In vivo modulation of androgen receptor by androgens

V. L. Kumar¹, P.K. Majumder¹, V. Kumar²

Asian J Androl 2002 Sep; 4: 229-231

Keywords: androgen receptor; androgens; antiandrogens

Abstract 

Aim: To study the effect of androgen and antiandrogen on the level of androgen receptor (AR) mRNA. Methods: The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated. Results: Androgenic deprivation produced by castration decreased the weight of the prostate and increased the levels of AR mRNA. Treatment of the castrated rats with testostrone increased the weight of prostate and decreased the levels of AR mRNA. Treatment of normal rats with flutamide decreased the weight of the gland and increased the levels of AR mRNA. Conclusion: Androgens produce proliferative effect on the prostate and negatively regulate the AR transcription.

1 Introduction

Androgens play a significant role in male physiology and pathology. Prostate is one of the targets for the action of androgens and the androgen receptor (AR) has been demonstrated in the human prostate whether normal, hypertrophied or carcinomatous. The level of androgen receptor is significantly higher in prostate cancer patients undergone endocrine treatment (Honvan^® or orchiectomy [1, 2]. The association of AR gene amplification with androgen deprivation therapy in prostate cancer patients has also been reported [3]. Androgen deprivation by castration, estradiol-, flutamide- or GnRH-treatment up-regulates the AR transcript [4-6]. In order to understand the regulatory mechanism of androgens and antiandrogens, we have investigated the effect of testosterone on the AR mRNA levels in the ventral prostate of castrated rats and compared it with that of flutamide.

2.1 Animals and experimental protocol

The study was carried out in male Wistar rats weighing 120-150 g. They were divided into groups of 10 animals each and were given different treatments for different time periods. The time course for the effect of castration on the weight of prostate and AR mRNA was studied at 1, 3, 5, 7 and 10 days. Group I - normal control; Group II-castrated control (5-day castration); Group III - Normal treated with flutamide (50 mg/kg body weight); Group IV - Castrated (5-day) and treated with testosterone (10 mg/kg). The drug treatment was given for 1 h, 4 h and 1, 3 and 7 days.

2.2 Slot blot analysis of RNA

The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated [4,7] .

The results were evaluated statistically using Student's t-test and values of P<0.05 were considered significant.

The regulation of AR mRNA was studied in rat ventral prostate. Castration of rats produced a significant decrease in the wet weight of prostate in five days (33.03.5 mg vs 100.37.7 mg in the normal controls, P< 0.01). Treatment of 5 day's castrated rats with testosterone produced a time-dependent increase in the wet weight of prostate and a 5-fold increase in the weight was observed after 3 days of treatment (181.144.2 mg). On the other hand, treatment of normal rats with flutamide for 7 days produced a significant decrease in the weight of prostate and the effect was comparable with that of castration (Figure 1). To study the effect of androgenic manipulation on the AR mRNA, the total RNA isolated from the prostate was blotted onto nylon membrane and hybridized with the AR cDNA probe. Figure 2 shows the representative autoradiograph of the slot blot analysis, where 1A was used as an internal control. Contrary to the changes in the weight of prostate, slot blot analysis showed that the levels of AR RNA increased following castration and the maximum level was achieved in 5 days. Thereafter, a decline in the level of AR RNA was observed. Treatment of 5 days castrated rats with testosterone produced a 2.5-fold decrease in the AR RNA levels within 24 hours of treatment followed by a gradual increase. On the other hand, treatment of normal rats with flutamide produced a marked increase in the AR mRNA (Figure 3).

Figure 1. Effect of castration and treatment with testosterone and flutamide on the weight of rat ventral prostate. : castration, : Flutamide treatment in normal rats, p: Testosterone treatment of 5-day castrated rats. h: indiicates the time at which testosterone treatment was started. The values are meanSEM, n=10 per treatment group. ^bP<0.05; ^cP<0.01.

Figure 2. Representative autoradiograph of a slot blot hybridization analysis of AR and 1A mRNA in rat prostate. A: Effect of castration; B: Effect of flutamide treatment of NC and testosterone treatment of CC. NC: normal control; CC: 5-day castrated control.

Figure 3. Effect of castration and testosterone and flutamide treatment on the level of AR mRNA.  : castration ,: Flutamide treatment in normal rats, p: Testosterone treatment in 5-day castrated rats. h: indicates the time at which testosterone treatment was started. . The values are in meanSEM, n=10 per treatment group. ^bP<0.05; ^cP<0.01.

In the present study we have investigated the effect of androgen and antiandrogen on the level of AR mRNA and weight of the rat ventral prostate. Both bilateral castration and flutamide treatment brought about a reduction in the weight of prostate, while the AR mRNA was up-regulated. Androgen deprivation has been shown to be associated with AR gene amplification and up-regulation of AR mRNA [3,4]. It has been proposed that AR gene amplification facilitates cell growth in low concentrations of androgens remaining in the serum after androgen deprivation therapy and therefore causes progression of human prostate cancer [3]. The hormone-refractory recurrent prostate cancer is associated with increased cell proliferation and decreased apoptosis [8]. Our study shows that androgenic supplementation of castrated rats produces a significant increase in the weight of prostate suggesting a proliferative action of testosterone on the prostate even though it is a negative regulator of AR transcription. A decrease in AR mRNA levels with testosterone was followed by a gradual increase which could have resulted due to its inhibitory effect on the adrenal gland [9]. Based on these findings, we propose that it may be necessary to control the levels of adrenal androgens along with orchiectomy for the management of prostate cancer.

Acknowledgment

This study was supported by a research grant from All India Institute of Medical Sciences, New Delhi.

References

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Correspondence to: Dr. Vijay L. Kumar, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India.
Tel: +91-11-659 3681 Fax: +91-11-686 2663
E-mail: kumarvl98@hotmail.com
Received 2002-02-01      Accepted 2002-06-23