neuroblastoma cell SHSY5 Y cultured in vitro with different concentrations(0,2.5,5,10,20ug/ml)of the cisplatin combined with autophagy inhibitor 3-MA(5mM)in logarithmic growth cells after 24 h,the colorimetric determination of cell viability by MTS,and calculated the IC50 value of cisplatin alone and combined with autophagy inhibitors.2 Observation By ordinary optical microscopy control group,conventional chemotherapy drug cisplatin(10ug/ml)group,autophagy inhibitor3-MA(5mM)group,autophagy inhibitor 3-MA(5mM)+ cisplatin(10ug/ml)chemotherapy group changes in cell morphology.3 After the treatment,the cells of the four groups were stained with PI,and the apoptosis and necrosis cells were observed under fluorescence microscope.4 Western blot method was used to detect the expression of autophagy related protein Beclin-1,LC3-II / I and apoptosis related protein Caspase-3 inthe above four groups.The experimental data calculated by statwastical softwwere13.0.Every date need to be Calculated by normality test.Enumeration data coincided with normal distribution showed by(x ąS).Enumeration data didn't coincided with normal distribution showed by(M(Q)).multiple groups of mean value which conform to normal dwastribution and equal variance were calculated by One-way ANOVA.Multiple comparison between groups were calculated by Student-Newman-Keuls test;non normal distribution data were analyzed by Kruskal-wallis H test,Mann-Whitney U test was used to comparison between any two means.Taking ? =0.05 as the test standard,P<0.05 as the criterion for distinguishing the difference.Result:1 The effect of cisplatin on human neuroblastoma cell line SH-SY5 Y was less than that of other groups after treated for 24 hours.The survival rate of SH-SY5 Y was decreased only at the concentration 10ug/ml of cisplatin(P<0.05).Cisplatin treatment of human neuroblastoma SH-SY5 Y cells after48 hours,when the cisplatin concentration reached 5ug/ml,decreased cell viability(P<0.01),when the cisplatin concentration reached 10ug/ml,cell viability decreased significantly(P<0.001).72 hours after cisplatin treatment of human neuroblastoma SH-SY5 Y cells,except for the concentration of1.25ug/ml group,the activity of neuroblastoma SH-SY5 Y cells in all the other groups decreased significantly,with statistical significance(P<0.05).Autophagy inhibitor 3-MA alone neuroblastoma SH-SY5 Y cells with different concentrations of 24 h,had no significant effect on cell viability,even if the concentration of 3-MA in the 10mmol/L,the cell survival rate is still no significant difference with the control group(P> 0.05).Respectively after cisplatin alone and combined with autophagy inhibitor 3-MA the IC50 value of cisplatin was 5.25ug/ml and 10.9ug/ml,and the IC50 value of cisplatin decreased by about 49%.2 Cell Morphological changes were observed under the microscope,found in the control group had more number of cells,adherent growth,morphology is consistent,fusiform or polygonal,small volume,has aggregated tendency,with less metabolites;autophagy inhibitor treatment group cells had no obvious decrease,but the cell protrusion is not obvious,a small amount is elliptic,cisplatin treatment group significantly reduced the number of cells,morphology significantly changed,can not see the long spindle shape cells,cytoplasmic shrinkage,round or oval together,the number of cell in autophagy inhibitor combined with cisplatin group decreased significantly,and the cells dispersed,do not see a significant cluster of cells,showing a large number of cell product and death cells in the culture dish the.3 Late apoptosis and necrosis of cells under fluorescence microscope were founded in the control group,there was almost no late apoptosis and necrosis and autophagy inhibitor group late apoptosis and necrosis of cells compared with the control group,no significant increase,in cisplatin group was significantly increased compared with control group and 3-MA group late apoptosis and necrosis,late apoptosis and necrosis of cisplatin combined with autophagy inhibitor 3-MA group was significantly higher than that of cisplatin group.Compared with the blank control group,cisplatin group and autophagy inhibitor 3-MA,the apoptosis rate and necrosis rate of cisplatin combined with autophagy inhibitor 3-MA group were Significantly higher under fluorescence microscope.4 Western blot results showed that cisplatin treatment of neuroblastoma SH-SY5 Y cells after 24 h,compared with the control group,expression of autophagy related protein Beclin-1,LC3-II/1 and apoptosis related protein Caspase-3 were increased,with autophagy inhibitor 3-MA,said compared to cisplatin alone group decreased significantly of autophagy related protein Beclin-1,LC3-II,but the expression of apoptosis related protein Caspase-3 is significantly increased,the difference was statistically significant(P<0.05).Conclusion:1 Cisplatin inhibited the proliferation of neuroblastoma SH-SY5 Y cells,in a range of certain concentration time and dose dependent;2 The application of autophagy inhibitor 3-MA24 hours alone,in a rangeof certain concentration did not significantly inhibit the Cell increment;3 Cisplatin combined with autophagy inhibitor 3-MA,can reduce the IC50 value of cisplatin,chemotherapy sensitization;4 Cisplatin induced apoptosis of neuroblastoma SH-SY5 Y cells,while autophagy increased,and autophagy plays a protective role in the process of apoptosis;5 Inhibition of autophagy can enhance cisplatin induced apoptosis of neuroblastoma SH-SY5 Y cells and enhance the effect of chemotherapy,suggesting that autophagy inhibitors may improve the therapeutic effect of conventional chemotherapy drug cisplatin on neuroblastoma cells,compared with conventional chemotherapy more value,provides new ideas and methods for clinical treatment of neuroblastoma cell tumor. |
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