Bryan: Estrogen- Friend or Foe?

[purecontrol]

Women don't have the levels of DHT or test that men do, and the women that do indeed have the hightend levels of DHT and Test have less hair.

The fact is that balding men have higher than normal/health levels of DHT and Estrogen, while having suppressed levels of Test.

If your DHT levels did not raise you would become more and more like a women, so inorder to keep that from happening the body increases levels of DHT to combat.

If you lower the levels of estrogen in the male the amount of DHT will decrease along with the amount and sensativity of the AR.

 

[michael barry]

Why did anyone have to dredge this bullshit up from the dead?


Hairs individually show differences in their response to androgens in test tube and ex vivo studies. The fault lies in your hair, and your shitty hair genetics. Thats why you are balding.

 

[Bryan]

If your DHT levels did not raise you would become more and more like a women, so inorder to keep that from happening the body increases levels of DHT to combat.

If you lower the levels of estrogen in the male the amount of DHT will decrease along with the amount and sensativity of the AR.

PROVE IT, GODDAMNIT!!

 

[docj077]

The fact is that balding men have higher than normal/health levels of DHT and Estrogen, while having suppressed levels of Test.

This is wrong. Men with male pattern baldness tend to have increased levels of DHT, but their lower than normal total testosterone is completely offset by their higher than normal levels of free testosterone. This is likely correlated with their tendency to have reduced levels of SHBG. I can find no information regarding elevated estrogen levels in men with male pattern baldness and if it is elevated, then the body is compensating appropriately.

 

[Bryan]

If your DHT levels did not raise you would become more and more like a women, so inorder to keep that from happening the body increases levels of DHT to combat.

If you lower the levels of estrogen in the male the amount of DHT will decrease along with the amount and sensativity of the AR.

HAHAHAH!! I just found some scientific evidence that blows your theory out of the water!

This is the study "Estrogen Reduction by Aromatase Inhibition for Benign Prostatic Hyperplasia: Results of a Double-Blind, Placebo-Controlled, Randomized Clinical Trial Using Two Doses of the Aromatase-Inhibitor Atamestane", Radlmaier et al, The Prostate 29:199-208 (1996).

The use of the aromatase inhibitor atamestane caused a reflexive INCREASE in serum androgens, including DHT! Here are the approximate numbers involved (I'm reading this off a graph they provide): after 48 weeks of therapy, the smaller dose of the drug raised serum DHT by about 23%, and the larger dose raised serum DHT by about 35%.

There was no question at all in my mind that reducing estrogen causes an increase in testosterone, because it's been thoroughly documented that estrogen plays an important role in the regulation of androgen synthesis; the only thing I wasn't completely sure about was whether or not it also raises DHT, although I felt it was highly likely that it would. It was just a little while ago that I remembered that they had probably tested for that in this study, and sure enough I was correct in my assumption, and YOU were WRONG: reducing estrogen with an aromatase inhibitor raises not only testosterone, but also DHT! How do you like them apples?

 

[CCS]

body builders inhibit their estrogen so they can make more testosterone. I'm going to do a cycle of that. I knew all along that that guy was full of BS.

 

[Matgallis]

Lets get it over with and chop off our nuts already!

 

[IBM]

so we all should produce more estrogen instead of testosterone. How we can do that?

 

[docj077]

If your DHT levels did not raise you would become more and more like a women, so inorder to keep that from happening the body increases levels of DHT to combat.

If you lower the levels of estrogen in the male the amount of DHT will decrease along with the amount and sensativity of the AR.

HAHAHAH!! I just found some scientific evidence that blows your theory out of the water!

This is the study "Estrogen Reduction by Aromatase Inhibition for Benign Prostatic Hyperplasia: Results of a Double-Blind, Placebo-Controlled, Randomized Clinical Trial Using Two Doses of the Aromatase-Inhibitor Atamestane", Radlmaier et al, The Prostate 29:199-208 (1996).

The use of the aromatase inhibitor atamestane caused a reflexive INCREASE in serum androgens, including DHT! Here are the approximate numbers involved (I'm reading this off a graph they provide): after 48 weeks of therapy, the smaller dose of the drug raised serum DHT by about 23%, and the larger dose raised serum DHT by about 35%.

There was no question at all in my mind that reducing estrogen causes an increase in testosterone, because it's been thoroughly documented that estrogen plays an important role in the regulation of androgen synthesis; the only thing I wasn't completely sure about was whether or not it also raises DHT, although I felt it was highly likely that it would. It was just a little while ago that I remembered that they had probably tested for that in this study, and sure enough I was correct in my assumption, and YOU were WRONG: reducing estrogen with an aromatase inhibitor raises not only testosterone, but also DHT! How do you like them apples?

Nice find. I've been curious about this, as well. The physiology makes sense.

 

[Bryan]

For what it's worth, I'll also mention here that there was no effect at all on BPH with either dose of the aromatase inhibitor in that study. That was surprising and unexpected, because of all the interest and speculation in recent years about the role of estrogen in the etiology of prostate disease. The authors of the study were quite open about their puzzlement and disappointment over the lack of results.

 

[wookster]

Topical estrogen might help with baldness but systemic absorption would not be a good thing ...for men

http://www.mf.uni-lj.si/acta-apa/acta-a ... oulos.html

In the mid-20th century, topical estrogens were used in a variety of dermatological disorders, including acne vulgaris, keratoderma climactericum, hidradenitis suppurativa, seborhea oleosa, male and female-pattern baldness, urogenital (vaginal/vulvar) atrophy and peri/postmenopausal vasomotor complaints

 

[CCS]

of course. I forgot. they also use topical testosterone for andropause. It absorbs and goes systemic very well.

But how much topical estrogen do we really need? How many mg does our body produce each day? What if we use enough to just raise our blood levels 5% with the maybe 20% that would be absorbed?

I think we have either 1000nanograms per deciliter, or 1000micrograms per deciliter, and 700 deciliters in our body, of testosterone, and maybe 5% that much estrogen. I bet docj0777 knows the numbers.

So that is 0.7mg or 700mg in our body, x5%, x5 which is the amount of estrogen we can have per daily application. So about 0.2mg or 200mg per day. I suspect it is the 0.2mg. So x30 = 6mg of the right birth control pill into 60mL of vodka applied in 2mL per day.

Think that would give systemic effects? I think the 20% absorption is very generous and gives a big safety margin. Also, by comparison to the 0.025% finasteride that worked, this solution is 0.02%. It is probably also very very cheap.

If we raise our systemic estrogen 5%, I bet scalp estrogen would be up at least 30% for an hour or so.

 

[Bryan]

If we raise our systemic estrogen 5%, I bet scalp estrogen would be up at least 30% for an hour or so.

The problem with all that theory is that an old study by Strauss, Kligman, and Pochi in the Journal of Investigative Dermatology ("The Effect of Androgens and Estrogens on Human Sebaceous Glands") clearly showed that at least in the specific experimental setup they used, topical estrogen worked EXCLUSIVELY through systemic absorption!

Here's an excerpt from the "Local Application of Estrogen" section (added emphasis is my own):

To determine whether estrogen acts directly on the target organ, the sebaceous gland, the experimental design of the topical androgen study was followed. The estrogen ointment was applied locally to one cheek, and the changes in the sebaceous glands of both cheeks were followed histologically. A control biopsy specimen was removed beforehand.

Six post-pubertal males, between the ages of 15 and 20 served as subjects. Four received 10 per cent ethynyl estradiol ointment once daily; two received 10 per cent beta-estradiol ointment once daily. The biopsies taken at six weeks revealed a marked and equal decrease in systemic action. Feminization was evident in all six subjects, obviously reflecting percutaneous absorption.

Since histologic examination is not suitable to serial follow-up, the more sensitive gravimetric technique was used in the remaining studies. The estrogen ointment was applied to one side of the forehead and sebum output was determined on both sides simultaneously at various intervals.

Ten per cent ethynyl estradiol in Hydrophylic Ointment, U.S.P., was applied to one side of the forehead of two adult males once daily. Three more received 5 per cent ethynyl estradiol ointment. These concentrations greatly exceed the threshold dose. In all of these subjects, there was an equivalent decrease in sebaceous secretion on both sides.

A threshold concentration of 1.0 per cent ethynyl estradiol ointment was applied to one side of the forehead of three adult males daily. In two subjects, sebum production decreased on the treated side. However, an equally great suppression of sebum output occurred on the untreated control side.

Comment

With neither the high (5 to 10 per cent) nor the threshold (1 per cent) concentration of ethynyl estradiol ointment was there any evidence of earlier or greater suppression of sebaceous secretion at the treated site. This result indicates that the effects obtained required systemic absorption prior to producing the observed results on sebum secretion.

