Evaluation of the Antioxidant and Antiangiogenic Activity of a Pomegranate Extract in BPH-1 Prostate Epithelial Cells

Abstract

Benign prostatic hypertrophy (BPH) is a noncancerous enlargement of the prostate gland that develops from hyper-proliferation of the stromal and epithelium region. Activation of pathways involving inflammation and oxidative stress can contribute to cell proliferation in BPH and tumorigenesis. Agricultural-waste-derived extracts have drawn the attention of researchers as they represent a valid and sustainable way to exploit waste production. Indeed, such extracts are rich in bioactive compounds and can provide health-promoting effects. In particular, extracts obtained from pomegranate wastes and by-products have been shown to exert antioxidant and anti-inflammatory effects. This study focused on the evaluation of the anti-angiogenic effects and chemopreventive action of a pomegranate extract (PWE) in cellular models of BPH. In our experimental conditions, we observed that PWE was able to significantly (p < 0.001) reduce the proliferation and migration rates (up to 60%), together with the clonogenic capacity of BPH-1 cells concomitantly with the reduction in inflammatory cytokines (e.g., IL-6, PGE2) and pro-angiogenic factor (VEGF-ADMA) release. Additionally, we demonstrated the ability of PWE in reducing angiogenesis in an in vitro model of BPH consisting in transferring BPH-1-cell-conditioned media to human endothelial H5V cells. Indeed, PWE was able to reduce tube formation in H5V cells through VEGF level reduction even at low concentrations. Overall, we confirmed that inhibition of angiogenesis may be an alternative therapeutic option to prevent neovascularization in prostate tissue with BPH and its transformation into malignant prostate cancer.

Keywords: BPH-1 cells; angiogenesis; bioactive compounds; by-products; oxidative stress; pomegranate.

Figure 1

HPLC profile of the pomegranate extract (PWE) detected at 378 nm. For the specific identification of the peaks, please refer to Table 1.

Figure 2

Assessment of PWE antioxidant activity using cell-free methods (A,B). Results are expressed as mean ± SEM.

Figure 3

Evaluation of PWE effect on BPH-1 cell viability (A). Measurement of RSH intracellular content (B). Representative images of single-cell clone proliferation, stained with crystal violet and colony quantification (C,D). Evaluation of HO-1 and TIGAR expression in BPH-1 cells treated with different concentrations of PWE (E–G). Results are expressed as mean ± SEM. (* p < 0.05; *** p < 0.0005 vs. CTRL).

Figure 4

PWE effect on cell migration rate at 24 and 48 h (A,B). Image magnification is 1000 μm. Results are expressed as mean ± SEM. (^### p < 0.0005 vs. CTRL 24 h; *** p < 0.0005 vs. CTRL 24 h, CTRL 48 h).

Figure 5

Evaluation of IL-6 (A), VEGF (C), and PGE2 (D) levels in BPH-1 cells after PWE treatment. Assessment of iNOS expression in BPH-1 cells following PWE treatment (B). iNOS levels are expressed as % of control (185.13 pg/mg prot). Results are expressed as mean ± SEM. (*** p < 0.0005 vs. CTRL).

Figure 6

Assessment of cell viability after PWE treatment in H5V cell culture at 24 h (A) and 48 h (B). Effect of CM collected from BPH-1 cells and PWE at different concentration on H5V viability after 24 h (C) and 48 h (D). The results are expressed as mean ± SEM. (*** p < 0.0005 vs. PWE 0).

Figure 7

Tube formation in H5V culture after incubation with conditioned media (CM) collected from BPH-1 cells and added with different concentrations of PWE (image magnification 400 μm) (A,B). Measurement of PGE2 (C), VEGF (D), and nitrite/nitrate levels (E) in H5V treated with CM from BPH-1 cells and PWE at different concentrations. The results are expressed as mean ± SEM. (^### p < 0.0005 vs. H5V control; * p < 0.05; ** p < 0.005; *** p < 0.0005 vs. PWE 0).

Figure 8

Graphic representation of BPH-1—H5V cells crosstalk and effect of PWE on inflammation and angiogenic processes.