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

Therapeutic Efforts Pharmaceutical
#121

Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells.

Normal prostate epithelial cell strains demonstrate no methylation. Exposure of AR-negative prostate cancer cell lines to 5-aza-2’ deoxycytidine, a demethylating agent, induces the reexpression of AR RNA in DuPro and TSU-PR1. This reexpression is associated with a demethylation of this region. Prostate-specific antigen, an androgen-responsive gene, is also specifically induced in these lines after AR reexpression. Therefore, in vitro DNA methylation of the 5’ CpG AR island may be associated with the loss of AR expression. Furthermore, our results demonstrate that treatment with demethylating agents may engender the reexpression and function of the androgen receptor in AR-negative cell lines.

ncbi.nlm.nih.gov/pubmed/9850055

#122

You are finding gold. !

#123

Of course not, it’s just for preventing too much excitement because:

I think you’re right, only trial and error will enable us to find out what’s going on.

#124

No worries Y-C, water under the bridge.

Moving along:

As we know, Procaine is the demethylating agent which is currently our number one candidate for treatment under this theory. This article discusses its ability to demethylate silenced genes but more importantly demonstrates that the combination of DNMT inhibitors and HDAC inhibitors appears to provide a synergistic effect in attempting to demethylate DNA. I believe this is something that should be given some consideration in a “demethylation based treatment plan” for PFS.

Procaine inhibits the proliferation and DNA methylation in human hepatoma cells

The viability of PCA-treated cells with or without trichostatin A (TSA)* was investigated…The viability of HLE, HuH7, and HuH6 cells was significantly decreased by PCA treatment. In these cells, the combination treatment with TSA and PCA exhibited stronger reduction of the viability.…All the genes transcriptionally suppressed by DNA hypermethylation were demethylated and reactivated with PCA treatment. PCA treatment led to partial demethylation and significant reduction in tumor volume in vivo.

*Trichostatin A = HDAC Inhibitor

ncbi.nlm.nih.gov/pmc/articles/PMC2716835/

#125

DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation

DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer…Recently, 3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation…Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs, sinefungin and adenosine dialdehyde, have similar effects as DZNep on H3K27me3. Intriguingly, chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor.

mct.aacrjournals.org/content/8/6/1579.full

#126

I had a read of that last paper and something caught me eye:

Demethylation induced by 5-Aza-CdR was also not completely stable and genes be- came resilenced following removal of the drug, although it took approximately 7 more days than cells treated with DZNep for the cell to return to its previous state.

Does this mean even if the drugs help us back on track then the positive effects will go away if we stop taking the drug?

#127

It does go on to say:

We think it would be interesting to study the kinetics of gene expression of cells treated with DZNep and 5-Aza-CdR or DZNep and a HDAC inhibitor to determine if the resilencing of genes can be blocked using these combination therapies.

So there are more options. Looks like you really have to demethylate the genes!

#128

Also as i slightly panicked when i found that:

biochemsoctrans.org/bst/032/0910/0320910.pdf

Indeed, orally administered zebularine has been shown to cause demethylation and reactivation of a silenced and hypermethylated p16 gene in human bladder tumour cells grown in nude mice [25]. Zebularine was also demonstrated to be minimally cytotoxic in vitro and in vivo [25]. In addition, zebularine can be given continuously at lower dose to maintain demethylation for a prolonged period which is only possible because of its low toxicity [26]. When cancer cells were transiently treated with 5-Aza-CdR and then by continuous treatment with zebularine, remethylation was hindered and gene expression was maintained, suggesting a combinatorial treatment method that would give a better response to the inhibition of DNA methylation

#129

Got this email reponse from Dr. Jacobs in regard to some of the research presented on this thread -

“The question here is what agent can we safely use to demethylate specific cell lines in vivo.”

#130

Well Procaine seems safe compared to the other commercially available drugs, which is why awor is trying it. Prehaps Zebularine too. Or a combination, a more powerful agent like Decetabine initially then one of the other two to prevent side effects but get the best results, as 19 correctly points out above.

In the past I believe awor used a HDAC inhibitor by itself (to prevent deacetylation and increase gene expression) with negative results. Anyone know why this would be?

Also, why dont we just get a test for methylation? This can be done. However we would need a scientist to order the test and interpret the results. Seems like a good idea?

#131

Bad news. This study (2005) suggests Procainamide would not be a useful demethylising agent.

mct.aacrjournals.org/content/4/10/1515.full

#132

Well its more saying that 5-aza-2′-deoxycytidine is a far superior demethylating agent.

Of the non-nucleoside agents tested, 200 μmol/L of procainamide reduced the methylation level of LINE repetitive elements in T24 cells by roughly 6%.

Other studies with Jurkat (16) and LnCAP (19) cell lines have shown apparent methylation inhibition activities of hydralazine and procainamide, respectively. Perhaps studies with different cell lines and/or a higher dose regimen will show the demethylating effect of these agents. However, we followed the treatment methods that were reported to be effective in previous studies for the non-nucleoside agents, and therefore we do not believe this to be the cause of the discrepancy (16, 17, 21). Nevertheless, longer treatments with these agents might be able to induce noticeable methylation inhibition (19).