 

[CCS]

The problem with that study is they used such high concentrations of estrogen. I'm sure if you dump in enough to saturate the body, of course you will see equal results on each side. They should have use 0.1% estrogen or lower.

The only reason estrogen would only work systemically is if it takes too long to complex before spreading. If that were the case, aromatase would not be located in specific cells.

 

[Bryan]

The problem with that study is they used such high concentrations of estrogen. I'm sure if you dump in enough to saturate the body, of course you will see equal results on each side. They should have use 0.1% estrogen or lower.

You missed the part about how the effect was also the same with the "threshold" levels of application!! Even when the biological effect was very minimal with only a small dose, there was the same minimal biological effect on the other, non-treated side, too! They were smart enough to anticipate your objection, which is why they tried it with both larges doses AND small doses.

The only reason estrogen would only work systemically is if it takes too long to complex before spreading. If that were the case, aromatase would not be located in specific cells.

I don't follow you.

 

[CCS]

The problem with that study is they used such high concentrations of estrogen. I'm sure if you dump in enough to saturate the body, of course you will see equal results on each side. They should have use 0.1% estrogen or lower.

You missed the part about how the effect was also the same with the "threshold" levels of application!! Even when the biological effect was very minimal with only a small dose, there was the same minimal biological effect on the other, non-treated side, too! They were smart enough to anticipate your objection, which is why they tried it with both larges doses AND small doses..

Yes, I missed that. Glad that is cleared up.

The only reason estrogen would only work systemically is if it takes too long to complex before spreading. If that were the case, aromatase would not be located in specific cells.

I don't follow you.

It means that estrogen takes time to bond to receptors at the application spot. And the time required is long enough for a lot of it to circulate to other parts of the body. I'm guessing the bonding is very reversible and transient.

But the point is, it won't work topically. Just like flutamide won't.

 

[Bryan]

I'm not too sure how much the speed of its binding really has to do with how successful it is at having a "local" effect; it may simply be that topical estrogen diffuses into blood capillaries before it even has much of a chance to get into skin cells, regardless of the binding speed.

But the main thing that puzzled me was your last sentence about aromatase being located (or NOT being located) in specific cells. I didn't understand that part. My assumption is that aromatase is located in estrogenic target cells, much like 5a-reductase is located in androgenic target cells.

But yes, regardless of whatever the exact mechanisms are that keep topical estrogen from having a noticeable "local" effect, it does seem to fail at that.

 

[purecontrol]

Try to keep it adult please, no reason to start a thead in an ignorant direction, thank you

Here are some studies for people to think about.

Effect of anti-estrogens on the androgen receptor activity and cell proliferation in prostate cancer cells.

Kawashima H, Tanaka T, Cheng JS, Sugita S, Ezaki K, Kurisu T, Nakatani T.

Department of Urology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan. hidenori@msic.med.osaka-cu.ac.jp

Although some anti-estrogens have been reported to inhibit the proliferation of prostate cancer cells, few studies on the mechanism by which they suppress the growth of prostate cancer have been reported. We investigated, for the first time, whether anti-estrogens modulate the transactivation activity of the androgen receptor (AR) in prostate cancer cells. In DU-145 cells transfected with AR, the transactivation activity of AR was inhibited by tamoxifen and toremifene, even in the presence of 10 nM of DHT. On the other hand, in LNCaP cells having an endogenous AR mutation at codon 877, the activity of AR was suppressed by faslodex in the presence of 10 nM DHT, whereas it was not inhibited by tamoxifen nor toremifene. In PC-3 cells, both the cell growth and the AR activity were remarkably inhibited by tamoxifen at 50 microM. Faslodex and toremifene inhibited AR activity to some extent, but they seemed to function as agonists at higher concentrations. In PC-3 cells, the inhibition of cell growth by flutamide, faslodex and toremifene was much less than their suppression of AR activity. We also demonstrated that a synthetic estrogen diethylstilbestrol and progesterone-related drugs such as chlormadinone acetate and allylestrenol dose-dependently inhibited the activity of AR in DU-145 and PC-3 cells. These results highlight the anti-androgenic aspect of anti-estrogens and estrogens in regard to the AR-mediated transcription of the relevant genes in prostate cancer.



Toremifene May Reduce Cancer Risk in Men with Precancerous Prostate Cells
May 17, 2005, 01:54


"This is the first time that a drug has shown promise for lowering the incidence of prostate cancer in men with PIN"



By American Society of Clinical Oncology, In a multicenter phase IIb study, the hormone drug toremifene (Acopodene) reduced the risk of prostate cancer development by nearly half in men with prostatic intraepithelial neoplasia (PIN), a precancerous condition that can progress to prostate cancer.

