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Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration.

It has long been realized that hematopoietic cells may have the capacity to trans-differentiate into non-lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans-differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast-like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type-1 collagen, and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum-free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut-offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony-stimulating factor (M-CSF). The mechanisms of M-CSF-inducing trans-differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA-1 and -3, OCT3/4, NANOG, and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M-CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications.

Summary There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1 H -indol-2-yl)-1 H -indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer. Mol Cancer Ther; 12(11); 2425–35. ©2013 AACR.

BACKGROUND Androgen deprivation therapy (ADT) and novel agents targeting the androgen synthesis axis (e.g., abiraterone acetate) are adjuvant therapies that are currently, or may in the future be, combined with radiotherapy to reduce the chance of disease relapse. Little is known about allelic loss or gain pertaining to genes associated with the androgen synthesis axis and whether this is prognostic in patients who receive localized radiotherapy. In this hypothesis generating study, we conducted an array comparative genomic hybridization (aCGH) analysis of 33 androgen synthesis genes to identify potential prognostic factors for radiotherapy outcome. METHODS aCGH analysis of tumor DNA prospectively derived from frozen needle biopsies of 126 men with intermediate-risk disease who underwent image-guided radiotherapy (IGRT) to a mean dose of 76.4 Gy was conducted. Statistical analyses were conducted for allelic loss or gain in genes as potential prognostic factors relative to prostate specific antigen, Gleason-score, and T-category. RESULTS We observed that allelic losses of loci containing the genes StAR and HSD17B2 were associated with increased genetic instability (as determined by percentage genome alteration). On multivariate analyses these loci were prognostic for biochemical disease-free relapse (StAR: HR = 2.84, 95% CI: 1.44–5.61, P = 0.00269; HSD17B2: HR = 1.97, 95% CI: 1.06–3.64, P = 0.031). The results were validated in a surgical cohort of 131 intermediate-risk patients. CONCLUSIONS Allelic losses of the loci containing StAR and HSD17B2 have significant prognostic value for intermediate-risk prostate cancer. With this hypothesis generating information future studies should test StAR and HSD17B2 losses as biomarkers of androgen response in combined modality protocols. Prostate 72:1295–1305, 2012. © 2011 Wiley Periodicals, Inc.

Abdelbaqi K, Lack N, Guns ET, Kotha L, Safe S, Sanderson JT. BACKGROUND : Cruciferous vegetables protect against prostate cancer. Indole-3-carbinol (I3C) and its major metabolite 3,3'-diindolylmethane (DIM), exhibit antitumor activities in vitro and in vivo. Several synthetic ring-substituted dihaloDIMs (ring-DIMs) appear to have increased anticancer activity. METHODS : Inhibition of LNCaP prostate cancer cell growth was measured by a WST-1 cell viability assay. Cytoplasmic and nuclear (...)

Dimethyl sulfoxide (DMSO) is currently being used by many people, both topically and orally, to treat inflammatory conditions as well as cancer. However, little is known about the efficacy, safety and mechanism of action of DMSO in humans. We therefore examined the effects of this polar solvent on whole human blood, stimulated or not, ex vivo, with E. coli. We found that, between 1 and 2%, DMSO markedly inhibited cytokine production without affecting cell viability. However, at greater than 5%, significant death of leukocytes was observed, suggesting that DMSO has only a narrow window of efficacy. The two in vivo generated metabolites of DMSO, dimethyl sulfide (DMS) and dimethylsulfone (DMSO2), required higher concentrations than DMSO to be anti-inflammatory. Mechanism of action studies using purified human monocytes revealed that DMSO's anti-inflammatory properties were due, at least in part, to inhibition of the ERK1/2, p38, PI3K and JNK pathways. In vivo mouse model studies revealed that topical admini...

Indoleamine 2,3 dioxygenase (Ido1), the first and rate-limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age-related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase-2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up-regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.

The human androgen receptor (AR) is a proven therapeutic target in prostate cancer. All current antiandrogens, such as Bicalutamide, Flutamide, Nilutamide, and Enzalutamide, target the buried hydrophobic androgen binding pocket of this protein. However, effective resistance mechanisms against these therapeutics exist such as mutations occurring at the target site. To overcome these limitations, the surface pocket of the AR called binding function 3 (BF3) was characterized as an alternative target for small molecule therapeutics. A number of AR inhibitors directly targeting the BF3 were previously identified by us ( J. Med. Chem. 2011 . 54 , 8563 ). In the current study, based on the prior results, we have developed structure-activity relationships that allowed designing a series of 2-((2-phenoxyethyl)thio)-1H-benzimidazole and 2-((2-phenoxyethyl)thio)-1H-indole as lead BF3 inhibitors. Some of the developed BF3 ligands demonstrated significant antiandrogen potency against LNCaP and Enzalutamide-resistant prostate cancer cell lines.

