SEARCH

SEARCH BY CITATION

Keywords:

  • catechol oestrogens;
  • oestogen metabolism;
  • prostatitis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

What's known on the subject? and What does the study add?

The role of oestrogen in prostatic inflammation has been extensively shown. The catechol oestrogens are known to be more potent oestrogenic moieties that not only aggravate the inflammatory response in situ, but are also believed to have oxidative stress and genotoxic effects.

The present study highlights a significant role of inflammation in oestrogen metabolism and, particularly, in generating ‘bad’ oestrogen metabolites. This finding may pave the way for new therapeutic methods for the treatment and/or prevention of prostate diseases.

OBJECTIVE

  • • 
    To investigate the impact of experimentally induced inflammation on oestrogen metabolism in rat prostate.

MATERIALS AND METHODS

  • • 
    Prostatitis was induced in normal and oestrogen-treated male rats by injecting 2% carrageenan solution into the ventral prostate. After 48 h, the rats were killed and the ventral prostate was collected.
  • • 
    Prostatic inflammation and proliferation were confirmed by gross visual evidence, histology and elevated tumour necrosis factor-α, prostaglandin E2 and cyclin-D1.
  • • 
    Expression of oestrogen-metabolizing enzymes was assessed using capillary electrophoresis, and oestrogen metabolites within prostate tissue were assayed using liquid chromatography mass spectrometry.

RESULTS

  • • 
    Animals exposed to carrageenan insult combined with oestrogen treatment showed the most prominent inflammatory and proliferative response.
  • • 
    Treatment of animals with oestrogen alone induced moderate inflammation and proliferation.
  • • 
    Oestrogen-metabolizing enzymes were overexpressed in animals with experimental prostatitis with sequential accumulation of catechol oestrogens within prostatic tissues.
  • • 
    Oestrogen receptor-α was underexpressed in the prostatitis with oestrogen group, while oestrogen receptor-β was overexpressed.

CONCLUSIONS

  • • 
    The present work provides experimental evidence that local inflammation enhances oestrogen synthesis and directs oestrogen metabolism to generate catechol oestrogens within prostatic tissues.
  • • 
    This may contribute, at least partly, to enhanced prostatic cell proliferation.

Abbreviations
ADT

androgen deprivation therapy

ER

oestrogen receptor

PGE2

prostaglandin E2

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

MRM

mixture reaction monitoring.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

Prostatic disorders such as prostatitis, BPH and prostatic cancers are considered to be the most common male disorders in elderly males. About 60% of 60-year-old males are diagnosed with BPH, and prostatic cancer is one of the most common male cancers in the USA [1–3]. Since <10% of prostatic disorders are attributed to genetic predispositions, the influence of hormones is receiving the most attention with regard to prophylactic and treatment approaches [4]. After the discovery of the androgenic role in prostatic growth and proliferation, androgen deprivation therapy (ADT) was the predominant approach for the treatment of prostatic disorders [5]; however, limited clinical results were obtained from ADT [6]. Androgen levels were found to be declining at a time of peak incidence of prostatic disorders [5]. Nevertheless, the great similarities between prostatic and breast tissues, not only from the gross endocrine structural point of view but also regarding their microenvironmental signalling traits, partially diverted the attention to oestrogen and their potential involvement in prostatic disorders [6]. Oestrogen receptor-α (ER-α) and oestrogen receptor-β (ER-β) were cloned from prostatic tissues [5,7]. Aromatase enzyme, the key link between androgenic and oestrogenic signalling, was found in prostatic tissue and affects the physiology and pathophysiology of prostate [5]. Aetiologically, serum oestrone and oestradiol are the highest in the African-American and lowest in the Japanese population which strongly correlates with their risk for prostatic cancer [4].

