Reversing silenced AR signal with demethylating agents - A promising treatment option?

Therapeutic Efforts Pharmaceutical
#317

Yes this test is very important. Please if you can try and persuade them.

endo.endojournals.org/cgi/content/abstract/137/4/1385

Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro

Accumulation of DNA is essential for muscle growth, yet mechanisms of androgen-induced DNA accretion in skeletal muscle are unclear. The purpose of this study was to determine whether androgen receptors (AR) are present in cultured skeletal muscle satellite cells and myotubes and examine the effects of testosterone on satellite cell proliferation and differentiation. Immunoblot analysis using polyclonal AR antibodies (PG-21) revealed an immunoreactive AR protein of approximately 107 kDa in porcine satellite cells and myotubes. Immunocytochemical AR staining was confined to the nuclei of satellite cells, myotubes, and muscle- derived fibroblasts. Administration of 10(-7) M testosterone to satellite cells, myotubes, and muscle-derived fibroblasts increased immunoreactive AR. In satellite cells and myotubes, AR increased incrementally after 6, 12, and 24 h of exposure to testosterone. Testosterone (10(-10) - 10(-6) M), alone or in combination with insulin- like growth factor I, basic fibroblast growth factor, or platelet- derived growth factor-BB, had no effect (P > 0.01) on porcine satellite cell proliferation, and testosterone pretreatment for 24 h did not alter the subsequent responsiveness of cells to these growth factors. Satellite cell differentiation was depressed (20-30%) on days 2-4 of treatment with 10(-7) M testosterone. This effect was not reversible within 48 h after treatment withdrawal and replacement with control medium. These data indicate that satellite cells are direct targets for androgen action, and testosterone administration increases immunoreactive AR protein
and reduces differentiation of porcine satellite cells in vitro.

onlinelibrary.wiley.com/doi/10.1111/j.1365-2605.1992.tb01125.x/abstract

Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS-treated animals.

In the ventral prostate, 5 days after EDS-treatment, a 4.0 ± 0.3–fold up-regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 ± 0.2–fold increase in actin mRNA. In the epididymis, a 2.0 ± 0.5–fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS-treated rats, the AR mRNA level was not changed (1.02 ± 0.17–fold of controls), and there was also no change in actin mRNA level at 5 days after EDS-treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis.

Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H-R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS-treated rats with testosterone-implants, remained unaltered compared to control rats (9.1 ± 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS-treated rats without testosterone-implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H-R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding).

toxsci.oxfordjournals.org/content/early/2011/03/21/toxsci.kfr063.abstract

Pharmacological doses of testosterone up-regulated androgen receptor (AR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase (3βHSD) and impaired Leydig cells steroidogenesis in adult rat

Anabolic androgenic steroids (AAS) are testosterone derivatives originally designed to enhance muscular mass and used for the treatment of many clinical conditions as well as in contraception. Despite popular interest and abuse, we still lack a broad understanding of effects of AAS on synthesis of steroid hormones on the molecular level. This study was designed to systematically analyze the effects of pharmacological/high doses of testosterone on steroidogenic machinery in Leydig cells. Two different experimental approaches were used: (1) In vivo experiment on groups of adult male rats treated with testosterone for one day, two weeks, and two months; (2) Direct in vitro testosterone treatment of Leydig cells isolated from intact rats. Result showed that prolonged in vivo treatment with testosterone decreased the expression of Scarb1 (Scavenger receptor class B type 1), Tspo (Translocator protein), Star (Steroidogenic Acute Regulatory protein), Cyp11a1 (Cholesterol side-chain cleavage enzyme); Cyp17a1 (17α-hydroxylase/17, 20 lyase) in Leydig cells. Oppositely, the expression of Hsd3b (3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase), Ar (androgen receptor) and Pde4a/b (cAMP-dependent phosphodiesterases) was increased. Androgenization for two weeks inhibited Cyp19 (aromatase) transcription, while two months exposure caused opposite effect. Direct in vitro testosterone treatment also decreased the expression of Cyp11a1, Cyp17a1 and Cyp19a1, while Hsd3b was up-regulated. The results of expression analysis were supported by declined steroidogenic capacity and activity of Leydig cells, while conversion of pregnenolone to progesterone was stimulated. The up-regulation of AR and 3βHSD in testosterone-impaired Leydig cells steroidogenesis could be the possible mechanism that maintain and prevent loss of steroidogenic function.

If the problem was downregulation of androgen expression increasing testosterone would help in all of us.

The problem must therefore lie after testosterone binds to the androgen receptor - aka the AR signal.

#318

To everyone having green tea - STOP!!! - its actually bad for us.

Yes it is can change DNA methylation but not in a way beneficial to us. In fact it helps more with estrogen silencing:

ncbi.nlm.nih.gov/pmc/articles/PMC2967543/

ncbi.nlm.nih.gov/pubmed/21177307

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer.

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC(50) 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.-Siddiqui, I. A., Asim, M., Hafeez, B. B., Adhami, V. M., Tarapore, R. S., Mukhtar, H. Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer.

nature.com/onc/journal/v19/n15/full/1203511a.html

Tea polyphenols down-regulate the expression of the androgen receptor in LNCaP prostate cancer cells

Androgens via their cognate receptor may be involved in the development and progression of prostate cancer. The aim of this study was to determine whether tea polyphenols have inhibitory effects on androgen action in an androgen-responsive, prostate cancer cell line, LNCaP. The tea polyphenol, EGCG, inhibited LNCaP cell growth and the expression of androgen regulated PSA and hK2 genes. Moreover, EGCG had a significant inhibitory effect on the androgenic inducibility of the PSA promoter. Immunoblotting detected a decrease in androgen receptor protein with treatments of the tea polyphenols EGCG, GCG and theaflavins. Northern blot analysis showed decreased levels of androgen receptor mRNA by EGCG. Transient transfections demonstrated that EGCG and theaflavins could repress the transcriptional activities of the androgen receptor promoter region. An Sp1 binding site in the androgen receptor gene promoter is an important regulatory component for its expression. This study suggests Sp1 is the target for the tea polyphenols because treatments of EGCG decreased the expression, DNA binding activity and transactivation activity of Sp1 protein. In conclusion, we have described a new property of tea polyphenols that inhibits androgen action by repressing the transcription of the androgen receptor

So STOP green tea - if you are hoping to nudge the silencing off into the right direction green tea is interfering with any recovery.

#319

Based on these studies I don’t think you can say Green Tea is bad for us. Here are some things that theanine, an amino acid in green tea does that are beneficial:

en.wikipedia.org/wiki/Theanine
This article also claims that green tea increases dht and testosterone, at least in mice

jn.nutrition.org/cgi/content/full/133/2/516#SEC2

#320

Interesting point.

In the prostate, testosterone is rapidly and irreversibly converted to a more biologically active metabolite, DHT, by catalysis of 5α-reductase. EGCG has been shown to inhibit the growth of prostate cancer cells in vitro (36 ) and in vivo (37 ) through mechanisms that might involve inhibition of type I 5α-reductase (37 ). In this study, black tea reduced serum levels of DHT (Fig. 2 D), suggesting that black tea may have bioactive components that inhibit the conversion of testosterone to DHT, presumably via inhibition of 5α-reductase in this SCID-LNCaP animal model. It is unclear whether black tea theaflavins, EGCG and/or other components are responsible for this function in vivo.

On the other hand, green tea did not reduce the serum level of DHT, but instead tended to increase it (P = 0.076) (Fig. 2 D), and we found that green tea treatment did not inhibit tumor growth (Fig. 1 A). Green tea contained more EGCG than black tea (Table 1) , and studies have shown that EGCG inhibits the activity of 5α-reductase (38 ). These results derived from our animal model suggest that, although EGCG may be a potent antitumor agent in green tea and inhibit 5α-reducatase activity, green tea contains other constituents that may counteract EGCG’s antitumor activity, in part by counteracting its modulation of 5α-reducatase activity. Further research is required to identify these constituents and study their effects and/or their interactions with other components on prostate cancer. Our results demonstrate the importance of evaluating the benefit of whole tea products, rather than just isolated tea catechins or EGCG, on prostate cancer prevention because other tea constituents may play important roles.

The way i see it green tea and tea can interact with androgen action by repressing transcription of the androgen receptor. It can boost testosterone and DHT but since I believe the problem is not our hormonal levels but the the AR receptor and signal inhibiting this component will have worse effects than the benefits a slight increase in DHT can give.

#321

There is a theory on the board that you can resensitize receptors of chemicals by promoting use of an antagonist of the receptor (viewtopic.php?f=27&t=3632). Based on this theory green tea polyphenol EGCG blunting androgen receptor function might actually be a good thing.

Add to this the fact that green tea is a known demethylating agent along with guru20 in the recoveries section (viewtopic.php?f=22&t=1524) who claimed he got better from green tea and DIM (also a known AR antagonist).

Anyway I usually feel better when I drink green tea, at least mentally so I’ll probably stick with it for now.

#322

I androgen receptors arent working why isnt estrogen dominating, and creating a large number of estrogen side effects?

#323

Who says E2 isn’t a factor for men with PFS issues? The drug is listed to cause gyno, and has been documented to alter the androgen/estrogen ratio to be in favor of estrogens.

I certainly have issues from E2 creeping up over the years:

  • Gynecomastia which manifested on drug and never resolved
  • Feminine fat deposits in thighs/buttocks
  • Feminine widening of hips
  • Thinned fingers/wrists/forearms/lower legs/calves/ankles where muscle used to be, now taking on more female proportions/loss of muscle

If androgen cannot exert its effects, it makes sense that E2 will predominate even at “normal” ranges.

#324

Finasteride can lead to clear E2 problems, I myself now have pseudo-gyno as a result of taking Propecia. But thats whilst using the stuff.

I have never read of anyone growing gyno after stopping. Thats important.

I am not saying androgens are working. I am saying this means androgens + estrogens arent working. The trend is towards the androgynous.

(Hell, maybe thyroid hormones have stopped working too which is why Im so cold all the time).

#325

Wouldn’t this idea suggest going back on finasteride? Which seems like the wrong things to do. Are you trying to say that by blocking transcriptional activity of the AR the body will try to compensate? Wouldn’t this lead to an even more hypersensitive receptor which awor thinks is the cause of our problems.

I see what you’re saying. It would be good if other people let us know how green tea affects them.

I think Mew is on the ball with the androgen/estrogen ratio being the issue for some people. If we are androgen resistant normal levels of estrogen can promote estrogenic signs. This ratio is fragile and our bodies are very sensitive to it. A simple thing like grapefruit juice has made people feel terrible. It made me feel different too. Normal people should not get put into a estrogenic state by a couple glasses of grapefruit juice. We are very sensitive to things.

viewtopic.php?f=27&t=4695&start=80

#326

and

I’ve seen you make this “observation” before and i REALLY don’t see where you’re getting it out of. Just because PFS victims appear “damaged” doesn’t lead to this conclusion.

We have said that many of us appear* to be " partially androgen resistent". But you’ve taken it upon yourself to suggest we’re numbed to estrogen, too? I see very little evidence of this on this board. Indeed, if it was the case i wouldn’t have developed worse sides as my estrogen got higher over the period of last year. Also, most of the sides Mew listed would surely abate in such a circumstance. Not to mention we’d actually show LOW estrogen symptoms which little or no of us actually exhibit, either…

*NB: When I say appear i don’t mean they are. The liver, adrenals and thryoids are potentially as far as we need to look…but noone is sure at this point.

Anyway, let’s not derail this thread. I wish Awor would update us.

#327

Mew grew gynecomastia in less than 11 months - this was due to increased levels of estrogens. Now the presumption is that the effects of androgens have been nulled, this would presumably mean only the effects of estrogens being felt.

Surely if that had happened his gyno would get worse and worse? (And many of the guys badly effected would report gyno after quitting?)

Surely if that had happened he would not have seen E2 effects ‘over the years’ (7 years) but over the course of a few months?

Its pretty clear at this point that testosterone has stopped working. But its not useful to get stuck on one idea. Im not saying I know anything, but I am simply reporting what are obvious facts (posistive and negative) from reading this forum.

This supports what Im saying.

No, gyno needs surgery.

YES WE DO. Especially if your saying testosterone no longer works.

#328

Conclusion: Either both androgens & estrogens are working or neither are working.

Its not as simple as just an AR problem.

#329

I disagree with you Oscar. As we’ve said if androgens are not working properly the estrogens which are still working will have dominance of the androgen/estrogen ratio. We experience hypogonadal symptoms which in itself can lead to to gynae problems which do not improve after stopping. You do not always needs surgery for this. Sometimes if it does not go away you do.

