Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells

Abstract

Benign prostatic hyperplasia (BPH) is a common disease in the male population, especially in elderly men. Vanillic acid (VA), a dihydroxybenzoic derivative used as a flavoring agent, is reported to have an anti-inflammatory effect. However, there are no reports of its effects on BPH to date. BPH was induced with a pre-4-week treatment of daily subcutaneous injections of testosterone propionate (TP), and the normal control group received injections of ethanol with corn oil instead. Six weeks of further injections were done with (a) ethanol with corn oil, (b) TP only, (c) TP + finasteride, and (d) TP + VA. Finasteride was used as a positive control group. VA had protective effects on the TP-induced BPH. In the VA treatment group, the prostate weight was reduced, and the histological changes including the epithelial thickness and lumen area were restored like in the normal control group. Furthermore, in the VA treatment group, two proliferation related factors, high molecular weight cytokeratin 34βE12 and α smooth muscle actin, were significantly down-regulated compared to the TP-induced BPH group. The expressions of dihydrotestosterone and 5α-reductase, the most crucial factors in BPH development, were suppressed by VA treatment. Expressions of the androgen receptor, estrogen receptor α and steroid receptor coactivator 1 were also significantly inhibited by VA compared to the TP-induced BPH group. In addition, we established an in vitro model for BPH by treating a normal human prostatic epithelial cell line RWPE-1 with TP. VA successfully inhibited proliferation and BPH-related factors in a concentration-dependent manner in this newly established model. These results suggest a new and potential pharmaceutical therapy of VA in the treatment of BPH.

Keywords: 5α-reductase; android receptor; benign prostatic hyperplasia (BPH); estrogen receptor α; vanillic acid.

Figure 1. Effect of VA on prostate weight and prostate index in TP-induced BPH rats

(A) Visual comparisons (upper panels) and area pixel density (lower panels) of the prostate tissues. (B) Total prostate, VP and DLP tissue weight, and (C) prostate indexes of rats. The prostate indexes were calculated dividing prostate weight (mg) by body weight (100 g). ^#P < 0.05 when compared to B; ^*P < 0.05 when compared to C. Total, total prostate; DLP, dorsolateral prostate; VP, ventral prostate; B, normal control group; C, TP-induced BPH group; VA, VA-treated BPH group; Fi, Fi-treated BPH group.

Figure 2. Effect of VA on histological changes of the prostate tissues in TP-induced BPH rats

(A) Representative photomicrograph of H&E stained prostate tissues (magnification ×200), epithelial thickness and relative lumen area of the prostate tissues. Representative photomicrograph and relative density of IHC stained prostate tissues with antibodies against (B) 34βE12 and (C) αSMA (arrows indicate immunostained cells, magnification ×400). ^#P < 0.05 when compared to B; ^*P < 0.05 when compared to C; ^***P < 0.001 when compared to C. B, normal control group; C, TP-induced BPH group; VA, VA-treated BPH group; Fi, Fi-treated BPH group.

Figure 3. Effect of VA on serum DHT and prostatic 5AR-2 in TP-induced BPH rats

(A) Serum ELISA for DHT. (B) Representative western blot bands and normalized relative PSA expression of each group. The differences in protein expressions were normalized to GAPDH. Values are the mean ± S.D. of the data from three or more separate experiments. ^#P < 0.05 when compared to B; ^*P < 0.05 when compared to C; ^**P < 0.01 when compared to C. B, normal control group; C, TP-induced BPH group; VA, VA-treated BPH group; Fi, Fi-treated BPH group.

Figure 4. Immunohistochemical analysis of AR, ERα and SRC1 in the prostate tissues of TP-induced BPH rats

Representative photomicrographs of the immunohistochemically stained prostate tissues (upper panels, magnification ×400) and relative density of the positively immunostained area (lower panels) of AR, ERα and SRC1 of each group. Values are the mean ± S.D. of the data from three or more separate experiments. ^#P < 0.05 when compared to B; ^*P < 0.05 when compared to C. B, normal control group; C, TP-induced BPH group; VA, VA-treated BPH group; Fi, Fi-treated BPH group.

Figure 5. Effect of VA on the protein expressions of AR, ERα and SRC1 in the prostate tissues of TP-induced BPH rats

Representative western blot bands (upper panels) and normalized relative expressions (lower panels) of AR, ERα and SRC1 of each group. The differences in protein expressions were normalized to GAPDH. Values are the mean ± S.D. of the data from three or more separate experiments. ^#P < 0.05 when compared to B; ^*P < 0.05 when compared to C. B, normal control group; C, TP-induced BPH group; VA, VA-treated BPH group; Fi, Fi-treated BPH group.

Figure 6. Effect of TP on proliferation and BPH-related factors in normal human prostatic epithelial RWPE-1 cells

(A) Effect of various concentrations of TP on cell proliferation in RWPE-1 cells. (B) Normalized cell index at the time points 12, 24 and 48 h. Representative photomicrographs of the (C) EdU assay and (D) IF staining of PSA and 5AR-2 (magnification ×200). Values are the mean ± S.D. of the data from three or more separate experiments. ^*P < 0.05 when compared to NC. NC, non-treated RWPE-1 cells.

Figure 7. Effect of VA on TP-induced proliferated RWPE-1 cells

(A) Effect of VA on the cell viability of RWPE-1 cells. (B) Effect of VA on cell proliferation in TP-induced proliferated RWPE-1 cells at the time points of 12 and 24 h. (D) Representative western blot bands (upper panels) and normalized relative expressions (lower panels) of AR, ERα and SRC1 of each group. (E) Effect of VA on Sdr5ar2 mRNA level in TP-induced proliferated RWPE-1 cells. Representative photomicrographs of the (F) EdU assay and (C) IF staining of PSA and 5AR-2 (magnification ×200). The differences in protein expressions were normalized to GAPDH. Values are the mean ± S.D. of data from three or more separate experiments. ^*P < 0.05 when compared to TP. TP, 0.5 μM TP-treated RWPE-1 cells.