The oestrogenic milieu has been an area of investigation of prostatic physiology and pathophysiology. The induction of prostate cancer in Noble rats required co-administration of oestrogen and androgen [8], and the use of a selective oestrogen receptor modulator reduced prostate proliferation and hyperplasia [9,10]. Oestrogenic signalling induces proliferation of prostate tissue [11]; however, the exact mechanism is unknown [12]. Some oestrogen metabolites, e.g. hydroxyestradiol and hydroxyestrone, are more potent relative to their parent oestrogens in inducing microsatellite instability and oxidative stress [13,14]; however, some oestrogen metabolites, e.g. methoxyestradiol and methoxyestrone, possess anti-proliferative properties [15]. The first report on oestrogen metabolism to catechol oestrogens in prostate tissue was published by Cavalieri et al. in 2002 [16]. Catechol oestrogens are known for their DNA-binding affinity and carcinogenic potential.

Recent interest has focused on the role that inflammation may play in the development of prostatic cancer [17,18]. In prostate tissue, the oestrogenic microenvironment induces prostatic proliferation and recruits inflammatory cells which, in turn, initiates inflammatory cascade [6]. Oestradiol has been used to induce non-bacterial prostatitis in an experimental rat model [19,20]. Aromatase knock-in mice showed extensive prostatitis with profound neutrophilic infiltration to prostatic stromal tissue; however, aromatase knock-out mice were protected from androgen-induced prostatic cancer [21]. The aromatase enzyme inhibitor, mepartracin, significantly improved clinical outcomes of prostatitis with no significant change in testosterone level [22]. Inflammatory cytokines, e.g. TNF-α and prostaglandin E2 (PGE2), promote the formation and accumulation of catechol oestrogens by misbalancing the expression of their metabolizing enzymes [23,24], but the potential influence of the inflammatory microenvironment on oestrogenic signalling within prostatic tissue is not known [12]. In the present study, the role of inflammation on oestrogen metabolism in experimentally induced prostatitis was investigated in rats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

CHEMICALS AND DRUGS

Carrageenan and 17β-hydroxyesradiol were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Oestrone, 4-hydroxyestradiol, 4-hydroxyestrone, 16α-hydroxyestradiol, 16α-hydroxyestrone, 2-methoxyestradiol, 2-methodxyestradiol, 4-methoxyestrone, and 4-methoxyestradiol were purchased from Steroids Inc. (Newport, RI, USA). Urethane was purchased from Biobasic Inc. (Toronto, Canada). B-Glucuronidase/Arylsulphatase (Helix pomatia, Type HP-2, ≥500 Sigma units β-glucuronidase and ≤37.5 units sulfatase activity) and dansyl chloride (Dns-Cl) 98% HPLC grade were purchased from Sigma-Aldrich Chemical Co. All other chemicals were of the highest available analytical grade.

ANIMALS AND TREATMENT

Male Sprague–Dawley rats (300 g weight) were acclimatized in the animal house facility of King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia, for at least 1 week before experimentation. Animals were kept at 20 ± 2°C and 65 ± 10% relative humidity for the duration of the experiment. Standard food pellets and water were supplied ad libitum. All experiments were performed between 8:00 and 10:00 a.m. Animal handling and treatment was approved by the bioethical and research committee of King Abdulaziz University.

Experimental prostatitis was induced in rats as previously described with minor modifications [25]. Briefly, animals were anaesthetized by i.p. injection of urethane (1 g/kg). A surgical V-shape incision was made in the supra pubic region in the rats, exposing the prostate gland. A volume of 50 µL of carrageenan solution (2%) was slowly injected into the ventral prostate and the wound was then surgically sutured and dressed with sterile povidone iodine solution. Sham operations were performed in animals of control groups without carrageenan injection. Animals were divided into four groups (n= 6). The control group was subjected to sham operation; the oestrogen-alone group was subjected to sham operation and received 17β-oestradiol in DMSO (5 mg/kg) via i.p. injection 3 h before being killed; the prostatitis group was subjected to intraprostatic carrageenan injection; and the oestrogen and prostatitis group was subjected to intraprostatic carrageenan injection and received 17β-oestradiol (5 mg/kg) via i.p. injection 3 h before being killed. Oestradiol treatment dose and duration were based on pilot experiments as well as the work of Cavalieri et al. [16]. All animals were allowed free access to food and water for 48 h after surgery then were killed by cervical dislocation. Prostatic tissues were collected post mortem and aliquots were distributed and snap frozen at −80°C until further analysis; portions of the prostatic tissues were fixed in buffered formalin solution (4%) for histological assessment.

