| Part oneTo investigate the expression of BRCA2 and clinical significance in epithelial ovarian cancerObjectiveTo detect the expression of BRCA2 in epithelial ovarian cancer tissues.To explore the relationship between the expression of BRCA2 and the clinicopathological features and clinical prognosis of patients with epithelial ovarian tumors.MethodsThe expression of BRCA2 in 63 specimens of epithelial ovarian cancer was detected by immunohistochemistry.The correlation of BRCA2 expression with clinicopathological characteristics and clinical prognosis was analyzed.Results(1)There was no association between the expression level and age,pathological type/grade,clinical stage,chemotherapy regimen/cycle,or the size of residual tumor.(2)Platinum resistant cancer had a higher BRCA2 level compared with sensitive cancer(with a high expression percentage of 87.5%(95% CI: 67.6–97.3%)vs 43.6%(95% CI: 27.8–60.4%),P = 0.001;and a score of 2.72 ± 1.24 vs 1.68 ± 1.54,P = 0.007).(3)Patients with a low level of BRCA2 in cancer tissues had longer PFS(with a median time of 28.0(95% CI: 18.2–37.8)vs 12.0(95% CI: 9.6–14.4)months,P < 0.001)and PFD(with a median time of 19.0(95% CI: 4.3–33.7)vs 5.0(95% CI: 3.0–7.0)months,P < 0.001),compared with those with a high level.ConclusionA low expression level of BRCA2 in cancer tissues indicated a better response to platinum.Part twoTo investigate the effect of silencing BRCA2 on the therapeutic effect of cisplatin on ovarian cancer cells in vitro and in vivoObjectiveTo explore the differences and characteristics of BRCA2 knockdown in treated with CDDP in ovarian cancer cells,and to investigate the efficacy of CDDP in ovarian tumors.Methods(1)The basal expression level of BRCA2 in five cell lines was determined by Western blotting to determine the appropriate cell line for gene silencing experiments.(2)The expression of BRCA2 and RAD51 in cisplatin-treated cells was detected by Western blotting after lentiviral infection of CAOV-3 and ES-2 cells.(3)DNA double-strand breaks were detected by neutral comet assay at 0h,2h,4h,and 8h after cisplatin treatment.(4)After the cells were treated with cisplatin,the formation of RAD51 foci was detected by cellular immunofluorescence.(5)Cytotoxicity of cisplatin after BRCA2 silencing in ovarian cancer CAOV-3 and ES-2 cell lines was detected by CCK-8 assay.(6)The cytotoxicity of cisplatin after BRCA2 silencing in ovarian cancer CAOV-3 and ES-2 cell lines was detected by plate cloning assay.(7)The effect of cisplatin treatment after BRCA2 silencing in CAOV-3 cells was observed in nude mice subcutaneous xenografts.(8)The expression levels of BRCA2 and RAD51 after transplantation of cisplatin were measured by immunohistochemistry.Results(1)Western blot demonstrated a higher level in ES-2,CAOV-3 and SKOV3 cell lines,and therefore ES-2 and CAOV-3(4.7 and 5.7 times higher than that in COC1,respectively)were selected.(2)The level of BRCA2 protein was decreased in sh BRCA2-infected CAOV-3 or ES-2 cells(P = 0.003,P = 0.034).CDDP did not affect the level of BRCA2 protein,but increased the level of RAD51 protein in both cell lines(P = 0.011,P = 0.012);this inductive effect was suppressed after sh BRCA2 transfection(P = 0.004,P = 0.030).(3)The neutral comet assay was performed to detect DSB.The percentage of comet-formed cells decreased gradually after CDDP removal,indicating repair.The value at 2–8 h in NC-transfected cells was less than that in sh BRCA2-transfected cells(P < 0.001 in CAOV-3;P = 0.001 in ES-2).80% DSB were repaired in NC-transfected cells after 8 h,but only 50% DSB were repaired in sh BRCA2-transfectedcells.