银杏酚酸通过ERK-JNK-AKT途径诱导前列腺癌细胞p53依赖性凋亡

Ginkgolic acid induces p53-dependent apoptosis of prostate cancer cells through ERK-JNK-AKT pathway

目的探究银杏酚酸对前列腺癌细胞增殖和凋亡的影响及机制。方法(1)将DU145细胞用不同浓度银杏酚酸(0,2.5,5,10,20,40,80,160μmol/L)培养48 h,通过MTT法检测细胞活力,通过AnnexinⅤ-FITC/PI染色法检测细胞凋亡。(2)将DU145细胞用0,80μmol/L的银杏酚酸培养48 h,作为对照组和80μmol/L银杏酚酸组(GA组)。通过Western blot检测凋亡相关蛋白(Bax、Bcl-2、cleaved-Caspase-3和P53)的表达。(3)将DU145细胞分为8组:正常对照组、银杏酚酸组(GA组)、PD98059组、银杏酚酸+PD98059组(GA+PD98059组)、SP600125组、银杏酚酸+SP600125组(GA+SP600125组)、LY294002组、银杏酚酸+LY294002组(GA+LY294002组)。对照组细胞不使用药物处理,GA组细胞使用80μmol/L的银杏酚酸培养48 h,PD98059组细胞使用50μmol/L的PD98059培养48 h,GA+PD98059组细胞使用80μmol/L的银杏酚酸和50μmol/L的PD98059共培养48 h,SP600125组细胞使用50μmol/L的SP600125培养48 h,GA+SP600125组细胞使用80μmol/L的银杏酚酸和50μmol/L的SP600125共培养48 h,LY294002组细胞使用50μmol/L的LY294002培养48 h,GA+LY294002组细胞使用80μmol/L的银杏酚酸和50μmol/L的LY294002共培养48 h,然后通过MTT法检测细胞活力,通过AnnexinⅤ-FITC/PI染色法检测细胞凋亡。(4)将BALB/c小鼠随机分为生理盐水组(NS组,n=6)、银杏酚酸低剂量组(GA-L组,10 mg/kg,n=6)、银杏酚酸中剂量组(GA-M组,50 mg/kg,n=6)、银杏酚酸高剂量组(GA-H组,100 mg/kg,n=6)。所有小鼠皮下接种DU145细胞(4×10;)建立荷瘤小鼠模型,接种第8天开始进行药物治疗。NS组小鼠腹腔注射生理盐水,GA-L组、GA-M组和GA-H组小鼠分别腹腔注射10,50,100 mg/kg的银杏酚酸,每周腹腔注射3次。每7 d用卡尺测量肿瘤大小。通过TUNEL染色检测肿瘤组织中的细胞凋亡。通过免疫组化染色检测肿瘤组织中Bax和Bcl-2的蛋白表达。通过Western blot检测肿瘤组织中Bax、Bcl-2、cleaved-Caspase-3、P53、p-ERK、p-JNK和p-AKT的蛋白表达。结果与0μmol/L比较,5,10,20,40,80,160μmol/L银杏酚酸处理后DU145细胞活力降低,细胞凋亡率升高(均P<0.05)。与对照组比较,GA组DU145细胞中Bax、cleaved-Caspase-3和P53蛋白表达水平升高,而Bcl-2、p-ERK、p-JNK和p-AKT蛋白表达水平降低(均P<0.05)。与对照组相比,GA组、PD98059组、SP600125组和LY294002组细胞活力降低,细胞凋亡率升高(P<0.05)。与NS组相比,GA-L组、GA-M组和GA-H组肿瘤体积降低,肿瘤组织中TUNEL阳性率升高(均P<0.05)。与NS组相比,GA-L组、GA-M组和GA-H组肿瘤组织中Bax、cleaved-Caspase-3和P53蛋白表达水平均升高,而Bcl-2、p-ERK、p-JNK和p-AKT蛋白表达水平均降低(P<0.05)。结论银杏酚酸可能通过ERK-JNK-AKT途径诱导前列腺癌细胞p53依赖性凋亡。