To assess the inflammatory status of the ventral prostate lobe, PGE2 and TNF-α within ventral prostatic tissue were quantified. Accurately weighted prostatic tissues were ice crushed in liquid nitrogen and then homogenized for 15 min in PBS (pH 7.4). The amounts of PGE2 and TNF-α were determined using Quantikine® rat immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Concentrations of PGE2 and TNF-α were normalized based on protein concentration.

To assess the expression of oestrogen-metabolizing enzymes, total RNA isolation from ventral prostatic tissues was performed using RNeasy Mini Kit® (Qiagen Inc. Valencia, CA, USA). Reverse transcription was undertaken to construct a cDNA library from different treatments using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). PCR reactions were then performed using a Taq PCR Master Mix Kit (Qiagen Inc.) and amplification products were assayed using QIAxcel System® capillary electrophoresis (Applied Biosystems). Primer sequences were as follows: aromatase forward primer GCT-TCT-CAT-CGC-AGA-GTA-TCC-GG and reverse primer CAA-GGG-TAA-ATT-CAT-TGG-GCT-TGG; catechol-O-methyltransferase (COMT) forward primer ATC-TTC-ACG-GGG-TTT-CAG-TG and reverse primer GAG-CTG-CTG-GGG-ACA-GTA-AG; nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 (NQO-1) forward primer CAG-GGT-CCT-TTC-CAG-AAT-AAG and reverse primer CTG-GTT-GTC-GGC-TGG-AAT-GGA-C; rat steroid sulphatase forward primer TAA-CCC-AGG-GAC-AAC-CTC-TG and reverse primer GGT-GTA-GCC-TTG-ACC-CTT-GA; 17 β-hydroxysteroid dehydrogenase type-I forward primer AGT-GCT-CAT-TAC-CGG-TTG-CT and reverse primer CTT-GCT-CAT-AAC-CAC-GCT-GA; 17 β-hydroxysteroid dehydrogenase type-II forward primer TTC-TCT-GCA-AAG-CCT-GGA-GT and reverse primer GGC-TCC-GAA-GAA-GTT-CAC-TG. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference housekeeping gene with forward primer AAA-CCC-ATC-ACC-ATC-TTC-CA and reverse primer GTG-GTT-CAC-ACC-CAT-CAC-AA.

Oestrogen metabolites were assessed in ventral prostatic tissues using the liquid chromatography mass spectrometry (LC-MS) method as described previously, with minor modifications [26]. On the day of analysis, samples were ice crushed in liquid nitrogen, homogenized and enzymatically hydrolysed using β-glucuronidase/arylsulphatase in 1 mL of 0.1 m acetate buffer (pH 5.5) for 24 h. Steroid content was extracted using 5 mL dichloromethane (three times). The combined organic layer was subjected to evaporation under a weak stream of nitrogen gas and the dried residue was completely dissolved in 100 µL Dns-Cl (1 mg/mL in acetone) and 100 µL 0.1 m sodium bicarbonate (pH 9.3). After shaking for 2 s, samples were heated at 60 °C for 5 min, cooled, and a volume of 20 uL was injected for LC-MS analysis using mixture reaction monitoring (MRM) mode. An Agilent 1200 series HPLC with Ion Trap 6320 MS/MS detector was used (Agilent Technology, Santa Clara, CA, USA). The analysis was performed on Agilent Zorbax Extend C18 column (150 mm × 46 mm, 5 microns) with a guard column Extend C18 (2.2 × 2.2 mm) at 35°C. The HPLC system was programmed to clean column with 100% acetonitrile for 10 min, followed by 100% water for 10 min, and allowed to equilibrate for 10 min with the mobile system. The ion-Trap detection was set as follows: MRM mode was selected and the M + H+ for Dns-Oestrogen were selected, range 450–850 m/z, target mass 500 m/z, max accumulation time 200 ms, ramp range 4500–1500 v. Peaks were verified by m/z and retention time.

