Toxicology and Applied Pharmacology 311 (2016) 52–60

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Toxicology and Applied Pharmacology

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Dapoxetine attenuates testosterone-induced prostatic hyperplasia in ratsby the regulation of inflammatory and apoptotic proteins

Rabab H. Sayed ⁎, Muhammed A. Saad, Ayman E. El-SaharDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

⁎ Corresponding author at: Department of PharmacolPharmacy, Cairo University, Kasr El-Aini Street, Cairo 1156

E-mail address: rabab.sayed@pharma.cu.edu.eg (R.H. S

http://dx.doi.org/10.1016/j.taap.2016.09.0240041-008X/© 2016 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 August 2016Revised 20 September 2016Accepted 24 September 2016Available online 27 September 2016

Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus anew selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathologicalchanges in the rat prostate. A dose response curvewas constructed between the dose of dapoxetine and prostateweight as well as relative prostate weight, then a 5mg/kg dose was used as a representative dose for dapoxetineadministration. Rats were divided into four groups; the control group that received the vehicle; the BPH-inducedgroup received daily s.c injection of 3 mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5 mg/kg/day p.o and BPH-induced group treated with dapoxetine 5 mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were bothreturned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also up-regulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testoster-one injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2(Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated Xprotein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of thechanges made by testosterone injection. In conclusion, the current study provides an evidence for the protectiveeffects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasingAR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH.

© 2016 Elsevier Inc. All rights reserved.

Keywords:Benign prostatic hyperplasiaDapoxetineProliferating cell nuclear antigenApoptosisInflammation

1. Introduction

Benign prostatic hyperplasia (BPH) is the most common urologicaldisorder in elderly men due to the unrestrained growth of the prostategland [1], which causes nocturia, urinary urgency, incomplete bladdervoiding and lower urinary tract symptoms [2]. The aetiology of BPHstill remains mysterious, despite its prevalence. Hormonal imbalanceand other growth regulatory factors disproportion, along with agingmay support cell proliferation and hyperplasia induction [3]. Prostateproliferation is an androgen-dependent process [4]. The enzyme 5α-re-ductase catalyzes the conversion of testosterone into the potent andro-gen dihydrotestosterone (DHT). Then, DHT binds to androgen receptors(AR) and promotes protein synthesis and cellular growth of the prostatecells [5]. In thismilieu, androgen/AR signaling plays an important role inthe pathogenesis of BPH and blockade of this signaling decreases BPHvolume and can relieve lower urinary tract symptoms [6].

Although aging and androgens are the two established risk factors inthe pathogenesis of BPH, other findings have highlighted the importantrole of inflammation [7]. Chronic inflammationmay contribute to tissue

ogy and Toxicology, Faculty of2, Egypt.ayed).

injury, directly through activating cytokines release and increasing theconcentration of growth factors, or alternatively through affectingapoptotic protein expression including the antiapoptotic factor B-celllymphoma-2 (Bcl-2); and the proapoptotic factor Bcl-2-associated Xprotein (Bax) [8,9].

Conventionally, the main drugs used in the clinical care for BPH in-volves α1-adrenoceptor antagonists and 5α-reductase inhibitors (e.g.,finasteride) [10]. However, these drugs are limited because of theirside effects, including gynecomastia, erectile dysfunction and low libido[11]. Therefore, it is important to find alternative pharmacologicalagents that can be used for treatment of this disease.

Although selective serotonin reuptake inhibitors (SSRIs) arefrequently used for psychiatric indications, depression and anxiety dis-orders [12], several studies revealed that SSRIs possess anti-inflamma-tory activity [13,14], as well as anti-cancer activity [15]. Dapoxetine isa short-acting SSRI which is used in treatment of men complainingfrompremature ejaculation [16]. Also, a recent report showed that sero-tonin may regulate the proliferation in prostate cells [17]. Therefore aquestion arises here if the use of dapoxetine can ameliorate the patho-genesis of prostatic hyperplasia beside retarding ejaculation or not. Inreviewing the literature, no data was found on the association betweendapoxetine administration and pathological changes in rat prostate instates of hypertrophy. Hence the current study was designed to

Table 1Primer sequences used for RT-PCR.