"This is the first time that a drug has shown promise for lowering the incidence of prostate cancer in men with PIN," said lead author David Price, MD, Director of Urologic Oncology and Clinical Research at Regional Urology, LLC in Shreveport, Louisiana. Toremifene, a hormonal therapy commonly used to treat women with advanced breast cancer, may work by blocking a particular estrogen receptor that has been implicated in prostate cancer development.
While PIN does not always lead to prostate cancer, prostate cancer usually develops in men with PIN. Approximately 10% of men who undergo prostate biopsies are diagnosed with PIN, and more than 30% of men with PIN will be diagnosed with prostate cancer within a year.

Currently, there is no effective treatment for PIN ­ men with this condition receive periodic biopsies, and many live with a fear of developing cancer. Prostate cancer is the most common cancer in men in the United States ­ more than 200,000 cases are expected to be diagnosed in 2005.

In this study, 514 men with PIN were randomly assigned to receive 20 mg, 40 mg, or 60 mg of toremifene or a placebo for one year, undergoing prostate biopsies at six and 12 months. The trial was completed in May 2004.

The cumulative incidence of prostate cancer was 31.2% in the placebo group at one year. Patients treated with 20 mg of toremifene for six months had a 22% reduction in prostate cancer, while patients who completed an entire year of treatment had a 48% reduction in prostate cancer risk (24.4% cumulative incidence). For the groups that received 40 mg and 60 mg of toremifene, the 12-month incidence of prostate cancer was also lower, but not statistically significant (18% and 25.3% risk reductions, respectively).

Toremifene was generally well tolerated, with the incidence of adverse events occurring at a similar rate in the toremifene-treated groups as the placebo group; the exception was fatigue, which occurred in 5% of patients treated with toremifene versus 3% of the placebo group.

"While these data are promising, more clinical trials are needed to determine whether toremifene should be widely prescribed to men with prostatic intraepithelial neoplasia," Dr. Price added.

 

[purecontrol]

Sex Hormone-Binding Globulin Mediates Prostate Androgen Receptor Action via a Novel Signaling Pathway
Victor D. H. Ding, David E. Moller, William P. Feeney, Varsha Didolkar, Atif M. Nakhla, Linda Rhodes, William Rosner and Roy G. Smith

Estradiol (E2) and 5-androstan-3,17ß-diol (3-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (RSHBG) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3-diol can amplify or substitute for androgens, with regard to activation of the AR via the RSHBG by a signal transduction pathway involving cAMP. Because both E2 and 3-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.


SEX hormone-binding globulin (SHBG) is a plasma glycoprotein that binds sex steroids with high affinity, thereby regulating their plasma concentrations (1). Also, by binding to a specific receptor (RSHBG) on prostate cell membranes, SHBG participates directly in cellular signaling pathways (2, 3, 4). Two steroids, 17ß-estradiol (E2) and 5-androstan-3,17ß-diol (3-diol), bind to the SHBG-RSHBG complex and stimulate cAMP production (3, 4, 5). Because cAMP transduces downstream signals implicated in modulation of cell growth (6, 7, 8, 9) and regulation of specific gene transcription and expression (10, 11, 12, 13, 14, 15, 16), activation of this pathway by E2 and 3-diol may prove to play a critical role in prostate function.

The androgen receptor (AR) is a tissue-specific transcription factor that is directly activated by binding to testosterone and 5-dihydrotestosterone (DHT). However, its indirect activation by FSH, by polypeptide growth factors, and through a cAMP pathway has been reported (17, 18, 19, 20, 21). Androgens cause an increase in the production of specific proteins in the prostate. For example, in humans, synthesis of prostate-specific antigen (PSA) is stimulated by DHT (22). In dogs, DHT increases prostate arginine esterase (AE) synthesis (23, 24, 25, 26, 27, 2.

The steroids, DHT, E2 and 3-diol, implicated in prostate growth, all bind to SHBG with high affinity; but only E2 and 3-diol activate SHBG-RSHBG in prostate tissue, causing rapid accumulation of intracellular cAMP (3, 4, 29). Traditionally, 3-diol has been thought to be a biologically inert metabolite of DHT; however, this metabolite was recently shown to exhibit hormonal activity (3, 30). Further, for more than a decade, it has been known that administration of 3-diol induces benign prostatic hyperplasia (BPH) in dogs (31, 32, 33, 34, 35). E2 also has been implicated in contributing to the pathogenesis of BPH. In castrated dogs, E2 prevents prostate regression; moreover, BPH can be induced experimentally by estrogens in intact dogs and monkeys (32, 33, 34, 35, 36). In humans, accumulation of E2 in the prostate is associated with advancing age (37). E2 also has been implicated in contributing to the growth and progression of human prostate cancer (3. In this regard, E2 binding to SHBG has been shown to stimulate cAMP accumulation in a human prostate cancer (LNCaP) cell line (5).

Prostate growth is known to be mediated by DHT activation of AR and consequent increase in the production of PSA (22). To address the mechanism by which E2 or 3-diol stimulation, via the SHBG-RSHBG pathway, might contribute to prostate growth in the dog, we asked whether the increase in cAMP was linked functionally to an androgen-responsive pathway in primary cultures of dog prostate. We selected the canine equivalent of PSA, AE as a marker of activation of the AR. As anticipated, AE was stimulated by DHT, but most importantly, in the presence (but not in the absence) of SHBG, both E2 and 3-diol mimicked the effect of DHT. While this work on normal prostate was in progress, similar observations were made using human BPH tissue, suggesting that this pathway has physiological relevance in the growth of both normal and hyperplastic tissue (39).


Chemicals and reagents
Tissue culture medium RPMI-1640, FBS, and sodium pyruvate were purchased from GIBCO-BRL (Gaithersburg, MD). All steroid compounds were obtained from Steraloids Inc. (Wilton, NH). Highly purified canine SHBG was prepared as previously described (3). All reagents and apparatus for electrophoresis and Western blot analysis were purchased from Novex (San Diego, CA). The enhanced chemiluminescence system for Western blot detection was obtained from Amersham Life Science (Arlington Heights, IL). 8-Br-cAMP, benzoyl arginine ethyl ester, forskolin, and isobutyl-methylxanthine were obtained from Sigma Chemical Company (St. Louis, MO). Hydroxyflutamide was obtained from the Merck Chemical Data collection. The anti-AE antiserum was a gift of Dr. J. Y. Dubé, Laboratory of Hormonal Bioregulation, Laval University Hospital Research Center, Sainte-Foy, Québec, Canada. AE protein standard was a gift of Dr. Alan Partin, Johns Hopkins University, Baltimore, MD. All other reagents were of analytical grade.

Prostatic tissue explants
Unless otherwise specified, prostate tissue was obtained from pure-bred male beagle dogs, 2–3 yr of age (3). Dogs were euthanized 7 days after surgical castration. All procedures for the humane handling, care, and treatment of research animals were done according to humane animal use procedures approved by the Merck Institutional Animal Care and Use Committee. The prostatic tissue was removed and brought to the laboratory under sterile conditions. It was divided into approximately 5-mm3 cubes and placed in 100-mm culture dishes (Corning Glass Works, Corning, NY) in RPMI-1640 medium with 5% FBS containing 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, and 0.25 mg/ml amphotericin B for 2 days at 37 C in an atmosphere of 95% air and 5% CO2. Tissue was then minced into 1–2 mm3 portions and transferred to 24-well plates in serum-free medium (0.5 ml/well) for approximately 18 h before beginning each experiment.

Tissue processing and AE activity determination
Prostatic minces were incubated in the presence or absence of steroid hormones for 2 days. To avoid having any SHBG in the culture medium, serum-free medium was used, as previously reported (3, 4). Fresh medium and hormones were replaced at 24 h. To saturate the SHBG receptor, prostatic minces were incubated with 50 nM purified dog SHBG (3) for 3 h. Subsequently, appropriate concentrations of steroids were added after washing to remove excess SHBG. Other treatments with hydroxyflutamide (100 nM), 2-methoxyestradiol (2MeOE2), were added 15 min before addition of steroids. All the steroids and the antagonists used in this study were initially dissolved in 100% ethanol. Tissue minces were harvested and homogenized using a polytron (Tekmar Co., Cincinnati, OH) for 30 sec in 0.5 ml cold PBS (10 mM, pH 7.2) containing 0.2% Triton X-100. The homogenates were centrifuged at 25,000 x g for 45 min at 4 C to remove particulate matter, and the supernatants were stored at -80 C. The protein concentration of the supernatant was determined using the Bio-Rad Protein microassay procedure. AE activity was determined as described previously (40, 41, 42, 43), with modifications. The substrate concentration was 1 mM, and the reaction was carried out at room temperature (24 C) in 1 ml 0.01 M Tris/HCl buffer at pH 8.0. Total protein amounts (5–20 µg) were used to determine the enzyme activity by following the increase in optical density at 253 nm upon the hydrolysis of benzoyl arginine ethyl ester (42). AE activity present in tissue homogenates was found to be stable after sample storage at -80 C and a single freeze/thaw cycle did not affect the enzyme activity.

