Synthesis of curcumin derivatives targeting androgen receptor for castration-resistant prostate cancer therapy.

In this work, a series of curcumin derivatives (1a-1h, 2a-2g, and 3a-3c) were synthesized for the suppression of castration-resistant prostate cancer cells. All synthesized compounds were characterized by 1H NMR, 13C NMR, HRMS, and melting point. The in vitro cytotoxicity study shows that compounds 1a, 1e, 1f, 1h, 2g, 3a, and 3c display similar or enhanced cytotoxicity against 22Rv1 and C4-2 cells as compared to ASC-J9, other synthesized compounds display reduced cytotoxicity against 22Rv1 and C4-2 cells as compared to ASC-J9. Molecular docking simulation was performed to study the binding affinity and probable binding modes of the synthesized compounds with androgen receptor. The results show that all synthesized compounds exhibit higher cdocker interaction energies as compared to ASC-J9. Compounds 1h, 2g, and 3c not only show strong cytotoxicity against 22Rv1 and C4-2 cells but also exhibit high binding affinity with androgen receptor. In androgen receptor suppression study, compounds 1f and 2g show similar androgen receptor suppression effect as compared to ASC-J9 on C4-2 cells, compound 3c displays significantly enhanced AR suppression effect as compared to ASC-J9, 1f and 2g. Compounds 1a, 1e, 1f, 1h, 2g, 3a and 3c prepared in this work have significant potential for castration-resistant prostate cancer therapy.

ASC-J9 Blocks Cell Proliferation and Extracellular Matrix Production of Keloid Fibroblasts through Inhibiting STAT3 Signaling.

Keloids are a fibrotic skin disorder caused by abnormal wound healing and featuring the activation and expansion of fibroblasts beyond the original wound margin. Signal transducer and activator of transcription 3 (STAT3) has been found to mediate the biological functions of keloid fibroblasts (KFs). Therefore, we aimed to demonstrate whether ASC-J9, an inhibitor of STAT3 phosphorylation, can suppress the activation of KFs. Western blotting results showed that ASC-J9 inhibited the levels of COL1A1 and FN1 proteins, which were upregulated in KFs, by decreasing the expression of pSTAT3 and STAT3. RNA sequencing and in vitro studies further demonstrated that ASC-J9 treatment of KFs reduced cell division, inflammation, and ROS generation, as well as extracellular matrix (ECM) synthesis. ELISA assays verified that ASC-J9 treatment significantly mitigated IL-6 protein secretion in KFs. Transmission electron microscopy images revealed that ASC-J9 induced the formation of multilamellar bodies in KFs, which is associated with autophagy-related signaling. These results suggested that inhibiting a vicious cycle of the ROS/STAT3/IL-6 axis by ASC-J9 may represent a potential therapeutic approach to suppress cell proliferation and ECM production in KFs.

Stiffness-tuneable nanocarriers for controlled delivery of ASC-J9 into colorectal cancer cells.

HYPOTHESIS: One of the main challenges in cancer therapy is the poor water solubility of many anticancer drugs which results in low bioavailability at the tumour sites and reduced efficacy. The currently available polymer-based anticancer drug delivery systems often suffer from low encapsulation efficiency, uncontrolled release, and lack of long-term stability. Herein, we report the development of novel stiffness-tuneable core-shell nanocarriers composed of naturally derived polymers silk fibroin (SF) and sodium alginate (SA) inside a liposomal shell for enhanced cellular uptake and controlled release of hydrophobic anticancer agent ASC-J9 (Dimethylcurcumin). It is anticipated that the stiffness of the nanocarriers has a significant effect on their cellular uptake and anticancer efficacy. EXPERIMENTS: The nanocarriers were prepared by thin film hydration method followed by extrusion and cross-linking of SA to obtain a uniform size and shape, avoiding harsh processing conditions. The structural transformation of SF in the nanocarriers induced by SA crosslinking was determined using Fourier transform infrared (FTIR) spectroscopy. The size, zeta potential, morphology and stiffness of the nanocarriers were measured using dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Drug loading and release were measured using UV-Vis spectrophotometry. The cellular uptake and anticancer efficacy of the nanocarriers were studied in HCT 116 human colorectal adenocarcinoma cells and 3D tumour spheroids using high content microscopy. FINDINGS: The synthesized nanocarriers had high encapsulation efficiency (62-78%) and were physically stable for up to 5 months at 4 ˚C. The release profile of the drug from the nanocarriers was directed by their stiffness and was easily tuneable by changing the ratio of SF to SA in the core. Furthermore, the designed nanocarriers improved the cellular uptake and anticancer activity of ASC-J9, and enhanced its tumour penetration in HCT 116 3D colorectal cancer spheroids. These findings suggest that the designed core-shell nanocarriers can be used as a highly efficient drug delivery system for cancer therapy.

