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1 Leptin influences estrogen metabolism and accelerates prostate2 cell proliferation

3 Christine N. Habib a, Ahmed M. Al-Abd b,c, Mai F. Tolba a,d, Amani E. Khalifa a, Alaa Khedr e,4 Hisham A. Mosli f, Ashraf B. Abdel-Naim a,⁎5 a Department of Pharmacology & Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt6 b Department of Pharmacology, Medical Division, National Research Center, Dokki, Cairo, Egypt7 c Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia8 d Biology Department, School of Science and Engineering, The American University in Cairo, New Cairo, Egypt9 e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia10 f Department of Urology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

a b s t r a c t1 1 a r t i c l e i n f o

12 Article history:13 Received 4 July 201414 Accepted 13 November 201415 Available online xxxx

16 Keywords:17 Obesity18 Leptin19 Prostate20 Estrogen metabolism

21Aim: The present study was designed to investigate the effect of leptin on estrogenmetabolism in prostatic cells.22Main methods: Malignant (PC-3) and benign (BPH-1) human prostate cells were treated with 17-β-23hydroxyestradiol (1 μM) alone or in combination with leptin (0.4, 4, 40 ng/ml) for 72 h. Cell proliferation24assay, immunocytochemical staining of estrogen receptor (ER), liquid chromatography–tandemmass spectrom-25etry method (LC–MS) and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were26used.27Key findings: Cell proliferation assay demonstrated that leptin caused significant growth potentiation in both28cells. Immunocytochemical staining showed that leptin significantly increased the expression of ER-α and29decreased that of ER-β in PC-3 cells. LC–MS method revealed that leptin increased the concentration 4-30hydroxyestrone and/or decreased that of 2-methoxyestradiol, 4-methoxyestradiol and 2-methoxyestrone.31Interestingly, RT-PCR showed that leptin significantly up-regulated the expression of aromatase and cytochrome32P450 1B1 (CYP1B1) enzymes; however down-regulated the expression of catechol-o-methyltransferase (COMT)33enzyme.34Significance: These data indicate that leptin-induced proliferative effect in prostate cells might be partly attribut-35ed to estrogen metabolism. Thus, leptin might be a novel target for therapeutic intervention in prostatic36disorders.

37 © 2014 Published by Elsevier Inc.

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42 Introduction

43 Obesity has become a critical health problem worldwide [5,22]. The44 number of deaths attributed to obesity in the United States, is estimated45 to be as high as 400,000 deaths per year [20]. Several studies have46 shown that obesity is a risk factor for prostate cancer (PCa) and benign47 prostatic hyperplasia (BPH) [21,37]. Positive correlation between the48 body mass index and the prostate tumor volume was observed in a49 large sized clinical trial in Italy [4].50 Several studies illustrated the relation of leptin to obesity [17,24,41].51 Leptin, a 16-kDa peptide hormone and the most well characterized52 adipokine, is involved in reproduction, decreases caloric intake and53 increases energy expenditure [19,28]. Previous studies suggested the54 involvement of leptin in rat prostate growth [23]. It is secreted mainly

55from adipocytes into the blood, with a fluctuating level according to56body mass. Lean individuals have an average of 4 ng/ml circulating57plasma leptin, while obese ones have a higher level, around 40 ng/ml58[10,32]. The expression of leptin receptors in the prostatewasfirst dem-59onstrated by Cioffi et al. [8]. The effect of leptin on malignant prostatic60cancer cells (DU145 and PC-3) was investigated in an in-vitro study,61and it illustrated that leptin causes significant growth potentiation in62these cell lines [34].63Successive studies showed that not only androgens are key players64in the development of prostatic cancer, but also estrogens play an im-65portant role [1,14]. The fact that the levels of androgen were found to66be decreasing in the age of peak incidence of prostatic disorders while67those of estrogen remained unchanged [39], led the scientists to consid-68er estrogen as an essential co-player in prostatic disorders. Confirming69this theory, was the finding that estrogen plays a significant role in70breast cancer [36], which is pathophysiologically similar to prostate71cancer to a great extent [30]. Prostate gland expresses two estrogen re-72ceptor subtypes (ER-α and ER-β). Activation of ER-α induces cell

Life Sciences xxx (2014) xxx–xxx

⁎ Corresponding author at: Faculty of Pharmacy, Ain Shams University, Abassia, Cairo,Egypt. Tel.: +20 2 01119998505; fax: +20 2 24051107.

