Does anyone have any background in fundraising or know how to start this? Would be great to start a fund and begin seeking grant money. I know grant money isnt going to be easy to come by.
I havent done it but unless you just trust someone to put the cash in their bank acct you should create a non profit with officials like a president, treasurer, etc. That way the money canât go wandering. Then you create a mission statement, legal entity if feasible, then go around with a tin cup selling the cause. You need a clear plan where the money is to go though.
We need a research team that believe in the project first. Then money to fund them.
The problem with research is that although we may find the area that is methylated we do not have the drugs to selectively demethylate. At the moment it is all or nothing. It will take a long time for new drugs to be approved for use.
Awor said in his previous post that he was already in touch with scientists who were interested in performing the study. There are a number of reasons why this study is important, if we can prove this is actually the cause, then that will provide further leverage in a law suit, which will provide money for further research. It will also give doctors a much clearer understanding of what we are dealing with. In regards to the fact that we canât specifically demethylate genes yet⌠yes that is true. But if i knew beyond a shadow of a doubt that it was my only chance at getting better, i would probably be willing to risk global demethylation.
The fact is that yes, it is a long road we have to travel but the sooner we start, the sooner we get there.
I totally agree with you.
Lets coordinate this then and not simply let it be a pipe dream. Awor if youâre reading this can you give us your thoughts? Which scientists are interested?
I am looking into the risks of global demethylation. The main risk of some of the drugs is myelotoxicity. I want to evaluate if global demethylation is a risk worth taking. There are lots of demethlyation agents - some we eat on a daily basis. Point is there are quite a few and if I can clear up my mental side effects and fatigue I would very happy. Azacitidine is no doubt the strongest but is 3 days of azacitidine going to have such detrimental effects on my epigenome? I doubt it. Patients with myelodysplastic syndrome take it on an outpatient basis for sometimes prolonged periods of time.
I wish Dr Irwig would get a move on. His study is a stepping stone to the medical community. Any news on this front?
19
I doubt this will be, or should be, publically revealed at this stage in case word gets back to âyou know whoâ.
As i understand in order to get grant money we would need the scientist who is wanting to undertake the study to put forward a grant proposal on our behalf. A grant proposal is basically an outline of the what, where, how, why etc. As i said in a previous post, persistent side effects are already recognized in Europe and as awor is in Europe this holds promise that we could get a study going there.
May 2009
December 2010
Dear Mew/Awor, are you able to give any hints or info on what exactly is going on âbehind the scenesâ, what if any progress has been made, and how others could help? thanks.
Maybe pm us if you want to keep it quiet.
By the way someone posted this article earlier
medical-hypotheses.com/article/S0306-9877(0900291-6/abstract
I bought it and I must say it is very convincing. It is not peer reviewed but the idea seems sound and explains the direct and indirect effect drugs can have and how they can persist. It does not specify finasteride but it talks about a lot of other drugs including isotretinoin. It costs 33 dollars but if youâre interested get it!
I am convinced our problem is epigenetic in nature.
This is a bad idea on the forum. Merck has a lot of motivation and a lot more cash than we do to suppress this, plus the have the motivation of making millions and millions of dollars.
I think a better way would be to create the fund and have someone involved write the research proposal requesting grant money, however it shouldnât be on here.
Nevirapine restores androgen signaling in hormone-refractory human prostate carcinoma cells both in vitro and in vivo.
Nevirapine induced extensive reprogramming of gene expression in vitro with up-regulation of genes that might be silenced during prostate tumor progression (i.e., K18, PSA and androgen receptor) and down-regulation of genes involved in the progression toward an androgen-independent phenotype (i.e., K5, EGFR1, EGF and VEGF-A).
Nerve growth factor induces the re-expression of functional androgen receptors and p75NGFR in the androgen-insensitive prostate cancer cell line DU145
The relevance of the re-expression of functionally
active ARs is underlined by the part played by ARs in
the prostate. Mechanisms by which prostate cancer
cells escape androgen ablation and become independ-
ent of the need for androgens are not yet well under-
stood. It has been proposed that development of the
androgen independency can occur by a variety of
mechanisms, such as environmental adaptation, multi-
focal origin or genetic instability (26). The vast
majority of androgen-independent prostate tumors
express ARs highly; many paradigms, indeed, relate
the ďŹnding of AR overexpression to the development
of hormone resistance, and it has been suggested that
ampliďŹcations or mutations, or both, of ARs may play
a part in altering sensitivity to androgens (27).
However, 20 â 30% of advanced metastatic hormone-
independent prostate cancer is characteriz ed by a
signiďŹcant loss of AR expression, as a result of
transcriptional silencing that does not comprise
either deletion or mutational mechanisms (27).
Both in clinical tumors and in prostate cancer cell
lines, the transcriptional silencing of ARs involves
hypermethylation of the AR promoter and histone
deacetylation, known as epigenetic mechanisms (28).
Results reported here lead to the hypothesis that
NGF treatment could modulate gene expression in
the DU145 prostate cancer cell line through the epige-
netic regulation of transcription. This suggestion is
supported by the ďŹnding that this phenomenon also
occurs in PC12 cells, in which NGF-mediated dif-
ferentiation induces downregulation of the enzyme
catalyzing the DNA methylation reaction, namely
DNA methyltransferase (29). Furthermore, the inhib-
ition of this enzyme restores androgen sensitivity in
DU145 cells, and it has been proposed that this ef fect
could be due to a partial demethylation of the AR
(30). A possible role of NGF in the epigenetic
mechanisms is also supported by results obtained in
the study of telomerase activity. NGF-treated DU145
cells had a strongly inhibited telomerase activity
through the reduction of hTERT mRNA, with no
modiďŹcations of hTR levels. In a recent paper by
Suenaga et al. (31), it was reported that hTERT
mRNA expression and telomerase activity could be
strongly decreased in prostate cancer cell lines when
exposed to drugs that inhibit histone deacetylation,
without an ef fect on hTR level. These results were
obtained in PC3 and LNCaP cell lines, and we have
found that both DU145 and PC3 cell lines are
responsive to NGF treatment, inducing inhibition of
telomerase activity (12), and also AR re-expression in
the PC3 cell line (unpublished results). The possible
role of NGF as a modulator of epigenetic mechanisms
in prostate cancer cell lines clearly deserves further
investigation.
Nice find. Its used to treat A.I.D.S. Half life is 45 hrs so it solves that problem. I wonder if it would be safe. In 2000, the FDA issued a black box label on nevirapine, warning that it could cause severe liver damage, including liver failure.
Just to make it clear solonjk you took procaine before you had problems with finasteride right? You said you took it ten years ago or so and its been about 6 years you being off finasteride.
Your problems sound more complicated due to your other issues.
Think i might get that book
thanks for that info could be helpful with fatigue side of things.
On plus note my sensitivity has been better than usual for about a week. Still on doxy but things have been better overall. Fatigue and vision still a big issue.
merry xmas!
Another one. I think Nevirapine is quite risky.
Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth.
Lin J, Haffner MC, Zhang Y, Lee BH, Brennen WN, Britton J, Kachhap SK, Shim JS, Liu JO, Nelson WG, Yegnasubramanian S, Carducci MA.
Chemical Therapeutics Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Abstract
BACKGROUND: The clinical success of the nucleoside analogs 5-aza-cytidine (5-azaC) and 5-aza-2âdeoxycytidine (5-aza-dC) as DNA methyltransferase (DNMT) inhibitors has spurred interest in the development of non-nucleoside inhibitors with improved pharmacologic and safety profiles. Because DNMT catalysis features attack of cytosine bases by an enzyme thiol group, we tested whether disulfiram (DSF), a thiol-reactive compound with known clinical safety, demonstrated DNMT inhibitory activity.
METHODS: Inhibition of DNMT1 activity by DSF was assessed using methyltransferase activity assays with recombinant DNMT1. Next, prostate cancer cell lines were exposed to DSF and assessed for: i) reduction of global 5-methyl cytosine ((5me)C) content using liquid chromatography/tandem mass spectrometry (LC-MS/MS); ii) gene-specific promoter demethylation by methylation-specific PCR (MSP); and iii) gene-reactivation by real-time RT-PCR. DSF was also tested for growth inhibition using prostate cancer cell lines propagated in vitro in cell culture and in vivo as xenografts in nude mice.
RESULTS: Disulfiram showed a dose-dependent inhibition of DNMT1 activity on a hemimethylated DNA substrate. In prostate cancer cells in culture, DSF exposure led to reduction of global genomic (5me)C content, increase in unmethylated APC and RARB gene promoters, and associated re-expression of these genes, but did not significantly alter prostate-specific antigen (PSA) expression. DSF significantly inhibited growth and clonogenic survival of prostate cancer cell lines in culture and showed a trend for reduced growth of prostate cancer xenografts.