ASC-J9® suppresses prostate cancer cell proliferation and invasion via altering the ATF3-PTK2 signaling.

BACKGROUND: Early studies indicated that ASC-J9®, an androgen receptor (AR) degradation enhancer, could suppress the prostate cancer (PCa) progression. Here we found ASC-J9® could also suppress the PCa progression via an AR-independent mechanism, which might involve modulating the tumor suppressor ATF3 expression. METHODS: The lentiviral system was used to modify gene expression in C4-2, CWR22Rv1 and PC-3 cells. Western blot and Immunohistochemistry were used to detect protein expression. MTT and Transwell assays were used to test the proliferation and invasion ability. RESULTS: ASC-J9® can suppress PCa cell proliferation and invasion in both PCa C4-2 and CWR22Rv1 cells via altering the ATF3 expression. Further mechanistic studies reveal that ASC-J9® can increase the ATF3 expression via decreasing Glutamate-cysteine ligase catalytic (GCLC) subunit expression, which can then lead to decrease the PTK2 expression. Human clinical studies further linked the ATF3 expression to the PCa progression. Preclinical studies using in vivo mouse model also proved ASC-J9® could suppress AR-independent PCa cell invasion, which could be reversed after suppressing ATF3. CONCLUSIONS: ASC-J9® can function via altering ATF3/PTK2 signaling to suppress the PCa progression in an AR-independent manner.

A review of the effects and molecular mechanisms of dimethylcurcumin (ASC-J9) on androgen receptor-related diseases.

Dimethylcurcumin (ASC-J9) is a curcumin analogue capable of inhibiting prostate cancer cell proliferation. The mechanism is associated with the unique role of ASC-J9 in enhancing androgen receptor (AR) degradation. So far, ASC-J9 has been investigated in typical AR-associated diseases such as prostate cancer, benign prostatic hypertrophy, bladder cancer, renal diseases, liver diseases, cardiovascular diseases, cutaneous wound, spinal and bulbar muscular atrophy, ovarian cancer and melanoma, exhibiting great potentials in disease control. In this review, the effects and molecular mechanisms of ASC-J9 on various AR-associated diseases are summarized. Importantly, the effects of ASC-J9 and AR antagonists enzalutamide/bicalutamide on prostate cancer are compared in detail and crucial differences are highlighted. At last, the pharmacological effects of ASC-J9 are summarized and the future applications of ASC-J9 in AR-associated disease control are discussed.

ASC-J9® increases the bladder cancer chemotherapy efficacy via altering the androgen receptor (AR) and NF-κB survival signals.

