Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.

Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.

Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.

Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33.

A multi-center study of ACE and the risk of late-onset Alzheimer's disease

A key pathological feature of late-onset Alzheimer's disease (LOAD) is the abnormal extracellular accumulation of the amyloid-ß (Aß) peptide. Thus, altered Aß degradation could be a major contributor to the development of LOAD. Variants in the gene encoding the Aß-degrading enzyme, angiotensin-1 converting enzyme (ACE) therefore represent plausible candidates for association with LOAD pathology and risk. Following Alzgene meta-analyses of all published case-control studies, the ACE variants rs4291 and rs1800764 showed significant association with LOAD risk. Furthermore ACE haplotypes are associated with both plasma ACE levels and LOAD risk. We tested three ACE variants (rs4291, rs4343, and rs1800764) for association with LOAD in ten Caucasian case-control populations (n = 8,212). No association was found using multiple logistic models (all p > 0.09). We found no population heterogeneity (all p > 0.38) or evidence for association with LOAD risk following meta-analysis of the ten populations for rs4343 (OR = 1.00), rs4291 (OR = 0.97), or rs1800764 (OR = 0.99). Although we found no haplotypic association in our complete dataset (p = 0.51), a significant global haplotypic p-value was observed in one population (p = 0.007) due to an association of the H3 haplotype (OR = 0.72, p = 0.02) and a trend towards an association of H4 (OR = 1.38, p = 0.09) and H7 (OR = 2.07, p = 0.08) although these did not survive Bonferroni correction. Previously reported associations of ACE variants with LOAD will be diminished following this study. At best, ACE variants have modest effect sizes, which are likely part of a complex interaction between genetic, phenotypic and pharmacological effects that would be undetected in traditional case-control studies.

Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cells

Despite recent therapeutic advances, the response rates to chemotherapy for patients with metastatic colon cancer remain at approximately 50% with the fluoropyrimidine, 5-fluorouracil (5-FU), continuing to serve as the foundation chemotherapeutic agent for the treatment of this disease. Previous studies have demonstrated that overexpression of thymidylate synthase (TS) is a key determinant of resistance to 5-FU-based chemotherapy. Therefore, there is a significant need to develop alternative therapeutic strategies to overcome TS-mediated resistance. In this study, we demonstrate that the histone deacetylase inhibitors (HDACi) vorinostat and LBH589 significantly downregulate TS gene expression in a panel of colon cancer cell lines. Downregulation of TS was independent of p53, p21 and HDAC2 expression and was achievable in vivo as demonstrated by mouse xenograft models. We provide evidence that HDACi treatment leads to a potent transcriptional repression of the TS gene. Combination of the fluoropyrimidines 5-FU or FUdR with both vorinostat and LBH589 enhanced cell cycle arrest and growth inhibition. Importantly, the downstream effects of TS inhibition were significantly enhanced by this combination including the inhibition of acute TS induction and the enhanced accumulation of the cytotoxic nucleotide intermediate dUTP. These data demonstrate that HDACi repress TS expression at the level of transcription and provides the first evidence suggesting a direct mechanistic link between TS downregulation and the synergistic interaction observed between HDACi and 5-FU. This study provides rationale for the continued clinical evaluation of HDACi in combination with 5-FU-based therapies as a strategy to overcome TS-mediated resistance.

Development of user-friendly, high throughput screening for ligands and inhibitors of carbohydrate modifying enzymes

This paper describes the impact of cloud computing and the use of GPUs on the performance of Autodock and Gromacs respectively. Cloud computing was applicable to reducing the ‘‘tail’’ seen in running Autodock on desktop grids and the GPU version of Gromacs showed significant improvement over the CPU version. A large (200,000 compounds) library of small molecules, seven sialic acid analogues of the putative substrate and 8000 sugar molecules were converted into pdbqt format and used to interrogate the Trichomonas vaginalis neuraminidase using Autodock Vina. Good binding energy was noted for some of the small molecules (~-9 kcal/mol), but the sugars bound with affinity of less than -7.6 kcal/mol. The screening of the sugar library resulted in a ‘‘top hit’’ with a-2,3-sialyllacto-N-fucopentaose III, a derivative of the sialyl Lewisx structure and a known substrate of the enzyme. Indeed in the top 100 hits 8 were related to this structure. A comparison of Autodock Vina and Autodock 4.2 was made for the high affinity small molecules and in some cases the results were superimposable whereas in others, the match was less good. The validation of this work will require extensive ‘‘wet lab’’ work to determine the utility of the workflow in the prediction of potential enzyme inhibitors.

The investigation of the reduction mechanism of the 5a-reductase enzyme of Penicillium decumbens /

The 5a-reductase of Penicillium decumbens ATCC 10436 was used as a model for the mammalian enzyme to investigate the mechanism of reduction of testosterone to 5adihydrotestosterone . The purpose of this study was to search for specific 5a-reductase inhibitors which antagonize prostate cancer . In a whole-cell biotransformation mode, this organism reduced testosterone (1) to 5a-dihydrosteroids (8) and 5aandrostane- 3, 17-dione (9) in yields of 28% and 37% respectively. Control experiments have shown that 5aandrostane- 3, 17-dione (9) can be produced from the corresponding alcohol (8) in a subsequent reaction separate from that catalysed by the 5a-reductase enzyme . Androst-4- ene-3, 17-dione (2) is reduced to give only (9) with a recovery of 80% The stereochemistry of the reduction was determined by 500 MHz ^H NMR analysis of the products resulting from the deuterium labelled substrates. The results were obtained by an analysis of the NOE difference spectra, double-quantum filtered phase sensitive COSY 2-D spectra, and ^^c-Ir 2-D shift correlation spectra of deuterium labelled products. According to the unambiguous assignment of the signals due to H-4a and H-4Ii in 5a-dihydro steroids, the NMR data show clearly that addition of hydrogen to the 4{5)K bond has occurred in a trans manner at positions 413 and 5a. To Study the reduction mechanism of this enzyme, several substrates were prepared as following; 3-methyleneandrost-4-en- 17fi-ol(3), androst-4-en-17i5-ol(5) , androst-4-en-3ii, 17fi-diol (6) and 4, 5ii-epoxyandrostane-3, 17-dione (7) . Results suggest that this enzyme system requires an oxygen atom at the 3-position of the steroid in order to bind the substrate. Furthermore, the mechanism of this 5a-reductase may proceed via direct addition of hydrogen at the 4,5 position without involvement of a carbonyl group as an intermediate.

Amélioration des résultats cliniques en chirurgie cardiaque

Des éléments contributifs à plusieurs facettes de la chirurgie cardiaque ont été étudiés dans la présente thèse. Le premier manuscrit adresse la problématique de l’accident cérébro-vasculaire (ACV) post-opératoire. Nous avons analysé de façon rétrospective la médication prise en pré-opératoire de 6813 patients nécessitant une chirurgie de revascularisation coronarienne. Le but étant d’établir si la présence d’une médication précise (aspirine, inhibiteur de l’enzyme de conversion de l’angiotensine, statine, bêta-bloqueur) peut agir en pré-opératoire pour diminuer le risque d’ACV. En analyse multivariée, la combinaison de la prise de bêta-bloqueurs avec une statine a produit un ratio de cote de 0,37, suggérant un effet protecteur très important. Dans le deuxième manuscrit, je présente une étude ciblant les patients avec insuffisance mitrale ischémique modérée. Trente et un patients furent randomisés entre un traitement par pontages seuls vs pontages et annuloplastie mitrale restrictive. L’insuffisance mitrale a disparu en post-opératoire immédiat en présence de l’annuloplastie alors qu’aucun effet immédiat de la revascularisation coronarienne n’était noté sur l’insuffisance mitrale. Un an suivant la chirurgie, une insuffisance mitrale légère est réapparue chez le groupe ayant subi l’annuloplastie alors que les patients du groupe pontages seuls ont remodelé leur ventricule gauche et diminué l’importance de leur insuffisance mitrale au même niveau que le groupe annuloplastie. Aucun des marqueurs d’évolution clinique, tant au niveau symptomatique qu’au niveau de la survie ne diffère entre les groupes. La troisième étude est un suivi sur 20 ans des patients ayant eu des remplacements valvulaires mitraux ou aortiques avec une prothèse mécanique Carbomedics. Cette étude démontre une excellente survie avec un taux de complications valvulaires hémorragiques, thrombotiques, thrombo-emboliques, et d’endocardite favorable comparé aux autres types de prothèse et une absence de bris mécanique.

Production of novel ACE inhibitory peptides from beta-lactoglobulin using Protease N Amano

Potent angiotensin l-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of beta-lactoglobulin (beta Lg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to beta Lg f(36-42) and had an IC50 value of 8 mu m, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine beta Lg reported so far. (C) 2008 Elsevier Ltd. All rights reserved.

Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

Background and Purpose: Calcitonin gene‐related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor‐like receptor (CLR) and receptor activity‐modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin‐converting enzyme‐1 (ECE‐1). However, it is not known if ECE‐1 regulates the resensitization of CGRP‐induced responses in functional arterial tissue. Experimental Approach: CLR, ECE‐1a‐d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA‐SMCs) and mesenteric arteries was analyzed by RT‐PCR and by immunofluorescence and confocal microscopy. CGRP‐induced signaling in cells was examined by measuring cAMP production and ERK activation. CGRP‐induced relaxation of arteries was measured by isometric wire myography. ECE‐1 was inhibited using the specific inhibitor, SM‐19712. Key Results: RMA‐SMCs and arteries contained mRNA for CLR, ECE‐1a‐d and RAMP1. ECE‐1 was present in early endosomes of RMA‐SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium‐independent relaxation of arteries. ECE‐1 inhibition had no effect on initial CGRP‐induced responses but reduced cAMP generation in RMA‐SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions and Implications: ECE‐1 regulates the resensitization of responses to CGRP in RMA‐SMCs and mesenteric arteries. CGRP‐induced relaxation does not involve endothelium‐derived pathways. This is the first report of ECE‐1 regulating CGRP responses in SMCs and arteries. ECE‐1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC50 value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Structure-activity relationships for a class of selective inhibitors of the major cysteine protease from Trypanosoma cruzi

Chagas` disease is a parasitic infection widely distributed throughout Latin America, with devastating consequences in terms of human morbidity and mortality. Cruzain, the major cysteine protease from Trypanosoma cruzi, is an attractive target for antitrypanosomal chemotherapy. In the present work, classical two-dimensional quantitative structure-activity relationships (2D QSAR) and hologram QSAR (HQSAR) studies were performed on a training set of 45 thiosemicarbazone and semicarbazone derivatives as inhibitors of T. cruzi cruzain. Significant statistical models (HQSAR, q2=0.75 and r2=0.96; classical QSAR, q2=0.72 and r2=0.83) were obtained, indicating their consistency for untested compounds. The models were then used to evaluate an external test set containing 10 compounds which were not included in the training set, and the predicted values were in good agreement with the experimental results (HQSAR, [image omitted]=0.95; classical QSAR, [image omitted]=0.91), indicating the existence of complementary between the two ligand-based drug design techniques.

Acridone alkaloids as potent inhibitors of cathepsin V

Cathepsin V is a lysosomal cysteine peptidase highly expressed in thymus, testis and corneal epithelium. Eleven acridone alkaloids were isolated from Swinglea glutinosa (Bl.) Merr. (Rutaceae), with eight of them being identified as potent and reversible inhibitors of cathepsin V (IC(50) values ranging from 1.2 to 3.9 mu M). Detailed mechanistic characterization of the effects of these compounds on the cathepsin V-catalyzed reaction showed clear competitive inhibition with respect to substrate, with dissociation constants (K(i)) in the low micromolar range (2, K(i) = 1.2 mu M; 6, K(i) = 1.0 mu M; 7, K(i) = 0.2 mu M; and 11, K(i) = 1.7 mu M). Molecular modeling studies provided important insight into the structural basis for binding affinity and enzyme inhibition. Experimental and computational approaches, including biological evaluation, mode of action assessment and modeling studies were successfully employed in the discovery of a small series of acridone alkaloid derivatives as competitive inhibitors of catV. The most potent inhibitor (7) has a K(i) value of 200 nM. (C) 2011 Elsevier Ltd. All rights reserved.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

Curso evolutivo e fatores de progressão da nefropatia diabética em pacientes com diabete melito tipo 2

A nefropatia diabética (ND) é uma complicação microvascular freqüente, que acomete cerca de 40% dos indivíduos com diabete melito (DM). A ND associa-se a significativo aumento de morte por doença cardiovascular. É a principal causa de insuficiência renal terminal em países desenvolvidos e em desenvolvimento, representando, dessa forma, um custo elevado para o sistema de saúde. Os fatores de risco para o desenvolvimento e a progressão da ND mais definidos na literatura são a hiperglicemia e a hipertensão arterial sistêmica. Outros fatores descritos são o fumo, a dislipidemia, o tipo e a quantidade de proteína ingerida na dieta e a presença da retinopatia diabética. Alguns parâmetros de função renal também têm sido estudados como fatores de risco, tais como a excreção urinária de albumina (EUA) normal-alta e a taxa de filtração glomerular excessivamente elevada ou reduzida. Alguns genes candidatos têm sido postulados como risco, mas sem um marcador definitivo. O diagnóstico da ND é estabelecido pela presença de microalbuminúria (nefropatia incipiente: EUA 20-199 μg/min) e macroalbuminúria (nefropatia clínica: EUA ≥ 200 μg/min). À medida que progride a ND, aumenta mais a chance de o paciente morrer de cardiopatia isquêmica. Quando o paciente evolui com perda de função renal, há necessidade de terapia de substituição renal e, em diálise, a mortalidade dos pacientes com DM é muito mais significativa do que nos não-diabéticos, com predomínio das causas cardiovasculares. A progressão nos diferentes estágios da ND não é, no entanto, inexorável. Há estudos de intervenção que demonstram a possibilidade de prevenção e de retardo na evolução da ND principalmente com o uso dos inibidores da enzima conversora da angiotensina, dos bloqueadores da angiotensina II e do tratamento intensivo da hipertensão arterial. Os pacientes podem entrar em remissão, ou até mesmo regredir de estágio. A importância da detecção precoce e da compreensão do curso clínico da ND tem ganhado cada vez mais ênfase, porque a doença renal do DM é a principal causa de diálise no mundo e está associada ao progressivo aumento de morte por causas cardiovasculares.

Effects and toxicity of Eugenia punicifolia extracts in streptozotocin-diabetic rats

Extracts and decoctions of Eugenia jambolana Lam., Eugenia uniflora L., and Eugenia punicifolia (Humb., Bonpl. & Kunt) DC. are used in traditional medicine to treat diabetes mellitus. Although there have been reports that Eugenia jambolana and Eugenia uniflora have antidiabetic effects, no study has yet been made on Eugenia punicifolia . We investigated the effects of aqueous, butanol, and methanol extracts of Eugenia punicifolia leaves administered by gavage to streptozotocin-diabetic rats for 26 to 29 days. Body weight, food and fluid intake, urine volume, and urinary glucose and urea were evaluated every 7 days. At the end of the experiment, we measured serum cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides and bilirubin, hepatic glycogen and serum marker-enzymes (alanine and aspartate aminotransferases, alkaline phosphatase, gamma-glutamyltransferase, L-lactate dehydrogenase, creatine kinase, alpha-amylase, and angiotensin I converting enzyme). We found that in rats treated with the aqueous extracts, food and liquid intake, urinary volume, and body weight were all reduced, while for rats treated with the methanol extract, not only were liquid intake, urinary volume and body weight reduced, but urinary glucose and urea also decreased. Rats treated with the butanol extract showed no significant alterations in any of the parameters measured. Chronic treatment with extracts had no effect on the marker enzymes nor on serum bilirubin levels. The results indicate that aqueous extracts of Eugenia punicifolia leaves produced an anorexic effect and that methanol extracts had a beneficial effect on the diabetic state by improving carbohydrate and protein metabolism without provoking hepatobiliary, microvascular, muscular, or pancreatic toxic effects.