E-mail address: abnaim@yahoo.com, abnaim@pharma.asu.edu.eg (A.B. Abdel-Naim).

LFS-14198; No of Pages 6

http://dx.doi.org/10.1016/j.lfs.2014.11.0070024-3205/© 2014 Published by Elsevier Inc.

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Please cite this article as: C.N. Habib, et al., Leptin influences estrogen metabolism and accelerates prostate cell proliferation, Life Sci (2014),http://dx.doi.org/10.1016/j.lfs.2014.11.007

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73 proliferation and inflammation, while ER-β mediates antiproliferative,74 anti-inflammatory and potentially anticarcinogenic effects [14]. In this75 context, it was reported that estrogenmetabolism and the profile of es-76 trogen metabolites have an impact on the biological effects of estrogen77 [15,27]. Some estrogen metabolites such as hydroxyestrogens are more78 potent than their parent estrogens in inducing carcinogenic effects [7].79 On the other hand,methoxyestrogens possess anti-proliferative proper-80 ties [31].81 However, the link between leptin and estrogenmetabolism remains82 unexplored. Therefore, the current study aimed to investigate the effect83 of leptin on estrogen metabolism in malignant (PC-3) and benign84 (BPH-1) human prostate cells.

85 Materials and methods

86 Chemicals

87 Human recombinant leptin was purchased from BioVision Chemical88 Company (Milpitas, California, USA). Sulforhodamine B (SRB), 17-β-89 hydroxyestradiol, dansyl chloride (Dns-Cl) 98% HPLC grade and90 β-glucuronidase/arylsulphatase (Helix pomatia, Type HP-2, ≥500 Sigma91 units β-glucuronidase and ≤37.5 units sulfatase activity) were pur-92 chased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).93 RPMI 1640 media, fetal bovine serum (FBS), antibiotics (100 μg/ml94 streptomycin, 100 units/ml penicillin) and phosphate-buffered saline95 (PBS; 0.15 M K2HPO4, 0.15 M Na2HPO4, 0.85% NaCl, pH 7.2) were pur-96 chased from Lonza Chemical Company (Walkersville, MD, USA). All97 other chemicals were of the highest grade commercially available.

98 Cell culture

99 Human prostate cancer cell line (PC-3) and benign prostate100 hyperplasic cells (BPH-1) were obtained from theNational Cancer Insti-101 tute (Cairo, Egypt). Cells were cultured in RPMI 1640media and supple-102 mented with 10% heat-inactivated FBS and antibiotics (100 μg/ml103 streptomycin, 100 units/ml penicillin). All cultures were maintained at104 37 °C in humidified atmosphere containing CO2 5% (v/v). Cellswere har-105 vested at 70–80% confluence andwashedwith PBS. Cells were detached106 using 0.25% trypsin in 0.1% EDTA, centrifuged at 1200 rpm for 10 min107 and re-suspended in growth medium in all experiments. Cell viability108 was checked using trypan blue exclusion assay.

109 Experimental design

110 Cells were divided into five groups for each cell line. Group I served111 as control group. Group II is considered as estrogen group and was ex-112 posed to 17-β-hydroxyestradiol (1 μM). Groups III, IV and V were co-113 treated with 17-β-hydroxyestradiol (1 μM) and 0.4, 4 and 40 ng/ml114 human recombinant leptin respectively.

115 Sulforhodamine B (SRB) proliferation assay

116 The SRB assay was used to assess cell proliferation as previously117 described [33] with minor modifications. Briefly, cells were seeded in118 96-well plates at a density of 500 cells/well then treated with 17-β-119 hydroxyestradiol and leptin as previously described for 72 h. Media120 was then discarded and cells were fixed by addition of 150 μl/well121 cold 10% trichloroacetic acid (TCA). The plate was incubated at 4 °C for122 1 h before being gently washed at least three times with tap water123 and then air-dried. The cells were stained by addition of 70 μl 4% SRB124 in 1% acetic acid for 10 min in the dark. The stain was removed and125 the cells were washed with 1% acetic acid, air-dried and 150 μl of126 10 mM aqueous Tris base was added to dissolve the dye. The plates127 were shaken for 5 min until the dye was uniformly distributed and128 then read on an ELISA microplate reader (ChroMate™ model 4300,129 USA) at 545 nm. Any alteration in the number of the viable cells results

130in a concomitant change in the amount of dye incorporated by the cells131in the culture.

132Immunocytochemical (ICC) detection of estrogen receptors α & β

133PC-3 and BPH-1 cells were seeded on cover slips overnight and sub-134sequently treated with 17-β-hydroxyestradiol and leptin for 72 h as135previously described. Cells attached to the cover slips were then fixed136with cold ethanol 70% for 1 h. Fixed cells were washed and immersed137in tris buffer saline (TBS). Permeabilization was then done by immers-138ing slides in 3% hydrogen peroxide for 10 min. Two drops of the ready139to use ER-α antibody (Novocastra Laboratories Ltd., Newcastle, UK)140were applied. The concentrated ER-β (AbD Serotec, USA) was prepared141according to manufacturer recommendation (1/50) and two drops142were applied to each slide. Subsequently, slides were incubated in the143humidity chamber overnight and poly horseradish peroxidase (HRP)144enzyme conjugate was applied to each slide for 30 min. Power Stain™1451.0 Poly HRP DAB Kit (Genemed Biotechnologies, CA, USA) was used146to visualize any antigen–antibody reaction in the cells. Diaminobenzi-147dine (DAB) chromogen was added to each slide for 2 min then rinsed,148after which counterstaining with Mayer Hematoxylin was performed149as the final step before slides were examined under the light micro-150scope. The number of DAB positive cells per high powerfieldwas count-151ed using the image analysis software (ImageJ, ver. 1.46a, NIH, USA).

152Assessment of estrogen and estrogen metabolites

153Cells were seeded in 6-well plates, incubated overnight and subse-154quently treated with 17-β-hydroxyestradiol alone or in combination155with leptin (0.4, 4, 40 ng/ml) for 72 h. Estrogen and estrogen metabo-156lites in cell media were analyzed using the liquid chromatography–tan-157demmass spectrometry (LC–MS) method as previously described [25].158Samples were hydrolyzed using β-glucuronidase/arylsulphatase. Di-159chloromethane was used to extract the steroid content then subjected160to evaporation. The dried residue was dissolved in Dns-Cl and sodium161bicarbonate. Samples were shaken and injected for LC–MS analysis162using mixture reaction monitoring (MRM) mode. An Ion Trap 6320163MS/MS detector was used.

164RNA extraction and semi-quantitative reverse transcriptase polymerase165chain reaction (RT-PCR)

166Total RNA isolation was performed by using RNeasy® Mini Kit167(Qiagen, Valencia, CA, USA) according to the supplier protocol. High-168capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster169City, CA, USA) was undertaken for constructing a cDNA library. PCR am-170plification reactions were then performed using a Taq PCR Master Mix171Kit (Qiagen, Valencia, CA, USA) and the following primers were used:172Catechol-O-methyltransferase (COMT) sense primer, 5′ CTA-CTG-GCT-173GAC-AAC-GTG-ATC-TG 3′ and the corresponding antisense primer, 5′174GTA-TTC-CAG-GAA-CGA-TTG-GTA-GTG-T 3′. Cytochrome P450 (CYP)175isoform 1B1 sense primer, 5′ TTT-CGG-CTG-CCG-CTA-CA 3′ and the cor-176responding antisense primer 5′ ACT-CTT-CGT-TGT-GGC-TGA-GCA 3′.177Aromatase sense primer, 5′ ATC-TCT-GGA-GAG-GAA-ACA-CTC-ATT-A1783′ and the corresponding antisense primer, 5′ CTG-ACA-GAG-CTT-179TCA-TAA-AGA-AGG-G 3′. Glyceraldehyde 3-phosphate dehydrogenase180(GAPDH) was used as reference housekeeping gene with sense primer,1815′ CAA-GGT-CAT-CCA-TGA-CAA-CTT-TG 3′ and antisense primer 5′182GTC-CAC-CAC-CCT-GTT-GCT-GTA-G 3′. All the primers were purchased183from (Metabion international AG, Martinsried, Germany). 40 cycles of184PCR amplification was performed, each consisted of a denaturation185step for 1 min at 94 °C, an annealing step for 30 s at 60 °C (COMT),18652 °C (CYP1B1), 58 °C (aromatase) and 58 °C (GAPDH). All PCR products187were resolved by 1.5% agarose gel electrophoresis and photomicro-188graphs were taken of the ethidium bromide-stained gels. Then gels189were scanned for quantification, and the pixel intensity for each band

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190 was determined using the image analysis software (ImageJ, ver. 1.46a,191 NIH, USA).