BACKGROUND: The current chemotherapy regimens may extend survival for patients with metastatic bladder cancer (BCa) for a few months, but eventually most patients succumb to disease because they develop resistance to their chemotherapy. METHODS: TCGA human clinical sample survey and urothelial tumor tissue microarrays (TMAs) were applied to investigate the expression of androgen receptor (AR) and NF-κB. Multiple BCa cell lines were used to test chemotherapy's efficacy via multiple assays including XTT, flow cytometry, TUNEL, and BrdU incorporation. The effects of the AR degradation enhancer, ASC-J9®, combined with various chemotherapy reagents were examined both in vivo and in vitro. RESULTS: We unexpectedly found that in muscle-invasive BCa (miBCa) the signals of both the AR and NF-κB were increased via a TCGA sample survey. Results from multiple approaches revealed that targeting these two increased signals by combining various chemotherapeutic agents, including Cisplatin, Doxorubicin or Mitomycin C, with ASC-J9® led to increase the therapeutic efficacy. The combined therapy increases the expression of the pro-apoptosis BAX gene and cell cycle inhibitor p21 gene, yet suppresses the expression of the pro-survival BCL2 gene in miBCa cells. Preclinical studies using an in vivo mouse model with xenografted miBCa cells confirmed in vitro cell line data showing that treatment with ASC-J9® combined with Cisplatin can result in suppressing miBCa progression better than Cisplatin alone. CONCLUSIONS: Together, these results support a novel therapeutic approach via combining Cisplatin with ASC-J9® to better suppress the progression of miBCa.

Targeting the androgen receptor (AR) with AR degradation enhancer ASC-J9® led to increase docetaxel sensitivity via suppressing the p21 expression.

Chemotherapy with docetaxel remains the effective therapy to suppress castration resistant prostate cancer (CRPC) in some patients. However, most chemotherapy with docetaxel eventually fails with the development of docetaxel resistance after 18-weeks of treatment. Here we found docetaxel treatment might have an adverse effect of increasing the androgen receptor (AR) protein level in the CRPC cells, and combining docetaxel with anti-AR therapy using AR-shRNA or the AR degradation enhancer ASC-J9® may increase docetaxel sensitivity to better suppress the CRPC cell growth. Mechanism dissection found docetaxel might have the adverse effect of increasing the AR protein stability via suppressing the AR ubiquitination due to the increased AR phosphorylation. The consequence of such increased AR protein may then lead to increase p21 expression via transcriptional regulation. Preclinical studies with in vitro cells lines also demonstrated that targeting AR with ASC-J9® led to suppressing the AR-increased p21 expression to improve the docetaxel sensitivity in the CRPC cells that already developed docetaxel resistance. Together, these results suggest that a combined therapy of docetaxel and ASC-J9® is a novel therapy to better suppress CRPC in patients that already developed docetaxel resistance.

ASC-J9® suppresses prostate cancer cell invasion via altering the sumoylation-phosphorylation of STAT3.

The androgen-deprivation therapy (ADT) to either reduce the androgen biosynthesis (for example, Abiraterone) or to prevent binding of androgen to the androgen receptor (AR), for example using Casodex or Enzalutamide, which may result in .decrease of the prostate cancer (PCa) cell growth, yet may also increase the PCa cell invasion. In contrast, the recently identified AR degradation enhancer ASC-J9® may function via degrading the AR protein to simultaneously suppress the PCa cell proliferation and invasion. The details of this unique mechanism, however, remain unclear. Here we found that ASC-J9® could suppress PCa cell invasion via inducing the sumoylation of STAT3, thereby inhibiting the STAT3 phosphorylation that led to suppress the EMT-SNAIL2 signals in both PCa DU145 and PC3 AR-negative cells. Mutation of lysine-679 on the sumoylation site of the STAT3 effectively blocked the ASC-J9®-suppressed PCa cell invasion in both in vitro cell lines and in vivo mouse models. These results suggest that in addition to degrading AR to suppress PCa cell proliferation, ASC-J9® can also function through an AR-independent mechanism via modulating the STAT3 sumoylation to alter the phospho-STAT3 status to suppress the PCa cell invasion. These dual functions of ASC-J9® to suppress PCa proliferation and invasion (via altering STAT3 sumoylation) may help us to develop a better anti-AR compound that may overcome the current antiandrogens' unwanted side-effect of increasing the metastasis to better suppress the castration-resistant PCa progression.

