Effects of Resveratrol on Benign Prostatic Hyperplasia by the

(1) BPH is a common disease in elderly men, with histological evidence of BPH in ..... G.; Pourreza , F.; Asgari , S. A.; Kamran , A. N. Urology 2008,...
3 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/jnp

Effects of Resveratrol on Benign Prostatic Hyperplasia by the Regulation of Inflammatory and Apoptotic Proteins Kyung-Sook Chung, Se-Yun Cheon, and Hyo-Jin An* Department of Pharmacology, College of Oriental Medicine, Sangji University, Gangwon-do 220-702, South Korea ABSTRACT: Resveratrol (1) is a natural polyphenolic compound that has cardioprotective, anticancer, and antiinflammatory properties. Although diverse biological studies of compound 1 have been conducted, no antiproliferative effects of 1 have been reported in benign prostatic hyperplasia (BPH). BPH is a progressive disease related to inflammation and an imbalance in cell growth and apoptosis. The aims of this study were to determine whether 1suppressed BPH progression in rats and to explore the underlying mechanisms related to regulation of inflammation and apoptosis. Compound 1 treatment decreased prostate weight and cell proliferation in this animal model and markedly decreased BPH-related upregulation of iNOS and COX-2 protein expression. In addition, 1 induced Bax expression and suppressed Bcl-2 and Bcl-xL expressions. Furthermore, 1 triggered caspase-3 activation and decreased levels of its substrate, PARP-1. These results suggested that 1 produced an antiproliferative effect by regulating the expression levels of proteins involved in inflammation and apoptosis during BPH.

B

cell growth and in the maintenance of tissue homeostasis.9 This form of programmed cell death comprises a series of molecular steps that culminate in the clearance of impaired and altered cells, while avoiding leakage of deleterious substances into the surrounding tissues.10 In mammalian cells, apoptosis proceeds via extrinsic and intrinsic pathways.11 The extrinsic pathway involves the binding of extracellular signaling factors to death receptors at the plasma membrane. The intrinsic pathway is activated by intracellular stress and is controlled by the mitochondria. This mitochondrial apoptotic pathway is regulated by the B-cell lymphoma-2 (Bcl-2) family, whose members are classified based on their structure and function; antiapoptotic proteins such as Bcl and Bcl-extra large (Bcl-xL) share three to four conserved Bcl-2 homology domains, while proapoptotic proteins such as Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak) share three such domains.12 Resveratrol (3,5,4′-trihydroxy-trans-stilbene, 1) is produced by several plants and is found in peanuts, mulberries, grapes, root extracts of the Polygonum cuspidatum Wild. ex Spreng (Polygonaceae) weed (used in traditional Chinese and Japanese medicine), etc. Compound 1 has been reported to have phytoestrogen activity, exerting both estrogenic and antiestrogenic effects by binding to the estrogen receptor (ER).13 Many researchers have demonstrated that 1 reduced cell viability and proliferation in ER-positive and ER-negative cells,13,14although the mechanism by which 1 inhibits cell proliferation is not clearly understood. Several subsequent studies have shown that

enign prostatic hyperplasia (BPH) is a nonmalignant enlargement of the prostate gland. This proliferative disorder affects the epithelial cells and smooth muscle within the transitional zone of the prostate gland.1 BPH is a common disease in elderly men, with histological evidence of BPH in approximately 50% of men in their 50s, a figure that increases up to 90% in men aged 80 years and above.2 BPH may lead to increased smooth muscle tone and resistance, as well as obstruction of the proximal urethra, leading to lower urinary tract symptoms (LUTS).3 The obstructive symptoms of BPH have an effect on urine storage (increased urinary frequency, nocturia, urinary incontinence), voiding (slow and/or weak stream, terminal dribble), and postmicturition symptoms (sense of incomplete emptying).4 From recent in vitro and in vivo studies, the notion emerged that chronic prostatic inflammation (CPI) could play a major role in the development of BPH.5 The correlation between CPI and hypertrophic prostate was first reported in 1968 and has been extensively studied since the early 1970s.6 In 2005, a subgroup analysis of the MTOPS (Medical Therapy of Prostatic Symptoms) study confirmed that CPI could be associated with a higher prostate volume and an increased risk of BPH complications such as acute urinary retention.7 Thus, inflammation may be a promising target for BPH management. Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) inhibitors have shown positive effects on LUTS attributable to BPH.8 However, these therapeutic agents may not be suitable for all or most BPH patients because of the gastrointestinal and cardiovascular side effects of these agents. BPH arises from an imbalance between cell growth and apoptosis. Apoptosis plays an important role in the control of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 29, 2014

