Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.

pressure, and total ICP (area under the curve). Organ-bath studies were performed on corpus cavernosum smooth muscle (CCSM) strips. Nitric oxide synthase (NOS) expression was determined immunohistochemically (IHC) for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. RESULTSRats with PBOO showed lower erectile responses than controls. Maximum electrical field stimulation-mediated and endothelium-dependent acetylcholineinduced relaxations and contractile responses to phenylephrine were significantly reduced in CCSM strips from the PBOO group. The NO donor sodium nitroprusside completely relaxed CCSM from rats in all groups. IHC analyses showed decreased expression of nNOS in PBOO groups compared with controls; by contrast, protein expression of eNOS and iNOS was increased. Alfuzosin-treatment partially attenuated functional and molecular changes in penises of PBOO rats. CONCLUSIONRats with PBOO show ED, most likely due to altered NOS expression and NO bioavailability. The α -adrenoreceptor antagonist alfuzosin reversed this ED by altering sympathetic tone, increasing NOinduced relaxation and augmenting blood flow in the penis. This study suggests a rationale for further clinical trials using combinations of α -adrenoceptor antagonists and phosphodiesterase-5 inhibitors in patients with ED and lower urinary tract symptoms. KEYWORDSrat model, partial bladder outlet obstruction, nitric oxide synthase, alfuzosin, erectile function Study Type -Therapy (RCT) Level of Evidence 1b OBJECTIVETo determine how partial bladder outlet obstruction (PBOO) in a rat model affects erectile function, and whether an uroselective α 1-adrenoceptor antagonist, alfuzosin (Sanofi-Aventis, Paris, France) attenuates any erectile dysfunction (ED). MATERIALS AND METHODSAdult male Sprague-Dawley rats (120) were randomized into four groups: 1, shamoperated; 2, alfuzosin-treated; 3, PBOO; and 4, alfuzosin-treated with PBOO. Groups 3 and 4 were subjected to PBOO for 6 weeks by ligation of the urethra, while groups 2 and 4 rats received daily oral alfuzosin (10 mg/day) for 6 weeks. In vivo erectile responses were monitored by evaluating ratios of intracavernosal pressure (ICP)/mean arterial

Thymoquinone (TQ) a well known anti‐oxidant is an active ingredient of black seed oil. It has been shown to inhibit growth of prostate cancer (PCa) cells by inhibiting expression of androgen receptor (AR) and E2F‐1 in parental LNCaP cells and its isogenic variant, hormone independent AR expressing C4‐2B cells. However, the precise mechanism of action of TQ remains unexplored, especially in AR naïve (PC‐3) cells as models of hormone refractory disease. Our results demonstrated that TQ inhibits growth of both androgen receptor positive (C4‐2B) and androgen receptor naïve (PC‐3) PCa cells with an IC50 value of 80 and 50 µM at 48 hr, respectively. TQ (100 µM) induced a 3 and 4 fold increase in ROS generation and a decrease in GSH levels by 35% and 45% in C4‐2B and PC‐3 cells, respectively. Treatment of PCa cells with TQ did not activate caspases but resulted in an increase in the levels of apoptosis inducing factor (AIF) indicating a caspase‐independent cell death mechanism. Treatment of PC‐3 cells with TQ (50 µM) up‐regulated GADD45α and down‐regulated BCL‐2 (2 fold) and BAG‐1 (1.5 fold). However, TQ‐induced inhibition of the expression and transactivational activity of AR in C4‐2B cells was not restored upon simultaneous co‐exposure with N‐acetylcysteine (NAC). In both cell types, (NAC) completely inhibited TQ‐induced ROS generation and protected the cells from cytotoxicity suggesting that ROS generation by TQ was primarily responsible for growth inhibition.