Reference standard calibration curves (n= 3) of each oestrogen metabolite in similar biomatrix were conducted and validated for accuracy and precision over a concentration range of 0.1–90 ng/mL.

Histological and immunohistochemical analysis for ventral prostatic tissues were performed according the laboratory routine protocol. Briefly, paraformaldhyde fixed tissues were embedded in paraffin wax. Cross-vertical sections (5 µm) were obtained and, after dewaxing and rehydration, sections were stained with haematoxylin and eosin (H&E).

Paraffin-embedded ventral prostate tissues were further used for immunohistochemical staining of cyclin-D1, ER-α and ER-β. Briefly, cross-vertical sections (5 µm) were obtained and, after dewaxing and rehydration, sections were incubated with 3% H2O2 for 30 min to eliminate the endogenous peroxidase activity. Non-specific binding sites were blocked with normal donkey serum for 30 min and then incubated overnight at 4°C in mouse antiserum (Abcam Inc., Cambridge, UK) against cyclin-D1 (dilution 1:100), ER-α (dilution 1:25) or ER-β (dilution 1:100). After rinsing in PBS, sections were incubated in peroxidase-conjugated donkey anti-mouse IgG (dilution 1:200; Jackson ImmunoResearch Lab, Inc. West Grove, PA, USA) for 1 h. For colouration, PBS-washed sections were incubated with a mixture of 0.05% 3,3’-diaminobenzidine containing 0.01% H2O2 at room temperature until a brown colour was visible, and then washed with PBS, counterstained with H&E, and mounted. Percent DAB-positive cells per high power field were counted and used for quantitative comparison [27]. Rat uterus samples were used as quality controls for ER immunostaining.

Data are presented as mean (sem). anova with a least significant difference post-hoc test was used to determine significance using SPSS® for Windows, version 17.0.0. A P value of <0.05 was considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

ASSESSMENT OF PROSTATIC INFLAMMATION AND PROLIFERATION

To induce prostatitis, carrageenan was administered by intraprostatic injection to the ventral prostate lobe. After 48 h, gross observation for the ventral prostate gland showed significant oedematous enlargement, and an increase in prostate/body weight ratios in rats with prostatitis treated with oestrogen was found compared with the control group. No significant prostatic enlargement was observed in animals with prostatitis or oestrogen treatment alone (Fig. 1A).

image

Figure 1. Evaluation of the prostatitis, Prostate tissues of normal and oestrogen-treated rats weighed and expressed relative to the whole animal body weight (A). Prostatic concentration of the inflammatory cytokines, PGE2 (B) and TNF-α (C). Data are expressed as mean (sem). * Significantly different from normal control group (P < 0.05); ** Significantly different from oestrogen-alone group (P < 0.05).

Download figure to PowerPoint

Further assessment for prostatitis was performed biochemically by measuring the tissue level of PGE2 and TNF-α in the ventral lobe. Intraprostatic PGE2 was significantly increased in the prostatitis group compared with the control group. In addition, oestrogen increased PGE2 level in the prostatitis group more than a hundredfold compared with the oestrogen-alone group. It is worth mentioning that oestrogen administration per se induced a significant increase in PGE2 within local prostatic tissues (Fig. 1B). Similarly, TNF-α level within prostatic tissue was significantly increased in rats with prostatitis and further doubled in rats with prostatitis and oestrogen co-administration compared with the corresponding controls. Oestrogen alone did not affect TNF-α level within prostatic tissue (Fig. 1C).

Histopathological examination of the ventral prostate in the oestrogen-alone group showed mild inflammation with mild to moderate inflammatory cells infiltration compared with the control group (Fig. 2A, B, E and F). Rats with prostatitis had significant inflammation with plasma cell infiltration, interstitial inflammatory cells, engorged blood vessels, luminal epithelial lining hyperplasia, intraluminal pseudo polyposis (Fig. 2C,D). Oestrogen administration to the prostatitis group induced extensive inflammation with intra- and extraluminal inflammatory cell infiltration and severe luminal epithelial lining hyperplasia with prominent nucleoli (Fig. 2G,H).

image

Figure 2. Histopathological evaluation for of carrageenan-induced prostatitis. The control group (A and B) showed normal endocrinal histological appearance. The prostatitis group (C and D) showed interstitial inflammatory cell infiltration, engorged blood vessels, luminal epithelial hyperplasia and intraluminal pseudo polyposis. The oestrogen-alone group (E and F) showed a similar but milder inflammatory response. Prostatitis groups treated with oestrogen (G and H) showed extensive inflammation with intra- and extraluminal inflammatory cell infiltration, severe luminal epithelial lining hyperplasia with prominent nucleoli. Scale bars = 20 µm in (A, C, E and G) and 5 µm in (B, D, F, and H).

