NDRG1过表达对去势抵抗性前列腺癌耐药细胞株C4-2/ENZA耐药性的影响及其机制

Effect of over-expression of NDRG1 on resistance of castration-resistant prostate cancer resistant cell line C4-2/ENZA and its mechanism

摘要

目的 探讨N-myc下游调节基因1（NDRG1）对去势抵抗性前列腺癌（CRPC）恩杂鲁胺（ENZA）耐药的影响，并阐明其作用机制。 方法 体外培养人CRPC C4-2细胞和ENZA耐药株C4-2/ENZA细胞，采用实时荧光定量PCR（RT-qPCR）法检测C4-2/ENZA细胞及其亲本C4-2细胞中NDRG1 mRNA表达水平，Western blotting法检测C4-2/ENZA细胞及其亲本C4-2细胞中NDRG1、雄激素受体（AR）和前列腺特异性抗原（PSA）蛋白表达水平，以验证细胞转染效率。将C4-2/ENZA细胞分为空白组（正常培养不进行处理）、阴性对照慢病毒（Lv-NC）组（转染Lv-NC）、Lv-NDRG1 组（转染Lv-NDRG1）、 Lv-NC+ENZA 组（转染 Lv-NC 后加入 ENZA 处理）、Lv-NDRG1+ENZA组（转染Lv-NDRG1后加入ENZA处理）、Lv-NDRG1+表皮生长因子（EGF）组（转染Lv-NDRG1后加入EGF处理）和Lv-NDRG1+EGF+ENZA组（转染Lv-NDRG1后加入EGF和ENZA处理）。采用噻唑蓝（MTT）法检测各组细胞半数抑制浓度（IC₅₀）、耐药指数（RI）和细胞增殖活性，流式细胞术检测各组细胞凋亡率，RT-qPCR法检测各组细胞中NDRG1 mRNA表达水平，Western blotting法检测各组细胞中NDRG1、AR、第213位丝氨酸磷酸化雄激素受体（p-ARSer²¹³）、第81位丝氨酸磷酸化雄激素受体（p-ARSer⁸¹ ）和PSA蛋白表达水平。 结果 与C4-2细胞比较，C4-2/ENZA细胞中NDRG1 mRNA和蛋白表达水平均明显降低（P<0.01），AR和PSA蛋白表达水平明显升高（P<0.01），提示ENZA耐药株C4-2/ENZA中NDRG1低表达；与Lv-NC组比较，Lv-NDRG1组细胞中NDRG1 mRNA和蛋白表达水平均明显升高（P<0.01），提示成功构建NDRG1基因过表达C4-2/ENZA耐药细胞株。MTT法，与C4-2细胞比较，C4-2/ENZA细胞IC₅₀明显升高（P<0.01），RI为17.78；与Lv-NC组比较，Lv-NDRG1组C4-2/ENZA细胞IC₅₀明显降低（P<0.01）。EGF处理24 h，与Lv-NC组比较，Lv-NC+EGF组C4-2/ENZA细胞IC₅₀明显升高（P<0.01）；与Lv-NDRG1组比较，Lv-NDRG1+EGF组C4-2/ENZA细胞IC₅₀明显升高（P<0.01）。与ENZA处理前比较，不同浓度ENZA处理24 h，C4-2和C4-2/ENZA细胞增殖活性均逐渐降低（F=223.80，P<0.01；F=81.46，P<0.01）。ENZA处理24 h，与Lv-NC组比较， Lv-NDRG1组C4-2/ENZA细胞增殖活性明显降低（P<0.01）。EGF处理24 h，与Lv-NC组比较， Lv-NC+EGF组C4-2/ENZA细胞增殖活性明显升高（P<0.01）， Lv-NDRG1+EGF组C4-2/ENZA细胞增殖活性明显降低（P<0.01）。选择10.000 μmol·L^－1 ENZA和干预24 h作为后续检测浓度及时间点。流式细胞术，ENZA处理24 h，与Lv-NC组比较，Lv-NDRG1组细胞凋亡率明显升高（P<0.01）；与Lv-NC+ENZA组比较，Lv-NDRG1+ENZA组细胞凋亡率明显升高（P<0.01）。EGF处理24 h，与Lv-NDRG1组比较，Lv-NDRG1+EGF组细胞凋亡率明显降低（P<0.01），Lv-NDRG1+ENZA组细胞凋亡率明显升高（P<0.01）；与Lv-NDRG1+ENZA组比较，Lv-NDRG1+EGF+ENZA组细胞凋亡率明显降低（P<0.01）。Western blotting法，ENZA处理24 h，与Lv-NC组比较，Lv-NDRG1组细胞中AR和PSA蛋白表达水平及p-ARSer²¹³/AR和p-ARSer⁸¹/AR比值均明显降低（P<0.05或P<0.01）。EGF处理24 h，与Lv-NC组比较， Lv-NC+EGF组细胞中AR和PSA蛋白表达水平及p-ARSer²¹³/AR和p-ARSer⁸¹/AR比值均明显升高（P<0.05或P<0.01）；与Lv-NDRG1组比较，Lv-NDRG1+EGF组细胞中AR和PSA蛋白表达水平及p-ARSer²¹³/AR和p-ARSer⁸¹/AR比值均明显升高（P<0.01）。 结论 NDRG1过表达可降低CRPC对ENZA的耐药性，其作用机制可能与抑制AR信号转导有关。

