| Objectives:The effect of quercetin(Que)combined with pacilitaxel(PTX)on the proliferation of prostatic carcinoma cell line PC-3,and whether the drug resistance of PC-3 to PTX was reversed by Que and its mechanism was also studied in this paper to provide the basis for the study of novel chemotherapy for prostatic cancer.Methods:(1)prostatic carcinoma cell line PC-3 cells were selected as the study object.The IC50 values of PTX and Que were calculated with CCK-8 detection.And the IC50 value of PTX+1/10 IC50 Que was calculated with CCK-8 detection.(2)According to IC50 values of the two drugs,PC-3 cells were treated with different concentrations of Que in combination with the same concentrations of PTX,the paclitaxel group as the internal control.Experimental groups were divided as follows:control group K,paclitaxel group A(PTX IC50),drug combination group(B:2/3 Que IC50+PTX IC50?C:Que IC50+PTX IC50?D:4/3Que IC50+PTX IC50?E:5/3 Que IC50+PTX IC50).The cell viability was detected by CCK-8 and Annexin V/PI double staining method was applied to detect apoptosis.The cell changes were observed by inverted microscope.PC-3 cells were treated with different concentrations of PTX in combination with the same concentrations of Que,with the Que group as the internal control.Experimental groups were divided as follows:control group k,quercetin group a(Que IC50),drug combination group(b:Que IC50+1/2 PTX IC50,c:Que IC50+PTX IC50,d:Que IC50+2 PTX IC50,e:Que IC50+4 PTX IC50).The cell viability and apoptosis were detected.The cell changes were observed by inverted microscope.(3)Drug-resistant PC-3DR cells were developed by the method of dose escalation.Experimental groups were divided as follows:Group X:PC-3DR cells were treated with Que(0?25?50?100?200?400?800?M).Group Y:PC-3DR cells were treated with PTX(0?25?50?100?200?400nM).Group Z:PC-3 DR cells were treated with PTX combined with 1/10 Que IC50.The drug resistance reversal fold(RF)was calculated.(4)Group J:PC-3DR cells,and group Q:PC-3DR cells treated with 1/10 Que IC50.Each group was detected for MRP1 mRNA by RT-qPCR,and P-glycoprotein(P-gp),glucose regulatory protein 78(GRP78)and ??-tubulin by Western blot(WB).(5)The experimental data was analyzed by SPSS version 13.0,and the results were expressed by mean value ± standard deviation(mean ±SD).Wilcoxon rank sum test was used for two independent samples,and Kruskal-Wallis H test was used for pairwise comparison between groups.*P<0.05 was defined as statistically significant difference.Results:(1)The PC-3 cells,Que IC50 was 180.21±0.27?M,PTX IC50 was 19.88±0.52nM.After adding low dose Que(1/10 Que IC50),the PTX IC50 of PC-3DR cells was 114.74±1.93nM,and the decreased concentration of PTX was observed.When the same inhibition rate was reached,the drug concentration of PTX could be significantly reduced,it.suggested that Que ccould increase the sensitivity-of PC-3,cells to PTX.(2)The cell viability of A,B,C,D,and E groups was 50.87 ± 1.06%,47.86 ±0.20%,33.61± 0.93%,25.31± 1.32%,14.72± 2.03%.respectively.The apoptosis rate was 50.14 ± 2.21%,62.28 ± 3.03%,68.97 ± 6.27%,75.87 ± 3.60%,85.13 ±4.19%,respectively.The cell viability and apoptosis rate were dependent on Que concentration.Under the inverted microscope,it could be seen that there was no significant change in cell morphology and the number of cells was significantly reduced after the effect of PTX combined with Que.The higher the concentration of Que,the more obvious.The cell activities of a,b,c,d and e groups was 50.98 ± 4.19%,47.09 ± 4.19%,34.91 ± 4.91%,21.32 ± 1.28%,14.72 ± 2.24%.respectively.The apoptosis rate was 50.80 ± 3.96%,55.94 ± 4.30%.67.31 ± 3.99%,74.97 ± 3.56%,86.48 ±2.27%,respectively.The cell viability and apoptosis rate were concentration dependent on with PTX.Under the inverted microscope,there was no significant change in the cell morphology and the number of cells after the treatment of Que combined with PTX.The higher the concentration of PTX,the more obvious.There was no significant difference between group B and group b,group D and group d,group E and group e in the inhibition rate and apoptosis rate of PC-3 cells(P>0.05).The concentration of PTX in group b was lower than that in group B,group D was lower than that in group d,and group E was lower than that in group e.Low dose concentrations of PTX suggested that the same inhibition and apoptosis effect could be achieved,and Que could reduce the use of PTX.(3)The PTX IC50 of PC-3DR cells was139.17 ± 2.45nM,the Que IC50 of PC-3DR cells was 2111.56 ± 2.39 ?M.After adding low dose Que,the PTX IC50 of PC-3DR cells was 114.74±1.93nM.RF=1.30.Que could increase the sensitivity of PC-3DR cells to PTX,and when combined,the decreased concertration of PTX was observed(4)The results of RT-qPCR showed that MRP1 mRNA in group J and group Q were 6.16±0.38 and 4.13±0.11,respectively,compared with parental PC-3 cells(1.06±0.08),P<0.01,suggested that MDR formation in PC-3DR cells was related to the elevation of MRP 1 mRNA.Compared with group J,MRP1 mRNA in group Q was decreased(P<0.05),suggested that Que reversed the resistance of PC-3DR cells to PTX may be related to the reduction of MRP1 mRNA.The WB testing results indicated that G-pg in group J and group Q was 1.24±0.07 and 1.17±0.03,respectively,P>0.05 compared with parental PC-3 cells(0.98±0.1),suggested that MDR of PC-3DR cells was not significantly related to G-pg expression.GRP78 in group J was 16.13±0.33,P<0.01 compared with PC-3 cells(6.83±0.08),suggested that MDR in PC-3DR cells may be related to GRP78 expression;GRP78 in group Q was 20.52±0.83,P<0.01 compared with PC-3 cells;P<0.05 compared with group P,suggested that Que increased PTX sensitivity may be related to GRP78 expression.The ? ?-tubulin in group J was 13.40±0.43,P<0.01 compared with PC-3 cells(5.68±0.39),suggested that MDR of PC-3DR cells was related to ? ?-tubulin expression;the ??-tubulin in group Q was 7.50±1.00,P<0.01 compared with PC-3 cells,but P<0.05 compared with group J,suggested that Que reversed the resistance of PC-3DR cells to PTX may be related to the decrease of ??-tubulin expression.Conclusions:(1)In this study Que could increase the sensitivity of PC-3R cells to PTX;(2)Que could reverse the resistance of PC-3 cells to PTX might be related to the regulation of MRP 1 mRNA,GRP78 and ??-tubulin in our study. |
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