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called Binding Function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in-silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and x-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its anti-androgen resistant forms.

Interest in developing androgen receptor (AR) inhibitors with novel mechanism of action is on the rise since the commercial anti-androgens (including recently approved drug, Enzalutamide) face clinical limitations. The current therapies fail over a period of time because they all target mutation-prone hormone binding pocket on AR to which the receptor has already developed effective resistance mechanisms. Hence, there is a pressing need for novel therapeutics that inhibit the AR through alternative modes of action. Recent studies have identified a novel binding pocket on the surface of AR called binding function-3 (BF3) that is critical for the AR transcriptional activity. In order to develop promising drug-like candidates, we performed structural optimization of our lead AR BF3 inhibitor, followed by a series of experimental validation of the synthetic analogues. As a result, we have discovered a chemical series of quinoline as new lead BF3 inhibitors. One of the most potent inhibitors identified, VPC-13566, demonstrated an IC50 of 0.071μM in AR eGFP transcriptional assay. Confirming it as a true BF3 binder, VPC-13566 displaced Bag-1L peptide, a co-regulator protein that binds to AR BF3 pocket. Additionally, the Biolayer Interferometry assay detected direct reversible interactions between the AR ligand binding domain and the inhibitor. To confirm VPC-13566 binding to the BF3 pocket, a mutagenesis study was performed. Results with mutants F673E, E837A and N833W confirmed that compound did not show any binding to the protein compared with the wild type in a BLI assay. VPC-13566 demonstrated strong anti-proliferative activity against LNCaP and Enzalutamide-resistant prostate cancer cell lines (MR49F) whereas it did not affect the growth of AR independent PC3 cell line. It also reduced prostate specific antigen (PSA) in both LNCaP and MR49F and reduced expression of AR target genes, PSA and TMPRSS2. These findings suggest that VPC-13566 exhibits AR BF3 specific mechanism of action. Furthermore, VPC-13566 significantly reduced AR-dependent growth of tumors and PSA levels in LNCaP and MR49F xenograft models. Based on these outcomes, it can be anticipated that such drug prototypes will lay a foundation for the development of alternative or supplementary small-molecule therapies capable of combating PCa even in its drug resistant forms. Because the emergence of castration resistance is the lethal end stage of the disease, we anticipate that the proposed research will eventually have a substantial impact on patient survival. Citation Format: Ravi Shashi Nayana Munuganti, Mohamed DH Hassona, Eric Leblanc, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. Targeting binding function-3 site on the androgen receptor to treat Enzalutamide-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3654. doi:10.1158/1538-7445.AM2015-3654

The androgen receptor (AR) is a hormone-activated transcription factor implicated in the development and progression of prostate cancer. The AR contains an N-terminal domain (NTD), followed by DNA binding (DBD) and ligand-binding (LBD) domains. Upon binding of androgens to the LBD, the AR moves into the nucleus where it interacts with target genes via the conserved AR-DBD. Prostate cancer treatment involves the use of small-molecules to block androgen production or to compete with androgens for binding to the AR-LBD. Drug-resistance occurs when LBD mutations render small-molecule treatment ineffective or when constitutively active AR splice variants, lacking the LBD, become overexpressed. We recently identified a surface pocket on the AR-DBD that may serve as an alternative drug-target site. The DBD-specific, small molecule compounds presented here block transcriptional activity of full-length and splice variant AR forms across multiple cell lines and show no cross-reactivity with related steroid nuclear receptors. The inhibition is lost when residues in the binding pocket are mutated or when the AR-DBD is replaced with a different DNA binding domain. Biophysical techniques were used to explore the interaction of these compounds with the purified, recombinant DBD protein. Gene expression analysis demonstrated the down-regulation of AR target genes following compound treatment, without any obvious expression signature associated with toxicity. The compounds affected DNA binding in vitro and also blocked interactions of the AR with chromatin inside the nucleus. Finally, the lead compound suppressed the growth of LNCaP tumor xenografts and was accompanied by down-regulation of AR target genes, such as PSA. These results suggest a mechanism whereby our developed inhibitory compounds interfere with DNA binding by the AR, potentially leading to effective treatment of advanced, castration-resistant prostate cancers. Citation Format: Kush Dalal, Mani Roshan-Moniri, Aishwariya Sharma, Huifang Li, Fuqiang Ban, Mohamed D. Hassona, Michael Hsing, Kriti Singh, Eric LeBlanc, Scott Dehm, Emma Tomlinson Guns, Artem Cherkasov, Paul S. Rennie. Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2015-1778