Abstract

Objective To discuss the effect of N-myc downstream-regulated gene 1 （NDRG1） on the enzalutamide （ENZA） resistance in the castration-resistant prostate cancer （CRPC）， and to clarify its mechanism. Methods The human CRPC C4-2 cells and ENZA-resistant strain C4-2/ENZA cells were cultured in vitro. The expression levels of NDRG1 mRNA in the C4-2/ENZA cells and their parental C4-2 cells were detected by real-time fluorescence quantitative PCR （RT-qPCR） method. The expression levels of NDRG1， androgen receptor （AR）， and prostate-specific antigen （PSA） proteins in the cells were detected by Western blotting method to verify the transfection efficiency of the cells. The C4-2/ENZA cells were divided into blank group （normally cultured without treatment）， negative control lentivirus （Lv-NC） group （transfected with Lv-NC）， Lv-NDRG1 group （transfected with Lv-NDRG1）， Lv-NC+ENZA group （transfected with Lv-NC followed by ENZA treatment）， Lv-NDRG1+ENZA group （transfected with Lv-NDRG1 followed by ENZA treatment）， Lv-NDRG1+epidermal growth factor （EGF） group （transfected with Lv-NDRG1 followed by EGF treatment）， and Lv-NDRG1+EGF+ENZA group （transfected with Lv-NDRG1 followed by EGF and ENZA treatment）. The half-maximal inhibitory concentration （IC₅₀）， resistance index （RI）， and proliferation activity of the cells were detected by MTT assay；the apoptotic rates of the cells in various groups were detected by flow cytometry； RT-qPCR method was used to detect the expression levels of NDRG1 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of NDRG1， AR， phosphorylated AR at serine²¹³ （p-ARSer²¹³）， phosphorylated AR at serine⁸¹ （p-AR^Ser81）， and PSA proteins in the cells in various groups. Results Compared with C4-2 cells， the expression levels of NDRG1 mRNA and protein in the C4-2/ENZA cells were significantly decreased （P<0.01） and the expression levels of AR and PSA proteins were increased （P<0.01）， indicating low expression of NDRG1 in the ENZA-resistant C4-2/ENZA strain. Compared with Lv-NC group， the expression levels of NDRG1 mRNA and protein in the cells in Lv-NDRG1 group were significantly increased （P<0.01）， indicating the successful construction of an NDRG1 gene over-expression strain of C4-2/ENZA resistant cells. The MTT assay results showed that compared with the C4-2 cells，the IC₅₀ of the C4-2/ENZA cells was increased （P<0.01） and the RI was 17.78； compared with Lv-NC group， the IC₅₀ of the C4-2/ENZA cells in Lv-NDRG1 group was decreased （P<0.01）. After 24 h of EGF treatment， compared with Lv-NC group， the IC₅₀ of the C4-2/ENZA cells in Lv-NC+EGF group was significantly increased （P<0.01）； compared with Lv-NDRG1 group， the IC₅₀ of the C4-2/ENZA cells in Lv-NDRG1+EGF group was increased （P<0.01）. Compared with before ENZA treatment， after 24 h of ENZA treatment， the proliferation activities of C4-2 and C4-2/ENZA cells were gradually decreased （F=223.80， P<0.01； F=81.46， P<0.01）. Compared with Lv-NC group， the proliferation activity in the C4-2/ENZA cells in Lv-NDRG1 group after 24 h of ENZA treatment was significantly decreased （P<0.01）. After 24 h of EGF treatment， compared with Lv-NC group， the proliferation activity of the C4-2/ENZA cells in Lv-NC+EGF group was significantly increased （P<0.01）， while the the proliferation activity of the C4-2/ENZA cells in Lv-NDRG1+EGF group was significantly decreased （P<0.01）. The chosen concentration and treatment duration for further testing were 10 000 μmol·L^-1 ENZA and the intervention time was 24 h. The flow cytometry results showed that after 24 h of ENZA treatment， compared with Lv-NC group， the apoptotic rate of the cells in Lv-NDRG1 group was significantly increased （P<0.01）； compared with Lv-NC+ENZA group，the apoptotic rate of the cells in Lv-NDRG1+ENZA group was significantly increased （P<0.01）. After 24 h of EGF treatment， compared with Lv-NDRG1 group， the apoptotic rate of the cells in Lv-NDRG1+EGF group was significantly decreased （P<0.01）， while the apoptotic rate of the cells in Lv-NDRG1+ENZA group was significantly increased （P<0.01）； compared with Lv-NDRG1+ENZA group， the apoptotic rate of the cells in Lv-NDRG1+EGF+ENZA group was significantly decreased （P<0.01）. The Western blotting results showed that after 24 h of ENZA treatment， compared with Lv-NC group， the expression levels of AR and PSA proteins and the ratio of p-ARSer²¹³/AR and p-ARSer⁸¹/AR in the cells in Lv-NDRG1 group were significantly decreased （P<0.05 or P<0.01）. After 24 h of EGF treatment， compared with Lv-NC group， the expression levels of AR and PSA proteins and the ratio of p-ARSer²¹³/AR and p-ARSer⁸¹/AR in the cells in Lv-NC+EGF group were significantly increased （P<0.05 or P<0.01）； compared with Lv-NDRG1 group， the expression levels of AR and PSA proteins and the ratio of p-ARSer²¹³/AR and p-ARSer⁸¹/AR in the cells in Lv-NDRG1+EGF group were significantly increased （P<0.01）. Conclusion Over-expression of NDRG1 can reduce the resistance of CRPC to ENZA， and its mechanism may be related to the inhibition of AR signaling.

关键词

去势抵抗性前列腺癌 / N-myc下游调节基因1 / 恩杂鲁胺 / 耐药性 / 雄激素受体

Key words

Castration-resistant prostate cancer / N-myc downstream regulatory gene 1 / Enzalumide / Drug resistance / Androgen receptor

Author summay

张 鹰（1987-），男，湖南省衡阳市人，主治医师，医学硕士，主要从事重症医学基础和临床方面的研究。