A Nutraceutical Strategy to Slowing Down the Progression of Cone Death in an Animal Model of Retinitis Pigmentosa

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by progressive degeneration of the visual cells and abnormalities in retinal pigment epithelium, the vision is lost slowly, and the final outcome is total blindness. RP primarily affects rods, but cones can also be affected as a secondary effect. Photoreceptor cell death is usually triggered by apoptosis, however the molecular mechanisms linking the rod degeneration to the secondary cone death are poorly understood. Possible causes of the secondary cone death are oxidative stress and/ or the release of toxic factors from dying rods. The aim of this study is to analyze the effect of nutraceutical molecules with antioxidant properties, on the progression of the disease in an established animal model of RP, and rd10 mice. We show that chronic treatment per os with a flavanone (naringenin) or a flavonol (quercetin) present in citrus fruits, grapes and apples, preserves retinal morphology, and ameliorates functionality. These actions are associated with a significant reduction of stress-oxidative markers, such as the detoxifying enzymes Sod1 and Sod2. In addition, naringenin and quercetin treatment reduces the levels of acrolein staining associated with a reduction of ROS in the cellular environment. The study demonstrates the beneficial effects of naringenin and quercetin, two molecules that possess antioxidant properties, limiting neurodegeneration, and thus preventing cone damage

Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2 h or 20 h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment

The administration of Enterococcus faecium SF68 counteracts compositional shifts in the gut microbiota of diet-induced obese mice

Microorganisms with probiotic properties are eliciting an increasing interest as coadjuvants in the prevention and treatment of obesity through modulation of the gut microbiota. In this study, a probiotic formulation based on Enterococcus faecium SF68 was administered to mice fed with a high-fat diet (HFD) to evaluate its efficacy in reducing body mass gain and in modulating the intestinal bacterial composition. Both stool and ileum samples were collected from untreated and treated mice and absolute abundances of specific taxa constituting the gut microbial consortium were evaluated. SF68 administration significantly reduced the HFD-induced weight gain. In these animals, the microbial gut composition shifted toward an enrichment in microbes positively correlated with mucus thickness, lower inflammation, lower glycemia levels, and SCFA production (i.e., Bifidobacterium, Akkermansia, and Faecalibacterium), as well as a depletion in bacterial phyla having a key role in obesity (i.e., Firmicutes, Proteobacteria). Our results demonstrate the efficacy of E. faecium SF68 in adjusting the composition of the dysbiotic microbiota of HFD-fed animals, thus ameliorating clinical conditions and exerting anti-obesity effects

The Adenosine System at the Crossroads of Intestinal Inflammation and Neoplasia

Adenosine is a purine nucleoside, resulting from the degradation of adenosine triphosphate (ATP). Under adverse conditions, including hypoxia, ischemia, inflammation, or cancer, the extracellular levels of adenosine increase significantly. Once released, adenosine activates cellular signaling pathways through the engagement of the four known G-protein-coupled receptors, adenosine A1 receptor subtype (A1), A2A, A2B, and A3. These receptors, expressed virtually on all immune cells, mitigate all aspects of immune/inflammatory responses. These immunosuppressive effects contribute to blunt the exuberant inflammatory responses, shielding cells, and tissues from an excessive immune response and immune-mediated damage. However, a prolonged persistence of increased adenosine concentrations can be deleterious, participating in the creation of an immunosuppressed niche, ideal for neoplasia onset and development. Based on this evidence, the present review has been conceived to provide a comprehensive and critical overview of the involvement of adenosine system in shaping the molecular mechanisms underlying the enteric chronic inflammation and in promoting the generation of an immunosuppressive niche useful for the colorectal tumorigenesis

Putative biomarkers for malignant pleural mesothelioma suggested by proteomic analysis of cell line secretome

BACKGROUND: Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. MATERIALS AND METHODS: The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. RESULTS: Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiver-operating characteristic analysis predicted a good diagnostic accuracy. CONCLUSION: A panel of the putative biomarkers represents a promising tool for MPM diagnosis

Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene

<div><p>Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR₁ in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR₁ was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR₁ over-expression compared to Met-5A cell line. Thrombin and PAR₁ activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR₁ signaling through G_q and G_12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR₁ signaling through G_i proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR₁ expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR₁ signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR₁ in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.</p></div

Neither β-catenin rescue nor deletion modify cell surface PAR₁ expression.

<p>NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR₁ expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR₁ expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).</p

Thrombin differently induces ERK1/2 activation in Met-5A and NCI-H28 cells.

<p>A, relative intensity of pERK1/2 immunoreactive bands quantified by densitometric scanning. Serum and growth factor starved Met-5A and NCI-H28 cells were incubated in the presence and absence of various thrombin concentrations ranging from 0.01 to 100 nM for 5 min. ERK1/2 activation was then determined using a specific anti-phospho-ERK1/2 antibody. Nitrocellulose membranes were then stripped and reprobed for total ERK1/2. Data (mean ± SEM) are expressed as fold-increase over Ctrl and are the averages of three independent experiments performed in duplicate. The differences in phosphorylated ERK1/2 level between Ctrl (vehicle treated Met-5A or NCI-H28 cells) and thrombin-treated cells were significant (*P≤0.05, **P≤0.01) by one-way ANOVA followed by Bonferroni’s multiple comparison test. B, a representative immunoblot.</p

NCI-H28 cells over-express PAR₁.

<p>A, relative expression levels of PAR₁ mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR₁ protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR₁ expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.</p