Gene Primer sequence

AR Forward primer 5′- CGGAAATGTTATGAAGCAGGG-3′Reverse primer 5′- GGAATCAGGCTGGTTGTTGTC-3′

PCNA Forward primer 5′ - GGTGCTTGGCGGGAGC-3′Reverse primer 5′- ATCGCTTGAGCCCAGAAGT-3′

GAPDH Forward: 5′- CTCCCATTCTTCCACCTTTG-3′Reverse: 5′- CTTGCTCTCAGTATCCTTGC-3′

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investigate the potential protective effect of dapoxetine against testos-terone-induced BPH in rats. In addition, the underlying molecularmechanismswere explored by assessing differentmarkers of inflamma-tion, proliferation and apoptosis along with the effect of dapoxetine onandrogen/AR signaling pathway.

2. Materials and methods

2.1. Animals

Adult male Wistar rats, weighing 200 ± 20 g, were obtained fromthe animal facility of Faculty of Pharmacy, Cairo University, Egypt. Ani-malswere housed under controlled environmental conditions: constanttemperature (25 ± 2 °C), humidity (60 ± 10%), and a 12/12-h light/dark cycle. Standard chow diet and water were allowed ad libitum.The investigationwas approved by the Ethics Committee for Animal Ex-perimentation of Faculty of Pharmacy, Cairo University (Permit Num-ber: PT 1635), and complied with the Guide for Care and Use ofLaboratory Animals published by the US National Institutes of Health(NIH Publication No. 85-23, revised 1996).

2.2. Experimental design

Apreliminary dose–response studywas conducted inwhich animalswere randomly divided into four groups (n=6per group); the BPH-in-duced group and three treatment groups with induced-BPH and dailyoral (p.o) administration of three different doses (1 mg/kg, 5 mg/kgand 10 mg/kg) of dapoxetine dissolved in distilled water. Benign pros-tatic hyperplasia was induced by daily subcutaneous (s.c) injection of3 mg/kg testosterone propionate dissolved in olive oil for four weeksas described by Ammar et al. [18]. Testosterone propionate anddapoxetine were purchased from Sigma-Aldrich Chemical Co (St Louis,MO, USA). Rats were sacrificed 72 h after the last testosterone injectionand the prostates were immediately removed and weighed, thenrelative prostate weight was calculated as the ratio of the prostateweight to body weight. Based on the results obtained from thedose–response study, the optimal dose of dapoxetine was chosenfor further investigations.

Afterwards, a mechanistic study was conducted in which animalswere randomly divided into four groups (n=8 per group); the controlgroup that received distilled water p.o with olive oil s.c.; the BPH-in-duced group; BPH-induced group treated with finasteride (5 mg/kg/day, p.o, Sigma–Aldrich, St. Louis, MO, USA) and BPH-induced grouptreated with dapoxetine 5 mg/kg/day p.o. The dose of finasteride fortreating BPH was determined based on previous studies [19,20]. Ratswere sacrificed 72 h after the last testosterone injection. The prostaticglandwas removed andweighed, then relative prostateweightwas cal-culated as the ratio of the prostate weight to body weight. Sections ofthe ventral lobes were fixed in 10% neutral buffered formalin and em-bedded in paraffin for both histological and immunohistochemical ex-aminations. The remainder of each prostate was stored at −70 °C andused for further analyses.

2.3. Serum concentrations of DHT

The serum concentrations of DHT were determined using an En-zyme-Linked Immunosorbent Assay (ELISA) kit according to themanufacturer's instructions (ALPCO Diagnostics, Salem, NH, USA).Values were expressed as pg/ml.