Western blotting analysis
Samples were resolved by electrophoresis through 4–20% SDS-polyacrylamide gradient gels, followed by electroblotting onto 100% methanol-treated PVDF membrane (0.45 µm, Immobilon P from Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk in PBS for 1 h. Immunostaining was performed by incubating with rabbit anti-AE polyclonal antibodies at 1:2,000 dilution in washing buffer (0.25% gelatin, 5 mM EDTA, 0.15 M NaCl, 0.05% Tween 20, 50 mM Tris/HCl, pH 7.4) for 1 h. The membranes were washed for 30 min, then incubated with horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham) at 1:3,000 dilution for 40 min. The Western blots were developed using enhanced chemiluminescence procedures similar to those described by Amersham. All steps were carried out at room temperature.

Measurement of cAMP level
Levels of cAMP were measured using commercial enzyme-linked immunosorbent assay kits (Oxford Biomedical Research, Inc., Oxford, MI), as described previously (3). All samples contained isobutyl-methylxanthine (100 µM).

Statistical analysis
The significance of differences between treatment and control groups was assessed using Student’s t test. Values are reported as the mean ± SEM (SE of the mean).

Stimulation of AE by DHT
As expected, AE activity increased as a function of the dose of DHT (Fig. 1A). The basal level of AE activity in castrated (7 days) dog prostate was approximately 3 µmol/min·mg protein. AE activity was not significantly stimulated by 1 nM DHT; however, at 10 nM and 100 nM DHT, AE activity was significantly increased. Thus, DHT stimulates AE activity in a dose-dependent manner; and although a concentration of 100 nM may not represent a maximal response, this concentration was used as a reference in subsequent experiments.


Figure 1. Effect of steroid hormones on AE activity. (A), DHT; (B), E2 or 3-diol ± SHBG; (), tissue incubated with dog SHBG for 3 h to saturate SHBG receptors before treatment with steroids; (), no SHBG treatment; **, P < 0.05, compared with the control; *, P < 0.05, compared with the same treatment in the absence of SHBG. Each data point represents the mean ± SEM of six determinations derived from two independent experiments.



Stimulation of AE through the SHBG receptor complex
After preincubation with SHBG, to allow formation of the SHBG-RSHBG complex, treatment with E2 or 3-diol led to an increase in AE activity similar to that observed with DHT (Fig. 1B). By contrast, in the absence of SHBG, E2 and 3-diol did not stimulate AE activity. Thus, the stimulation of AE, caused by E2 and 3-diol, required the SHBG-RSHBG complex.
The effects on AE protein were measured by Western blot using a specific anti-AE antibody. AE migrated as a 29-KDa molecular mass species on a 4–20% polyacrylamide gel under nonreducing conditions. The AE protein in prostate tissue treated with DHT, or SHBG plus E2, was increased substantially, relative to the respective controls (Fig. 2). In tissue treated with SHBG or E2 alone, the AE activity increased only slightly, compared with control.



Figure 2. Western blot of prostate tissue homogenates treated with steroid hormones. Samples were resolved under nonreducing conditions on a 4–20% linear gradient polyacrylamide gel, followed by electrophoretic blotting on a PVDF membrane. AE, arginine esterase standard; lane 1, control; lane 2, DHT (100 nM); lane 3, DHT + hydroxyflutamide; lane 4, SHBG; lane 5, SHBG + E2; lane 6, E2 (lanes 1–6, 200 ng protein of tissue homogenate were loaded in each well). Similar results were obtained in two additional experiments.




Effect of AR antagonists on SHBG-mediated stimulation of AE activity.
Hydroxyflutamide (100 nM), a specific AR antagonist, inhibited the DHT-mediated increase in AE (Fig. 3). Similarly, the increased activity after treatment with SHBG+E2 or SHBG+3-diol also was blocked by hydroxyflutamide (Fig. 3). Western blotting revealed parallel results in AE protein.