Objective To explore the effect of ginkgolic acid on the proliferation and the apoptosis of prostate cancer cells and its mechanism.Methods(1)DU145 cells were cultured with 0,2.5,5,10,20,40,80 and 160μmol/L ginkgolic acid for 48 h,and then the cell viability was detected by MTT assay,and the cell apoptosis was detected by AnnexinⅤ-FITC/PI staining method.(2)DU145 cells were divided into control group and 80μmol/L ginkgolic acid group(GA group).DU145 cells were cultured with 0,80μmol/L ginkgolic acid for 48 h,respectively.The expression levels of apoptosis-related proteins(Bax,Bcl-2,cleaved-Caspase-3 and P53)were detected by Western blot.(3)DU145 cells were divided into 8 groups,namely normal control group,ginkgolic acid group(GA group),PD98059 group,ginkgolic acid+PD98059 group(GA+PD98059 group),SP600125 group,ginkgolic acid+SP600125 group(GA+SP600125 group),LY294002 group,Ginkgolic acid+LY294002 group(GA+LY294002 group).The cells in control group were not treated with drugs,the cells in GA group were cultured with 80μmol/L ginkgolic acid for 48 h,the cells in PD98059 group were cultured with 50μmol/L PD98059 for 48 h,and the cells in GA+PD98059 group were co-cultured with 80μmol/L ginkgolic acid and 50μmol/L PD98059 for 48 h,the cells in SP600125 group were cultured with 50μmol/L SP600125 for 48 h,and the cells in GA+SP600125 group were co-cultured with 80μmol/L ginkgolic acid and 50μmol/L SP600125 for 48 h,the cells in LY294002 group were cultured with 50μmol/L LY294002 for 48 h,and the cells in GA+LY294002 group were co-cultured with 80μmol/L ginkgolic acid and 50μmol/L LY294002 for 48 h.Then the cell viability was detected by MTT assay,and the apoptosis was detected by AnnexinⅤ-FITC/PI staining.(4)BALB/c mice were randomly divided into normal saline group(NS group,n=6),low-dose ginkgolic acid group(GA-L group,10 mg/kg,n=6),medium-dose ginkgolic acid group(GA-M group,50 mg/kg,n=6),high-dose ginkgolic acid group(GA-H group,100 mg/kg,n=6).Mice in all groups were subcutaneously inoculated with DU145 cells(4×10~5)to establish a tumor-bearing mouse model,and were treated from the eighth day after the inoculation.The mice in NS group were intraperitoneally injected with normal saline,and the mice in GA-L group,GA-M group and GA-H group were intraperitoneally injected with 10,50,100 mg/kg ginkgolic acid three times a week,respectively.Tumor size was measured with calipers every 7 d.Cell apoptosis in tumor tissues was detected by TUNEL staining.The protein expression levels of Bax and Bcl-2 in tumor tissues were detected by immunohistochemical staining.The protein expression levels of Bax,Bcl-2,cleaved-Caspase-3,P53,p-ERK,p-JNK and p-AKT in tumor tissues were detected by Western blot.Results Compared with 0μmol/L,the cell viability of DU145 was decreased after treatment with 5,10,20,40,80 and 160μmol/L GA,while the apoptosis rate was increased(P<0.05).Compared with control group,the protein expression levels of Bax,cleaved-Caspase-3 and P53 in DU145 cells were increased in GA group,while the protein expression levels of Bcl-2,p-ERK,p-JNK and p-AKT were decreased(P<0.05).Compared with control group,the cell viability was decreased in GA group,PD98059 group,SP600125 group and LY294002 group,while the apoptosis rate was increased(P<0.05).Compared with NS group,the tumor volume was decreased in GA-L group,GA-M group and GA-H group,while the positive rate of TUNEL in the tumor tissue was increased(P<0.05).Compared with NS group,the protein expression levels of Bax,cleaved-Caspase-3 and P53 in the tumor tissues were all increased in GA-L,GA-M and GA-H groups,while the protein expression levels of Bcl-2,p-ERK,p-JNK and p-AKT were decreased(P<0.05).Conclusion Ginkgolic acid may induce the p53-dependent apoptosis of prostate cancer cells through ERK-JNK-AKT pathway.