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Year : 2019  |  Volume : 62  |  Issue : 1  |  Page : 99-102
Association of androgen and estrogen receptor expression with prostate volume in benign prostatic hyperplasia


1 Department of Biochemistry, JIPMER, Puducherry, India
2 Department of Urology, JIPMER, Puducherry, India
3 Department of Pathology, JIPMER, Puducherry, India

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Date of Web Publication31-Jan-2019
 

   Abstract 


Background: The expression of androgen (AR) and estrogen receptors (ER-A, ER-B) in Prostate cancer is well documented, but there are limited data about the same in patients with BPH. Hence the present study was designed to analyse the gene and protein expression of androgen and estrogen receptors in patients with BPH. Materials and Methods: Prostatic tissues were obtained from 27 BPH patients aged between 55 to 85 years by transurethral resection of prostate. Based on prostate volume, BPH patients were divided into two groups, Group A (≤30mL) and Group B (>30mL). The mRNA and protein expression of AR, ER-A and B were assessed by Quantitative real time PCR, Western blotting and Immunohistochemistry. Results: AR gene (P < 0.05) and protein expression (P = 0.03) and ER-A gene (P < 0.05) and protein expression (P = 0.02) was significantly higher in BPH patients with larger prostate size compared to smaller prostate size. Immunohistochemistry showed that AR expression was predominate in ductal cells of larger volume prostate tissues while AR expression in stromal tissue was the dominant finding in patients with smaller prostate size. Also serum estradiol was significantly increased in patients with larger prostate size (P = 0.03). Conclusion: Androgen and Estrogen receptor expression increases with increase in prostate volume in BPH cases.

Keywords: Androgen receptor, benign prostatic hyperplasia, estrogen receptor

How to cite this article:
Sreenivasulu K, Nandeesha H, Dorairajn LN, Rajesh NG. Association of androgen and estrogen receptor expression with prostate volume in benign prostatic hyperplasia. Indian J Pathol Microbiol 2019;62:99-102

How to cite this URL:
Sreenivasulu K, Nandeesha H, Dorairajn LN, Rajesh NG. Association of androgen and estrogen receptor expression with prostate volume in benign prostatic hyperplasia. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Feb 21];62:99-102. Available from: http://www.ijpmonline.org/text.asp?2019/62/1/99/251248





   Introduction Top


Benign prostatic hyperplasia (BPH) is a clinical condition associated with Lower urinary tract symptoms (LUTS) and its prevalence increases with age.[1] In normal prostate development, androgen/estrogen balance and stromal-epithelial cell interactions are known to be important and this interaction is strategic in the development and progression of BPH.[2]

Androgen receptor (AR) and Estrogen receptor (ER) are nuclear hormone receptor superfamily. ARs are found to be in the nuclei of mostly all luminal epithelial cells and stromal cells of both normal human prostates and BPH specimens.[3],[4] Interaction between AR and their ligands (testosterone or dihydrotestosterone) are important for cellular growth and function.[5]

Among different forms of estrogen, the role of estradiol (E2) has been evaluated in prostate research.[6] Estrogen receptors (ER-α and ER-β) are ligand-modulated nuclear transcription factors. Studies have reported that ER-α stimulation in the prostate results in inflammation, hyperplasia and squamous metaplasia.[7],[8] Experimental studies have shown that rats treated with testosterone in combination with the ER-α agonist developed prostate intraepithelial neoplasia (PIN), whereas rats treated with ER-β agonist prevented the onset of PIN lesions.[9] Since the AR and ERs are involved in the cell proliferation and cell survival, the present study was designed to analyse the gene and protein expression of AR and ERs in prostate tissues and their association with prostate volume in patients with BPH.


   Materials and Methods Top


Study subjects

27 BPH patients who were admitted in urology ward for transurethral resection of prostate surgery were recruited in the study. Subjects with prostate cancer and other medical co morbidities were excluded from the study. Written informed consent was obtained from all the subjects prior to the study. This study was approved by institute advisory committee and ethics committee. 5 ml of fasting venous blood samples were collected from the subjects and used for the analysis of PSA, Testosterone and Estrogen (Calbiotech Inc., Spring Valley, California) by using ELISA.

Prostate tissue collection and processing

After TURP surgery, prostate tissue from transition zone of the prostate was collected and transferred immediately to the laboratory in liquid nitrogen container, tissues were aliquoted and stored at -80°C. Based on their prostate size BPH patients were divided into two groups: Group A with prostate size less than or equal to 30mL (n = 13), Group B with prostate size by greater than 30mL (n = 14).

