Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia

Abstract

Background and purpose: Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid-based 'dual inhibitor' of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH.

Experimental approach: Rats were treated daily with testosterone propionate (3 mg·kg(-1) s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg(-1) , i.p.) or flavocoxid (20 mg·kg(-1) , i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells.

Key results: Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE(2) and leukotriene B(4) (LTB(4) ), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth.

Conclusion and implications: Our results show that a 'dual inhibitor' of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism.

Figure 1

A. Sham prostate from vehicle-treated rats showing regular acini with cuboidal and low cylindrical epithelium with round nuclei showing basal alignment. Fine stroma and a continuous basal layer are also noted. Haematoxylin and eosin staining; original magnification, ×20. B. Sham prostate from flavocoxid-treated rats showing regular architecture comparable to sham animals treated with vehicle. Haematoxylin and eosin staining; original magnification, ×20. C. BPH prostate showing irregular acinar shape with papillary projection into the lumen and foci of piling-up hyperplastic nodules are evident. The epithelium is high cylindrical, multilayered and round/ovoid nuclei are irregularly aligned. There are abundant stroma and a focal interruption of basal layer surrounding the acini. Haematoxylin and eosin staining; original magnification, ×20. D. BPH prostate treated with flavocoxid displaying histological features of normal prostate structure. Haematoxylin and eosin staining; original magnification, ×20.

Figure 2

Western blot analysis of COX-2 (A) and 5-LOX (B) in prostate from sham BPH and BPH rats treated with either vehicle or flavocoxid. PGE₂ (C) and LTB₄ (D) levels in homogenates of samples of prostate from sham BPH and BPH rats treated with either vehicle or flavocoxid. §P < 0.01 significantly different from Sham-BPH; P < 0.05 significantly different from BPH.

Figure 3

Expression of mRNA for Bax (A), Bcl-2 (B), EGF (C) and VEGF (D) in samples of prostate from sham-BPH and BPH rats treated with either vehicle or flavocoxid. §P < 0.01 significantly different from Sham-BPH; *P < 0.05 significantly different from BPH.

Figure 4

Western blot analysis for caspase-9 (A), BAX (B) and Bcl-2 (C) in samples of prostate from sham BPH and BPH rats treated with either vehicle or flavocoxid. §P < 0.01 significantly different from Sham-BPH; P < 0.05 significantly different from BPH.

Figure 5

Effect of incubation with flavocoxid (256-1000 µg mL^-1) on PC3 cell cultures. The graphs represent cell viability (A) and cell growth, as number of live cells in each field (B). *P < 0.01 significantly different from CTRL.

Figure 6

Bax (A), Bcl-2 (B), mRNA and BAX, Bcl-2 and caspase-9 protein (C, D and E, respectively) expression in PC3 cells treated with vehicle, flavocoxid (500 µg·mL⁻¹) or the pan-caspase inhibitor Z-VAD-FMK and flavocoxid. *P < 0.01 significantly different from CTRL.

Figure 7

Annexin V (A), PARP (B) and cytochrome c protein (C) expression in PC3 cells treated with vehicle, flavocoxid (500 µg ml⁻¹) or the pan-caspase inhibitor Z-VAD-FMK and flavocoxid. *P < 0.01 significantly different from CTRL.

Figure 8

Expression of mRNA for EGF (A) and VEGF (B) in PC3 cells treated with vehicle or flavocoxid (500 µg·mL⁻¹) *P < 0.01 significantly different from CTRL

Figure 9

The effect of flavocoxid on specific markers of apoptosis is shown by ↓ (decrease) and ↑ (increase). Inducible NOS (iNOS); inhibitor of caspase-3-activated DNase (ICAD) is a caspase-3 substrate; caspase-3-activated DNase; heat shock factor 1 (HSF1); heat shock element (HSE); Bcl-2–associated X protein(Bax); B-cell lymphoma 2(Bcl-2).