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Mercury is a significant volcanic volatile species from effusive and explosive activity1. Understanding its emission to the atmosphere from volcanic activity, aids our understanding of the global mercury cycle and its environmental impacts. Sedimentary and ice core records can be archives of these mercury enrichments2,3.The Laki 1783-84 AD fissure eruption in Iceland was significant due to its voluminous outpouring of basaltic lava, copious sulphur emissions and widespread environmental effects locally and across the Northern Hemisphere4,5. Extreme weather events were recorded in Europe and North America, owing to a veil of sulphur dioxide that remained at the tropopause for over a year5. Due to the phreatomagmatic and thus explosive nature of Laki, a significant eruption plume was produced4. As such, cryptotephra shards have been located at distal locations from Iceland including ice cores in Svalbard and Greenland6,7 and in an Irish woodland peat at Brackloon Wood, Co. Mayo8. There is evidence to suggest significant heavy metal emission to the atmosphere during the Laki eruption, however these records are currently restricted to Greenland ice cores9. Previous heavy metal findings linked to Laki do not include mercury, despite its significance as a volcanic volatile, and a potentially environmentally damaging heavy metal. Therefore, to expand our knowledge of the Laki 1783-84 AD eruption plume, its associated emissions, and environmental consequences we returned to the woodland peat site in Brackloon Wood, Co. Mayo, Ireland.Analysis of a 50 cm peat core using an AMA 254 mercury analyser was combined with a novel technique to find tephra using BSE (back scattered electron) imaging and geochemical discrimination using SEM-EDX (scanning electron microscopy-energy dispersive x-ray). The Laki tephra is successfully located using this method and coincides with a visible enrichment in mercury relative to background concentrations and organic matter. An age-depth model developed using the tephra layer and two radiocarbon dates indicate a strong likelihood that such enrichments are a product of volcanic emission. Such a finding can expand our understanding of heavy metal deposition during Laki 1783-84 AD away from the poles and to our knowledge, demonstrates the first direct exploration of mercury enrichment in distal peat for this eruption. As a secondary test of volcanic volatile enrichment, trace element analysis of the same bulk peat will be conducted to explore enrichments in other volcanic volatiles such as sulphur, cadmium, lead, copper and zinc. 1. Pyle, D. M. & Mather, T. A. Atmos. Environ. 37, 5115–5124 (2003).2. Schuster, P. F. et al.,. Environ. Sci. Technol. 36, 2303–2310 (2002).3. Roos-Barraclough, F. et al.,. Earth Planet. Sci. Lett. 202, 435–451 (2002).4. Thordarson, T. & Self, S. Bull. Volcanol. 55, 233–263 (1993).5. Thordarson, T. & Self, S., J. Geophys. Res 108, 4011 (2003).6. Kekonen, T. et al., Polar Res. 24, 33–40 (2005).7. Wei, L. et al., Geophys. Res. Lett. 35, L16501 (2008).8. Reilly, E. & Mitchell, F. J., Holocene 25, 241–252 (2015).9. Hong, S. et al., Earth Planet. Sci. Lett. 144, 605–610 (1996).

PDF file, 162K, AR mRNA levels are unaffected upon treatment with VPC-3022 and VPC-3033. Total RNA was extracted after treatment of LNCaP and MDV3100-resistant cells with VPC-3022 and VPC-3033 for 24 hours at the indicated concentrations in the presence of 0.1 nM R1881. Quantitative RT-PCR analysis of AR expression was performed and normalized to 18S rRNA levels. Error bars indicate standard deviation.

PDF file, 122K.

PDF file, 2136K, (A) Effect of VPC-3022 on AR activity in HeLa-AR cells in the presence of cyclohexamide. HeLa-AR cells were treated with serially-diluted concentrations of VPC-3022 (0 - 100 ?M) with or without 10 ?M of cyclohexamide for 8 hours, and then lysed with passive lysis buffer. (B) AR degradation caused by VPC-3031 and VPC-3041 in HeLa-AR cells. HeLa-AR cells were treated by VPC-3031 and VPC-3041 at different concentrations in the presence of 0.1 nM R1881.

PDF file, 1976K, (A) Inhibition of in vitro polymerization of tubulin at 37 {degree sign}C by various concentrations of VPC-3033; turbidity was recorded at 360 nM.The steady-state tubulin assembly level in the absence of inhibitor was set at 100%. (B) IC50 values were determined by sigmoidal fitting of the plot of the steady state levels of tubulin assembly (taken after 45 min) aginst drug concentration and represent the concentration for 50% inhibition of the maximum tubulin polymerization level.

PDF file, 3480K, The binding pose of DHT (A) and VPC-3022 with AR by molecular docking (B) and binding pose of VPC-3022 with AR after 10 ns molecular dynamics (C); RMSD values of the protein backbone, protein-ligand, ligand in the 10 ns molecular dynamics for AR-DHT (D) and AR-VPC-3022 (E).

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.

PDF file, 109K, Inhibition of VPC-3022 against wild-type AR in HeLa-AR cells. HeLa-AR cells transfected with ARR3tk-luciferase reporter were treated with VPC-3022 for 24 hours in various concentrations in the presence of 0.1 nM R1881. Error bars indicate standard deviation.

The persistence of Archaean cratons for >2.5Ga was aided by thick, mechanically strong, and cool lithospheric mantle keels up to 250km deep. It is widely accepted that the cratonic mantle, dominated by depleted harzburgite, lherzolite and dunite, was formed by extensive melt extraction from originally fertile mantle peridotite. Models seeking to explain the formation of deep cratonic mantle in the garnet and diamond stability fields, initially sought to answer how such rocks could form in-situ at high temperatures and pressures and envisaged large-scale thermochemical plume upwellings. More recently, mineralogical and geochemical observations, namely the high Cr content of garnet and low whole rock HREE concentrations in cratonic harzburgites, have led to the conclusion that the deep cratonic mantle couldn’t have originally melted in the garnet stability field. Mechanical stacking of shallowly depleted oceanic lithosphere was therefore proposed to have thickened the depleted lithosphere cratonic roots. In this process, the spinel facies minerals are envisaged to transform into the garnet stability field.Here we present the first results of combined thermodynamic and geochemical modelling at temperatures high enough to reconcile the very refractory residues. We found that the requirement for initially shallow melting is no longer supported. Deep (150-250km), ultra-hot (>1800°C), incremental melting can produce the mineralogical and geochemical signatures of depleted cratonic harzburgites. The modelling also implies a link between areas of extreme depletion in the deep lithospheric mantle and the genesis of Earth’s hottest lavas (Al-enriched komatiite) by re-melting depleted harzburgite. Diamond inclusion minerals have a well-documented skew to the most refractory compositions found in cratonic peridotite. We propose that these ultra-depleted, highly reducing regions of the lithospheric root possess the highest diamond formation and preservation potential.

Category: Midfoot/Forefoot; Trauma; Other Introduction/Purpose: Metatarsal fractures account for over 35% of all foot fractures, and of these 68% specifically involve the fifth metatarsal [1],[2]. Subgroups of fractures affecting the fifth metatarsal base may be at higher risk of nonunion and therefore benefit from early surgical fixation, but traditional predictive models focus on the location of the fracture and little else. In this study, we aimed to determine predictive factors associated with non-union of fifth metatarsal fractures to assist surgeons and patients, alike, in identifying those at higher risk of nonunion. Methods: A retrospective machine learning-based analysis of 1,000 patients, >=18 y/o, diagnosed with a fifth metatarsal fracture at three tertiary medical centers was conducted. The fifth metatarsal base fracture was confirmed radiographically. We gathered imaging and narrative data including demographics (age, height, weight, BMI, gender, race, smoking habits, activity level), medications, chronic conditions, and fracture status (fracture zone, displacement, treatment method, healing status, and healing time). Non-union was described as failing to heal within 180 days of initial injury [3]. A machine learning analysis together with Pearson's correlation test and T-test were utilized where applicable. Five imputation methods were used to fill in missing datapoints. PResults: Overall, this cohort of patients demonstrated a non-union rate of 22.4%. When divided by fracture zone, Zone 2 fractures results in a statistically significant increased rate of delayed union (17.2%) and non-union (8.6%), when compared to Zone 1 (10.8% and 5.8%, respectively) and Zone 3 (9.7%, 2.3%). Analysis of correlation between demographics data and union rates found no correlation with age, gender, race, or BMI. Our machine learning algorithm outcomes showed a significant correlation between nonunion and seven chronic diseases: diabetes, thyroid disease, hypertension, gastroesophageal reflux disease (GERD), irritable bowel syndrome (IBS) obstructive sleep apnea (OSA), and glaucoma. In terms of medications, significant correlation with nonunion was demonstrated with the use of levothyroxine, lisinopril, aspirin, steroids, and acetaminophen. Conclusion: Zone 2 fifth metatarsal base fractures have a significantly higher rate of nonunion as compared to other zones. Comorbid conditions including diabetes, thyroid disease, OSA, and glaucoma as well as medications, may also play a role, though their mechanism or correlative precipitants are yet to be determined. Although our results demonstrated correlation, causality can only be assessed using studies with limited confounding factors, such as clinical trials. Our outcomes suggest that physicians should pay attention not only to the location of the fracture, but also past medical history and medication use when making treatment decisions and discussing prognosis with patients.

Emma Tomlinson MA VetMB MRCVS, Tomlinson Equine outlines what owners need to consider before breeding from their mares

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.

// Valentina R. Minciacchi 1 , Sungyong You 1 , Cristiana Spinelli 1 , Samantha Morley 2 , Mandana Zandian 1 , Paul-Joseph Aspuria 3 , Lorenzo Cavallini 1,4 , Chiara Ciardiello 1,5 , Mariana Reis Sobreiro 1 , Matteo Morello 1 , Geetanjali Kharmate 6 , Su Chul Jang 7 , Dae-Kyum Kim 7 , Elham Hosseini-Beheshti 6 , Emma Tomlinson Guns 6 , Martin Gleave 6 , Yong Song Gho 7 , Suresh Mathivanan 8 , Wei Yang 1 , Michael R. Freeman 1,2 and Dolores Di Vizio 1,2 1 Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA 2 The Urological Diseases Research Center, Boston Children’s Hospital, Boston, MA, Department of Surgery, Harvard Medical School, Boston, MA, USA 3 Women’s Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA 4 Department of Experimental and Clinical Biomedical Science, University of Florence, Florence, Italy 5 Experimental Pharmacology Unit, Department of Research, IRCCS-Istituto Nazionale Tumori G. Pascale, Naples, Italy 6 Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, BC, Canada 7 Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea 8 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia Correspondence to: Dolores Di Vizio, email: // Keywords : Extracellular Vesicles, SILAC Proteomics, Cancer metabolism, Tumor progression, Amoeboid blebbing Received : January 05, 2015 Accepted : February 22, 2015 Published : March 14, 2015 Abstract Large oncosomes (LO) are atypically large (1-10µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep TM ) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5 th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

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.

Ureteral stents are fraught with problems. A conditioning film attaches to the stent surface within hours of implantation; however, differences between stent types and their role in promoting encrustation and bacterial adhesion and colonization remain to be elucidated. The present work shows that the most common components do not differ between stent types or patients with the same indwelling stent, and contain components that may drive stent encrustation. Furthermore, unlike what was previously thought, the presence of a conditioning film does not increase bacterial adhesion and colonization of stents by uropathogens. Genitourinary cytokeratins are implicated in playing a significant role in conditioning film formation. Overall, stent biomaterial design to date has been unsuccessful in discovering an ideal coating to prevent encrustation and bacterial adhesion. This current study elucidates a more global understanding of urinary conditioning film components. It also supports specific focus on the importance of physical characteristics of the stent and how they can prevent encrustation and bacterial adhesion.

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 (...)

Post-translational protein modification by the covalent conjugation of ubiquitin, originally implicated as a signal for proteolytic degradation by 26S proteasome, has now been realised to play important roles in the regulation of almost all biological processes in eukaryotes. In order to understand these processes in greater detail there is a requirement for techniques that can purify mixtures of ubiquitin-conjugated proteins, as a prerequisite to their identification and characterisation. Here we review the methods that have been applied to the bulk purification of ubiquitinated proteins and discuss their applications in proteomic analyses of the 'ubiquitome'.

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.

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.

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.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.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.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.

BACKGROUND Docetaxel is one of the few chemotherapeutic drugs that are considered highly effective when used to treat prostate cancer patients that have relapsed and/or metastatic disease, it is therefore reasonable to expect further improvements in treatment outcomes when it is combined with other therapeutic agents active in prostate cancer. This study assesses the combination of well tolerated and orally bioavailable formulations of ginsenoside Rh2 or its aglycone aPPD with docetaxel. METHODS The in vitro activity of Rh2, aPPD, and docetaxel was determined in four prostate cancer cell lines: PC-3, LNCaP, DU145, and C4-2. Combinations of Rh2 or aPPD with docetaxel were assessed using the constant ratio combination design. Combination Indices (CI) and Dose Reduction Indices (DRI) were subsequently estimated using Calcusyn™. In vivo efficacy studies and Immunohistochemical analyses (PC-3 model) were also evaluated. RESULTS In PC-3, DU145 and C4-2 prostate cancer cells combinations of Rh2 or aPPD with docetaxel were predominantly additive or synergistic. Combinations of Rh2 + docetaxel and aPPD + docetaxel caused established PC-3 tumors to regress from their initial size by 15% and 27%, respectively. Tumor cell proliferation rate (measured by Ki-67 positive cells) was significantly lower for combinations of Rh2 + docetaxel and aPPD + docetaxel, compared to animals treated with docetaxel alone. CONCLUSIONS Rh2 and aPPD can be combined with docetaxel to yield additive or synergistic activity in vitro and in vivo. Pending further assessment of toxicity and pharmacodynamic behavior, this study supports testing of combinations of ginsenoside Rh2 or its aglycone aPPD with docetaxel in a clinical setting. Prostate 70: 1437–1447, 2010. © 2010 Wiley-Liss, Inc.

This study assesses the pharmacokinetics, biodistribution and efficacy of ginsenoside Rh2 as a single agent administered in a novel oral dosage formulation.A novel oral dosage formulation of Rh2 has been described. Rh2 levels in blood and tissues following administration to nu/nu nude mice were determined by high performance liquid chromatography tandem mass spectroscopy. Efficacy was determined in an established PC-3 human prostate cancer model.Rh2 administered at a dose of 120 mg/kg exhibited a peak plasma concentration of 19.0 +/- 2.0 microg/ml. Rh2 levels were measurable in prostate and tumor tissues, with as much as 0.3% of the administered dose being detected in tumors. This formulation exhibited no measurable toxicity as judged by weight loss or changes in serum levels of aspartate aminotransferase, alanine aminotransferase, and creatinine. This dose engendered a significant delay in PC-3 tumor growth, an increase in apoptotic index, and a decrease in tumor cell proliferation.Rh2 is a stable compound that can be formulated for oral gavage. Pharmacokinetics studies demonstrate its ability to be absorbed following oral administration. Future studies will assess the pharmacokinetics of Rh2 when administered in combination with docetaxel.

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

e16120 Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. Leech saliva contains a large number of peptides and pr...

The androgen receptor (AR) is one of the most validated therapeutic targets in prostate cancer (PCa). Conventional anti-androgens lose effectiveness as cancer therapeutics because anti-androgen resistance usually develops after long term treatment. The challenge is that the current therapeutics bind to the same site of the AR (hormone binding pocket) and act via the same mode, to which the receptor has already developed effective resistance mechanisms. Hence, there is a pressing need for novel therapeutics that inhibit the AR through novel, alternative modes of action. Recent studies have identified a novel binding pocket on the surface of AR called binding function 3 (BF3) that is important for the AR transcriptional activity. In order to identify compounds that specifically bind to BF3 site and inhibit the AR, we conducted a systematic in silico screen (that included large-scale docking, in-site rescoring, and consensus voting procedures) followed by experimental validation of the identified hit molecules. As a result, we have discovered a novel chemical series of indoles as lead BF3 inhibitors. One of the most potent inhibitors identified, VPC-13163, demonstrated an IC50 of 0.31µM in AR eGFP transcriptional assay. Confirming it as a true BF3 binder, VPC-13163 neither displaced the co-activator from an alternative coactivator binding site, activation function 2 site, nor androgen from the hormone binding pocket. Additionally, the Biolayer Interferometry assay detected direct reversible interactions between the AR ligand binding domain and the inhibitor. VPC-13163 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 inhibits prostate specific antigen (PSA) in both LNCaP and MR49F and reduces expression of AR target genes, PSA and TMPRSS2. These findings suggest that VPC-13163 exhibits AR BF3 specific mechanism of action. Furthermore, VPC-13163 reduces AR-dependent growth of xenograft tumors in vivo. 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, Fuqiang Ban, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. The development of anti-androgens with a novel mechanism of action for treatment of 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 2508. doi:10.1158/1538-7445.AM2014-2508

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