摘要
目的:探讨姜黄素对人前列腺癌C4-2细胞和LNCaP细胞增殖、迁移及侵袭的影响,并阐明其可能的作用机制.方法:采用慢病毒转染系统分别转染C4-2 细胞和LNCaP细胞,作为shCD147-C4-2组和shCD147-LNCaP组.采用RNA干扰技术制备沉默CD147基因细胞,以转入空载体的细胞作为阴性对照,分为shNC-C4-2组(shNC-C4-2细胞)和shNC-LNCaP组(shNC-LNCaP细胞).取生长对数期C4-2、LNCap、shCD147-C4-2和shCD147-LNCaP细胞,加入 20 μmol·L-1 姜黄素,处理 0和 24 h时,显微镜观察各组细胞形态表现.噻唑蓝(MTT)法检测各组细胞增殖活性,细胞划痕实验检测各组细胞迁移率,Western blotting法检测各组细胞中凋亡、侵袭和迁移相关蛋白表达水平.结果:与C4-2组比较,沉默CD147基因后shCD147-C4-2组细胞中CD147蛋白表达量明显减少;与LNCaP组比较,沉默CD147基因后shCD147-LNCaP组细胞中CD147蛋白表达量明显减少.与处理0 h比较,20 μmol·L-1姜黄素处理24 h后C4-2组和LNCaP组部分细胞出现凋亡征象,且有典型凋亡小体存在;shCD147-C4-2组和shCD147-LNCaP组细胞凋亡现象减弱.MTT法检测,与C4-2+0 μmol·L-1 姜黄素组比较,C4-2+20 μmol·L-1 姜黄素组、C4-2+40 μmol·L-1 姜黄素组、C4-2+60 μmol·L-1 姜黄素组和 C4-2+80 μmol·L-1 姜黄素组细胞增殖活性均明显降低(P<0.01);与LNCaP+0 μmol·L-1姜黄素组比较,LNCaP+20 μmol·L-1姜黄素组、LNCaP+40 μmol·L-1姜黄素组、LNCaP+60 μmol·L-1 姜黄素组和LNCaP+80 μmol·L-1 姜黄素组细胞增殖活性均明显降低(P<0.01);与shNC-C4-2组比较,shNC-C4-2+20 μmol·L-1姜黄素组细胞增殖活性明显降低(P<0.01);与shNC-C4-2+20 μmol·L-1姜黄素组比较,shCD147-C4-2+20 μmol·L-1 姜黄素组细胞增殖活性明显升高(P<0.01);与shNC-LNCaP组比较,shNC-LNCaP+20 μmol·L-1姜黄素组细胞增殖活性明显降低(P<0.01);与shNC-LNCaP+20 μmol·L-1 姜黄素组比较,shCD147-LNCaP+20 μmol·L-1姜黄素组细胞增殖活性明显升高(P<0.01).细胞划痕愈合实验检测,姜黄素处理24 h,与C4-2组比较,C4-2+20 μmol·L-1 姜黄素组和C4-2+40 μmol·L-1 姜黄素组细胞迁移率均明显降低(P<0.01);与LNCaP组比较,LNCaP+20 μmol·L-1 姜黄素组和LNCaP+40 μmol·L-1 姜黄素组细胞迁移率均明显降低(P<0.01);与shNC-C4-2组比较,shNC-C4-2+20 μmol·L-1姜黄素组细胞迁移率明显降低(P<0.01);与shNC-C4-2+20 μmol·L-1 姜黄素组比较,shCD147-C4-2+20 μmol·L-1 姜黄素组细胞迁移率明显升高(P<0.05);与shNC-LNCaP组比较,shNC-LNCaP+20 μmol·L-1 姜黄素组细胞迁移率明显降低(P<0.01);与shNC-LNCaP+20 μmol·L-1姜黄素组比较,shCD147-LNCaP+20 μmol·L-1姜黄素组细胞迁移率明显升高(P<0.05).Western blotting法检测,与C4-2组比较,C4-2+20 μmol·L-1姜黄素组和C4-2+40 μmol·L-1姜黄素组细胞中B细胞淋巴瘤2(Bcl-2)相关X蛋白(Bax)、裂解的含半胱氨酸的天冬氨酸蛋白酶3(cleaved Caspase-3)和聚二磷酸腺苷(ADP)-核糖聚合酶1(PARP1)蛋白表达水平均明显升高(P<0.01),Bcl-2蛋白表达水平均明显降低(P<0.05或P<0.01);与LNCaP 组比较,LNCaP+20 μmol·L-1 姜黄素组和 LNCaP+40 μmol·L-1 姜黄素组细胞中 Bax、cleaved Caspase-3和PARP1蛋白表达水平均明显升高(P<0.01),LNCaP+40 μmol·L-1 姜黄素组Bcl-2蛋白表达水平明显降低(P<0.01);与shNC-C4-2组比较,shNC-C4-2+20 μmol·L-1 姜黄素组细胞中Bax、cleaved Caspase-3和PARP1蛋白表达水平均明显升高(P<0.01),Bcl-2蛋白表达水平明显降低(P<0.05);与shNC-C4-2+20 μmol·L-1姜黄素组比较,shCD147-C4-2+20 μmol·L-1姜黄素组细胞中Bax和cleaved Caspase-3 蛋白表达水平均明显降低(P<0.01).与shNC-LNCaP组比较,shNC-LNCaP+20 μmol·L-1 姜黄素组细胞中Bax、cleaved Caspase-3 和PARP1 蛋白表达水平均明显升高(P<0.05或P<0.01),Bcl-2蛋白表达水平明显降低(P<0.05);与shNC-LNCaP+20 μmol·L-1姜黄素组比较,shCD147-LNCaP+20 μmol·L-1 姜黄素组细胞中Bax、cleaved Caspase-3和PARP1蛋白表达水平均明显降低(P<0.05或P<0.01),Bcl-2蛋白表达水平明显升高(P<0.05).与C4-2组比较,C4-2+20 μmol·L-1 姜黄素组和C4-2+40 μmol·L-1姜黄素组细胞中E-钙黏蛋白(E-cadherin)蛋白表达水平均明显升高(P<0.01),神经钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)蛋白表达水平均明显降低(P<0.01);与LNCaP组比较,LNCaP+20 μmol·L-1姜黄素组和LNCaP+40 μmol·L-1姜黄素组细胞中E-cadherin蛋白表达水平均明显升高(P<0.01),LNCaP+40 μmol·L-1姜黄素组细胞中N-cadherin和Vimentin蛋白表达水平均明显降低(P<0.01);与shNC-C4-2组比较,shNC-C4-2+20 μmol·L-1姜黄素组细胞中N-cadherin和Vimentin蛋白表达水平均明显降低(P<0.01);与shNC-C4-2+20 μmol·L-1姜黄素组比较,shCD147-C4-2+20 μmol·L-1姜黄素组细胞中E-cadherin蛋白表达水平明显降低(P<0.01),N-cadherin和Vimentin蛋白表达水平均明显升高(P<0.01);与shNC-LNCaP组比较,shNC-LNCaP+20 μmol·L-1姜黄素组细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin和Vimentin蛋白表达水平均明显降低(P<0.01);与shNC-LNCaP+20 μmol·L-1姜黄素组比较,shCD147-LNCaP+20 μmol·L-1姜黄素组细胞中E-cadherin蛋白表达水平明显降低(P<0.01),N-cadherin表达水平明显升高(P<0.05).结论:姜黄素对体外前列腺癌细胞增殖、迁移和侵袭有抑制作用,并诱导细胞凋亡,沉默CD147基因可在一定程度上降低其抑制作用和诱导细胞凋亡能力.
Abstract
Objective:To discuss the effect of curcumin on the proliferation,migration,and invasion of the human prostate cancer C4-2 and LNCaP cells,and to clarify its possible mechanism.Methods:The lentiviral transfection system was used to transfect the C4-2 and LNCaP cells,regarded as shCD147-C4-2 group and shCD147-LNCaP group.RNA interference technology was used to prepare the CD147-silenced cells;the cells transfected with an empty vector were regarded as negative control and divided into shNC-C4-2 group(shNC-C4-2 cells)and shNC-LNCaP group(shNC-LNCaP cells).The C4-2 and LNCaP cells at logarithmic growth phase,as well as shCD147-C4-2 and shCD147-LNCaP cells,were treated with 20 μmol·L-1 curcumin.The morphology of the cells in various groups was observed under microscope at 0 and 24 h of treatment;MTT method was used to detect the proliferation activities of the cells in various groups;cell scratch assay was used to detect the migration rates of the cells in various groups;Western blotting method was used to detect the expression levels of apoptosis,invasion,and migration-related proteins in the cells in various groups.Results:Compared with C4-2 group,the expression of CD147 protein in the cells in shCD147-C4-2 group was significantly decreased after CD147 gene silenting.Compared with LNCaP group,the expression level of CD147 protein in the cells in shCD147-LNCaP group was significantly decreased after CD147 gene silenting.Compared with 0 h of treatment,some cells in C4-2 and LNCaP groups after 24 h of treatment with 20 μmol·L-1 curcumin,showed apoptosis signs with the presence of typical apoptotic bodies.The apoptotic phenomena in shCD147-C4-2 and shCD147-LNCaP groups was reduced.The MTT assay results showed that compared with C4-2+0 μmol·L-1 curcumin group,the proliferation activities of the cells in C4-2+20 μmol·L-1 curcumin group,C4-2+40 μmol·L-1 curcumin group,C4-2+60 μmol·L-1 curcumin group,and C4-2+80 μmol·L-1 curcumin group were decreased(P<0.01).Compared with LNCaP+0 μmol·L-1 curcumin group,the proliferation activity of the cells in LNCaP+20 μ mol·L-1 curcumin group,LNCaP+40 μmol·L-1 curcumin group,LNCaP+60 μmol·L-1 curcumin group,and LNCaP+80 μmol·L-1 curcumin group were decreased(P<0.01).Compared with shNC-C4-2 group,the proliferation activity of the cells in shNC-C4-2+20 μmol·L-1 curcumin group was decreased(P<0.01).Compared with shNC-C4-2+20 μmol·L-1 curcumin group,the proliferation activity of the cells in shCD147-C4-2+20 μmol·L-1 curcumin group was increased(P<0.01).Compared with shNC-LNCaP group,the proliferation activity of the cells in shNC-LNCaP+20 μmol·L-1 curcumin group was decreased(P<0.01);compared with shNC-LNCaP+20 μmol·L-1 curcumin group,the proliferation activity of the cells in shCD147-LNCaP+20 μmol·L-1 curcumin group was significantly increased(P<0.01).The cell scratch healing assay results showed that compared with C4-2 group,the migration rates of the cells in C4-2+20 μmol·L-1 curcumin group and C4-2+40 μmol·L-1 curcumin group after 24 h of treatment were decreased(P<0.01);compared with LNCaP group,the migration rates of the cells in LNCaP+20 μmol·L-1 curcumin group and LNCaP+40 μmol·L-1 curcumin group were increased(P<0.01);compared with shNC-C4-2 group,the migration rate of the cells in shNC-C4-2+20 μmol·L-1 curcumin group was decreased(P<0.01);compared with shNC-C4-2+20 μmol·L-1 curcumin group,the migration rate of the cells in shCD147-C4-2+20 μmol·L-1 curcumin group was significantly increased(P<0.05);compared with shNC-LNCaP group,the migration rate of the cells in shNC-LNCaP+20 μmol·L-1 curcumin group was decreased(P<0.01);compared with shNC-LNCaP+20 μmol·L-1 curcumin group,the garation rate of the cells in shCD147-LNCaP+20 μmol·L-1 curcumin group was significantly increased(P<0.05).The Western blotting results showed that compared with C4-2 group,the expression levels of Bcl-2-associated X protein(Bax),cleaved Caspase-3,and poly ADP-ribose polymerase 1(PARP1)proteins in the cells in C4-2+20 μmol·L-1 curcumin group and C4-2+40 μmol·L-1 curcumin group were significantly increased(P<0.01),and the expression levels of Bcl-2 protein was significantly decreased(P<0.05 or P<0.01);compared with LNCaP group,the expression levels of Bax,cleaved Caspase-3,and PARP1 proteins in the cells in LNCaP+20 μmol·L-1 curcumin group and LNCaP+40 μmol·L-1 curcumin group were significantly increased(P<0.01),and the expression level of Bcl-2 protein in the cells in LNCaP+40 μmol·L-1 curcumin group was decreased(P<0.01);compared with shNC-C4-2 group,the expression levels of Bax,cleaved Caspase-3,and PARP1 proteins in the cells in shNC-C4-2+20 μmol·L-1 curcumin group were significantly increased(P<0.05 or P<0.01),and the expression level of Bcl-2 protein was significantly decreased(P<0.05);compared with shNC-C4-2+20 μmol·L-1 curcumin group,the expression levels of Bax and cleaved Caspase-3 proteins in the cells in shCD147-C4-2+20 μmol·L-1 curcumin group were significantly decreased(P<0.01);compared with shNC-LNCaP group,the expression levels of Bax,cleaved Caspase-3,and PARP1 proteins in the cells in shNC-LNCaP+20 μmol·L-1 curcumin group were significantly increased(P<0.05 or P<0.01),and the expression level of Bcl-2 protein was significantly decreased(P<0.05);compared with shNC-LNCaP+20 μmol·L-1 curcumin group,the expression levels of Bax,cleaved Caspase-3,and PARP1 proteins in the cells in shCD147-LNCaP+20 μmol·L-1 curcumin group were significantly decreased(P<0.05 or P<0.01),and the expression level of Bcl-2 protein was significantly increased(P<0.05).Compared with C4-2 group,the expression levels of E-cadherin protein in the cells in C4-2+20 μmol·L-1 curcumin group and C4-2+40 μ mol·L-1 curcumin group were significantly increased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins were significantly decreased(P<0.01);compared with LNCaP group,the expression levels of E-cadherin protein in the cells in LNCaP+20 μmol·L-1 curcumin group and LNCaP+40 μmol·L-1 curcumin group were significantly increased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins in the cells in LNCaP+40 μmol·L-1 curcumin group were significantly decreased(P<0.01);compared with shNC-C4-2 group,the expression levels of N-cadherin and Vimentin proteins in the cells in shNC-C4-2+20 μmol·L-1 curcumin group were significantly decreased(P<0.01);compared with shNC-C4-2+20 μmol·L-1 curcumin group,the expression level of E-cadherin protein in the cells in shCD147-C4-2+20 μmol·L-1 curcumin group was significantly decreased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins were significantly increased(P<0.01);compared with shNC-LNCaP group,the expression level of E-cadherin protein in the cells in shNC-LNCaP+20 μmol·L-1 curcumin group was significantly increased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins were significantly decreased(P<0.01);compared with shNC-LNCaP+20 μmol·L-1 curcumin group,the expression level of E-cadherin protein in the cells in shCD147-LNCaP+20 μmol·L-1 curcumin group was significantly decreased(P<0.01),and the expression level of N-cadherin was significantly increased(P<0.05).Conclusion:Curcumin inhibits the proliferation,migration,and invasion of the prostate cancer cells in vitro and induces the apoptosis;silencing the CD147 gene partially reduces its inhibitory effect and its ability to induce the apoptosis.
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