Download figure to PowerPoint

To assess cellular proliferation status within prostatic tissues, immunohistochemistry for the proliferation marker cyclin-D1 within ventral prostate tissue was examined and percent of positive cells was calculated (Fig. 3). In the control group, cyclin-D1 was moderately expressed (6.3[0.9]%) by cells confined to the prostatic interstitial compartment (Fig. 3A,E). In both the prostatitis and oestrogen-alone groups, cyclin-D1 was significantly overexpressed in both the epithelial lining and interstitial compartments (28.8[4.6] and 26.6[2.8]%, respectively) of the prostatic tissue (Fig. 3B, C and E). Oestrogen administration to the prostatitis group induced marked overexpression for cyclin-D1 (63.2[5.4]%) in epithelial and interstitial compartments of the prostate which indicates profound proliferation (Fig. 3D,E).

image

Figure 3. Assessment of cyclin-D1 expression within prostatic tissue. Prostatic proliferation was assessed immunehistochemically by cyclinD1 staining in paraffin sections of prostatic tissues. Percent of positive DAB cells were counted in 10 different high power fields of control (A), prostatitis (B), oestrogen alone (C) and prostatitis groups treated with oestrogen (D). Percent positive cells were compared between different treatments (E). Data are expressed as mean (sem). Scale bar = 5 µm. *Significantly different from normal control (P < 0.05); **Significantly different from oestrogen control group (P < 0.05).

Download figure to PowerPoint

GENE EXPRESSION OF OESTROGEN-METABOLIZING ENZYMES IN PROSTATE TISSUE

To examine the effect of prostatitis on oestrogen metabolism in situ, the expression levels of several key enzymes in oestrogen metabolism were measured within ventral prostatic tissue using the Qiaxel system. Aromatase, steroid sulphatase and rat 17 β-hydroxysteroid dehydrogenase type-2 enzymes were significantly overexpressed (2–4fold compared with the control group) in the prostatitis group with oestrogen co-administration, while no significant change in their level in the remaining groups was found. COMT gene was significantly down-regulated to 60% of its original expression level in the prostatitis group with oestrogen administration. Inflammation and oestrogen-alone administration showed a nonsignificant decrease in the expression level of COMT. NQO-1 was significantly overexpressed in the prostatitis group. Oestrogen administration counteracted inflammation-induced NQO-1 overexpression, bringing its level back to normal control. Oestrogen alone did not show any change in NQO-1 expression level in prostatic tissue (Fig. 4).

image

Figure 4. Expression of oestrogen-metabolizing enzymes within prostate tissue. Data are expressed as mean (sem). *Significantly different from corresponding control (P < 0.05).

Download figure to PowerPoint

QUANTIFICATION OF OESTROGEN AND OESTROGEN METABOLITES IN PROSTATE TISSUE

Free and conjugated oestrogen and oestrogen metabolites were quantified in ventral prostate tissue. Several key moieties in oestrogen metabolism (oestradiol, oestrone, 4-hydroxyestradiol, 4-hydroxyestrone, 16α-hydroxyestradiol, 16α-hydroxyestrone, 2-methoxyestradiol, 2-methodxyestrone, 4-methoxyestrone, and 4-methoxyestradiol) were determined using LC-MS. The linearity of the method was found to be acceptable (R2 ranges from 0.9394–0.9877) in the concentration range (0.1–45 ng/mL) for all metabolites. Accuracy and precision were found to be within 10% in the linear range and 15% at the lower limit of quantification (data not shown).