CONCLUSIONS: Disulfiram is a non-nucleoside DNMT1 inhibitor that can reduce global (5me)C content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines. Prostate Š 2010 Wiley-Liss, Inc.
Another one looking at azacitidine.
ukpmc.ac.uk/articles/PMC2939768/
Safety and tolerability
In early trials, higher IV doses (100â400 mg/m2) of azacitidine were associated with significant nausea, vomiting, infusion reactions, in addition to severe hematologic toxicities.21â24 Additionally, in previous studies evaluating azacitidine in patients with metastatic tumors or with concurrent cirrhosis, severe hepatic toxicity was seen. As a result, azacitidine is contraindicated in patients with significant hepatic involvement by malignancy.12,30
With current dosing schemes, azacitidine is generally well-tolerated. Interestingly, a stringent evaluation of adverse effects is made difficult by similar adverse events reported in both patients receiving azacitidine and those patients in supportive care arms who suffered from sequelae due to the natural history of their MDS.30 The documented adverse effects are primarily hematologic. Patients receiving azacitidine on the AZA-001 trial frequently experienced grade 3 or 4 neutropenia (91%), thrombocytopenia (85%), or anemia (57%) during their course of care compared with the patients in conventional care arms at 76%, 80%, and 68%, respectively.11 Given the inherent biology of MDS, attribution of cytopenias to azacitidine vs MDS itself is challenging. Patients treated with azacitidine generally do have an increase in transfusion requirements during their first cycles of therapy, although this effect disappears in those with a positive response to therapy.9 In addition, the important effect noted in the AZA-001 trial was that azacitidine therapy did not result in an increased risk of infection.11 For patients who discontinued therapy early due to adverse effects, the reason was generally due to hematologic toxicities.10,11,30 Additionally, in CALGB studies, there was no increased risk of bleeding events in patients on azacitidine.10 In summary, cytopenias inherent to both therapy and MDS did not lead to increased risk of bleeding or infection in those patients treated with azacitidine, and for those who responded to therapy, resolution of cytopenias occurred with a median time to response of 3 months.
Certain nonhematologic toxicities commonly documented in patients receiving azacitidine included nausea, vomiting, constipation, diarrhea, anorexia, fatigue, arthralgias, headache, hepatic function abnormalities, and injection site reactions (with anecdotal reports suggesting primrose oil to ameliorate the SQ injection site reactions).10,11,30,45 Also, adverse effects are more frequently reported within the initial 2 cycles of therapy.30 Although nausea and vomiting were a dose-limiting toxicity in the 1960sâ1980s, presumably modern-day antiemetic therapy has played a role in improving the tolerability of azacitidine.
ncbi.nlm.nih.gov/pubmed/11074602
Inactivation of the human androgen receptor gene is associated with CpG hypermethylation in uterine endometrial cancer.
Sasaki M, Oh BR, Dharia A, Fujimoto S, Dahiya R.
Department of Urology, University of California, and VA Medical Center, San Francisco, California 94121, USA.
Abstract
Androgens mediate their effects through the androgen receptor (AR) and have antiproliferative effects on uterine endometrial cells. In this report, we investigated methylation status and the expression of the AR gene in normal endometrium and uterine endometrial cancer (UEC) tissues using methylation-specific polymerase chain reaction (MSP) and immunohistochemical staining. Seventy of 89 cancer samples were AR negative, although 39 of 46 normal samples were AR positive by immunohistochemistry. By MSP, 64 of 89 cancer samples showed only methylated AR alleles, although all normal tissues showed both unmethylated and methylated AR alleles. To determine whether similar changes occurred in methylation status in the UEC carcinogenesis, we studied AR methylation using pairs of cancerous and normal samples from 28 patients. Twenty-three of 28 cancer samples showed only methylated AR alleles, although all normal samples showed both unmethylated and methylated alleles. All of the 23 cancer samples that lost unmethylated alleles were negative for AR by immunohistochemical analysis. Reverse transcription-polymerase chain reaction was performed by using UEC cell lines with and without treatment by the demethylating reagent 5-aza-2â-deoxycytidine. No AR expression was found in any of the UEC cell lines, except for MFE-296 without 5-aza-2â-deoxycytidine. Treatment with 5-aza-2â-deoxycytidine restored AR expression in all of the UEC cell lines that showed no AR expression before treatment. This study is the first to report that the possible mechanism of AR inactivation in endometrial cancer is through hypermethylation of the AR gene CpG islands.
ncbi.nlm.nih.gov/pubmed/11140692
Epigenetic regulation of androgen receptor gene expression in human prostate cancers.