There is no evidence to suggest problems with low estrogen. There have been problems with high estrogen but not low. Also if estrogens were not working things like the grapefruit juice wouldn’t have any effect.

The problem is with the effects produced by androgens. Everything else is secondary.

#330

Been thinking of other ideas. DNA methlyation is one thing we need to rule out.

From wiki:

DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene, and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histones, thereby forming compact, inactive chromatin, termed silent chromatin. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome; and methyl-CpG-binding domain protein 2 (MBD2) mediates the transcriptional silencing of hypermethylated genes in cancer.

However, the possibility of the problem to be after this process is also valid and in this idea the problem is not with the DNA, genes and epigenome but with the proteins - the things the genes make. This would be termed a protein post translational modification.

premierbiosoft.com/glycan/glossary/post-translational-modifications.html

Ways in which it happens:

Glycosylation: Many proteins, particularly in eukaryotic cells, are modified by the addition of carbohydrates, a process called glycosylation. Glycosylation in proteins results in addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine. Software for studying glycosylation by glycan structure prediction.

Acetylation: the addition of an acetyl group, usually at the N-terminus of the protein.

Alkylation: The addition of an alkyl group (e.g. methyl, ethyl).

Methylation: The addition of a methyl group, usually at lysine or arginine residues. (This is a type of alkylation.)

Biotinylation: Acylation of conserved lysine residues with a biotin appendage.

Glutamylation: Covalent linkage of glutamic acid residues to tubulin and some other proteins.

Glycylation: Covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail of the amino acid sequence.

Isoprenylation: The addition of an isoprenoid group (e.g. farnesol and geranylgeraniol).

Lipoylation: The attachment of a lipoate functionality.

Phosphopantetheinylation, The addition of a 4’-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis.

Phosphorylation, the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine.

Sulfation: The addition of a sulfate group to a tyrosine.

Selenation

C-terminal amidation

In terms of research we should start at the genetic level and then work up to see where the problem is. Although I’m in favour with a systems view of biology this is the only realistic way to look at it.

The more I think about it this could be VERY important!

#331

Hi everyone

Just wanted to drop a line to let everyone know I’m still alive. As I said before, this is not a recovery thread. But given that I let people know that demethylation has helped me, I think it’s time to give you all an update.

As stated previously, a demethylation therapy using intravenous procaine did wonders for me. So much, that I decided to dump all other medication (mainly two antidepressants). This led to a further improvement in my condition, including sexual sides (fuller penis, usable erection, libido). Antidepressants work by increasing neurosteroid levels. They achieve this (just about all of them), by increasing induction of 3a-HSD. This, unfortunately, causes a reduction in androgens at the cellular level.

So dumping my antidepressants had the effect of giving me an androgen boost, which made me feel better for about a week or two following quitting. In my case, increasing androgens eventually always makes me feel worse. So going off the SSRI’s eventually caused me to crash again and I was feeling pretty shitty for quite a few months. Since about a month, I am starting to feel better again. Some libido, often good morning erections. But if I do anything to raise testosterone (ejaculation, too much exercise), I feel bad, depressed and get shrinkage. So not really useful from a sexual perspective.

Since 6 months I am completely off any medication besides TRT. The procaine therapy seemed to have weakened my immune system because I was coughing almost all winter. This appears to have gotten better now. Global demethylation is not the magic bullet because it messes with too many other mechanisms in the body and can cause other, serious problems. My personal focus is still on getting scientists interested in working the basic science side of things. We need a more detailed understanding of what’s wrong in order to assist with a more targeted therapy. I hope we can start initiating some molecular level studies in the foreseeable future.

Take care all.

#332

Thanks for the update.

I am not sure I agree with your statement on global demethylation as the majority of the epigenome would be far more resilient to demethylating therapies than the ones that have been altered. It is not like people who have taken azacitadine suddenly find their bodies acting in a completely different manner. Most of the more fixed gene expressions will return to their normal function by process of remethylation or would not necessarily demethylate in the first place. Given this and the fact you improved on procaine wouldn’t it suggest that it could help. If I’m wrong about this could you point to examples where demethylation has messed things up permanently for people. Also have you retained any benefit from the procaine therapy or do you think you suffered from remethylation? If so again what reasoning and evidence do you have to suggest a longer therapy would cause more harm than good?