Total oestrogen within ventral prostatic tissue was increased fivefold in the prostatitis group receiving oestrogen compared with the oestrogen-alone group, which might be attributed to the elevated level of aromatase enzyme. Oestradiol, oestrone and the hydroxyl metabolites 16α-hydroxyestrone and 4-hydroxyestradiol were markedly increased within prostatic tissue of the prostatitis and oestrogen group, showing more than 10 times their concentration in their corresponding oestrogen-alone group. However, 4-hydroxyestrone and the methoxy oestrogen metabolites (2-methoxyestradiol, 2-methodxyestrone, 4-methoxyestrone, and 4-methoxyestradiol) did not show any significant change in their tissue concentration (Fig. 5). It is worth mentioning that the prostatic level of oestrogen and oestrogen metabolites in the ventral lobes of the control and prostatitis groups were beyond the lower limit of quantification of the method of analysis used in the present study.

image

Figure 5. Estimation of oestrogen and oestrogen metabolites within prostate tissue. Data are expressed as mean (sem). *Significantly different from corresponding oestrogen control (P < 0.05).

Download figure to PowerPoint

EXPRESSION LEVELS OF ER-A AND ER-B IN THE PROSTATE TISSUE

The classic nuclear receptors for oestrogenic signalling in different tissues, including prostatic tissue, are ER-α and ER-β. The expression of ER-α in the epithelium and stroma tissues and ER-β in the epithelium of prostatic tissues was determined in the present study using immunohistochemical staining. ER-α was not significantly affected in the oestrogen-alone group compared with the control group. Inflammation markedly down-regulated ER-α in both the prostatitis and prostatitis with oestrogen groups compared with their corresponding controls (Fig. 6); however, oestrogen per se significantly up-regulated the expression of ER-β compared with the control group (Fig. 7). Inflammation significantly up-regulated the expression level ER-β in both the prostatitis and prostatitis with oestrogen groups compared with their corresponding controls (Fig. 7).

image

Figure 6. Expression level of ER-α in prostatic tissue. Percent of positive DAB cells were counted in 10 different high power fields of normal (A), prostatitis (B), oestrogen-alone (C) and prostatitis groups treated with oestrogen (D). Percent positive cells were compared between different treatments (E). Data are expressed as mean (sem). Scale bar = 5 µm. ** Significantly different from oestrogen control group (P < 0.05).

Download figure to PowerPoint

image

Figure 7. Expression level of ER-β with in prostatic tissue. Percent of positive DAB cells were counted in 10 different high power fields of normal (A), prostatitis (B), oestrogen alone (C) and prostatitis groups treated with oestrogen (D). Percent positive cells were compared between different treatments (E). Data are expressed as mean (sem). Scale bar = 5 µm. *Significantly different from normal control (P < 0.05); **Significantly different from oestrogen control group (P < 0.05).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

The role of oestrogen and its metabolites in prostate physiology and pathophysiology has been growing rapidly [5,16]. Prostatitis has been linked to other prostate diseases such as BPH and prostate cancer [28]. In the present work, the impact of inflammation on oestrogen metabolism in experimentally induced prostatitis was investigated in rats’ ventral prostates. Injection of intraprostatic carrageenan resulted in profound prostatitis manifested by gross visual, biochemical and histological evidence which was significantly aggravated by oestrogen administration. Exposure of prostatic tissue to carrageenan resulted in a comprehensive inflammatory environment in vivo. Oestrogen administration dramatically increased the inflammatory markers TNF-α and PGE2. TNF-α, and other inflammatory cytokines directly influence key steps in steroidogenesis in various endocrine cell types [29]. TNF-αper se has been used to show the effect of inflammation on oestrogen metabolism and signalling in isolated endometrial cells [23]. Further, PGE2 has been shown to significantly increase endogenous oestrogen biosynthesis in prostate stromal cells as well as in breast fibroblast cells secondary to aromatase enzyme overexpression [24]. Two days after induction of inflammation, the proliferation marker, cyclin-D1 was overexpressed in inflamed prostate tissues and further aggravated by oestrogen administration. As in the present study, oestrogen has been shown to increase rat prostatic proliferation by modulating cell cycle transition proteins such as cyclin-D1, which remained highly expressed for 5 days [30]. Thus, the observed enhancement of prostatic proliferation and cyclin-D1 overexpression might be attributed to auto-augmentation interaction between inflammation and oestrogenic activity.