The human androgen receptor (AR) represents a well-established drug target for prostate cancer treatment. All clinically used antiandrogens, such as Bicalutamide and Enzalutamide, possess similar chemical structures and bind to the AR in the androgen binding site (ABS). These AR antagonists are initially effective, but resistance invariably developed in the castration-resistant prostate cancer (CRPC). Even in late-stage patients, the AR activity is still persistent in the progression of the disease. Thus, there is a continuing need for novel chemical classes of AR antagonists that could overcome the drug resistance. In this study, we performed a virtual screening against the AR ABS, and identified novel AR antagonists with chemical structures completely different from existing antiandrogens. A 10-(4-Hydroxybenzylidene)anthracen-9(10H)-one compound was discovered that not only effectively inhibits AR transcription and strongly displaces androgen in the ABS, but induces AR degradation in prostate cancer cells. Starting from the initial hit compound, a series of 10-benzylidene-10H-anthracen-9-ones were synthesized and evaluated by in vitro assays. A close analogue with enhanced potency was identified as a lead compound, which demonstrated strong androgen displacement potency, effective AR transcriptional inhibition, and a profound ability to cause degradation of AR. Notably, it exhibited significant activity against MDV3100-resistant prostate cancer cells. This lead compound was evaluated in both non-castrated and castration-resistant LNCaP xenograft models, and demonstrated significant effect on inhibiting tumor growth and reducing prostate specific antigen (PSA). This series of compounds were predicted to adopt a different binding mode from current antiandrogens, which may circumvent the resistance rendered by identified gain-of-function mutations. Further development of this series of AR antagonists may have great therapeutic potential in CRPC. Citation Format: Huifang Li, Mohamed DH Hassona, Nathan A. Lack, Peter Axerio-Cilies, Eric Leblanc, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. Discovery of a novel series of androgen receptor antagonists with potential therapeutic applications in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2014-1632

In the development and progression of prostate cancer, the androgen receptor (AR) plays a critical role and its hormone/ligand-binding site is the primary therapeutic target for all antiandrogens (eg Bicalutamide, MDV3100) currently used to treat advanced, metastatic forms of this disease. While treatment with these AR inhibitors initially suppresses prostate tumor growth, resistance to these drugs invariably emerges. Accordingly, there exists a clinical need for the development of new AR antagonists with different chemical structures and different mechanisms of action for targeting and inhibiting AR transactivation. To this end, we have focused on a surface pocket on the AR called Binding Function 3 (BF3). Applying iterative in silico screening to millions of chemicals in the ZINC database, coupled with biological screening for AR binding and activity, we identified several compounds that can bind directly to the BF3 pocket and inhibit AR transactivation. Cell-based screening assays for inhibition of a fluorescent ARE-reporter and PSA expression, together with Biolayer Interferometry analysis for measuring binding to the AR, revealed several chemically distinct BF-3 binders that showed significant IC50 inhibitions of the AR in the nano-molar to low micro-molar range. Validation for binding to the BF3 site was performed by x-ray crystallography. Furthermore, none of these compounds were able to displace DHT from the hormone binding site even at concentrations more than 10-fold higher than their IC50 values. In addition to wild type LNCaP prostate cancer cells, these BF3 binders effectively inhibited cell proliferation (MTS assay) and PSA expression in derived MDV3100-resistant prostate cancer cell lines, but had no effect on cell viability of AR-negative prostate cancer cell lines. In summary, we have identified an entirely new class of anti-androgens that bind to the BF3 surface site of the AR and inhibit its transactivation by a mechanism which does not involve binding to its hormone/ligand binding site and thereby bypasses the treatment resistance seen with conventional antiandrogens. (Supported by funds from the PCF, Canada Safeway, and CIHR). Citation Format: Ravi Munuganti, Fuqiang Ban, Huifang Li, Eric LeBlanc, Peter Axerio, Kate Frewin, Emma Tomlinson Guns, Artem Cherkasov, Paul S. Rennie. New potent inhibitors of the androgen receptor that target its BF3 surface binding site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2013-1070