There is still reason to be optimistic, and it makes sense to try procain before moving on to substances like 5-aza-2′-deoxycytidine.

#133

Its not terrible news but very useful to know. Thanks. Procainamide is not procaine though.

#134

A few questions:

  1. Does/can the human body demethylate androgen receptors naturally?

  2. If so, by which mechanism?

  3. If answers above are known, can we increase this process via drugs/supplements?

  4. Is there a feedback loop for demethylation/methylation. And if so, what regulates it?

#135

Answer/Partial Answer to questions 1 & 2:

http://www.labmeeting.com/paper/26324821/metzger-2005-lsd1-demethylates-repressive-histone-marks-to-promote-androgen-receptor-dependent-transcription

LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription.

Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor. Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues. Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcription. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.

I know the above was stated before, but I wanted to reiterate that it appears LSD1 may be specifically what needs to be targeted.

#136

http://books.google.com/books?id=mEgckDNkonUC&pg=PA47&lpg=PA47&dq=LSD1+androgens&source=bl&ots=1C8zjXzhSf&sig=b19O-HwrCXosKrU1xZktyHug1xU&hl=en&ei=KEgCTcHyKsP98Aad6Ij5Ag&sa=X&oi=book_result&ct=result&resnum=1&ved=0CBUQ6AEwADgK#v=onepage&q=LSD1%20androgens&f=false

Page 47-48

#137

It seems like SARMS (Selective Androgen Receptor Modulators) may be of interest.

#138

The possible answer to question 4:

http://www.cell.com/molecular-cell/abstract/S1097-2765(05)01569-8

Regulation of LSD1 Histone Demethylase Activity by Its Associated Factors
Yu-Jiang Shi1, 4, Caitlin Matson1, 3, Fei Lan1, 3, Shigeki Iwase2, Tadashi Baba2 and Yang Shi1, ,

1 Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115
2 Graduate School of Life and Environmental Sciences and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan

Ph: (617) 432-4318, F: (617) 432-6687

3 These authors contributed equally to this work.

4 Present address: Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, EBRC 222A, Boston, Massachusetts 02115.

Summary

LSD1 is a recently identified human lysine (K)-specific histone demethylase. LSD1 is associated with HDAC1/2; CoREST, a SANT domain-containing corepressor; and BHC80, a PHD domain-containing protein, among others. We show that CoREST endows LSD1 with the ability to demethylate nucleosomal substrates and that it protects LSD1 from proteasomal degradation in vivo. We find hyperacetylated nucleosomes less susceptible to CoREST/LSD1-mediated demethylation, suggesting that hypoacetylated nucleosomes may be the preferred physiological substrates. This raises the possibility that histone deacetylases and LSD1 may collaborate to generate a repressive chromatin environment. Consistent with this model, TSA treatment results in derepression of LSD1 target genes. While CoREST positively regulates LSD1 function, BHC80 inhibits CoREST/LSD1-mediated demethylation in vitro and may therefore confer negative regulation. Taken together, these findings suggest that LSD1-mediated histone demethylation is regulated dynamically in vivo. This is expected to have profound effects on gene expression under both physiological and pathological conditions.

#139

http://www.regrowth.com/hairloss-topic/18750

Read what the poster says about double and triple methylation.

#140

http://www.ncbi.nlm.nih.gov/books/NBK22523/

“14. Demethylation. Methyl amines are often demethylated by cytochrome P450 enzymes.”

http://en.wikipedia.org/wiki/Cytochrome_P450

“Cytochrome P450 enzymes are present in most tissues of the body, and play important roles in hormone synthesis and breakdown (including estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism. Cytochrome P450 enzymes also function to metabolize potentially toxic compounds, including drugs and products of endogenous metabolism such as bilirubin, principally in the liver.”

"Other specific CYP functions

Steroidogenesis, showing many of the enzyme activities that are performed by cytochrome P450 enzymes. HSD: Hydroxysteroid dehydrogenase
A subset of cytochrome P450 enzymes play important roles in the synthesis of steroid hormones (steroidogenesis) by the adrenals, gonads, and peripheral tissue:
CYP11A1 (also known as P450scc or P450c11a1) in adrenal mitochondria effects “the activity formerly known as 20,22-desmolase” (steroid 20α-hydroxylase, steroid 22-hydroxylase, cholesterol side-chain scission).
CYP11B1 (encoding the protein P450c11β) found in the inner mitochondrial membrane of adrenal cortex has steroid 11β-hydroxylase, steroid 18-hydroxylase, and steroid 18-methyloxidase activities.
CYP11B2 (encoding the protein P450c11AS), found only in the mitochondria of the adrenal zona glomerulosa, has steroid 11β-hydroxylase, steroid 18-hydroxylase, and steroid 18-methyloxidase activities.
CYP17A1, in endoplasmic reticulum of adrenal cortex has steroid 17α-hydroxylase and 17,20-lyase activities.
CYP21A1 (P450c21) in adrenal cortex conducts 21-hydroxylase activity.
CYP19A (P450arom, aromatase) in endoplasmic reticulum of gonads, brain, adipose tissue, and elsewhere catalyzes aromatization of androgens to estrogens."

I know p450 has been discussed before, but who knows…