2.4. Quantitative real time polymerase chain reaction (qRT-PCR) analysis

Total RNA was extracted from the frozen tissue samples using theRNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer'sprotocol. The RNA concentration and purity were spectrophotometri-cally determined at OD 260/280 nm. The RNA integrity was assessed

using agarose gel electrophoresis and ethidium bromide staining.Equal amounts of RNA (2 mg) were reverse transcribed into comple-mentary DNA (cDNA) using Superscript Choice systems (Life Technolo-gies, Breda, Netherlands) according to themanufacturer's guidelines. Toassess the expression of androgen receptor (AR) and proliferating cellnuclear antigen (PCNA) genes, quantitative real-time PCR was per-formed using SYBR Green PCR Master Mix (Applied Biosystems, FosterCity, CA, USA) as described by themanufacturer. Briefly, in a 25-μl reac-tion volume, 5 μl of cDNAwas added to 12.5 μl of 2× SYBRGreenMasterMix and 200 ng of each primer. The sequences of primers are describedin Table 1. PCR reactions included 10 min at 95 °C for activation ofAmpliTaq Gold DNA polymerase, followed by 40 cycles at 95 °C for15 s (denaturing) and 60 °C for 1 min (annealing/extension). The rela-tive expression of target genes was obtained using the 2−ΔΔCT formulaas described previously by [21] using Glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) as a housekeeping gene.

2.5. Western blot analysis

Tissue proteins were extracted and its concentrations were estimat-ed by Bradford kit (Bio-Rad, HemelHempstead, UK) using bovine serumalbumin as a standard. An equal volume of sample buffer (2% sodiumdodecyl sulphate (SDS), 10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol and 50 mM Tris HCl, pH 7.2) was added to 50 μg pro-tein from each sample loaded on SDS-polyacrylamide gel and electro-phoresed at 150 V for 90 min. The proteins were transferred to apolyvinylidene difluoride membrane (PVDF; Immobilon-P 0.45 μMpore size; Merck Millipore, Darmstadt, Germany). Immunodetection ofWestern blots was conducted by incubating the membranes at roomtemperature for 2 h with blocking solution comprised of 5% nonfatdried milk in 10 mM Tris-Cl, pH 7.5, 100 mM NaCl, and 0.1% Tween20.Membraneswere incubated overnight at 4 °Cwith the indicated pri-mary antibodies (bax, bcl2 and beta-actin supplied by Thermo Scientif-ic) and then incubated with a mouse anti-rabbit secondary monoclonalantibody conjugated to horseradish peroxidase at room temperature for2 h. After each incubation, themembraneswerewashed four timeswith10 mM Tris-Cl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20 at room tem-perature Chemiluminescence detection was performed with theAmersham detection kit according to the manufacturer's protocols.The amount of studied proteinwas quantified by densitometric analysisusing a scanning laser densitometer (Biomed Instrument Inc., USA). Re-sults were expressed as arbitrary units after normalization for β-actinprotein expression.

2.6. Enzyme linked immunosorbent assay of prostatic caspase 3 activity andtumor necrosis factor alpha (TNF-α) content

Tissue TNF-α was assayed using commercial ELISA assay kits(RayBiotech Inc., Norcross, Georgia, USA). Likewise, prostatic caspase-3 activity was estimated according to the manufacturer's instructionsprovided by the ELISA assay kits (USCN Life Science Inc.,Wuhan, China).

2.7. Histopathological examination

Ventral prostate tissues were embedded in 10% formalin and proc-essed for paraffin sections of 4 μm thickness. After de-waxing and

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rehydration, sections were stained with hematoxylin and eosin (H&E)for routine histopathological examination using light microscopy.

2.8. Immunohistochemical detection of cyclooxygenase II (COX II) and in-ducible nitric oxide synthase (iNOS)

Paraffin embedded tissue sections of 4 mm thickness, taken fromthree animals per group, were deparaffinized first with xylene then hy-drated in graded ethanol solution and heated in citrate buffer (pH 6.0)for 5min. After that, the sectionswere blockedwith 5% bovine serumal-bumin (BSA) in tris buffered saline (TBS) for 2 h. The slides were thenincubated overnight at 4 °C with one of the following primary antibod-ies; COX II rabbit polyclonal antibody (Cat#RB-9072-R7, Thermo FischerScientific) and iNOS rabbit polyclonal antibody (Cat#RB-9242-R7, Ther-mo Fisher Scientific). The slides were rinsed with TBS and incubated for10 min in a solution of 0.02% diaminobenzidine containing 0.01% H2O2.Counter stainingwas carried out using hematoxylin and the slides werevisualized under a light microscope. The immunohistochemical quanti-tation was performed by Image J1.45 F image analysis system (NationalInstitute of Health, USA) to determine the optical density of the immu-nostaining. It was represented as the optical density of positive cellsacross ten different fields for each rat section.

