androgen receptor (AR) degradation enhancer
Dimethylcurcumin (also referred to as ASC-J9 in the literature) is an androgen receptor (AR) degradation enhancer [1]

In a range of human PCa cells, dimethylcurcumin (ASC-J9) can degrade fAR and AR3 in a dose-dependent manner. In CWR22Rv1-fARKD cells, dimethylcurcumin (ASC-J9) can also efficiently block genes that are targeted by AR. In all three PCa cell lines, dimethylcurcumin (ASC-J9) (5 or 10 µM) effectively reduced DHT-induced cell proliferation. Dimethylcurcumin (ASC-J9) breaks down fAR and ectopic AR3 in C81 and C4-2 cells, which suppresses the development of cells and genes targeted by AR [1]. Dimethylcurcumin (ASC-J9) breaks off the connection between AR and AR coregulators, hence preferentially promoting the degradation of AR. Cells accumulate less AR when ASC-J9 AR-112Q is present. SBMA PC12/AR-112Q cell aggregation of AR-112Q is inhibited by dimethylcurcumin (ASC-J9) [2].

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Dimethylcurcumin (ASC-J9) degraded both full-length androgen receptor (fAR) and the splice variant AR3 (AR-V7) in a dose-dependent manner in various human castration-resistant prostate cancer (CRPC) cell lines, including C81, C4-2, and CWR22Rv1 cells, as shown by Western blot analysis after 24-hour treatment [1].
Treatment with 10 µM Dimethylcurcumin suppressed the mRNA expression of canonical AR target genes (PSA, TMPRSS2, FKBP5) induced by 1 nM dihydrotestosterone (DHT) in C81, C4-2, and CWR22Rv1 cells, as measured by quantitative real-time PCR (qPCR) [1].
Dimethylcurcumin (10 µM) also suppressed the expression of AR3-specific target genes (Akt1 and c-Myc) in CWR22Rv1 and CWR22Rv1-fARKD (fAR knockdown) cells [1].
In luciferase reporter assays (MMTV-Luc and ARE4-Luc), 10 µM Dimethylcurcumin suppressed AR transcriptional activity induced by 1 nM DHT in C81, C4-2, and CWR22Rv1 cells [1].
Dimethylcurcumin (5 µM and 10 µM) significantly suppressed cell growth (proliferation) induced by 1 nM DHT in C81, C4-2, and CWR22Rv1 cells, as measured by MTT assay. It also suppressed the growth of CWR22Rv1-fARKD cells in the absence of DHT [1].
The anti-proliferative effect of Dimethylcurcumin in CWR22Rv1 cells was associated with increased protein levels of cell cycle inhibitors p21 and p27, as shown by Western blot [1].
In C81 and C4-2 cells with ectopic AR3 overexpression (C81/AR3, C4-2/AR3), Dimethylcurcumin (10 µM) degraded both endogenous fAR and overexpressed AR3, suppressed DHT-induced AR target gene expression, and inhibited cell growth [1].
Treatment with 5 µM Dimethylcurcumin did not inhibit the growth of AR-negative prostate cancer cell lines (PC-3 and DU-145) [1].
Subcellular fractionation in CWR22Rv1 cells showed that 1 nM DHT promoted fAR nuclear translocation, and co-treatment with 10 µM Dimethylcurcumin inhibited this nuclear translocation. AR3 was predominantly nuclear, and its level was also reduced by Dimethylcurcumin treatment [1].
Dimethylcurcumin (10 µM) treatment did not significantly alter the mRNA levels of fAR or AR3 in CWR22Rv1 cells, indicating its action is primarily at the protein degradation level [1].
The effects of Dimethylcurcumin were compared with other antiandrogens. Casodex (bicalutamide) showed little suppressive effect on the growth of CWR22Rv1 and C4-2/AR3 cells. MDV3100 failed to degrade fAR and AR3 proteins in CWR22Rv1 cells, whereas Dimethylcurcumin effectively degraded both. In CWR22Rv1 cells, Dimethylcurcumin showed better growth inhibition than MDV3100 [1]

In xenograft tumors, dimethylcurcumin (ASC-J9) (75 mg/kg, i.p.) degrades fAR and AR3, and tumors treated with SC-J9 exhibit a significant reduction in Ki67-positive cells [1]. In AR-97Q mice, intraperitoneal injections of 50 mg/kg dimethylcurcumin (ASC-J9) every 48 hours markedly reduced the symptoms of SBMA and enhanced neuromuscular pathology. Serum testosterone concentrations in SBMA animals treated with dimethylcurcumin (ASC-J9) are comparatively normal [2]. When compared to mice getting traditional ADT/castration with low serum androgen levels, mice treated with ASC-J9 showed noticeably reduced prostate tumor sizes [3].