Thanks.

#333

But they dont, thats what Im saying.

No. Gynecomastia always needs surgery. Look it up.

If testosterone isnt working there must be low estrogen effects too - else it would be obvious from the symptoms reported.

[Size=4]NAME ONE.[/size]

This supports what Im saying. If E is working but T isnt why should a person even need to boost there estrogen levels before they feel E sides? (Anyway provide some proof grapefruit juice does this).

There is no “everything else” - THERE SHOULD BE - this is the point that Im making.

I welcome a counter arguement. Otherwise the conclusion must be as follows: Either both androgens & estrogens are working or neither are working. Its not as ‘simple’ as just an AR problem.

#334

Your tone is very confrontational. I hope you are ok.

To answer your questions. Gynecomastia can be go away by itself if the hormonal abnormality is corrected, or through medication. However, if left untreated it can persist then surgery is an option. You obviously have not looked this up. Even wikipedia gives this info.

No testosterone and estrogen do not bind to the same receptors. Therefore one can work when the other doesn’t if hormonal levels are normal.

One problem of high estrogen - gynecomastia which Mew has had issues with. Not everyone will have these issues as not everyone has complete inactivation of their AR signal. For example some people can still have morning erections.

Grapefruit juice - maturitas.org/article/0378-5122(9490012-4/abstract

I hope this helps. And please you’re going to wind a lot of people up if you continue in your confrontational manner. Present your findings in a fair manner. Let the science speak for itself.

#335

Ok. The main point is it doesnt go away without intervention. But gynecomastia, which Mew has, as opposed to pseudogynecomastia - always needs surgery. Either way - it IS NOT a sign of high estrogen.

en.wikipedia.org/wiki/Gynecomastia

Who said they did?

So…why isnt it?

This developed on the drug, has not got worse, and would not necessarily resolve. Therefore in the whole forum NOT ONE EXAMPLE OF HIGH ESTROGEN AFTER QUITTING!

But they should - an inactivation of AR (even partial – as you say) would cause a massive estrogen dominance.

You have provided no argument to counter what I am saying, nothing scientific, nothing at all. Unless you can find an example of high estrogen after stopping and not simply something that developed on the drug and never went away, I must be correct. Its only unfair to deny such blatant and obvious facts. Pull yourself out of your mindset.

#336

Again with the aggressive manner. Your logic doesn’t add up as there are people with high estrogen.

I will answer your questions:

First off. Gynecomastia is a range of disorders ranging from whether adipose tissue is formed or breast tissue is -pseudo is primarily adipose tissue. It may improve upon resolving the hormonal issue:

Treating the underlying cause of the gynecomastia may lead to improvement in the condition. Patients should talk with their doctor about revising any medications, such as risperdal, that are found to be causing gynecomastia. Often, an alternative medication can be found that avoids gynecomastia side-effects while still treating the primary condition (e.g., in place of taking spironolactone the alternative eplerenone can be used). Selective estrogen receptor modulator medications, such as tamoxifen and clomiphene, or androgens (typically testosterone) or aromatase inhibitors such as Letrozole are medical treatment options, although they are not universally approved for the treatment of gynecomastia. Endocrinological attention may help during the first 2–3 years. After that window, however, the breast tissue tends to remain and harden, leaving surgery (either liposuction, gland excision, skin sculpture, reduction mammoplasty, or a combination of these surgical techniques) as the only treatment option.

Secondly show me evidence that estrogen isn’t working or is low in individuals. My estrogen was in normal values. Why do you think it is so important? What you are saying is that if androgens are not functioning estrogen would dominate completely and cause a cascade of symptoms. However, you do not see this happening. What are you implying? Estrogen resistance as well? Then why the response to estrogenic substances? It doesn’t add up. Keep in mind we still have normal androgen levels and a varying degree of function of the AR and what happens after it.

This guys estrogen was raised and you yourself told him so.

Check out: viewtopic.php?f=4&t=4827

Also: viewtopic.php?f=4&t=4406

I don’t know why you’ve come up with this idea. Show me evidence it is the case and then maybe I’ll believe you. But at the moment all you tell me is I’m categorically wrong which is not only unconstructive but is unprofessional.