2.9. Statistical analysis

The data are presented as means ± S.E. Data were analyzed usingone-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. The GraphPad Prism software(version 5; GraphPad Software, Inc., San Diego, CA, USA) was usedto perform the statistical analysis and creat the graphical presenta-tions. The level of significance was fixed at P b 0.05, with respectto all statistical tests.

3. Results

3.1. Relationship between dapoxetine dose and prostate weight as well asrelative prostate weight for the identification of the dose used in the study

In Fig. 1, there is a clear trend of decreasing in the prostate weightupon increasing the dose of dapoxetine, but therewas no significant dif-ference between the effect of the 5 and 10 mg/kg doses on the prostateweight. The more surprising correlation is in the U-shaped curve whenplotting the dose of dapoxetine and the relative prostate weight. The10 mg/kg dose returned the relative prostate weight back to the

Fig. 1.Dose response relationship between dose of dapoxetine and prostate weight in grams (AP b 0.05. b: significantly different from the testosterone-treated group at P b 0.05. c: significantlymean of 6 experiments ± SEM.

response of the 1 mg/kg dose of dapoxetine. Taken together, theseresults suggest that there is an association between dapoxetinedose and prostate weight, and that the 5 mg/kg dose is the optimaldose, thus it was chosen as a representative dose for dapoxetineadministration.

3.2. Prostate weight and relative prostate weight in rats having BPH in-duced by testosterone injection after administration of finasteride anddapoxetine

Table 2 presents that testosterone propionate (3 mg/kg, s.c) injec-tionmarkedly elevated the prostateweight and relative prostateweightto attain 262% and 248%, respectively of the respective control group.Neither finasteride nor dapoxetine regained the normal prostateweightback to the normal value but both almost reduced it to about half thetestosterone group. Along similar lines, finasteride reduced the relativeprostate weight by 44% and dapoxetine decline it by 45% of the testos-terone-treated rats.

3.3. Androgen receptor gene expression and serum DHT concentrationfollowing finasteride and dapoxetine administration

There was a significant positive correlation between the injection oftestosterone and the expression of AR and the serum DHT concentration,whichwere elevated by five folds and three folds of the control group, re-spectively after the injection of testosterone propionate (3 mg/kg, s.c).Dapoxetine administration declined the AR expression to about 70% ofthe testosterone group, but was still higher than the standard finasteridetreated rats who reached 50% of the testosterone-treated rats. On theother hand, the reduction in the DHT level following dapoxetine adminis-tration was more severe than that following finasteride injection, as itreduced the DHT level by 35% and 17% of the respective testosteronegroup, respectively (Fig. 2).

3.4. Proliferating cell nuclear antigen gene expression

The results, as shown in Fig. 3 demonstrate that there was about 5.5times elevation in the PCNA gene expression in prostate homogenate oftestosterone treated rats. Also, bothfinasteride and dapoxetine declinedit to reach 41% and 48% of the respective testosterone-treated rats, re-spectively without any significant difference between the comparedtreatments, but they didn't attain the control level.

) and as a percent from bodyweight (B). a: Significantly different from the control group atdifferent from the standardfinasteride-treated group at P b 0.05. Valueswere expressed as

Table 2Effect of finasteride (5 mg/kg, p.o) and dapoxetine (5 mg/kg, p.o) administration on theprostate weight and relative prostate weight in testosterone-induced BPH in rats: valueswere expressed asmean±SEMof eight rats. Statistical analysiswas performed by ANOVAfollowed by Tukey's Post-hoc test.

Parameter Prostate weight (g) Relative prostate weight (%)

Group

Control 0.342 ± 0.039 0.153 ± 0.015Testosterone 0.898a ± 0.079 0.380a ± 0.021Testosterone + finasteride 0.437b ± 0.072 0.210b ± 0.031Testosterone + dapoxetine 0.453b ± 0.055 0.208b ± 0.021

a Significantly different from the control group at P b 0.05.b Significantly different from the testosterone-treated group at P b 0.05.