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In an orthotopic xenograft model using castrated male nude mice, intraperitoneal injection of Dimethylcurcumin (ASC-J9) at 75 mg/kg every other day for 4 weeks significantly suppressed the growth of implanted CWR22Rv1 tumors compared to vehicle-treated control [1].
Western blot analysis of harvested tumors showed decreased levels of both fAR and AR3 proteins in the Dimethylcurcumin-treated group [1].
Immunohistochemical analysis of the xenograft tumors revealed reduced AR protein intensity, a significant decrease in Ki67-positive proliferating cells, and an increase in TUNEL-positive apoptotic cells in the Dimethylcurcumin-treated group compared to the control [1]

Western Blot Analysis, Quantitative Real-time Polymerase Chain Reaction, and Luciferase Reporter Assay [1]
Cells were cultured and treated with or without ASC-J9 for 24 hours in 10% charcoal-dextran-stripped fetal bovine serum (CD-FBS) media. Cell lysates were harvested and subjected to Western blot analysis. Quantitative real-time polymerase chain reaction (qPCR) was performed in triplicate with a Bio-Rad iCycler system (Bio-Rad, Hercules, CA); and messenger RNA (mRNA) levels of PSA, TMPRSS2, FKBP5, and GAPDH were measured. Cells were transiently transfected with mouse mammary tumor virus luciferase reporter (MMTV-Luc) or ARE4-Luc plus pRL-TK as internal control. Luciferase activities were measured using GloMax 20/20 Luminometer (Promega, Madison, WI).

Cell Growth Assay[1]
Cells were treated with vehicle, 1 nM dihydrotestosterone (DHT), 5 µM Casodex, and 5 or 10 µM ASC-J9 in 10% CD-FBS medium. The media were replenished every other day, and we followed the standard MTT assay protocol.

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For Western blot analysis to detect AR protein degradation, cells (e.g., CWR22Rv1, C4-2, C81) were cultured and treated with or without Dimethylcurcumin (ASC-J9) at indicated concentrations (e.g., 5, 7.5, 10 µM) in medium containing 10% charcoal-dextran-stripped fetal bovine serum for 24 hours. Cell lysates were harvested, subjected to SDS-PAGE, and transferred to membranes. Membranes were probed with anti-AR and anti-GAPDH antibodies for detection and loading control [1].
For quantitative real-time PCR (qPCR) analysis of gene expression, cells were treated as indicated. Total RNA was extracted, reverse transcribed into cDNA, and subjected to qPCR using gene-specific primers (e.g., for PSA, TMPRSS2, FKBP5, Akt1, c-Myc, GAPDH). Reactions were performed in triplicate, and mRNA levels were normalized to GAPDH [1].
For cell growth/proliferation assays (MTT assay), cells were seeded and treated with vehicle, 1 nM DHT, Dimethylcurcumin (e.g., 5 or 10 µM), or other compounds in 10% charcoal-dextran-stripped fetal bovine serum medium. Media containing compounds were replenished every other day. At indicated time points, MTT reagent was added to the wells, incubated, and the resulting formazan crystals were dissolved. Absorbance was measured to determine relative cell viability/growth [1].
For subcellular fractionation, CWR22Rv1 cells were treated, and nuclear and cytoplasmic fractions were separated using a commercial extraction kit. The fractions were then analyzed by Western blot using anti-AR, anti-PARP-1 (nuclear marker), and anti-α-tubulin (cytoplasmic marker) antibodies [1].
For the examination of AR3 knockdown effects, CWR22Rv1 and CWR22Rv1-fARKD cells were infected with lentivirus carrying short hairpin RNA specific for AR3 (shAR3) or a scrambled control. Knockdown efficiency and subsequent effects on gene expression and cell growth were assessed by Western blot, qPCR, and MTT assay [1]

In Vivo Tumor Growth Assay [1]
Animal procedures were conducted in accordance with the protocol approved by the University of Rochester Committee on Animal Resources. CWR22Rv1 cells (1 x 106 cells per site) were injected into both anterior prostates (orthotopic) of castrated nude mouse after 2 weeks of implantation. The mice were randomly divided into two groups (four mice/eight tumors each group) and either received 75 mg/kg ASC-J9 intraperitoneal injection or vehicle control every other day. After 4 weeks of treatment, all mice were killed to examine the tumor growth. Body weights and mice activity were measured weekly.