Nakayama T, Watanabe M, Suzuki H, Toyota M, Sekita N, Hirokawa Y, Mizokami A, Ito H, Yatani R, Shiraishi T.
Second Department of Pathology, Mie University School of Medicine, Japan.
Abstract
Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5â CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5â-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2â-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.
ncbi.nlm.nih.gov/pubmed/15156193
Androgen receptor level controlled by a suppressor complex lost in an androgen-independent prostate cancer cell line.
Wang LG, Ossowski L, Ferrari AC.
Department of Medicine, Division of Hematology/Oncology, Mount Sinai School of Medicine, Box 1129, 1 Gustave Levy Place, New York, NY 10029, USA.
Abstract
Androgen receptor (AR) overexpression is one of the characteristics of prostate cancer (PC) that progresses to hormone independence. An androgen-independent (AI) derivative, with much higher AR-mRNA and protein levels than the parental LNCaP cell line, whose proliferation was androgen dependent (AD), was used to explore the mechanism of AR overexpression. We found that a suppressor element (ARS), previously identified in mouse AR and located in the 5â-untranslated region of human AR gene, malfunctions in AI cells. Transfection of constructs that included ARS element into AD cells reduced the transactivating activities of both AR promoter and a heterologous SV40 promoter. The deletion of ARS resulted in an eightfold increase in AR-promoter activity in AD cells, but had no effect in AI cells. Moreover, the nuclear extracts of AD cells contained proteins that produced a specific, ARS-binding complex, while this complex appeared to have been lost from AI cells. Most importantly, treatment of AI cells with a demethylating agent or histone deacetylase inhibitors restored the lost ARS-binding complex. The restoration of the complex coincided with a reduced expression of AR-mRNA and protein and a reduced rate of AR-gene transcription, determined by nuclear run-on experiment. Thus, epigenetic transcriptional silencing of the suppressor protein(s) may be responsible for AR overexpression in AI cells, and its reversal in hormone-independent PC may normalize AR levels and restore their hormone dependence.
Think we may have had this one before
ncbi.nlm.nih.gov/pubmed/16079795
LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription.
Metzger E, Wissmann M, Yin N, MĂźller JM, Schneider R, Peters AH, GĂźnther T, Buettner R, SchĂźle R.
Universitäts-Frauenklinik und Zentrum fßr Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg, Germany.
Abstract
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.
finally a bit about Dr Jay Goldstein now no longer practicing.
prohealth.com/library/showarticle.cfm?libid=9210
Relates to CFS mainly but may be of interest.
Hey man, great work. I was very interested in that article on Disulfiram. It seems to have one of the better safety profiles of the drugs we have looked at so far. I did a bit more digging and came across the following study.
Development of disulfiram as a novel DNMT1 inhibitor in prostate cancer.
DNA methylation and histone modifications have been implicated in prostate cancer (PCa) carcinogenesis. These epigenetic marks may be therapeutically reversible and are targets for both PCa treatment. Disulfiram was found to be a potent DNMT1 inhibitor in vitro and is one of the most potent inhibitors for PCa growth in vitro by screening the Johns Hopkins Drug Library. We hypothesized that Disulfiram is a potent DNMT inhibitor which can alter DNA methylation pattern and thereby reverse silencing of tumor suppresser genes in PCa.
MSP showed that disulfiram induced demethylation of known highly methylated APC, RARâ gene promotors in prostate cancer cells.
Expression data were further corroborated by Western blot analysis and revealed re-expression of androgen receptor (AR) in DU145 cells after disulfiram treatment. The AR gene restoration was correlated with limited CpG island demethylation in bisulfite sequencing analysis.
Disulfiram is a novel DNMT1 inhibitor which induces re-expression of several tumor suppressor genes in prostate cancer. Based on these preclinical studies, a phase II clinical trial is underway to evaluate the efficacy of disulfiram in patients with recurrent prostate cancer.
asco.org/ascov2/Meetings/Abstracts?&vmview=abst_detail_view&confID=64&abstractID=20452
Two more:
These are about the risks of hypomethylation.
Hypomethylation is seen in cancers as well as hypermethylation. The cause could be simply as a by product or it could be contributory. Havenât bought these articles so not sure about their conclusions.