A

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

To find out whether this effect of 1 was associated with altered levels of the 5α-reductase 2 protein, 5α-reductase 2 mRNA level was examined. As shown in Figure 2B, 1 treatment significantly reduced 5α-reductase 2 mRNA level in prostate tissue of BPH-induced rats. Resveratrol (1) Attenuated Prostate Hyperplasia in BPH-Induced Rats. In the control group, one layer of lowcolumnar epithelial cells formed a secretory lumen that was filled with thin acidophilic materials. Undeveloped epithelial cells forming the prostate gland were arranged as a single layer (Figure3A). In contrast, the epithelial cells in the BPH-induced rats were arranged in several uneven layers, and the gland was excessively developed. In the 1-treated group, columnar epithelial cells were arranged as multiple layers, and both the proliferation of epithelial cells and the number of glands were increased compared with the control group. However, in comparison with the BPH-induced group, 1-treated animals showed suppressed prostate cell proliferation and gland development. As shown in Figure 3B, the thickness of epithelium tissue from prostate (TETP) was significantly higher in the BPH-induced group than in the control group. In the 1-treated group, although TETP was higher than that recorded for the control group, it was significantly lower than that observed in the BPH-induced group, suggesting a marked recovery of prostate hyperplasia. To evaluate proliferation of prostatic epithelial cells, we examined the protein expression of proliferating cell nuclear antigen (PCNA) in the prostatic tissue of BPH-induced rats. As shown in Figure 3C, Western blot analysis indicated an increase in PCNA protein expression in the BPH-induced group, as compared with the levels in the control group. In comparison with the BPH-induced group, however, finasteride- and 1-treated groups reduced the increased expression of PCNA protein, indicating antiproliferation effects in BPH. Resveratrol (1) Attenuated iNOS and COX-2 Protein Expression in BPH-Induced Rats. Clinical observations also indicate that chronic inflammation correlates with the clinical progression of BPH, as BPH tissues often have infiltrating lymphocytes and macrophages around the glandular elements.22 This suggests that chronic inflammation could be a causative factor in the pathogenesis of BPH.23 Immunohistochemical analysis of prostate tissues from BPH patients has shown increased CD68+ macrophages in both the epithelium and the stromal area. It has been shown that nitric oxide (NO) and COX-2 may play an important role in the association between inflammation and prostate growth.24 As shown in

1 prevented or slowed the progression of a wide range of illnesses, including cancer,15 cardiovascular disease,16 diabetes,17 ischemic injuries,18 and age-related diseases.19 In this study, the molecular mechanism(s) underlying the effects of 1 on BHP were investigated using an in vivo approach, in order to assess the protective potential of 1 in this condition.

■

RESULTS AND DISCUSSION Resveratrol (1) Reduced Prostate Weight in a BPHInduced Rat Model. Chemical stimulation with testosterone induced BPH, and after 4 weeks of treatment, the prostate weight in the BPH-induced group was significantly higher than that observed in any other study group (Figure 1A). The prostate weight in the BPH-induced group was 1.74 times higher than that in the control group. A lower prostate weight is identified significantly in the finasteride-treated group and 1treated group as compared with the BPH group (Figure 1B). Resveratrol (1) Suppressed Dihydrotestosterone (DHT) Production and Expression of 5α-Reductase 2 mRNA in BPH-Induced Rats. The presence of testesproducing androgens is a known risk factor for BPH because androgens, including testosterone and DHT, play a central role in regulating cell proliferation and death in the prostate. In particular, the level of DHT, which is the most active form produced from testosterone by 5-alpha reductase, is correlated with the occurrence and progression of BPH.20 Finasteride is a 5α-reductase inhibitor that is approved for the treatment of BPH, and it reduces the conversion of testosterone to DHT.21 To demonstrate whether 1 attenuated production of DHT, the effect of 1 on serum DHT levels in the BPH-induced rats was examined. As shown in Figure 2A, a significant increase in serum DHT concentration was found in the BPH-induced group, as compared with the control group. In both the finasteride and 1 treatment groups, however, serum DHT concentrations were significantly lower than those observed in the BPH-induced group. Compound 1 and finasteride treatment suppressed DHT production by 50.48% and 56.78%, respectively.