RNA extraction, cDNA synthesis, and real-time PCR

RNA was isolated and converted into cDNA by using commercial kits (Qiagen and Applied Biosystems Warrington, UK). The gene-specific oligonucleotide primers (IDT) were used as listed in [Table 1]. Gene expression levels were analysed by using SYBR green method (Applied Biosystems Warrington, UK) followed by melt curve analysis. Signals were normalized to the housekeeping gene β-actin as the endogenous internal control. To assess the variance, mRNA levels derived from the group A (<30mL) tissues were arbitrarily taken as control group. Fold change was calculated by using 2–ΔΔCt method. The gene expression data were represented in arbitrary units.
Table 1: Primers used for real time polymerase chain reaction

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Western blot analysis

Proteins were extracted from prostate tissues and separated through SDS-PAGE, Proteins were transferred to nitrocellulose membranes (Advantec, Japan). The membranes were then blocked with the 5% Bovine serum albumin, followed by overnight incubation at 4°C with appropriate primary antibodies (Rabbit polyclonal anti-AR, anti-ER-α (Abclonal, Woburn, United states) and Beta actin (Biolegend, San Diego, USA). After that, secondary antibody incubation at 25°C for one hour. Blots were projected with a chemiluminescence blotting substrate (Roche, Germany) kit under Chemidoc system.

Immunohistochemical analysis

Representative blocks of paraffin-embedded BPH tissues were cut at a 4 μm thickness, deparaffinised and rehydrated, followed by incubation for 30 min with a solution of 0.5% H2O2 in 50% methanol for blocking endogenous peroxidase. Antigen retrieval was done by microwaving sections in 10 mM citrate buffer (pH 6.0). Further processing was done as per standard protocol (Vector Laboratories) using ImmPRESS™ universal reagent and ImmPACT™ DAB Peroxidase Substrate. Then sections were counterstained with hematoxylin and slides were dehydrated over an ethanol series to xylene and mounted. The visualizations were done using a light microscope.

The slides were visualized in imaging system and scored by pathologist. Immuno staining was semi-quantitatively scored using a 4 point scale scoring system by which pathologist assigns a value to each immunostain. Intensity is scored as follows: 0-represents no staining, 1-represents a faint staining, 2-represents as a moderate staining, 3-represents as a strong staining. The proportion of cells showing positive stain is recorded as 0 (≈<10%), 1 (10%-25%), 2 (26%-50%), and 3 (>50%).

Statistical analysis

Student's t-test was used to relate the differences between two groups and P value < 0.05 considered as statistically significant.


   Results Top


[Table 2] shows the mean and standard deviation of age and biochemical parameters in serum of BPH patients with prostate size less than 30 ml and more than 30 ml. Estradiol and PSA levels are significantly increased in serum of larger prostate size group. There is no significant difference in Testosterone levels.
Table 2: Mean and standard deviation or median (range) of age, prostate size and biochemical parameters among benign prostatic hyperplasia groups

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[Figure 1] shows the gene expression levels of androgen receptor and estrogen receptors in BPH patients with different prostate size. AR and ER-α expression was relatively high in patients with larger prostate size when compared with lesser prostate size, but there was no significant difference of ER-β expression between both the groups.
Figure 1: Relative fold change of androgen (AR) and estrogen (ESR A and B) receptors gene expression in prostate tissues of BPH patients (*P < 0.05 compared to group A)

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[Figure 2] and [Figure 3] shows the protein expression of AR and ER-α in prostate tissues. AR and ER-α expression, relative ratio of AR/Beta actin and ER-α/Beta actin was higher in large prostate size group when compared with small prostate size group.
Figure 2: (a) Relative ratio of AR/Beta actin protein expression in prostate tissues of BPH patients (*P = 0.03 compared to group A). (b) First line (C1) represent Group A (<30 mL prostate size), next 4 lines (S1, S2, S3, S4) represents Group B (>30 mL prostate size)

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Figure 3: (a) Relative ratio of ESR-A/Beta actin protein expression in prostate tissues of BPH patients. Data were expressed in Mean ± SEM, *Indicates statistically significant, P value-0.02. (b) First line (C1) represent Group A (<30 mL prostate size), next 4 lines (S1, S2, S3, S4) represents Group B (>30 mL prostate size)

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Immunohistochemical analysis of Androgen receptor [Figure 4] shows nuclear staining in ductal epithelial cells as well as in stromal cells. However, the intensity in ductal epithelium was much stronger than in the stromal nuclei [Figure 4]c. When the percentage of AR staining in the tissue of these two groups was compared, a greater number of ductal cells were stained with AR in large volume prostate tissues when compared to small volume of prostate tissues [Figure 4]b. Similarly in most of the small volume BPH patients, the staining pattern in stromal cells were higher than the AR expression in ductal cells [Figure 4]a. This indicates the stimulus for cell proliferation is high in ductal cells of benign prostatic tissues which attain a larger size.
Figure 4: (a) Sections from patients with small volume BPH show increased stromal nuclear expression of AR with tract to negative expression in ducts (x400 view) (b) Section from patients with large volume BPH show intense nuclear expression of AR in ducts compared with negative expression in stroma (x400 view) (c) Increased ductal expression of AR in large volume prostate of BPH patient (x400 view)