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3.5. Western blot analysis of the apoptotic biomarkers Bax, Bcl2 and Bax/Bcl2 ratio

It is apparent from Fig. 4 that protein expression of Baxwas reducedin testosterone treated rats to about half of the normal control rats,while Bcl2 expression was elevated more than the double of thecontrol group. Both finasteride and dapoxetine elevated the pro-ap-optotic Bax protein expression by 91% and 95%, respectively of thetestosterone group. On the other hand, the expression of theantiapoptotic marker Bcl2 were declined to about half of the testos-terone-treated rats following treatment with finasteride anddapoxetine. Additionally, Bax/Bcl2 ratio showed a marked declineafter testosterone injection to reach 16% of the respective controlgroup. It can be also seen from the data in Fig. 4 that finasteride

Fig. 3. The effect of finasteride and dapoxetine on PCNA gene expression in testosterone-inducefrom the control group at P b 0.05. b: significantly different from the testosterone-treated group

Fig. 2. The effect of finasteride and dapoxetine on prostatic AR gene expression (A) and DHT seexperiments± SEM. a: Significantly different from the control group at P b 0.05. b: significantlystandard finasteride-treated group at P b 0.05.

and dapoxetine administration elevated this ratio, but failed toregain the control value.

3.6. Caspase 3 activity and TNF-α content in prostate homogenate

Fig. 5 demonstrates that TNF-α content was induced after testoster-one injection to reach 622% of the control group. Neither finasteride nordapoxetine could return the elevated TNF-α back to the normal value,but both could reduce it to about half the testosterone-treated rats. Onthe other side, caspase 3 showed a reduced activity following testoster-one administration to reach about one fourth of the control group. Fi-nasteride administration elevated caspase 3 activity to about 199% ofthe testosterone group, but didn't reach the control value. The singlemost striking observation emerging from the data comparison wasthat dapoxetine administration elevated the prostatic activity of caspase3 to more than five times the testosterone injected rats and reached alevel even more than the control rats.

3.7. Effect of finasteride and dapoxetine on the histopathological examina-tion of the ventral prostate

Fig. 6 shows a photomicrograph of ventral prostate of the normalcontrol rats showing variable size packed prostatic acini, while ratstreatedwith testosterone propionate showedwidely separated prostat-ic acini with papillary projections and desquamated epithelial lining inthe lumen with thick fibromuscular stroma and vacuolated epitheliallining. From the same figure, we can see that treatment of rats havingBPH with finasteride showed normal prostatic acini and reducedfibromuscular stroma. Dapoxetine also returned the histopathological

d BPH. Values were expressed as mean of 8 experiments ± SEM. a: Significantly differentat P b 0.05. c: significantly different from the standardfinasteride-treated group at P b 0.05.

rum concentration (B) in testosterone-induced BPH. Values were expressed as mean of 8different from the testosterone-treated group at P b 0.05. c: significantly different from the

Fig. 4. The effect of finasteride and dapoxetine on protein expression of Bax (A), Bcl2 (B) as well as their ratio “Bax/Bcl2” (C) in rats suffering from BPH through testosterone injection.Values were expressed as mean of 8 experiments ± SEM. a: Significantly different from the control group at P b 0.05. b: significantly different from the testosterone-treated group atP b 0.05. c: significantly different from the standard finasteride-treated group at P b 0.05.

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structure of prostatic acini back to the normal state and reducedfibromuscular stroma.

3.8. Immunohistochemical analysis of the inflammatory proteins (COX IIand iNOS)

From Figs. 7 and 8, it can be seen that there was no expressionfor both COX II and iNOS in the control group as evidenced by the neg-ative immunohistochemical reaction. On the other hand, both proteinswere highly expressed in the testosterone group, as shown by thestrong brown colour in the immunohistochemical staining. The resultsobtained from the preliminary analysis of COX II and iNOS protein

Fig. 5. The effect of finasteride and dapoxetine on prostatic inflammatory and apoptoticmarkersSignificantly different from the control group at P b 0.05. b: significantly different from the testtreated group at P b 0.05.

expression after treatment with the standard finasteride and the testeddrug dapoxetine revealed a weaker positive expression significantlylower than that of the testosterone group. Both proteins were returnedback to the normal level after treatmentwith the aforementioned drugs,except for COX II expression in the dapoxetine groupwhich didn't attainthe normal value of the control group.

