Effect of over-expression of NDRG1 on resistance of castration-resistant prostate cancer resistant cell line C4-2/ENZA and its mechanism
10.13481/j.1671-587X.20240315
- VernacularTitle: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
- Author:
Ying ZHANG
1
;
Zhaohui WAN
;
Xianxun JIANG
Author Information
1. 南华大学衡阳医学院附属第二医院重症医学科,湖南 衡阳 421001
- Keywords:
Castration-resistant prostate cancer;
N-myc downstream regulatory gene 1;
Enzalumide;
Drug resistance;
Androgen receptor
- From:
Journal of Jilin University(Medicine Edition)
2024;50(3):708-717
- CountryChina
- Language:Chinese
-
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-ARSer81),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.
