Chemopreventive effects of angiotensin II receptor type 2 agonist on prostate carcinogenesis by the down-regulation of the androgen receptor

Abstract

We recently reported that angiotensin II receptor blockers (ARBs) have chemopreventive and chemotherapeutic potential against prostate cancer via the reduction of androgen receptor (AR) expression. In this study, we investigated the effects of the angiotensin II receptor type 2 (AT2R) agonist Compound 21 (C21), which is expected to play similar roles to an ARB, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model previously established in our laboratory. In vitro analyses of the cell growth, Western blotting and reporter gene assays were performed using LNCaP cells. TRAP rats at 6 weeks of age were randomly divided into 3 groups of 12 animals each and treated with C21 at 1 or 2 mg/kg/day in drinking water for 12 weeks. C21 reduced the proliferation activity of prostate cancer cells and down-regulated the PSA promoter activity and the AR protein expression. We discovered that C21 inhibited the progression of prostate carcinogenesis in TRAP rats and decreased the incidence of adenocarcinoma in the lateral prostate. A significant increase in the apoptotic index with activation of caspase 3 and 7 were observed by immunohistochemistry and Western blotting analyses. C21 also down-regulated the expression of AR significantly in TRAP rat prostate. C21 decreased the expression of AR and reduced the proliferation activity effectively in prostate cancer cells and TRAP rat prostate. These findings suggest that AT2R agonist may be a candidate novel chemopreventive agent against human prostate cancer.

Keywords: RAS; angiotensin II receptor type 2; compound 21; prostate cancer.

Figure 1. C21 suppressed cell proliferation and down-regulated the expression of AR in LNCaP and 22RV1 cells

(A) An MTT assay was performed, and the cells were counted after daily treatment with 100 nM, 1 or 10 µM of C21. LNCaP or 22RV1, 3 × 10³ cells/well were seeded in a 96-well plate, and all experiments were repeated 3 times. Data are presented as means ± SD, n = 6 per group, ^*P < 0.05, ^**P < 0.005, ^***P < 0.0005 vs. the control group. (B) A luciferase reporter assay was performed. LNCaP and 22RV1 cells, 10³ cells/well were seeded in a 24-well plate. The cells were treated with 1 or 10 µM of C21 with or without 1 nM of DHT, and all experiments were repeated 3 times. Data are presented as means ± SD, ^*P < 0.05, ^**P < 0.005, ^***P < 0.0005 vs. the control group. (C) A Western blot assay of AR and β-actin in LNCaP cells. LNCaP cells were incubated with 10 µM of C21. The data are representative of three independent experiments. (D) The intensity of the Western blot band was measured using the ImageJ software program and normalized to β-actin.

Figure 1. C21 suppressed cell proliferation and down-regulated the expression of AR in LNCaP and 22RV1 cells

(A) An MTT assay was performed, and the cells were counted after daily treatment with 100 nM, 1 or 10 µM of C21. LNCaP or 22RV1, 3 × 10³ cells/well were seeded in a 96-well plate, and all experiments were repeated 3 times. Data are presented as means ± SD, n = 6 per group, ^*P < 0.05, ^**P < 0.005, ^***P < 0.0005 vs. the control group. (B) A luciferase reporter assay was performed. LNCaP and 22RV1 cells, 10³ cells/well were seeded in a 24-well plate. The cells were treated with 1 or 10 µM of C21 with or without 1 nM of DHT, and all experiments were repeated 3 times. Data are presented as means ± SD, ^*P < 0.05, ^**P < 0.005, ^***P < 0.0005 vs. the control group. (C) A Western blot assay of AR and β-actin in LNCaP cells. LNCaP cells were incubated with 10 µM of C21. The data are representative of three independent experiments. (D) The intensity of the Western blot band was measured using the ImageJ software program and normalized to β-actin.

