Small interfering RNA targeting N-cadherin regulates cell proliferation and migration in enzalutamide-resistant prostate cancer

Abstract

Enzalutamide is one of the options for treating patients with castration-resistant or metastatic prostate cancer. However, a substantial proportion of patients become resistant to enzalutamide after a period of treatment. Cells in these tumors typically exhibit increased proliferative and migratory capabilities, in which N-cadherin (CDH2) appear to serve an important role. In the present study, by up- and downregulating the expression of CDH2, the possible effects of CDH2 on the prostate cancer cell line LNCaP were investigated. Male sex hormone-sensitive LNCaP cells treated with 10 µM enzalutamide were named LNCaP enzalutamide-resistant (EnzaR) cells. Reverse transcription-PCR, western blotting and immunofluorescence staining were used to measure CDH2, E-cadherin, α-SMA, Snail and Slug expression. Transfection with the pCMV-CDH2 plasmid was performed for CDH2 upregulation, whilst transfection with small interfering RNA (siRNA)-CDH2 was performed for CDH2 downregulation. MTT and Cell Counting Kit-4 assays were used to evaluate the proportion of viable cancer cells. Subsequently, gap closure assay was performed to evaluate the migratory capability of both LNCaP and LNCaP EnzaR cell lines. CDH2 expression was found to be increased in LNCaP EnzaR cells compared with that in LNCaP cells. CDH2 overexpression increased cell viability and migration in both LNCaP and LNCaP EnzaR cell lines. By contrast, the opposite trend was observed after CDH2 expression was knocked down. CDH2 expression also showed a high association with that of four epithelial-mesenchymal transition markers, which was confirmed by western blotting. Based on these results, it was concluded that knocking down CDH2 expression using siRNA transfection mediated significant influence on LNCaP EnzaR cell physiology, which may be a potential therapeutic option for prostate cancer treatment.

Keywords: N-cadherin; enzalutamide resistance; migration; proliferation; prostate cancer.

Figure 1.

Expression of CDH2 in human PCa cell lines. CDH2 expression was higher in the LNCaP EnzaR cell line compared with that in the LNCaP cell line. (A) RT-PCR analysis of total RNA isolated from LNCaP and LNCaP EnzaR cells. GAPDH served as a loading control. (B) Protein expression of CDH2 in LNCaP and LNCaP EnzaR cells. β-actin served as a loading control. (C) Immunofluorescence assay was used to measure CDH2 expression in LNCaP and LNCaP EnzaR cells. Scale bars, 20 µm. CDH2, N-cadherin; PCa, prostate cancer; EnzaR, enzalutamide-resistant; RT, reverse transcription.

Figure 2.

Overexpression of CDH2 increases LNCaP and LNCaP EnzaR cell viability. The OD values are expressed as a percentage of the total number of cells. (A) Cell viability increased significantly in both cell lines overexpressing CDH2 as observed by MTT assay. (B) LNCaP and LNCaP EnzaR cells overexpressing CDH2 also showed increased cell viability. *P<0.05 vs. Control and ^#P<0.05 vs. pCMV-GFP. CDH2, N-cadherin; PCa, prostate cancer; EnzaR, enzalutamide-resistant; GFP, green fluorescent protein.

Figure 3.

CDH2 overexpression increases LNCaP and LNCaP EnzaR cell migration. Ibidi insert gap closure assays were performed in (A) LNCaP and (B) LNCaP EnzaR cells, (C) which were also quantified. The initial 0 h area was used as a 100% control. Scale bars, 50 µm. *P<0.05 vs. Control; and ^#P<0.05 vs. pCMV-GFP. CDH2, N-cadherin; EnzaR, enzalutamide-resistant; GFP, green fluorescent protein.

Figure 4.

Measurement of EMT markers in LNCaP and LNCaP EnzaR cells overexpressing CDH2. Western blot analysis measuring E-cadherin, α-SMA, Snail and slug expression in (A) LNCaP cells and (B) LNCaP EnzaR cells. Transfection with pCMV-CDH2 may increase EMT but not in cells transfected with pCMV-GFP. (C) The expression of E-cadherin was significantly decreased whereas that of α-SMA, Snail and Slug was significantly increased in both cell lines overexpressing CDH2 compared with those in cells transfected with pCMV-GFP. *P<0.05 vs. Control; and ^#P<0.05 vs. pCMV-GFP. EMT, epithelial-mesenchymal transition; CDH2, N-cadherin; α-SMA, α-smooth muscle actin; EnzaR, enzalutamide-resistant; GFP, green fluorescent protein.

Figure 5.

CDH2 knockdown reduces LNCaP and LNCaP EnzaR cell viability. The OD values of MTT and CCK-8 assays are expressed as percentages of the total number of cells. (A) After transfection with siRNA-CDH2, LNCaP cell viability was significantly decreased as according to results from MTT assay. (B) Cell viability was also reduced after transfection with siRNA-CDH2 according to results from CCK-8 assays. *P<0.05 vs. Control; and ^#P<0.05 vs. siRNA-control. CDH2, N-cadherin; CCK-8, cell counting kit-8; siRNA, small interfering RNA; EnzaR, enzalutamide-resistant.

Figure 6.

Knocking down CDH2 expression inhibits LNCaP and LNCaP EnzaR cell migration. After transfection with siRNA-CDH2, images were obtained at 0 and 24 h after wounding. Ibidi insert gap closure assay was performed in (A) LNCaP and (B) LNCaP EnzaR cells, (C) which were then quantified. The 0 h area was used as a 100% control. Scale bars, 50 µm. *P<0.05 vs. Control; and ^#P<0.05 vs. siRNA-control. CDH2, N-cadherin; siRNA, small interfering RNA; EnzaR, enzalutamide-resistant.

Figure 7.

Measurement of epithelial-mesenchymal transition markers in PCa cells with CDH2 knockdown. Western blot analysis measuring E-cadherin, α-SMA, Snail and slug expression in (A) LNCaP cells and (B) LNCaP EnzaR cells, (C) which were then quantified. α-SMA, Snail and slug protein expression in cells transfected with siRNA-CDH2 was reduced, whereas E-cadherin expression was increased compared with that in cells transfected with siRNA-control or the control group. *P<0.05 vs. Control; and ^#P<0.05 vs. siRNA-control. PCa, prostate cancer; CDH2, N-cadherin; α-SMA, α-smooth muscle actin; EnzaR, enzalutamide-resistant; siRNA, small interfering RNA