192 Data analysis

193 Data are presented asmean± SEM.Multiple comparisonswere per-194 formed using one-way ANOVA followed by Dunnett as a post-hoc test;195 the estrogen group is considered as the control group. The 0.05 level196 of probability was used as the criterion for significance. All statistical197 analyses were performed using Instat version 3 software package.198 Graphs were sketched using GraphPad Prism (ISI® software, USA)199 version 5 software.

200 Results

201 Assessment of cell proliferation

202 To evaluate the potential proliferative effect of leptin in malignant203 (PC-3) and benign (BPH-1) human prostate cells, SRB assay was used204 after 72 h of exposure to 17-β-hydroxyestradiol (1 μM) alone or in com-205 bination with serial concentrations of leptin. It was found that leptin206 promoted proliferation of both cell-lines. Exposure of PC-3 cells to 0.4207 ng/ml leptin didn't change cell proliferation significantly compared to208 the estrogen group. However, exposure to 4 and 40 ng/ml leptin signif-209 icantly increased cell proliferation by 21.9% and 22.2% respectively,210 compared to the estrogen group (Fig. 1.A). Regarding BPH-1 cell line,

211exposure to 0.4 and 4 ng/ml leptin didn't induce any significant prolifer-212ative pattern, while exposure to 40 ng/ml leptin significantly (P b 0.05)213induced cell proliferation by 50.9% compared to cells treatedwith estra-214diol alone (Fig. 1.B).

215Determining the expression level of estrogen receptors α & β

216To assess the effect of leptin on the expression level of ER-α & ER-β217in PC-3 and BPH-1 cell lines, immunocytochemical staining was per-218formed. In PC-3 cells, 17-β-hydroxyestradiol (1 μM) alone significantly219increased the number of cells expressing ER-α by 54.7%, compared to220the control group (Fig. 2.A). Treatment with 0.4 & 4 ng/ml leptin didn't221show significant difference in the number of cells expressing ER-α,222compared to the estrogen group (Fig. 2.A). However, exposure of PC-3223cells to 40 ng/ml leptin showed significant elevation in the number of224cells expressing ER-α by 72.2% compared to the estrogen group. On225the other hand, leptin decreased the number of cells expressing ER-β.226Exposure of PC-3 cells to 0.4 ng/ml leptin didn't show any significant227difference, even though exposure to 4 & 40 ng/ml leptin significantly228decreased the number of cells expressing ER-β (P b 0.05) by 28.4%229and 62.5% respectively compared to the estrogen group (Fig. 2.B). The230immunocytochemical staining was quantified as number of positive231cells/high power field, and the figures are represented in (Fig. 2.C & D).

232Quantification of estrogen & estrogen metabolites

233To investigate the effect of leptin on estrogen metabolism within234PC-3 and BPH-1 cell lines, several key metabolites (estradiol, 2-235methoxyestradiol, 4-methoxyestradiol, 2-methoxyestrone, 2-236hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone) were237measured in culture media using LC–MS. In the current work, exposure238of PC-3 cells to leptin did not significantly change the levels of estradiol,2392-methoxyestrone and 4-hydroxyestrone compared to the estrogen240group (Fig. 3.A). The level of 4-hydroxyestrone in the control group241was under the detection limit of the method used for analysis. Howev-242er, exposure to 4 and 40 ng/ml leptin significantly decreased the con-243centration of 2-methoxyestradiol by 37.4% and 45.3% respectively244compared to the estrogen group. In addition, exposure to 0.4, 4245and 40 ng/ml leptin significantly decreased the concentration of2464-methoxyestradiol (P b 0.05) by 65.34%, 65.37% and 74.39% respec-247tively compared to the estrogen group. On the other hand, 40 ng/ml lep-248tin significantly decreased estradiol levels in BPH-1 cells by 34.9%249compared to the estrogen group (P b 0.05) (Fig. 3.B). Leptin didn't sig-250nificantly change the concentration of 2-methoxyestradiol compared251to the estrogen group. Exposure of BPH-1 cells to 4 and 40 ng/ml leptin252significantly decreased the concentration of 4-methoxyestradiol by 62%253and 68.4% respectively compared to the estrogen group. Moreover, lep-254tin decreased the concentration of 2-methoxyestrone in a dose related255manner. Exposure of BPH-1 cells to 0.4, 4 and 40 ng/ml leptin decreased256the concentration of 2-methoxyestrone by 96%, 99% and 99.3%257respectively compared to the estrogen group. Interestingly 0.4, 4 and25840 ng/ml leptin significantly increased the concentration of 4-259hydroxyestrone (P b 0.05) in BPH-1 cells by 2, 3.9 and 4.1 fold respec-260tively compared to the estrogen group (Fig. 3.B). The levels of 2-261hydroxyestradiol and 4-hydroxyestradiol were under the detection262limit of the method used for analysis.

263Assessment of gene expression

264To explain the changes in the concentrations of some estrogen265metabolites, essential enzymes involved in estrogen metabolism have266been measured using RT-PCR in PC-3 and BPH-1 cell lines. Our data re-267vealed that exposure of PC-3 cells to 0.4, 4 and 40 ng/ml leptin signifi-268cantly increased aromatase expression in a non-dose related manner269by 0.76, 2.6 and 1 fold compared to the estrogen group (Fig. 4.A).270Furthermore, exposure to 0.4, 4 and 40 ng/ml leptin significantly up-

Fig. 1. Effect of leptin on PC-3 (A) and BPH-1 (B) cell proliferation. Cells were treatedwith17-β-hydroxyestradiol and serial concentrations of human recombinant leptin for 72 h.Cell proliferation was determined using SRB assay. Data are expressed as mean ± SEM.*Significantly different from estrogen group at P b 0.05 using one-way ANOVA followedby Dunnett as a post hoc test.

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271 regulated CYP1B1 expression (P b 0.05) by 0.5, 1.3 and 1.7 fold272 compared to the estrogen group. On the other hand, the highest concen-273 tration of leptin (40 ng/ml) significantly down-regulated COMT expres-274 sion by 48% compared to the estrogen group (Fig. 4.A). Regarding BPH-1275 cells, 0.4 ng/ml leptin didn't significantly change aromatase and CYP1B1276 expression. Exposure to 4 and 40 ng/ml leptin significantly increased277 aromatase expression by 1.5 and 3.3 fold respectively, while the expres-278 sion of CYP1B1 was increased by 32% and 99% respectively (P b 0.05) as279 compared to the estrogen group (Fig. 4.B). Interestingly, estradiol280 (1 μM) significantly down-regulated COMT expression by 26.3%281 compared to the untreated cells. This down-regulation was even more282 profound by co-treatment with leptin, where the highest concentration283 of leptin (40 ng/ml) induced 60.5% suppression of COMT expression as284 compared to the estrogen group (Fig. 4.B).

285 Discussion

286 Obesity is implicated in the progression of prostatic diseases such as287 prostate cancer (PCa) and benign prostatic hyperplasia (BPH) [13]. It is288 associated with greater risk for PCa-specific mortality [3,12]. Moreover,289 there is a growing body of evidence on the impact of estrogen and its290 metabolites in prostate pathophysiology. 17-β-Hydroxyestradiol is291 metabolized into several compounds that are different in their carcino-292 genic potentials. Catechol estrogens are involved in prostate cancer293 development and progression [7], while methoxyestrogens possess294 anti-proliferative properties [31]. The link between leptin and estro-295 gen metabolism has not been yet clearly demonstrated. Therefore,296 this study was designed to examine the effect of leptin on estrogen297 metabolism in malignant (PC-3) and benign human prostate cells298 (BPH-1).299 Our results demonstrated that leptin significantly stimulated the300 proliferation of both PC-3 and BPH-1 cells, which are in accordance301 with several studies indicating that leptin had a mitogenic effect on

302prostate cancer cells (PC-3 and DU145) [16,34,35]. It is worth mention-303ing that treatment of BPH-1 cells with 17-β-hydroxyestradiol alone304caused an inhibition in cell proliferation. This finding is in agreement305with previous studies illustrating that estrogens had growth inhibitory306effects on some cells [2,38,42].307To characterize the proliferative effects of leptin on prostate cells,308PC-3 and BPH-1 cell lines were analyzed for expression of the estrogen309receptor subtypes (ER-α and ER-β). ER-α is responsible for mediating310the effect of estrogen in enhancing cellular proliferation. In contrast,311ER-β activation results in anti-proliferative effects [1,14]. The312current study illustrated that leptin significantly increased ER-α ex-313pression whereas decreased ER-β expression in PC-3 cells. This was314in line with previous studies which indicated that leptin enhanced315the estradiol-induced activation of ER-α in MCF-7 and HeLa cells316[6] and reduced ER-β gene expression in rat prostate ventral lobe317[9]. This can partly explain the proliferative effect of leptin which318might be attributed to the overexpression of ER-α and down-319regulation of ER-β.320Moreover, the current study revealed that leptin increased the con-321centration of the proliferative metabolite 4-hydroxyestrone and/or de-322creased that of methoxyestradiol and methoxyestrone. The disturbance323in balance between hydroxyestrogens and methoxyestrogens might324elucidate the proliferative effect of leptin on PC-3 and BPH-1 cells.325This was in accordance with our previous study demonstrating that326catechol estrogens could neoplastically transform BPH-1 cells [26]. To327further substantiate the changes in the concentration of estrogen me-328tabolites, the effect of leptin on the expression of some key enzymes in-329volved in estrogen metabolism was examined. The expression levels of330aromatase, CYP1B1 and COMT enzymes were of special importance.331Since, aromatase is responsible for the conversion of dihydrotestoster-332one (DHT) into 17-β-hydroxyestradiol while, CYP1B1 is responsible333for the transformation of 17-β-hydroxyestradiol into the delirious334catechol estrogen 4-hydroxyestradiol [40]. We previously reported

Fig. 2. Immunocytochemical staining of ER-α (A) and ER-β (B) in PC-3 cell line (magnification ×100). Arrows indicate positive cells for ER-α or ER-β antibodies. C and D: Quantitativeimage analysis for immunocytochemical staining of ER-α and ER-β respectively expressed as number of positive DAB cells across 6 different high power fields for each group. Data areexpressed as mean ± SEM. *Significantly different from estrogen group at P b 0.05 using one-way ANOVA followed by Dunnett as a post hoc test.

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335 that thismetabolite can induce proliferation andmalignant transforma-336 tion of prostate cells [26]. COMT is of equal significance as it is the en-337 zyme responsible for the detoxification of this noxious metabolite [40]338 . Leptin significantly increased aromatase expression. At the same339 time, it significantly increased the expression level of CYP1B1 which340 might explain the increase in the proliferative estrogen metabolite 4-341 hydroxyestrone in BPH-1 cells. Our results are in accordance with an342 earlier study that showed that leptin increases the expression level of343 CYP1B1 in breast cancer cell lines [29]. On the other hand, leptin signif-344 icantly decreased the expression level of COMTwhichmight explain the345 decrease in the formation of the anti-proliferative metabolites (2-346 methoxyestradiol, 4-methoxyestradiol and 2-methoxyestrone).

347 Conclusion

348 In summary, the current study demonstrated that leptin directed es-349 trogenmetabolism to generate catechol estrogens and/or decreased the350 formation of methoxyestrogens in malignant (PC-3) and benign (BPH-351 1) human prostate cells. This disturbance in balance between352 hydroxyestrogens andmethoxyestrogensmight explain the role of lep-353 tin in the progression of prostatic disorders. Further studies are needed354 to elucidate that leptin suppression could be a novel way to improve the355 efficacy of prostate disease treatments.

356Conflict of interest statement

357The authors declare that there is no conflict of interest.

358Authors' contribution

359CN: practical part andwriting themanuscript. AMA,MFT: idea of re-360search and practical part. AK: analysis of estrogen metabolites. AEK,361HAM, ABA: idea of the research and writing the manuscript.

362Uncited references Q1

363[11,18]

364Acknowledgments

365This project was funded by the Deanship of Scientific Research366(DSR), King Abdulaziz University, Jeddah, under grant No. 57/140/32.

Fig. 3. Estimation of estrogen and estrogen metabolites in the culture media from PC-3(A) and BPH-1 (B) cells treated with 17-β-hydroxyestradiol (1 μM) with or withoutvarious concentrations of leptin (0.4, 4, 40 ng/ml). Data are expressed as mean ± SEM.*Significantly different from the corresponding estrogen group at P 0.05 using one-wayANOVA followed by Dunnett as a post hoc test.

Fig. 4. Effect of leptin on mRNA expression of some estrogen metabolizing enzymes(aromatase, catechol-o-methyltransferase, and cytochrome P 450 1B1) in PC-3 (A) andBPH-1 (B) cell lines. Cells were incubated with 17-β-hydroxyestradiol (1 μM) with orwithout serial concentrations of leptin for 72 h. Total RNA was extracted and subjectedto reverse transcription PCR. Data were normalized to GAPDH and expressed asmean ± SEM. *Significantly different from estrogen group at P b 0.05 using one-wayANOVA followed by Dunnett as a post hoc test.

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367 The authors, therefore, acknowledge with thanks DSR technical and368 financial support.

369 References

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Please cite this article as: C.N. Habib, et al., Leptin influences estrogen metabolism and accelerates prostate cell proliferation, Life Sci (2014),http://dx.doi.org/10.1016/j.lfs.2014.11.007