Androgen receptor (AR) degradation enhancer ASC-J9® in an FDA-approved formulated solution suppresses castration resistant prostate cancer cell growth.

ASC-J9® is a recently-developed androgen receptor (AR)-degradation enhancer that effectively suppresses castration resistant prostate cancer (PCa) cell proliferation and invasion. The optimal half maximum inhibitory concentrations (IC50) of ASC-J9® at various PCa cell confluences (20%, 50%, and 100%) were assessed via both short-term MTT growth assays and long-term clonogenic proliferation assays. Our results indicate that the IC50 values for ASC-J9® increased with increasing cell confluency. The IC50 values were significantly decreased in PCa AR-positive cells compared to PCa AR-negative cells or in normal prostate cells. This suggests that ASC-J9® may function mainly via targeting the AR-positive PCa cells with limited unwanted side-effects to suppress the surrounding normal prostate cells. Mechanism dissection indicated that ASC-J9® might function via altering the apoptosis signals to suppress the PCa AR-negative PC-3 cells. Preclinical studies using multiple in vitro PCa cell lines and an in vivo mouse model with xenografted castration-resistant PCa CWR22Rv1 cells demonstrated that ASC-J9® has similar AR degradation effects when dissolved in FDA-approved solvents, including DMSO, PEG-400:Tween-80 (95:5), DMA:Labrasol:Tween-80 (10:45:45), and DMA:Labrasol:Tween-20 (10:45:45). Together, results from preclinical studies suggest a potential new therapy with AR-degradation enhancer ASC-J9® may potentially be ready to be used in human clinical trials in order to better suppress PCa at later castration resistant stages.

Testosterone regulates 3T3-L1 pre-adipocyte differentiation and epididymal fat accumulation in mice through modulating macrophage polarization.

Low testosterone levels are strongly related to obesity in males. The balance between the classically M1 and alternatively M2 polarized macrophages also plays a critical role in obesity. It is not clear whether testosterone regulates macrophage polarization and then affects adipocyte differentiation. In this report, we demonstrate that testosterone strengthens interleukin (IL) -4-induced M2 polarization and inhibits lipopolysaccharide (LPS)-induced M1 polarization, but has no direct effect on adipocyte differentiation. Cellular signaling studies indicate that testosterone regulates macrophage polarization through the inhibitory regulative G-protein (Gαi) mainly, rather than via androgen receptors, and phosphorylation of Akt. Moreover, testosterone inhibits pre-adipocyte differentiation induced by M1 macrophage medium. Lowering of serum testosterone in mice by injecting a luteinizing hormone receptor (LHR) peptide increases epididymal white adipose tissue. Testosterone supplementation reverses this effect. Therefore, our findings indicate that testosterone inhibits pre-adipocyte differentiation by switching macrophages to M2 polarization through the Gαi and Akt signaling pathways.

Sorafenib with ASC-J9® synergistically suppresses the HCC progression via altering the pSTAT3-CCL2/Bcl2 signals.

Sorafenib is currently used as a standard treatment to suppress the progression of hepatocellular carcinoma (HCC), especially in advanced stages. However, patients who receive Sorafenib treatment eventually develop resistance without clear mechanisms. There is a great need for better efficacy of Sorafenib treatment in combination with other therapies. Here, we demonstrated that the treatment combining Sorafenib with ASC-J9® could synergistically suppress HCC progression via altering cell-cycle regulation, apoptosis and invasion. Mechanism dissection suggests that while Sorafenib impacts little or even slightly increases the activated/phosphorylated STAT3 (p-STAT3), a key stimulator to promote the HCC progression, adding ASC-J9® significantly suppresses the p-STAT3 expression and its downstream genes including CCL2 and Bcl2. Interrupting these signals via constitutively active STAT3 partially reverses the synergistic suppression of Sorafenib-ASC-J9® combination on HCC progression. In vivo studies further confirmed the synergistic effect of Sorafenib-ASC-J9® combination. Together, these results suggest the newly developed Sorafenib-ASC-J9® combination is a novel therapy to better suppress HCC progression.