Figure 1. Effect of resveratrol (1) administration on prostate weight in BPH-induced rats. Changes in prostate total weight (A) and relative prostate weight ratio (B). The data shown represent mean ± SEM, n = 6 rats per group. #p < 0.05 vs Con group, **p < 0.01 vs BPH group; ***p < 0.001 vs BPH group. B

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Effect of resveratrol (1) administration on the levels of serum DHT and prostate tissue 5α-reductase 2 mRNA in BPH-induced rats. (A) Serum concentrations of DHT, determined by ELISA. (B) Expression of 5α-reductase 2 mRNA in prostate tissue, analyzed by quantitative real-time PCR. The data shown represent mean ± SEM, n = 6 rats per group. #p < 0.05 vs Con group; ***p < 0.001 vs BPH group.

Figure 3. Effect of resveratrol (1) administration on the prostatic cell proliferation. Hematoxylin and eosin staining of prostate tissue from BPHinduced rats (A) and TETP (B); original magnification 40×. (B) The expression level of PCNA protein was determined by Western blotting using specific antibodies. β-Actin was used as internal control. Densitometric analysis was performed using Biorad Quantity One software. The data shown represent mean ± SEM, n = 6 rats per group. #p < 0.05 vs Con group; **p < 0.01 vs BPH group.

Figure 4. Effect of resveratrol (1) administration on the expression of iNOS and COX-2 in prostate tissue from BPH-induced rats. The levels of iNOS and COX-2 proteins were determined by Western blotting using specific antibodies. β-Actin was used as an internal control. Densitometric analysis was performed using Bio-Rad Quantity One software. The data shown represent the mean ± SEM, n = 6 rats per group. #p < 0.05 vs Con group; ***p < 0.001 vs BPH group.

C

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 5. Effect of resveratrol (1) administration on the expression of apoptosis-related proteins in prostate tissue from BPH-induced rats. The expression levels of (A) PARP-1 and procaspase-3 and (B) Bcl-2 family proteins were determined by Western blotting using specific antibodies. βActin was used as an internal control. (C) Densitometric analysis of Bax and Bcl-2 bands was performed using Biorad Quantity One software, and the data (relative density normalized to β-actin) were plotted as the Bax/Bcl-2 ratio. The data shown represent the mean ± SEM, n = 6 rats per group. #p < 0.05 vs Con group; ***p < 0.001 vs BPH group.

of caspases: the initiator/apical caspases (caspase-8, caspase-9, and caspase-10) and the effector/executioner caspases (caspase3, caspase-6, and caspase-7). Effector caspases generally contain only a small pro-domain and cleave diverse cellular substrates such as PARP and lamin A/C, events that also lead to apoptosis. Evidence suggests that initiator caspases are capable of autocatalytic activation, while effector caspases require activation by initiator caspase cleavage. As shown in Figure 5A, in comparison with the BPH-induced group, the 1-treated group showed decreased protein expression of procaspase-3 and PARP-1 (an endogenous substrate of caspase-3). Inflammation mediated by elevated COX-2 enzyme levels may play an important role in disrupting cell homeostasis. Indeed, COX-2 is known to induce inhibition of apoptosis via

Figure 4, Western blot analysis revealed an increase in iNOS and COX-2 protein expression in the BPH-induced group, as compared with the levels in the control group. In comparison with the BPH-induced group, however, the 1-treated group showed reduced iNOS and COX-2 protein expression, indicating anti-inflammatory effects in BPH. Resveratrol Induced Apoptosis via Regulating the Expression of Bcl-2 Family Proteins in BPH-Induced Rats. An imbalance between prostate cell growth and apoptosis has been linked to BPH.24 In this regard, the caspase family of aspartate-specific cysteine proteases plays an important role in apoptosis triggered by a range of proapoptotic signals.25 Caspases are expressed as inactive precursors that form active oligomers after initiating cleavage events. There are two groups D