Figure 3. Effect of hydroxyflutamide on AE activity. Prostate minces treated with steroid only or with 100 nM hydroxyflutamide (HF) plus steroid. The SHBG receptor of prostate tissue was saturated with dog SHBG before treatment with E2 and 3-diol. (), tissue treated in the absence of HF; (), tissue treated in the presence of HF; control, vehicle only; *, P < 0.05, compared with the same treatment in the absence of HF. Each data bar represents the mean ± SEM of six determinations derived from two independent experiments.




Effect of an SHBG-cAMP pathway antagonist
In confirmation of our previous studies (3), intracellular cAMP levels were stimulated markedly by the combination of SHBG, E2, and 3-diol (Fig. 4A). In a dose-dependent manner, 2MeOE2 inhibited the accumulation of cAMP stimulated by E2 or 3-diol (Fig. 4A). Importantly, the stimulation of AE by E2 and 3-diol also was blocked by 2MeOE2 (Fig. 4B). Thus, 2MeOE2 blocks both the accumulation of intracellular cAMP and the stimulation of AE activity that is initiated by E2 or 3-diol and suggests that cAMP is a necessary intermediator between E2- or 3-diol-SHBG-RSHBG and AE stimulation. Indeed, both the cAMP analog, 8-Br-cAMP, and the inducer of cAMP forskolin stimulated AE activity (Fig. 5).


Figure 4. Effect of 2MeOE2 treatment on cAMP levels present in prostate tissue. (A), Concentration dependent on 2MeOE2 (nM) inhibition of cAMP accumulation induced by E2 and 3-diol in the presence of SHBG. (B), Effect of 2MeOE2 on AE activity. Prostate tissue minces saturated with SHBG, were treated either with steroid hormone alone () or in the presence of 1 µM 2MeOE2 (). Control, tissue AE activity without treatment; SHBG only, tissue saturated with SHBG; SHBG+E2, SHBG-saturated tissue treated with 100 nM E2 alone () or in the presence of 2MeOE2 (); SHBG+3-diol, SHBG-saturated tissue treated with 3-diol alone () or in the presence of 2MeOE2 (); *, P < 0.05, compared with the same treatment in the absence of 2MeOE2. Each data bar represents the mean ± SEM of six determinations derived from two independent experiments.




Figure 5. Effect of stimulating the cAMP pathway on AE activity. AE activity was measured in the homogenates of prostate tissue treated with 100 nM DHT and compared with the effects of 50 µM forskolin (Fsk), a stimulator of cAMP production, or 50 µM 8-Br-cAMP. **, P < 0.05, compared with the control. Each data bar represents the mean ± SEM of six determinations derived from two independent experiments.




Androgens are important mediators of prostate growth, but although androgens require ARs to mediate their effects, the converse may not be true. Both in transformed cell lines and in appropriately transfected cells, evidence has been presented showing that the AR can be activated by substances other than steroids (19, 20, 21). However, the relevance of these observations to actual physiology has been problematical. The old observations that E2 and 3-diol induced prostate hyperplasia in dogs (31, 32, 33, 34, 35), together with our demonstration that both these steroids caused increases in cAMP in dog prostate, led us to hypothesize that these events might be connected (3). We speculated that perhaps ARs in the prostate were being activated by a cAMP-mediated pathway.
To test our hypothesis, we investigated the effects of E2 and 3-diol on an androgen-responsive protein in the dog prostate. AE is a dog prostate-specific protein under androgenic control that can be employed as a prostate marker, analogous to the use of PSA in humans (23, 24, 25, 26, 27, 2. We first confirmed that AE in dog prostate tissue was stimulated by DHT and that this effect could be antagonized by the specific AR antagonist, hydroxyflutamide. We then demonstrated that E2 and 3-diol caused AE increase in prostate in the presence (but not in absence) of SHBG. The effects were not mediated by the estrogen receptor because, in the absence of SHBG, E2 alone had no effect, and 3-diol does not bind to the estrogen receptor. The possible conversion of 3-diol to DHT does not explain the effects of 3-diol, because increases in AE required preincubation of prostate tissue with SHBG; 3-diol alone was ineffective. 2MeOE2 a competitive inhibitor of E2 and 3-diol binding to SHBG, blocked stimulation of cAMP by E2 and 3-diol and prevented increases in AE caused by these steroids, suggesting that cAMP was involved in increasing AE. That 8-Br-cAMP and an inducer of cAMP (forskolin) also increased AE is consistent with this explanation. Because the AR selective antagonist, hydroxyflutamide, blocks E2, 3-diol, and cAMP induction of AE, it seems that these effects are mediated through ARs. Furthermore, these results support the notion that ARs can be activated, even in the absence of androgens, by natural steroids that activate the cAMP pathway. Blockage of ligand-independent AR activation by antiandrogen is in agreement with several other recent reports.