New therapy with ASC-J9® to suppress the prostatitis via altering the cytokine CCL2 signals.

Prostatitis is a common disease contributing to 8% of all urologist visits. Yet the etiology and effective treatment remain to be further elucidated. Using a non-obese diabetes mouse model that can be induced by autoimmune response for the spontaneous development of prostatitis, we found that injection of the ASC-J9® at 75 mg/Kg body weight/48 hours led to significantly suppressed prostatitis that was accompanied with reduction of lymphocyte infiltration with reduced CD4+ T cells in prostate. In vitro studies with a co-culture system also confirmed that ASC-J9® treatment could suppress the CD4+ T cell migration to prostate stromal cells. Mechanisms dissection indicated that ASC-J9® can suppress CD4+ T cell migration via decreasing the cytokine CCL2 in vitro and in vivo, and restoring CCL2 could interrupt the ASC-J9® suppressed CD4+ T cell migration. Together, results from in vivo and in vitro studies suggest that ASC-J9® can suppress prostatitis by altering the autoimmune response induced by CD4+ T cell recruitment, and using ASC-J9® may help us to develop a potential new therapy to battle the prostatitis with little side effects.

ASC-J9(®) suppresses castration resistant prostate cancer progression via degrading the enzalutamide-induced androgen receptor mutant AR-F876L.

Androgen deprivation therapy (ADT) with the newly developed powerful anti-androgen enzalutamide (Enz, also known as MDV3100) has promising therapeutic effects to suppress castration resistant prostate cancer (CRPC) and extending patients' lives an extra 4.8 months. However, most Enz therapy eventually fails with the development of Enz resistance. The detailed mechanisms how CRPC develops Enz resistance remain unclear and may involve multiple mechanisms. Among them, the induction of the androgen receptor (AR) mutant AR-F876L in some CRPC patients may represent one driving force that confers Enz resistance. Here, we demonstrate that the AR degradation enhancer, ASC-J9(®), not only degrades wild-type AR, but also has the ability to target AR-F876L. The consequence of suppressing AR-F876L may then abrogate AR-F876L mediated CRPC cell proliferation and metastasis. Thus, developing ASC-J9(®) as a new therapeutic approach may represent a novel therapy to better suppress CRPC that has already developed Enz resistance.

ASC-J9(®), and not Casodex or Enzalutamide, suppresses prostate cancer stem/progenitor cell invasion via altering the EZH2-STAT3 signals.

Early studies suggested that prostate cancer (PCa) stem/progenitor (S/P) cells might play key roles to promote the tumor initiation and metastasis. Yet their linkage to the failure of androgen deprivation therapy (ADT), however, remains unclear. Here we demonstrated that the ADT with anti-androgens Casodex (also known as Bicalutamide) and Enzalutamide (also known as MDV3100), but not the newly identified AR degradation enhancer, ASC-J9(®), increased PCa S/P population, which might then lead to enhance the PCa cell invasion. Targeting AR with ASC-J9(®), and not targeting androgens with Casodex or Enzalutamide, led to suppress PCa S/P cell invasion. Mechanism dissection revealed ASC-J9(®) could suppress S/P cell invasion via altering the EZH2/STAT3 and/or AKT/EZH2/STAT3 signals. Together, these results suggest that targeting PCa S/P cells with ASC-J9(®) or inhibitors to interrupt the EZH2/STAT3 and/or Akt/EZH2/STAT3 signals may become a new therapy to overcome the unwanted side effects of Casodex or Enzalutamide to further suppress the PCa metastasis.

Targeting fatty acid synthase with ASC-J9 suppresses proliferation and invasion of prostate cancer cells.