(4)CDDP induced the formation of RAD51 foci,which was decreased after sh BRCA2 transfection(P = 0.007 in CAOV-3;P = 0.004 in ES-2);in NC-and sh BRCA2-transfected cells,values were 46.3 ± 6.5 vs 26.3 ± 1.5 in CAOV-3 cells,and 54.3 ± 7.1 vs 27.7 ± 2.5 in ES-2 cells,respectively.(5)CDDP treatment resulted in a less survival percentage in sh BRCA2-transfected cells in comparison with NC-transfected cells(P < 0.001 in CAOV-3;P < 0.001 in ES-2),with IC50 values of 1.28 vs 2.97 ?M in CAOV-3 cells,and 1.37 vs 2.63 ?M in ES-2 cells,respectively.(6)sh BRCA2 transfection did not impact on colony formation in both cell lines.Clone inhibition due to CDDP was enhanced after silencing BRCA2,decreasing the clone number(37.7 ± 2.5 vs 8.0 ± 2.0 in CAOV-3,P = 0.010;34.0 ± 7.9 vs 8.3 ± 1.5 in ES-2,P = 0.001).(7)Tumor volume and mass in group sh BRCA2 + CDDP were less than those in group NC + CDDP,with volumes of 327.7 ± 68.4 and 125.7 ± 21.4 mm3,and masses of 0.42 ± 0.05 and 0.24 ± 0.03 g,in groups NC + CDDP and sh BRCA2 + CDDP,respectively(P < 0.001,P < 0.001).(8)A much lower level of BRCA2 protein was detected in tumors originated from sh BRCA2-transfected cells(3.40 ± 0.55 vs 1.00 ± 0.00,P < 0.001),demonstrating effective inhibition.CDDP treatment improved the level of RAD51 protein in tumors formed from both NC-and sh BRCA2-transfected cells,and this inductive effect was suppressed after sh BRCA2 transfection,with scores of 1.40 ± 0.55,4.00 ± 0.00,1.00 ± 0.00 and 1.60 ± 0.55 in groups NC,NC + CDDP,sh BRCA2 and sh BRCA2 + CDDP,respectively(P < 0.001).ConclusionSilencing BRCA2 inhibited the expression of BRCA2 and RAD51 proteins,and decreased DNA repair;silencing BRCA2 enhanced the action of CDDP;silencing BRCA2 improved the anticancer effect of CDDP in vivo.Part threeTo investigate the effects of silencing BRCA2 on the therapeutic effect of cisplatin combined with chloroquine on ovarian cancer cells in vitro and in vivoObjectiveTo investigate the effect of silencing BRCA2 on autophagy induced by cisplatin in ovarian tumor cells and the effect of BRCA2 silencing on the therapeutic effect of cisplatin combined with chloroquine on ovarian tumor cells in vitro and in vivo.Methods(1)The expression of LC3 was detected by Western blot treated with CDDP and CQ in CAOV-3 and ES-2 cells,and the CQ concentration was screened.(2)CCK-8 assay was used to test the cytotoxicity of CQ-treated CAOV-3 and ES-2 cells,and the CQ concentration was screened.(3)The protein expression of LC3 was detected by Western blotting after EBSS and CQ treatment.(4)Fluorescence confocal microscopy was used to detect the autophagy after EBSS and CQ treatment in CAOV-3 and ES-2 ovary cancer cells.(5)Western blotting was used to detect autophagy after CDDP and CQ treatment in sh BRCA2-transfected cells.(6)The autophagic flux was detected by fluorescence confocal after CDDP and CQ treatment in sh BRCA2-transfected cells.(7)Comet assay was used to detect DNA damage and repair after CDDP and CQ treatment in sh BRCA2-transfected cells.(8)Cellular immunofluorescence was used to detect the status of RAD51 foci following the treatment of CDDP and CQ in sh BRCA2-transfected cells.(9)CCK-8 assay was used to detect the cell death after treatment of CDDP and CQ in sh BRCA2-transfected cells.(10)Flow cytometry was used to detect the apoptosis of BRCA2-silenced cells treated with CDDP and CQ.