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

kg i.p. 1. To eliminate the influence of intrinsic testosterone, all rats inthe BPH-induced groups underwent bilateral orchiectomies, which were performed 3 days prior to the induction of BPH. Prostate hyperplasia was induced in the BPH groups by daily intramuscular injections of testosterone propionate (10 mg/kg) for 4 weeks. The finasteride or resveratrol was administered daily for 4 weeks at a dose of 1 mL per 100 g of body weight. Twenty-four hours after the last administration, the rats were bled to death and the prostate was taken out and weighed.30 Serum DHT Analysis. Serum concentrations of DHT were determined using a commercial enzyme-linked immunosorbent assay (ELISA; ALPCO Diagnostics, Salem, NH, USA). Assays were performed according to the manufacturer’s instructions. Histological Analysis. The prostatic tissue from rats in each group was fixed in 4% buffered formalin, embedded in paraffin, and cut into 4 μm sections. The sections were stained with hematoxylin and eosin (H&E) prior to histological examination. Images were acquired using an SZX10 microscope (Olympus, Tokyo, Japan). Histological analysis was performed, and the TETP was measured by professional pathologists (Seo-Na Jang and Jae-Hak Park) from Korean Experimental Pathology Inc. (Seoul, Republic of Korea) (sample number: XP13-0087). Quantitative Real-Time (RT) PCR Analysis. Prostatic tissue from each animal was homogenized, and total RNA was isolated using EasyBlue Reagent (Intron Biotechnology Inc., Gyeongi-do, Republic of Korea) according to the manufacturer’s instructions. Total RNA was quantified using an Epoch microvolume spectrophotometer system (BioTek Instruments, Inc. Winooski, VT, USA). Total RNA from the prostate was converted to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed in the presence of SYBR green (Applied Biosystems). The oligonucleotide primers for rat 5α-reductase2 was ATG GGG ACC CTG ATC CTG TG (forward) and CGA CAC CAC AAA GGA AGG CA (reverse), and those for rat GAPDH, analyzed as a house-keeping gene, were TGA TTC TAC CCA CGG CAA GT (forward) and AGC ATC ACC CCA TTT GAT GT (reverse). Quantitative RT-PCR was carried out using a thermocycler (Gene Amp PCR system 9700, Applied Biosystems), and the results were expressed relative to GAPDH. Western Blot Analysis. Prostatic tissue from each animal was homogenized in a commercial lysis buffer (PRO-PREP; Intron Biotechnology Inc.) and incubated for 25 min on ice to induce cell lysis. Tissue extracts were centrifuged at 13 000 rpm at 4 °C for 20 min, and the supernatants were transferred to clean tubes. The protein concentration was determined using the Bio-Rad protein assay reagent, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Aliquots of each protein sample (30 μg) were separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% skim milk at 4 °C for 1 h. The membranes were incubated overnight with primary antibodies, which included the antiiNOS, anti-COX-2, anti-PARP, anticaspase-3, anti-Bcl-xL, anti-Bax, and anti-β-actin antibodies. Blots were washed three times with Tween-20/tris-buffered saline (TTBS), followed by incubation with the corresponding secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Blots were again washed three times with TTBS, and immunoreactive protein bands were visualized using enhanced chemiluminescence and exposure to X-ray film (Amersham, Piscataway, NJ, USA). StatisticalAnalysis. All values are expressed as mean ± the standard error of the mean (SEM) for data from 6 rats. The data were analyzed using one-way analysis of variance with Dunnett’s test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS; version 19.0).