Figure 2. AT2R agonist supplementation suppressed the progression of prostate carcinogenesis and induced apoptosis in the TRAP model

(A) Representative histological findings of VP and LP of TRAP. Representative histological findings of adenocarcinoma (arrow), HG-PIN (arrowhead), and LG-PIN (asterisk). (B, C) Labeling indices for Ki67- (B) and TUNEL- (C) positive cells in VP and LP. Hematoxylin was used as a nuclear counterstain. Data are presented in a box plot, n = 12 per group, ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001 vs. the control group. (D, E) A Western blotting analysis for Erk 1/2, phospho-Erk 1/2, p38 Mapk, phosphor-p38 Mapk, Cyclin D1, caspase 3 and 7, cleaved (cl)-caspase 3 and 7, and β-actin in VP (D) and LP (E) of TRAP. Western blotting was repeated at least three times, and each lane represents an individual rat (n = 4 per group).

Figure 3. AT2R agonist down-regulated AR signaling

(A) Immunohistochemical staining for AR and SV40 T antigen in the prostate lobes of TRAP rats. Representative photographs for each staining of LP (left). Hematoxylin was used as a nuclear counterstain. The percentages of positive cells for each staining (right). Data are presented as the mean ± SD, n = 12 per group, ^**P < 0.01, ^***P < 0.001 vs. the control group. (B) A Western blotting analysis for AR, SV40 T antigen, and β-actin in VP and LP of TRAP. The intensity of each band was measured and normalized by β-actin. Western blotting was repeated at least three times, and each lane represents an individual rat. Data are presented the relative value for a Control (Ventral: lane 3, Lateral: lane 2) as the mean ± SD, n = 4 per group. (C, D) A quantitative RT-PCR analysis for AR (C), AT1R, and AT2R (D) in LP of TRAP rats. The average results were calculated from five rats per group. Each PCR was repeated 4 times. The data was normalized with Gapdh as an endogenous control gene. (E) RT-PCR array analysis of the effect of C21 on the expression of 84 androgen-regulated genes in LP of TRAP rats. RNA samples from all rats in the Control (n = 12) and the 2 mg/kg/day C21 (n = 12) groups were analyzed. The data was normalized to the Actb gene. Top left, scatter plot: Green dots represent genes that were down-regulated. The center line indicates no changed in gene expression, while the boundary dotted lines represent a 2-fold change in expression. Top right, clustergram: non-supervised hierarchical clustering of the entire data set to display a heat map indicating gene alteration across groups (83 out of every 84 genes). Red shows relatively high expression level (n = 7), green shows relatively low expression level (n = 76) as compared with the other group. Bottom, List of genes down-regulated more than double by C21 treatment.

Figure 3. AT2R agonist down-regulated AR signaling

(A) Immunohistochemical staining for AR and SV40 T antigen in the prostate lobes of TRAP rats. Representative photographs for each staining of LP (left). Hematoxylin was used as a nuclear counterstain. The percentages of positive cells for each staining (right). Data are presented as the mean ± SD, n = 12 per group, ^**P < 0.01, ^***P < 0.001 vs. the control group. (B) A Western blotting analysis for AR, SV40 T antigen, and β-actin in VP and LP of TRAP. The intensity of each band was measured and normalized by β-actin. Western blotting was repeated at least three times, and each lane represents an individual rat. Data are presented the relative value for a Control (Ventral: lane 3, Lateral: lane 2) as the mean ± SD, n = 4 per group. (C, D) A quantitative RT-PCR analysis for AR (C), AT1R, and AT2R (D) in LP of TRAP rats. The average results were calculated from five rats per group. Each PCR was repeated 4 times. The data was normalized with Gapdh as an endogenous control gene. (E) RT-PCR array analysis of the effect of C21 on the expression of 84 androgen-regulated genes in LP of TRAP rats. RNA samples from all rats in the Control (n = 12) and the 2 mg/kg/day C21 (n = 12) groups were analyzed. The data was normalized to the Actb gene. Top left, scatter plot: Green dots represent genes that were down-regulated. The center line indicates no changed in gene expression, while the boundary dotted lines represent a 2-fold change in expression. Top right, clustergram: non-supervised hierarchical clustering of the entire data set to display a heat map indicating gene alteration across groups (83 out of every 84 genes). Red shows relatively high expression level (n = 7), green shows relatively low expression level (n = 76) as compared with the other group. Bottom, List of genes down-regulated more than double by C21 treatment.