Fatty acid synthase (FASN) is the key enzyme for the control of fatty acid synthesis that contributes significantly to the prostate cancer (PCa) progression. It was reported that androgens were able to induce FASN expression in PCa, and addition of the anti-androgen Casodex might suppress the androgen-induced FASN expression. However, here we found androgen-deprivation-therapy (ADT) with anti-androgens Bicalutamide (Casodex) or Enzalutamide (MDV3100) had little effect to suppress FASN expression and FASN-mediated cell growth and invasion during the castration resistant stage when the androgen concentration is 1 nM DHT (dihydrotestosterone). In contrast, the newly developed androgen receptor (AR) degradation enhancer ASC-J9® suppressed FASN expression and FASN-mediated cell growth and invasion in various PCa cell lines at 1 nM DHT. Mechanism dissection found ASC-J9® could suppress significantly the FASN expression and FASN-mediated PCa progression via the AR-dependent pathway involving AR→SREBP-1→FASN signaling in AR-positive C4-2 and LNCaP cells and via the AR-independent pathway involving the modulation of PI3K/AKT→SREBP-1→FASN signaling in AR-negative PC-3 and DU145 cells. Together, these results suggest that FASN is one of the important mechanism why the current ADT eventually fails. ASC-J9® might represent a new potential therapeutic approach to suppress FASN-mediated PCa progression via both AR-dependent and AR-independent pathways during the castration resistant stage of PCa. © 2016 Wiley Periodicals, Inc.

Determination of androgen receptor degradation enhancer ASC-J9(®) in mouse sera and organs with liquid chromatography tandem mass spectrometry.

A novel androgen receptor (AR) degradation enhancer ASC-J9(®) has displayed beneficial effects during the in vitro and in vivo studies for treatment of prostate cancer, liver cancer, bladder cancer and spinal and bulbar muscular atrophy (SBMA). It works mainly via the degradation of AR with minimal side effects on the tested mice. Here we developed a fast, robust and more sensitive method for the quantification of ASC-J9(®) in 100µL of mouse serum by using liquid chromatography tandem mass spectrometry (LC-MS/MS). The limit of quantification (LOQ) was found to be 5nM for ASCJ9(®). This method was successfully applied to investigate the pharmacokinetics of ASC-J9(®) in mice serum samples and also the distribution of the drug in various mice organs after single dose injection with results showing that ASC-J9(®) could be quickly absorbed in vivo and had a relatively slow elimination half-life of 5.45h. The ASC-J9(®) also exhibited a higher tendency to accumulate in organs such as liver, testes and prostate.

An electrospun scaffold loaded with anti-androgen receptor compound for accelerating wound healing.

Current dermal regenerative scafolds provide wound coverage, and structural support and guidance for tissue repair, but usually lack enough bio-signals needed for speeding up skin cell growth, migration, wound closure, and skin regeneration. In this study, an androgen receptor (AR) inhibitor called ASC-J9 is used to demonstrate the concept and feasibility of fabricating drug-loaded scafolds via electrospinning. Inhibition of androgen is known to promote skin wound healing. The novel ASC-J9 - loaded porous scafold was fabricated for skin wound repair using electrospun fibers of collagen and polycaprolactone (PCL) blend. Our preliminary results indicated that ASC-J9 - loaded scafolds facilitated more efficient attachment and ingrowth of dermal fbroblasts, compared to the control collagen-PCL scafold. A signifcant increase of cell proliferation was observed with the drug-loaded scafold over a 28-day period. The drug-loaded scafold also accelerated keratinocyte migration and wound closure in a contraction-inhibited mouse wound model over 21 days. The data indicated a sustained release of ASC-J9 from the scafold and its potential to accelerate wound healing by promoting cell proliferation and migration over an extended period of time. More importantly, our results proved the concept and feasibility of fabricating drug-releasing or bioactive dermal scaffolds for more efective wound healing.