A bioavailable cathepsin S nitrile inhibitor abrogates tumor development

BACKGROUND: Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models. METHODS: Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K(i) values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors. RESULTS: We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis. CONCLUSIONS: In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12943-016-0513-7) contains supplementary material, which is available to authorized users

Atrial Natriuretic Peptide: Binding and Cyclic GMP Response in Cultured Vascular SmoothMuscle Cells From Spontaneously Hypertensive Rats

Atrial natriuretic peptide (ANP) is vasodilatory and natriuretic, but whereas increased plasma ANP levels occur in spontaneously hypertensive rats, their elevated vascular resistance suggests inappropriate target tissue responsiveness to ANP. This study examines ANP-receptor binding properties (at 25 °C and 4°C) in cultured vascular aortic smooth muscle cells from spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) rats. [I125]-human ANP saturation (0.0625-12.0 nmol) profiles were analyzed using nonlinear regression (LIGAND). Vascular smooth muscle cells from WKY possessed both high affinity (KD1 0.3 nmol; R1 33 fmol/105 cells) and low affinity (KD2 15 nmol; R2 400 fmol/105 cells) binding sites for ANP. In contrast, for smooth muscle cells from SHR, two receptor forms could not be resolved using identical analytical protocols. Parameter estimates at 25 °C and 4°C were not different for either SHR or WKY. The number of receptors for SHR (Bmax ~ 100 fmol/105 cells) was lower than the total number of receptors for WKY (high plus low affinity ~ 430 fmol/105 cells). The intermediary KD value (~1.0 nmol) for ANP binding in SHR suggests an ANPreceptor interconversion from high affinity to low affinity in smooth muscle cells from SHR. Competition-binding experiments also revealed a decreased affinity for ANP in SHR-derived smooth muscle cells. The cyclic GMP response (intracellular accumulation and extracellular levels) was decreased in SHR smooth muscle cells compared to WKY, although this difference was evident only after prolonged (one hour) stimulation with ANP. Our data indicate a reduced sustained vascular responsiveness to ANP in hypertension. Am J Hypertens 1989; 2:32-3

Small-particle Inhaled Corticosteroid as First-line or Step-up Controller Therapy in Childhood Asthma

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Peer reviewedPublisher PD

Increased Oxidative Stress in Injured and Ill Elite International Olympic Rowers

Identifying strategies that reduce the risk of illness and injury is an objective of sports science and medicine teams. No studies have examined the relationship between oxidative stress (OS) and illness or injury in international athletes undergoing periods of intensified training and competition. Purpose: We aimed to identify relationships between illness, injury and OS. Methods: A longitudinal, observational study of elite male rowers (n=10) was conducted over 18-weeks leading into World Championships. Following a recovery day and a 12-hour fast, hydroperoxides (FORT) and total anti-oxidant capacity (FORD) were measured in venous blood, with the ratio calculated as the oxidative stress index (OSI). At all study time points, athletes were independently dichotomized as ill or not ill, injured or not injured. OS data were compared between groups using independent t-tests. A Cox proportional hazard model was used to assess the association of OS with injury and illness while adjusting for age and body mass index. Results: FORD was lower (p<0.02) and OSI was higher (p<0.001) with illness than without illness. FORT and OSI were higher with injury than without injury (p<0.001). FORD exerts a protective effect on illness with 0.5 mmol•L-1 increase related to a 30.6% illness risk reduction (p=0.014), and OSI exerts a harmful effect on illness risk with a 0.5 unit increase in OSI related to an 11.3% increased risk (p=0.036). Conclusion: OS is increased in injured and ill athletes. Monitoring OS may be advantageous in assessing recovery from, and in reducing injury and illness risk given the association

Cardiovascular risks in smokers treated with nicotine replacement therapy : a historical cohort study

The authors would like to thank Julie von Ziegenweidt, Daina Lim, and Muzammil Ali, who assisted with the analysis. Many thanks to Alison Chisholm for contribution to the study design and critical review of the manuscript, Derek Skinner for preparation of data for analysis, Rosalind Bonomally, and Martina Stagno d’Alcontres for medical writing. The study data were provided by the CPRD without charge (via a Medical Research Council study grant). The analysis was conducted by the Observational and Pragmatic Research Institute Pte Ltd, in collaboration with the Respiratory Effectiveness Group (REG), and funded by the Observational and Pragmatic Research Institute Pte Ltd. Manuscript costs were covered by the REG.Peer reviewedPublisher PD

Performance of database-derived severe exacerbations and asthma control measures in asthma : responsiveness and predictive utility in a UK primary care database with linked questionnaire data

Peer reviewedPublisher PD

Research priorities in advanced heart failure: James Lind alliance priority setting partnership.

OBJECTIVE: To determine research priorities in advanced heart failure (HF) for patients, carers and healthcare professionals. METHODS: Priority setting partnership using the systematic James Lind Alliance method for ranking and setting research priorities. An initial open survey of patients, carers and healthcare professionals identified respondents' questions, which were categorised to produce a list of summary research questions; questions already answered in existing literature were removed. In a second survey of patients, carers and healthcare professionals, respondents ranked the summary research questions in order of priority. The top 25 unanswered research priorities were then considered at a face-to-face workshop using nominal group technique to agree on a 'top 10'. RESULTS: 192 respondents submitted 489 responses each containing one or more research uncertainty. Out-of-scope questions (35) were removed, and collating the responses produced 80 summary questions. Questions already answered in the literature (15) were removed. In the second survey, 65 questions were ranked by 128 respondents. The top 10 priorities were developed at a consensus meeting of stakeholders and included a focus on quality of life, psychological support, the impact on carers, role of the charity sector and managing prognostic uncertainty. Ranked priorities by physicians and patients were remarkably divergent. CONCLUSIONS: Engaging stakeholders in setting research priorities led to a novel set of research questions that might not have otherwise been considered. These priorities can be used by researchers and funders to direct future research towards the areas which matter most to people living with advanced HF

Ion-channel function and cross-species determinants in viral assembly of nonprimate hepacivirus p7

Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified