双调控DD3-ZD55条件增殖腺病毒荷载核基质结合区结合蛋白1短发卡RNA联合化疗对激素敏感性前列腺癌LNcap细胞的杀伤作用

DD3-ZD55 double regulation of replication-competent adenovirus loaded special AT rich sequence binding protein-1 short hairpin RNA combined with Docetaxel for treatment of prostate cancer in vitro

目的观察DD3启动子、E1B55KD缺失双调控并荷载核基质结合区结合蛋白1(SATB1)短发卡RNA(shRNA)的条件增殖腺病毒DD3-ZD55-SATB1联合多西他赛(Docetaxel, DTX)对激素敏感性前列腺癌LNcap细胞的杀伤效果并探讨作用机制。方法 LNcap细胞来源于上海中科院细胞库。将LNcap细胞分为4组进行干预, 分别为联合组(DD3-ZD55-SATB1+DTX, 5 MOI+1 ng/ml)、病毒治疗组(DD3-ZD55-SATB1, 10 MOI)、多西他赛组(DTX, 2 ng/ml)、磷酸盐缓液组(PBS)。细胞计数试剂盒(CCK-8)检测细胞增殖;Transwell检测细胞迁移和侵袭;Hoechst-33258检测细胞凋亡;蛋白质印迹法(Western blot)检测SATB1、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-2(MMP-2)、Caspase-3与Caspase-8的表达。采用单因素方差分析进行多组件比较。结果 CCK-8结果, 联合组吸光度值为1.95±0.12、DD3-ZD55-SATB1组为2.33±0.03(t=5.43, P<0.01)、DTX组为2.68±0.09(t=8.42, P<0.01), PBS组为3.49±0.04(t=21.71, P<0.01), 联合组的吸光度值显著低于单一治疗组。Transwell结果:联合组穿透小室膜的细胞数为49.25±6.24、DD3-ZD55-SATB1组为79.25±7.41(t=6.19, P<0.05)、DTX组为80.75±4.57(t=8.15, P<0.05), PBS组为118.75±5.38(t=16.88, P<0.01), 联合组小室底膜细胞数较单一治疗组明显减小。Hoechst-33258显示:联合组凋亡率分别为(55.54±5.43)%, DD3-ZD55-SATB1组为(41.23±3.28)%(t=5.29, P<0.01), DTX组为(35.15±2.47)%(t=8.19, P<0.01), PBS组为(9.62±2.69)%(t=17.49, P<0.01), 联合组与病毒或化疗药物单一治疗组比较, 细胞凋亡更加明显。Western blot结果, 以PBS组内的蛋白表达为标准计算为1.00, 联合组、DD3-ZD55-SATB1组和DTX组内, SATB1相对表达量分别为0.48±0.04、0.63±0.05和0.79±0.04, 联合组内SATB1表达较单一治疗组下调更为显著, 差异有统计学意义(t=4.06、10.38, P<0.01)。E-cadherin、Vimentin和MMP-2蛋白在联合组、DD3-ZD55-SATB1组和DTX组内相对表达量分别为2.25±0.14、0.48±0.07、0.45±0.04, 1.83±0.12、0.69±0.07、0.65±0.05和1.60±0.06、0.79±0.04、0.76±0.05。与PBS组比较, 治疗组内E-cadherin表达上调, Vimentin和MMP-2的表达下调, 联合组内E-cadherin的上调与Vimentin和MMP-2的下调更为显著, 与单一治疗组比较, 差异有统计学意义(t=3.94、3.79、5.48, P<0.05;t=7.43、6.99、8.88, P<0.01)。凋亡相关蛋白Caspase-8和Caspase-3的相对表达量分别为2.38±0.14、2.44±0.16, 1.84±0.07、1.90±0.05和1.70±0.13、1.67±0.07。各治疗组内Caspase-8和Caspase-3的表达较PBS组明显上调。联合组内Caspase-3和Caspase-8的上调更为显著, 与单一治疗组比较, 差异有统计学意义(t=5.89、5.75, P<0.01;t=6.26、7.91, P<0.01)。结论 DD3-ZD55-SATB1联合DTX在体外可有效抑制LNcap细胞的增殖、迁移和侵袭, 并诱导肿瘤细胞凋亡, 且优于单独使用DD3-ZD55-SATB1或DTX, 其作用机制为抑制上皮-间充质转化进程和上调Caspase-8和Caspase-3诱导肿瘤凋亡有关。

Objective To observe the therapeutic effect of DD3-ZD55 double regulation of oncolytic adenovirus loaded special AT rich sequence binding protein-1(SATB1)short hairpin RNA(shRNA)combined with Docetaxel(DTX)on hormone-dependent prostate cancer LNcap cells in vitro,and to preliminarily explore the action mechanism.Methods Lncap cell was provided by the Institute of Biochemistry of Chinese Academy of Science.Prostate cancer LNcap cells were divided into 4 groups:virus plus DTX group(DD3-ZD55-SATB1+DTX,5 MOI+1 ng/ml);virus treatment group(DD3-ZD55-SATB1,10 MOI);DTX group(DTX,2 ng/ml);phosphate buffer group(PBS).Cell counting kit-8(CCK-8)assay was used to detect cell proliferation.Transwell assay was used to detect the change of cell migration and invasion ability.Hoechst-33258 test was used to detect the cell apoptosis.Western blotting was used to detect the expression of SATB1,invasion-related proteins E-cadherin,Vimentin and matrix metalloproteinase-2(MMP-2),apoptosis-related proteins Caspase-8 and Caspase-3.One-way ANOVA was used for comparison among multiple groups.Results CCK-8 assay results showed that the absorbance values of DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group,DTX group and PBS group were 1.95±0.12,2.33±0.03(t=5.43,P<0.01),2.68±0.09(t=8.42,P<0.01)and 3.49±0.04(t=21.71,P<0.01)respectively.The absorbance value of the DD3-ZD55-SATB1+DTX group was significantly lower than that of the single treatment group.Transwell results showed that the number of cells penetrating the ventricle membrane in the DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group,DTX group and PBS group was 49.25±6.24,79.25±7.41(t=6.19,P<0.05),80.75±4.57(t=8.15,P<0.05)and 118.75±5.38(t=16.88,P<0.01),respectively.The number of ventricular fundus cells in the DD3-ZD55-SATB1+DTX group was significantly less than that in the single treatment group.Hoechst-33258 results showed that the apoptosis rate of DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group,DTX group and PBS group was(55.545.43)%,(41.233.28)%(t=5.29,P<0.01),(35.152.47)%(t=8.19,P<0.01)and(9.622.69)%(t=17.49,P<0.01)respectively.Apoptosis was more pronounced in the DD3-ZD55-SATB1+DTX group than in the viral or chemotherapeutic monotherapy group.Western blotting results showed that the protein expression in PBS group was calculated as 1.00.The relative expression of SATB1 in DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group and DTX group was 0.48±0.04,0.63±0.05 and 0.79±0.04,respectively.The SATB1 expression in the DD3-ZD55-SATB1+DTX group was down-regulated more significantly than that in the monotherapy group,and the difference was statistically significant(t=4.06,P<0.05;t=10.38,P<0.01).The relative expression levels of e-cadherin,Vimentin and MMP-2 proteins in DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group and DTX group were 2.25±0.14,0.48±0.07,0.45±0.04;1.83±0.12,0.69±0.07,0.65±0.05 and 1.60±0.06,0.79±0.04,0.76±0.05,respectively.As compared with the PBS group,the expression of E-cadherin in the treatment group was up-regulated,while the expression of Vimentin and MMP-2 was down-regulated.In the DD3-ZD55-SATB1+DTX group,the expression of E-cadherin was up-regulated and that of Vimentin and MMP-2 was down-regulated more significantly than the monotherapy group,and the difference was statistically significant(t=3.94,3.79,5.48,P<0.05;t=7.43,6.99,8.88,P<0.01).The relative expression levels of apoptosis related proteins Caspase-8 and Caspase-3 in DD3-ZD55-SATB1+DTX group,DD3-ZD55-SATB1 group and DTX group were 2.38±0.14,2.44±0.16;1.84±0.07,1.90±0.05 and 1.70±0.13,1.67±0.07,respectively.The expression of Caspase-8 and Caspase-3 in each treatment group was more significantly upregulated than that in the PBS group,and the difference was statistically significant(t=5.89,5.75,P<0.01;t=6.26,7.91,P<0.01).Conclusion DD3-ZD55-SATB1 combined with DTX can effectively inhibit the proliferation,migration and invasion of LNcap cells and induce cell apoptosis in vitro,and the effect is better than DD3-ZD55-SATB1,ZD55-SATB1 or DTX alone by one of the mechanisms in which epithelial-mesenchymal transition in tumor cells is inhibited and tumor apoptosis is induced by up-regulating Caspase-8 and Caspase-3.