Based on our findings, we propose a novel mechanism whereby E2 and 3-diol can increase the production of androgen-responsive proteins in a physiologically relevant system. The AR, like other steroid hormone receptors, is a phosphoprotein (44, 45), and its activation state can potentially be modulated by phosphorylation-dephosphorylation, resulting in augmented or ligand-independent activation (17, 19, 21, 46, 47, 48, 49). The phosphorylation state of the AR has been shown to be increased upon hormone binding; however, changes in cAMP levels and activation of protein kinase A also have recently been implicated in causing ligand-independent activation of transfected ARs when expressed in either CV-1 or human prostate (PC-3) cells (17, 20, 21). Consistent with our results in normal prostate, it is therefore plausible that activation of the SHBG-RSHBG pathway can lead to androgen-independent AR activation via stimulation of protein kinase A. It is also possible that up-regulation of AR expression might occur via a cAMP response element present within the regulatory region of the AR gene (10, 13). This could potentially augment AE expression, causing an increase in AR concentrations and a corresponding increase in basal transcription.

A number of lines of evidence have implied a role for E2 in the pathogenesis of BPH or in androgen-independent progression of prostate cancer (31, 32, 33, 34, 35, 36, 37, 3. These studies indicate that E2 synergizes with 3-diol, but not DHT, in induction of canine BPH. Because E2 and 3-diol are the only two known steroids that activate the SHBG-RSHBG pathway in prostate tissue (3), and we have shown that both are capable of activating pathways normally considered androgen responsive, antagonism of the pathway by which SHBG leads to the induction of androgen-responsive genes may be a valuable therapeutic target for the treatment or prevention of BPH or prostate cancer. To our knowledge, this is the first demonstration in a physiologically relevant system that androgenic events can be observed in the complete absence of exogenous androgens.

 

[purecontrol]

J Clin Invest. 2002 July 15; 110(2): 219–227.
doi: 10.1172/JCI0215552.
Copyright © 2002, American Society for Clinical Investigation
Signaling through estrogen receptors modulates telomerase activity in human prostate cancer
Simona Nanni,1,2 Michela Narducci,1 Linda Della Pietra,1,3 Fabiola Moretti,1,4 Annalisa Grasselli,1,4 Piero De Carli,1 Ada Sacchi,1 Alfredo Pontecorvi,1,2 and Antonella Farsetti1,4
1Molecular Oncogenesis Laboratory and Urology Division, Regina Elena Cancer Institute, Experimental Research Center, Rome, Italy2 Institute of Medical Pathology, Catholic University, Rome, Italy3 University of Rome, La Sapienza, Rome, Italy4 Institute of Neurobiology and Molecular Medicine, National Research Council, Rome, Italy
Address correspondence to: Antonella Farsetti, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Via delle Messi D’Oro 156, 00158 Rome, Italy. Phone: (39-06) 5266-2531; Fax: (39-06) 4180526; E-mail: farsetti@ifo.it.
Received March 28, 2002; Accepted June 5, 2002.


Our hypothesis favoring an etiopathogenetic role of ERs in prostate tumorigenesis may appear at odds with the well-known androgen dependency of prostate tumors. However, the age-dependent decline of androgens-to-estrogens ratio has been already suggested as a pathogenetic factor for prostate tumor development (7). In addition, local conversion of androgens to estrogens by aromatase may provide significant amounts of intracellular estrogens, leading to activation of endogenous ERs. Although molecular and cellular mechanisms responsible for estrogen formation in the prostate, and their physiological and clinical relevance, are currently under investigation, it should be emphasized that in breast cancer aromatase inhibitors have been proposed as second-line drugs in the hormonal therapy of this disease to achieve a complete estrogen deprivation (40). Indeed, in our study, the combination of T and letrozole resulted in a consistent inhibition of telomerase activity. In this regard we hypothesize that the androgen effects on the prostate gland may be mediated, at least in part, by ERs rather than by ARs, following aromatization of androgen to estrogen. The recent identification of the T metabolite 5α-androstane-3β,17β-diol as a specific ligand of ER-β in rat ventral prostate (15) further supports our hypothesis of a relevant role of ER signaling in prostate pathophysiology.



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