4. Discussion

Despite significant progress in its diagnosis and treatment, BPHremains the most common prostatic disease affecting older men [1].Hence a dose of 5 mg/kg of dapoxetine was administered orally for

; TNF-α (A) and caspase 3 (B). Valueswere expressed asmean of 8 experiments± SEM. a:osterone-treated group at P b 0.05. c: significantly different from the standard finasteride-

Fig. 6. A& B: sections of the ventral prostate taken from the control group showing variable size packed prostatic acini. C, D, E & F: sections taken from testosterone-treated group showingwidely separated prostatic acini with papillary projections (small arrow), desquamated epithelial lining in the lumen (large arrow) and thick fibromuscular stroma (arrow head in C andlarge arrow in D), aswell as papillary projections and vacuolated epithelial lining (E & F). G, H & I: sections taken from the testosterone+ finasteride group showing normal prostatic aciniwith papillary projections (arrow) and reduced fibromuscular stroma (arrow head). J, K & L: sections taken from the testosterone + dapoxetine group showing normal prostatic acini(arrow), papillary projections (arrow) and reduced fibromuscular stroma.

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four weeks to investigate its protective potential against testosterone-induced BPH in rats. Dapoxetine administration in ourwork significant-ly inhibited testosterone mediated increase in the prostate weight andrelative prostate weight as compared to the lower dose (1 mg/kg),while the highest dose (10 mg/kg) showed no significant benefit overthe middle dose (5 mg/kg). Based on these results, the dose 5 mg/kghas proven efficacy in protecting against testosterone-induced BPHand was used for further mechanistic investigations.

Dapoxetine belongs to a class of drugs called SSRIs commonlyused for treatment of depression. Their primarymechanism of actioninvolves the inhibition of serotonin reuptake into pre-synaptic nerveterminals, enabling higher serotonin levels in the synaptic cleft with-in the central nervous system [22]. On the other side, it is known thatandrogen/AR signaling pathway plays a key role in development ofBPH and that targeting androgen/AR signaling could be a major ther-apeutic approach for BPH [6]. Binding of DHT to AR leads to activa-tion of numerous genes that promotes prostatic growth [4]. It hasbeen reported that testosterone-treated rats show over-expressionof AR [23,24]. In the present study, testosterone significantly in-creased serum DHT concentration, up-regulated gene expression ofAR and induced histopathological alterations in the prostate tissue.On the other hand, co-treatment with dapoxetine attenuated thesetestosterone-induced changes. These effects were compared to fi-nasteride, a 5α-reductase inhibitor that is approved for the treat-ment of BPH [25]. Along similar lines, Carvalho-Dias et al. [17]demonstrated that the elevated serotonin inhibits prostate growth

in BPH via down-regulating the AR gene expression, which agreeswith our data.

To evaluate proliferation of prostatic epithelial cells, we examinedthe gene expression of PCNA, which is a proliferation marker, inthe prostatic tissue of testosterone-treated rats. Co-treatment withdapoxetine reduced the gene expression of PCNA induced by testos-terone. Hence the observed correlation between dapoxetine admin-istration and amelioration of prostatic hyperplasia might be throughthe antiproliferative effect that dapoxetine has.

Patients with chronic inflammation and BPH have been shown tohave larger prostate volumes and more severe lower urinary tractsymptoms [26]. This suggests that chronic inflammation plays a keyrole in the pathogenesis of BPH [9]. It has been reported that nitricoxide and COX II may play an important role in determining the associ-ation between inflammation and prostate growth [27]. In all prostaticinflammatory cells, iNOS is the principal factor activating reactive nitro-gen that can damage cells [28]. Nitric oxide also potentiates COX II activ-ity generating pro-inflammatory prostaglandins [4]. The infiltratingcells have the ability to secrete growth factors and pro-inflammatory cy-tokines such as TFN-α [29].

In the current study, iNOS and COX II proteins expression in epithe-lial cells was assessed using immunohistochemistry. Overexpression ofiNOS and COX II was demonstrated in the epithelial cells of testoster-one-treated rats. However, co-treatment with dapoxetine showed astrong anti-inflammatory activity through attenuation of the testoster-one-induced increase in iNOS and COX II proteins expression and TNF-

Fig. 7. Immunohistochemical staining of COX II in the prostate tissues. A: COX II expression in the prostate epithelial cells of the control group showing no expression. B: Expression of COXII in prostate epithelial cells of testosterone-treated rats showing strong positive expression. C: COX II expression in prostate epithelial cells of rats treatedwith testosterone+ finasterideshowingweak positive expression. D: Expression of COX II in prostate epithelial cells of rats treatedwith testosterone+dapoxetine showingweak positive expression. (Immunopositivityindicated by brown colour) (×100). E: Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fields for each rat section.Values were expressed as mean of 8 experiments ± SEM. a: Significantly different from the control group at P b 0.05. b: significantly different from the testosterone-treated group atP b 0.05. c: significantly different from the standard finasteride-treated group at P b 0.05.

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α level in the prostate tissue. These data are in harmonywith Taler et al.and Walker [13,14] who have demonstrated the anti-inflammatoryproperties of SSRIs.

Apoptosis refers to the death of cells, which occurs as a normal andcontrolled part of the growth of an organism [30]. COX II expression inprostate epithelium can increase cellular proliferation and decrease ap-optotic activity through induction of Bcl-2 [31]. The Bcl-2 family hasbeen postulated as one of the critical mechanisms for determining apo-ptosis. Bcl-2 family members are divided into those that protect cellsfrom apoptosis (e.g. Bcl-2), and those that induce apoptosis (e.g. Bax)[32]. The ratio of the pro-apoptotic Bax protein and the anti-apoptoticBcl-2 seems to be amain determinant of relative resistance to apoptosis[33]. They trigger opposing effects on the mitochondria; Bcl-2 blockswhile Bax induces the release of cytochrome cwhich subsequently acti-vates the executioner caspase-3 which is critical for apoptosis [34].

Our data showed that dapoxetine markedly decreased protein ex-pression of the anti-apoptotic Bcl-2, but increased expression of thepro-apoptotic Bax, as compared with the BPH-induced group. Thus,rats treated with dapoxetine showed an increased Bax to Bcl-2 ratio

and increased caspase-3 activity, suggesting that dapoxetine-inducedapoptosis played a role in the effects of this compound on BPH andthat this was mediated via regulation of the expression of anti- andpro-apoptotic Bcl-2 family proteins. In harmony with our results, theantiproliferative and pro-apoptotic properties of SSRIs in different ma-lignant cells were previously reported [15,35].

In conclusion, the current study provides an evidence for the protec-tive effects of dapoxetine against testosterone-induced BPH in rats. Thiscan be attributed, at least in part, to decreasing AR expression, and theanti-proliferative, anti-inflammatory and pro-apoptotic activities ofdapoxetine in BPH. However, more clinical research on this topicneeds to be undertaken before the association between dapoxetineand the prevention of prostatic hyperplasia be more clearly understoodand practically used.

Funding

This research received no specific grant from any funding agency inthe public or commercial.

Fig. 8. Immunohistochemical staining of iNOS in theprostate tissues. A: Expression of iNOS in the prostate epithelial cells of the control group showing no expression. B: Expression of iNOSin prostate epithelial cells of testosterone-treated rats showing strong positive expression. C: iNOS expression in prostate epithelial cells of rats treated with testosterone + finasterideshowing weak positive expression. D: Expression of iNOS in prostate epithelial cells of rats treated with testosterone + dapoxetine showing weak positive expression.(Immunopositivity indicated by brown colour) (×100). E: Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fieldsfor each rat section. Values were expressed as mean of 8 experiments ± SEM. a: Significantly different from the control group at P b 0.05. b: significantly different from thetestosterone-treated group at P b 0.05. c: significantly different from the standard finasteride-treated group at P b 0.05.

59R.H. Sayed et al. / Toxicology and Applied Pharmacology 311 (2016) 52–60

Conflict of interest

None.

Transparency document

The Transparency document associated with this article can befound, in the online version.

Acknowledgements

None.

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