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.

Key words: Castration-resistant prostate cancer, N-myc downstream regulatory gene 1, Enzalumide, Drug resistance, Androgen receptor