(11)Silencing BRCA2 and/or ATG7,a cytotoxic effect was detected by CCK-8.(12)The efficacy of CDDP combined with CQ in sh BRCA2-transfected ovarian tumor cells was detected in nude mice.Results(1)CQ with a concentration of 9.69 ?M caused no cytotoxicity(the percentage of dead cells was < 10%),and was therefore used to inhibit autophagy.Basal autophagy was noted in both cell lines,and EBSS-induced autophagy can be blocked by CQ.(2)CDDP caused autophagy in BRCA2-intact cells(P < 0.001 in CAOV-3;P < 0.001 in ES-2);silencing BRCA2 enhanced CDDP-induced autophagy,increasing the LC3-? level(P = 0.004 in CAOV-3;P = 0.008 in ES-2).(3)More RFP+GFP+ and RFP+GFP-puncta were detected after CDDP exposure,and the number of RFP+GFP+ puncta was less than that of RFP+GFP-ones(P < 0.001),confirming the autophagy flux.The number of RFP+GFP+ dots was remarkably increased after adding CQ(P < 0.001),demonstrating a blockage of the fusion of autophagosomes and autolysomes.(4)DNA repair was decreased in sh BRCA2-transfected cells,which was exacerbated by CQ(P = 0.003 in CAOV-3;P = 0.043 in ES-2);percentages of comet-formed cells at 8 h were 20.3% and 34.3% in CAOV-3 cells,and 19.0% and 32.7% in ES-C cells,without and with CQ,respectively.(5)CQ enhanced the decrease in RAD51 foci due to silencing BRCA2(P < 0.001 in CAOV-3;P = 0.002 in ES-2);the number was 26.3 ± 1.5 vs 13.3 ± 1.5 in CAOV-3 cells,and 27.7 ± 2.5 vs 12.3 ± 2.5 in ES-2 cells,respectively.(6)CDDP led to a higher percentage of dead cells in sh BRCA2-transfected cells compared with NC-transfected cells.CQ enhanced CDDP against sh BRCA2-transfected cells,resulting in the highest cell death percentage(P = 0.002 in CAOV-3;P = 0.001 in ES-2);values at 8 ?M CDDP were 53.6% vs 69.9% in CAOV-3 cells,and 58.3% vs 68.5% in ES-2 cells,without and with CQ,respectively.The enhancement was not detected in NC-transfected cells.Percentages of dead cells at 8 ?M CDDP were 34.6% vs 39.0% in CAOV-3 cells,and 38.4% vs 41.2% in ES-2 cells,without and with CQ,respectively.(7)CDDP induced apoptosis.The addition of CQ increased the percentage of apoptotic cells in sh BRCA2-transfected cells,which did not occur in NC-transfected cells(P = 0.005 in CAOV-3;P = 0.003 in ES-2).In NC-transfected cells,values at 8 ?M CDDP were 10.2% vs 11.6% in CAOV-3 cells and 12.0% vs 12.1% in ES-2 cells,without and with CQ,respectively;levels were increased to 21.6% vs 36.6%,and 23.3% vs 36.4% in sh BRCA2-transfected cells.(8)BRCA2 and ATG7 proteins were downregulated after si RNA transfection(P < 0.001,P < 0.001);silencing ATG7 inhibited CDDP-induced autophagy,decreasing the LC3-? level(P < 0.001);ATG7 silence did not improve the percentage of dead cells in BRCA2-intact cells,but the value was increased in BRCA2-silenced cells(P = 0.001).(9)CQ alone did not inhibit tumor growth;CDDP inhibited tumors;the combination of CDDP and CQ led to the smallest tumors(P < 0.001,P < 0.001),with volumes of 293.6 ± 35.7 and 83.6 ± 28.0 mm3 in groups CDDP and CDDP + CQ,respectively.The pattern of LC3-? demonstrated intratumoral autophagy in group CDDP + CQ(P = 0.041).ConclusionSilencing BRCA2 can enhance CDDP-induced autophagy;CQ can enhance the suppression of DNA repair due to silencing BRCA2;CQ enhanced cell death and apoptosis attributable to CDDP in BRCA2-silenced cells;CQ modulated the cells' sensitivity via the autophagy pathway;CQ enhanced the effect of CDDP against BRCA2-silenced tumors,where an increase in autophagy played an important role. |
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