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For the in vivo tumor growth assay, male nude mice were surgically castrated. Two weeks after castration, CWR22Rv1 cells were orthotopically injected into both anterior prostate lobes of each mouse. Mice were randomly divided into treatment and control groups. Dimethylcurcumin (ASC-J9) was administered via intraperitoneal injection at a dose of 75 mg/kg every other day for 4 weeks. The control group received vehicle injection. Body weights and activity were monitored weekly. After 4 weeks, all mice were euthanized, and orthotopic tumors were harvested, weighed, and measured for volume calculation. Tumor tissues were processed for histology, immunohistochemistry, and Western blot analysis [1]

In the described in vivo studies, mice treated with dimethylcurcumin (75 mg/kg, intraperitoneally, every other day for 4 weeks) had comparable body weight to control mice treated with the carrier, indicating no significant side effects or toxicity under these experimental conditions [1].

[1]. ASC-J9 suppresses castration-resistant prostate cancer growth through degradation of full-length and splice variant androgen receptors. Neoplasia. 2012 Jan;14(1):74-83.

[2]. ASC-J9 ameliorates spinal and bulbar muscular atrophy phenotype via degradation of androgen receptor. Nat Med. 2007 Mar;13(3):348-53.

[3]. New therapy targeting differential androgen receptor signaling in prostate cancer stem/progenitor vs non-stem/progenitor cells. J Mol Cell Biol. 2012 Jul 24.

[4]. Targeting androgen receptor with ASC-J9 attenuates cardiac injury and dysfunction in experimental autoimmune myocarditis by reducing M1-like macrophage. Biochem Biophys Res Commun. 2017 Apr 15;485(4):746-752. doi: 10.1016/j.bbrc.2017.02.123.

Dimethylcurcumin (ASC-J9) has been described as a novel androgen receptor degradation enhancer. Its mechanism of action is to promote the degradation of AR proteins by disrupting the interaction between the androgen receptor (AR) and selective AR co-regulatory factors, which differs from traditional antiandrogens (e.g., cassodex, MDV3100) that primarily act as ligand-binding domain antagonists [1]. This study suggests that targeting castration-resistant prostate cancer with dimethylcurcumin to degrade full-length AR and constitutively active AR splice variants (e.g., AR3) is a potential therapeutic strategy, especially in cases where splice variants lead to treatment resistance [1]. Previous studies (cited in the introduction/discussion of the provided literature) have shown that dimethylcurcumin has little effect on other steroid receptors (e.g., glucocorticoid receptor, estrogen receptor α), and mice treated with dimethylcurcumin maintained normal sexual function and fertility [1].

C23H24O6

396.43306

396.157

C, 69.68; H, 6.10; O, 24.21

52328-98-0

6477182

Light yellow to red solid powder

1.2±0.1 g/cm3

588.6±50.0 °C at 760 mmHg

129-130 °C

201.8±23.6 °C

0.0±1.7 mmHg at 25°C

1.608

4.05

1

6

9

29

596

0

O=C(/C=C(O)/C=C/C1=CC=C(OC)C(OC)=C1)/C=C/C2=CC=C(OC)C(OC)=C2

ZMGUKFHHNQMKJI-CIOHCNBKSA-N

InChI=1S/C23H24O6/c1-26-20-11-7-16(13-22(20)28-3)5-9-18(24)15-19(25)10-6-17-8-12-21(27-2)23(14-17)29-4/h5-15,24H,1-4H3/b9-5+,10-6+,18-15-

(1E,4Z,6E)-1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one

ASC-J9; ASC-J-9; ASC J9; GO-Y025; GO-Y 025; GO Y025;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~126.13 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (5.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.2 mg/mL (5.5 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21.7 mg/mL of DMSO stock solution and add tO + 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution).
Preparation of saline: Dissolve 0.9 g of sodium chloride in ddH ₂ O and make up to 100 mL to obtain a clear and transparent saline solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)