an increased expression of the antideath protein, Bcl-2, and by stimulating production of several growth factors.26 Cells undergoing apoptosis are rapidly and specifically recognized and removed by phagocytes such as macrophages or immature dendritic cells.27 This process is generally considered to be immunologically silent due to the release of anti-inflammatory mediators and the suppression of local inflammation.28 Since Bcl-2 family proteins are known to control apoptosis via maintaining a balance between pro- and antiapoptotic members, we examined the effects of 1 on the levels of Bcl-2 family members in the BPH-induced animals. In this study, the 1-treated group showed decreased protein expression of the antiapoptotic Bcl-2 and Bcl-xL, but increased expression of the proapoptotic Bax, as compared with the BPH-induced group (Figure 5B). Most cancer tissues, including those in prostate cancer, generally overexpress Bcl-2,29 thereby reducing apoptosis. Bcl-2 forms a heterodimer with the apoptotic protein Bax, neutralizing its apoptotic effects. The ratio of these proteins determines whether a cell will undergo apoptosis. Thus, the ratio of Bcl-2 to Bax was significantly decreased after treatment with 1, suggesting involvement of the Bcl-2 family of proteins, an important mediator of the apoptosis pathway in 1-induced apoptosis during BPH (Figure 5C). These findings suggested that 1-induced apoptosis played a role in the effects of this compound on BPH and that this was mediated via regulation of the expression of anti- and proapoptotic Bcl-2 family proteins. Altogether, the results of present study showed, for the first time, that 1 had the ability to decrease prostate weight and DHT production in BPH-induced rats. These effects may be due to the anti-inflammatory and apoptotic effects of 1. Accordingly, these results supported the notion that resveratrol (1) should be further explored as a potent candidate for the treatment of BPH.

■

EXPERIMENTAL SECTION

Chemicals and Reagents. Compound 1 (≥99%), testosterone, phenylmethylsulfonyl fluoride, Triton X-100, propidium iodide, nonidet P-40, and protein inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan), and finasteride was obtained from Merck & Co., Inc. (NJ, USA). Oligonucleotide primers for 5α-reductase 2 and glyceraldehyde 3phosphate dehydrogenase (GAPDH) were purchased from Bioneer (Daejeon, Republic of Korea), and SYBR Premix Ex Taq was purchased from Takara (Shuzo, Shiga, Japan). Antibodies for inducible nitric oxide synthase (iNOS; M-19), COX-2 (C-20), poly(ADPribose) polymerase-1 (PARP-1; F-2), caspase-3 (E-8), Bcl-2 (C-2), Bcl-xL (H-5), Bax (B-9), and β-actin (ACTBD11B7) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anantibody for PCNA was purchased from BD Biosciences, Pharmingen (San Diego, CA, USA). Animals. Ten-week-old male Sprague−Dawley rats (200 ± 20 g) were purchased from DaehanBiolink (Daejeon, Korea). The animals were housed under conditions that were in accordance with the guidelines for the care and use of laboratory animals, adopted and promulgated by the Institutional Animal Care Committee of Sangji University (Reg. No. 2014-07). The rats were acclimatized to the laboratory conditions for 2 weeks before starting the experiment. They were provided free access to food and water for 4 weeks and were maintained under a 12 h light/12 h dark cycle at a constant temperature (22 ± 2 °C) and relative humidity (55 ± 9%) throughout the experiment. The rats were randomly assigned to one of four groups (n = 6 per group): the control group (Con); the BPH-induced group (BPH); and two additional treatment groups with induced BPH and daily injections of either 5 mg/kg p.o. finasteride (Fina) or 1 mg/

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +82337387503. Fax:+82 33 730 0679. E-mail: [email protected]. kr. E

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Notes

(30) Guo, Q. L.; Ding, Q. L.; Wu, Z. Q. Biol. Pharm. Bull. 2004, 27, 333−337.

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This research was supported by Sangji University Research Fund, 2014. REFERENCES

(1) Auffenberg, G. B.; Helfand, B. T.; McVary, K. T. Urol. Clin. North Am. 2009, 36 (4), 443−459 v, vi. (2) Schwarz, S.; Obermuller-Jevic, U. C.; Hellmis, E.; Koch, W.; Jacobi, G.; Biesalski, H. K. J. Nutr. 2008, 138, 49−53. (3) McVary, K. T.; Roehrborn, C. G.; Avins, A. L.; Barry, M. J.; Bruskewitz, R. C.; Donnell, R. F.; Foster, H. E., Jr.; Gonzalez, C. M.; Kaplan, S. A.; Penson, D. F.; Ulchaker, J. C.; Wei, J. T. J. Urol. 2011, 185, 1793−1803. (4) Coyne, K. S.; Sexton, C. C.; Thompson, C. L.; Milsom, I.; Irwin, D.; Kopp, Z. S.; Chapple, C. R.; Kaplan, S.; Tubaro, A.; Aiyer, L. P.; Wein, A. J. BJU Int. 2009, 104, 352−360. (5) Schenk, J. M.; Kristal, A. R.; Neuhouser, M. L.; Tangen, C. M.; White, E.; Lin, D. W.; Thompson, I. M. Prostate 2009, 69, 1303−1311. (6) Hubmer, G. Z. Urol. Nephrol. 1968, 61, 801−804. (7) Roehrborn, C. G. Rev. Urol 2005, 7 (Suppl8), S43−51. (8) Falahatkar, S.; Mokhtari, G.; Pourreza, F.; Asgari, S. A.; Kamran, A. N. Urology 2008, 72, 813−816. (9) Alison, M. R.; Sarraf, C. E. J. R. Coll. Physicians London 1992, 26, 25−35. (10) Sebastiano, C.; Vincenzo, F.; Tommaso, C.; Giuseppe, S.; Marco, R.; Ivana, C.; Giorgio, R.; Massimo, M.; Giuseppe, M. Front. Biosci. (Elite Ed.) 2012, 4, 195−204. (11) Gonzalvez, F.; Schug, Z. T.; Houtkooper, R. H.; MacKenzie, E. D.; Brooks, D. G.; Wanders, R. J.; Petit, P. X.; Vaz, F. M.; Gottlieb, E. J. Cell Biol. 2008, 183, 681−696. (12) Walensky, L. D. Cell Death Differ. 2006, 13, 1339−1350. (13) Gehm, B. D.; McAndrews, J. M.; Chien, P. Y.; Jameson, J. L. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14138−14143. (14) Lu, R.; Serrero, G. J. Cell Physiol. 1999, 179, 297−304. (15) Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W.; Fong, H. H.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J. M. Science 1997, 275, 218−20. (16) Bradamante, S.; Barenghi, L.; Villa, A. Cardiovasc. Drug Rev. 2004, 22, 169−188. (17) Lagouge, M.; Argmann, C.; Gerhart-Hines, Z.; Meziane, H.; Lerin, C.; Daussin, F.; Messadeq, N.; Milne, J.; Lambert, P.; Elliott, P.; Geny, B.; Laakso, M.; Puigserver, P.; Auwerx, J. Cell 2006, 127, 1109− 1122. (18) Wang, Q.; Xu, J.; Rottinghaus, G. E.; Simonyi, A.; Lubahn, D.; Sun, G. Y.; Sun, A. Y. Brain Res. 2002, 958, 439−447. (19) Rogina, B.; Helfand, S. L.; Frankel, S. Science 2002, 298, 1745. (20) Carson, C., 3rd; Rittmaster, R. Urology 2003, 61, 2−7. (21) Altavilla, D.; Minutoli, L.; Polito, F.; Irrera, N.; Arena, S.; Magno, C.; Rinaldi, M.; Burnett, B. P.; Squadrito, F.; Bitto, A. Br. J. Pharmacol. 2012, 167, 95−108. (22) Di Silverio, F.; Gentile, V.; De Matteis, A.; Mariotti, G.; Giuseppe, V.; Luigi, P. A.; Sciarra, A. Eur. Urol. 2003, 43, 164−175. (23) Chughtai, B.; Lee, R.; Te, A.; Kaplan, S. Curr. Urol. Rep. 2011, 12, 274−277. (24) Sciarra, A.; Mariotti, G.; Salciccia, S.; Autran Gomez, A.; Monti, S.; Toscano, V.; Di Silverio, F. J. Steroid Biochem. Mol. Biol. 2008, 108, 254−260. (25) Favaloro, B.; Allocati, N.; Graziano, V.; Di Ilio, C.; De Laurenzi, V. Aging (Albany N.Y.) 2012, 4, 330−349. (26) Nakanishi, Y.; Kamijo, R.; Takizawa, K.; Hatori, M.; Nagumo, M. Eur. J. Cancer 2001, 37, 1570−8. (27) Savill, J.; Dransfield, I.; Gregory, C.; Haslett, C. Nat. Rev. Immunol. 2002, 2, 965−975. (28) Kono, H.; Rock, K. L. Nat. Rev. Immunol. 2008, 8, 279−289. (29) Ricci, M. S.; Zong, W. X. Oncologist 2006, 11, 342−57. F

DOI: 10.1021/np500810c J. Nat. Prod. XXXX, XXX, XXX−XXX