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   Discussion Top


Among hormones, androgens play a prominent and essential role in normal and hyperplastic prostate growth.[10] The androgen dependence of the prostate is highlighted by androgen deprivation, which leads to induction of apoptosis in vitro and in vivo.[11],[12]Visakorpi et al. have reported an increase in the amplification of the AR gene during androgen deprivation therapy and concluded that the proliferation of cancer cells in an environment with a low concentration of androgens may involve the overexpression of the AR.[13] In the present study, we also observed increased AR gene and protein expression in BPH patients with larger prostate size when compared with lesser prostate size. These findings were supported by other investigators who have revealed amplification of the AR at the genomic level, RNA level and protein level in androgen-independent prostate tumours.[14],[15]

Estrogen receptors (ER-α, ER-β) are expressed in the human prostate. Several studies have hypothesised that ER-β has a predominantly protective effect in PCa, while ER-α is oncogenic.[8],[9] Our findings showed over expression of ER-α in larger size prostate tissues when compared with lesser prostate size, indicating ER-α may involve in the cell proliferation and BPH development. ER-β gene expression was found to be reduced but it was not statistically significant. These findings were supported by increase in serum estradiol levels in BPH patients with larger size prostate tissues when compared with lesser prostate size.

When Immunohistochemistry analysis was done, we found an interesting and novel observation that AR expression was higher in the ductal epithelial cells in patients with larger prostate size, whereas in patients with smaller prostate size, AR expression was higher in stromal cells, thus pointing to a complex intercellular and intracellular homeostasis in the pathogenesis of the disease.

The main limitation of the present study is less sample size due to exclusion of BPH cases associated with diabetes and hypertension. Since the current research work is a short term student project, we could not increase the sample size owing to financial constraints.

The translation value of this finding is that, at present BPH is treated mainly by the drugs targeting androgens and androgen receptor. Based on our findings, we recommend that further studies can be undertaken to investigate whether drugs targeting estrogen receptors are useful in reducing the symptoms of BPH.


   Conclusion Top


We conclude that the gene and protein expression of androgen and estrogen receptors are increased with an increase in prostate size in patients with BPH. The increased expression of ER-α co-existing with the increased AR expression in the large volume BPH patients highlight their synonymous role in the pathogenesis of the disease and may be a potential therapeutic target.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgements

This work was supported by a grant from JIPMER intramural fund sanctioned to the corresponding author.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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2.
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Ricke WA, Wang Y, Cunha GR. Steroid hormones and carcinogenesis of the prostate: The role of estrogens. Differentiation 2007;75:871-82.  Back to cited text no. 6
    
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Ricke WA, McPherson SJ, Bianco JJ, Cunha GR, Wang Y, Risbridger GP. Prostatic hormonal carcinogenesis is mediated by in situ estrogen production and estrogen receptor alpha signaling. FASEB J 2008;22:1512-20.  Back to cited text no. 8
    
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Attia DM, Ederveen AG. Opposing roles of ERα and ERβ in the genesis and progression of adenocarcinoma in the rat ventral prostate. Prostate 2012;72:1013-22.  Back to cited text no. 9
    
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Khvostova EP, Otpuschennikov AA, Pustylnyak VO, Gulyaeva LF. Gene expression of androgen metabolising enzymes in benign and malignant prostatic tissues. Horm Metab Res 2015;47:119-24.  Back to cited text no. 10
    
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Mercader M, Sengupta S, Bodner BK, Manecke RG, Cosar EF, Moser MT, et al. Early effects of pharmacological androgen deprivation in human prostate cancer. BJU Int 2007;99:60-7.  Back to cited text no. 11
    
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Hu M, Xin D, Chen J, Sun G, Wang Y, Na Y. Changes in the androgen levels in the ventral prostate of spontaneously hypertensive rats after castration. BJU Int 2009;104:406-11.  Back to cited text no. 12
    
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Linja MJ, Savinainen KJ, Saramäki OR, Tammela TL, Vessella RL, Visakorpi T. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 2001;61:3550-5.  Back to cited text no. 13
    
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Edwards J, Krishna NS, Grigor KM, Bartlett JM. Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer. Br J Cancer 2003;89:552-6.  Back to cited text no. 14
    
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Latil A, Bièche I, Vidaud D, Lidereau R, Berthon P, Cussenot O, et al. Evaluation of androgen, estrogen (ER alpha and ER beta), and progesterone receptor expression in human prostate cancer by real-time quantitative reverse transcription-polymerase chain reaction assays. Cancer Res 2001;61:1919-26.  Back to cited text no. 15
    

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Correspondence Address:
Hanumanthappa Nandeesha
Department of Biochemistry, JIPMER, Puducherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_315_18

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