The role of oestrogen has been studied extensively with respect to its effect on prostate physiology and pathophysiology [31], but, the effect of oestrogen metabolites on prostate pathophysiology has not been fully explored [16,23]. Recently, the potential significance of urinary oestrogen metabolites has been studied as a prostate cancer risk predictor [32]. The oestrogen metabolites, catechol oestrogens, are more potent in inducing oxidative stress and microsatellite instability [13]. The present study showed elevation in 2-hydroxyl and 4-hydroxy-oestrogen metabolites in alignment with exaggerated inflammatory response within prostate tissue. Enzymes responsible for the synthesis and activation of oestrogen and catechol oestrogen, e.g. aromatase, steroid sulphatase and HSD17β-II, were significantly overexpressed in the combined inflammation with the oestrogen group. The enzymes responsible for the detoxification of catechol oestrogen, COMT and NQO-1, were either down-regulated or unchanged, respectively. Sequentially, a significant accumulation of oestrone, oestradiol and catechol oestrogens (16-α hydroxy oestrone and 4-hydroxy oestradiol) was detected in the inflamed prostatic tissues. No compensatory change was detected in the prostatic level of methoxy oestrone or methoxy oestradiol, which might be attributed to the observed decreased expression of COMT. The lack of COMT- and NQO-1-dependent catechol oestrogen detoxification would aggravate their proliferative effects in prostate tissue. Selective oestrogen receptor modulators attenuate glandular inflammation in non-bacterial prostatitis and possess anti-inflammatory properties in other tissues [10,33]. Several reports provided accumulating evidence for an oestrogenic role in prostatitis and prostatic proliferation [5,19,28]. Reciprocally, local inflammation potentiates the oestrogenic components within different hormone-dependent tissues [17,24,28,34]. In the present study, we found experimental evidence for the influence of inflammation on oestrogen metabolism in that more reactive oestrogen metabolites with potentially proliferative properties were generated. Chemically induced inflammation directed oestrogen metabolism to generate more potent oestrogen metabolites which, in turn, are believed to aggravate the inflammatory and proliferative status within prostatic tissue in an auto-amplification vicious cycle.

Oestrogen receptors were identified in prostatic tissues and are thought to mediate oestrogenic effects within the prostate [5]. The role of ERs in prostate physiology and pathophysiology has been previously investigated [27]. Classically, ER-α possesses inflammatory, proliferative and pre-malignant properties while ER-β counteracts ER-α effects [31]. In the present study, ER-α was down-regulated in prostatitis and further depressed by oestrogen co-administration. Conversely, ER-β was overexpressed in the inflammation group and further elevation was observed as a result of oestrogen co-administration. This cannot be fully explained, but several previous reports indicated controversial relevance between ER expression and prostatic disorders. Overexpression of ER-β was observed in prostatic cancer, with no significant alteration in ER-α[35]. By contrast, Fujimura et al. [27] reported a significant decrease in ER-β positive cells and a significant increase in ER-α immune-reactive cells in prostate cancer [36]. Accordingly, alteration in the expression level of ERs in the progression of different prostatic disorders is controversial and warrants further investigation. Given that oestrogen metabolite-induced effects are believed to be receptor-independent [23], the key element in aggravating prostatic inflammation would be catechol oestrogen metabolites rather than oestrogen per se.

In conclusion, the present study provides experimental evidence that local inflammation enhances oestrogen synthesis and directs oestrogen metabolism to generate catechol oestrogens within prostatic tissues. These may contribute, at least partly, to enhanced prostatic cell proliferation.

ACKNOWLEDGEMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

This work was supported by Sheikh Ahmed H. Fetaihi Chair for Research on Prostatic Diseases, King Abdulaziz University, Jeddah, Saudi Arabia. We would like to thank Dr. Nabila Salah, Department of Pathology, National Research Center, Egypt, for her help in the histological studies.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES