Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetateinduced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.This work was supported by the Fundação para a Ciência e Tecnologia (FCT) research project PTDC/BIA-BCM/69448/2006 and FCT PhD grants for SA (SFRH/BD/64695/2009) and CO (SFRH/BD/77449/2011). This work was also supported by FEDER through POFC—COMPETE, and by national funds from FCT through the project PEst-C/BIA/UI4050/2011

Surface plasmon resonance imaging of cells and surface-associated fibronectin

<p>Abstract</p> <p>Background</p> <p>A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells.</p> <p>Results</p> <p>Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm²of protein was deposited by cells in 24 h.</p> <p>Conclusion</p> <p>SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.</p

SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds

Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism

Diagnostic approach and management of cow's milk protein allergy in infants and children

OBJECTIVES:: This position paper provides recommendations for the diagnosis and management of suspected cow's milk protein allergy (CMPA) in Europe. It presents a practical approach with a diagnostic algorithm and is based on recently published evidence based guidelines on CMPA. DIAGNOSIS:: If CMPA is suspected by history and examination, strict allergen avoidance is initiated. In certain circumstances (e.g. a clear history of immediate symptoms or a life threatening reaction with a positive test for cow's milk protein (CMP) specific IgE) the diagnosis can be made without a milk challenge. In all other circumstances, a controlled oral food challenge (open or blind) under medical supervision is required to confirm or exclude the diagnosis CMPA. TREATMENT:: In breast-fed infants, the mother should start a strict CMP-free diet. Non-breast fed infants with confirmed CMPA should receive an extensively hydrolyzed protein based formula with proven efficacy in appropriate clinical trials; amino acids based formulae are reserved for certain situations. Soy protein formula, if tolerated, is an option beyond 6 months of age. Nutritional counseling and regular monitoring of growth is mandatory in all age groups requiring CMP exclusion. RE-EVALUATION:: Patients should be re-evaluated every 6-12 months to assess whether they have developed tolerance to CMP. This is achieved in >75% by three years and >90% by six years of age. Inappropriate or overly long dietary eliminations should be avoided. Such restrictions may impair the quality of life of both child and family, induce improper growth, and also incur unnecessary healthcare costs

Outcome measures for clinical trials in paediatric IBD: an evidence-based, expert-driven practical statement paper of the paediatric ECCO committee.

OBJECTIVE: Although paediatric-onset IBD is becoming more common, few medications have a registered paediatric indication. There are multiple hurdles to performing clinical trials in children, emphasising the importance of choosing an appropriate outcome measure, which can facilitate enrolment, and thereby also drug approval. The aim of this consensus statement is to highlight paediatric specific issues and key factors critical for the optimal conduct of paediatric IBD trials. DESIGN: The Paediatric European Crohn's and Colitis Organisation (ECCO) committee has established an international expert panel to determine the best outcome measures in paediatric IBD, following a literature search and a modified Delphi process. All recommendations were endorsed by at least 80% agreement. RESULTS: Recognising the importance of mucosal healing (MH), the panel defined steroid-free MH as primary outcome measure for all drugs of new category with one or two postintervention endoscopies per trial (at 8-12 weeks and/or 54 weeks). Since endoscopic evaluation is a barrier for recruitment in children, trials with medications already shown to induce MH in children or adults, could use paediatric-specific disease activity scores as primary outcome, including a modified Paediatric Crohn's Disease Activity Index in Crohn's disease and the Paediatric Ulcerative Colitis Activity Index in UC. Secondary outcomes should include safety issues, MR enterography-based damage and inflammatory scores (in Crohn's disease), faecal calprotectin, quality of life scales, and a patient-reported outcome. CONCLUSIONS: It is crucial to perform paediatric trials early in the development of new drugs in order to reduce off-label use of IBD medication in children. The thoughtful choice of feasible and standardised outcome measures can help move us towards this goal

Myo5b knockout mice as a model of microvillus inclusion disease

Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches