NMR-Based Metabolomic Approach Tracks Potential Serum Biomarkers of Disease Progression in Patients with Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia associated with alterations in carbohydrate, lipid, and protein metabolism. The prognosis of T2DM patients is highly dependent on the development of complications, and therefore the identification of biomarkers of T2DM progression, with minimally invasive techniques, is a huge need. In the present study, we applied a H-1-Nuclear Magnetic Resonance (H-1-NMR)-based metabolomic approach coupled with multivariate data analysis to identify serum metabolite profiles associated with T2DM development and progression. To perform this, we compared the serum metabolome of non-diabetic subjects, treatment-naive non-complicated T2DM patients, and T2DM patients with complications in insulin monotherapy. Our analysis revealed a significant reduction of alanine, glutamine, glutamate, leucine, lysine, methionine, tyrosine, and phenylalanine in T2DM patients with respect to non-diabetic subjects. Moreover, isoleucine, leucine, lysine, tyrosine, and valine levels distinguished complicated patients from patients without complications. Overall, the metabolic pathway analysis suggested that branched-chain amino acid (BCAA) metabolism is significantly compromised in T2DM patients with complications, while perturbation in the metabolism of gluconeogenic amino acids other than BCAAs characterizes both early and advanced T2DM stages. In conclusion, we identified a metabolic serum signature associated with T2DM stages. These data could be integrated with clinical characteristics to build a composite T2DM/complications risk score to be validated in a prospective cohort

Complete disaggregation of MCF-7-derived breast tumour spheroids with very low concentrations of \u3b1-mangostin loaded in CD44 thioaptamer-tagged nanoparticles

Background: \u3b1-Mangostin (\u3b1MG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free \u3b1MG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, \u3b1MG was encapsulated into lipidic nanoparticles (NPs), conjugated or not to a CD44 thioaptamer, and the anticancer action evaluated against MCF-7 breast MCTSs. Methods: NPs containing \u3b1MG were formulated with a core of polylactic-co-glycolyc acid. Some of them were decorated with a CD44 thioaptamer using as catalysts 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Both size and density of MCF-7-derived MCTSs were monitored during 72 h of treatment with NPs carrying 0.1, 0.5 and 1.0 \ub5g/ml final concentrations of \u3b1MG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment. Results: The NPs without thioaptamer and conveying 0.1 \ub5g/ml \u3b1MG caused a significant dissociation of the MCTSs grown in gelatine after 24 h of treatment (p < 0.01). The most significant disaggregation of MCTSs was obtained using NPs carrying 0.5 \ub5g/ml \u3b1MG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 \u2013 72 h. By contrast, only concentrations over 1.0 \ub5g/ml of free \u3b1MG were able to provoke a damage to MCTSs, consisting in a substantial reduction in their size (p < 0.05). Since the MCTS dissociation induced by \u3b1MG-loaded NPs occurred only in the presence of Matrigel or gelatine, an impairment of cell contacts to collagen fibres was likely responsible of this effect. Finally, the treatment of MCTSs with \u3b1MG-loaded NPs that were conjugated to the CD44 thioaptamer caused a similar decrease in density but a lower expansion of the spheroid, suggesting that a significant number of cells were died or arrested in cycle. Conclusion: Very low concentrations of \u3b1MG delivered by lipidic NPs are sufficient to provoke a substantial disaggregation of MCF-7 MCTSs that involves cell-to-collagen contacts. Similarly, the treatment of MCTSs with NPs conjugated to a CD44 thioaptamer leads to MCTS dissociation but through a more damaging action that causes also a reduction in cell number

Effects od alpha-mangostin on cell growth, viability and metabolic activity evaluated in a hypoxic model of breast cancer spheroids

Introduction. Mangosteen xantones exert anti-tumorigenic effects, however their toxicity was never investi-gated in a 3D model mimicking the hypoxic regions far from the vasculature that cannot be easily reached by drugs. Here we tested a-mangostin toxicity against breast cancer spheroids at doses useful to kill cancer cells under 2D conditions. Methods. Cancer spheroids of radius > 250 um were produced by seeding MCF7 breast cancer cells at the density of 1.5x103 cells per well onto 96-well round-bottomed plates and culturing them for 3 days. a-Man-gostin (5, 10, 20 ug/ml) was then added for 4 days and the spheroid radius measured by Image-J. Resazurin reduction assay allowed the evaluation of redox metabolism while accutase-induced cell dissociation was performed to count living cells that resisted to drug toxicity. Results. Spheroid cells became dishomogeneous and showed irregular frayed edges after drug treatment. These looser contacts were presumably responsible of the increased radius observed with the lowest dose of a-mangostin vs. control (507 vs. 415 um, respectively). By contrast, the radius decreased at higher doses (384 and 307 um for 10 and 20 ug/ml, respectively). The effects of 10 and 20 ug/ml a-mangostin also correlated with a 75% and 90% reduction in cell number, respectively. Redox metabolism was 90% reduced after admi-nistration of the lowest dose and decrease by 99% with 10 or 20 ug/ml a-mangostin vs. control.Conclusions. Although the most relevant effects of a-mangostin on MCF7-derived spheroids was related to the decrease of the redox status, a few viable cells were dissociated indicating that they were not affected by drug toxicity suggesting that a very low cell component inside big-sized spheroids can resist to a-mangostin and that might generate cancer stem cells.Research support: Fondazione del Monte di Bologna e Ravenna. Project: \u201cDistruzione selettiva di cellule sta-minali tumorali ipossiche mediante uso di nanoparticelle bifunzionali\u201d

Molecular mechanisms of ischemic preconditioning and postconditioning as putative therapeutic targets to reduce tumor survival and malignancy

In tumors intermittent hypoxia has been reported to be more representative than normoxia or continuous exposure to low oxygen concentrations. Intermittent hypoxia is thought to increase tumor resistance against both anti-cancer therapy and the sustained ischemia that randomly occurs because of the dynamic nature of tumor vasculature. Here, we hypothesize that the molecular mechanisms underlying intermittent hypoxia in tumor cells share some triggers, modulators, and end-effectors of the intermittent episodes of ischemia and reperfusion that characterize ischemic preconditioning and postconditioning. These are among the most effective maneuvers protecting cells from ischemia-reperfusion injury. If this hypothesis were confirmed, several well-investigated molecular mediators of pre/post-conditioning could be explored as therapeutic targets against tumor malignancy. For examples, drugs that completely block the cardioprotection induced by ischemic preconditioning, such as mitochondrial potassium ATP channel inhibitors or mitochondrial permeability transition pore openers, could be extraordinarily efficient in counteracting the adaptations of tumor cells and cancer stem cells to intermittent hypoxia. As a consequence, this strategy should be effective in blunting tumor capacity to progress toward malignancy and survive in ischemic conditions

Comparison between stem cells harvested from wet and dry lipoaspirates

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p<0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 7 10(3) SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of ASC in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies

An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells

At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes

An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand-alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly-reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells 'trained' over 1\u2009week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures

Anti-TNF-\u3b1 treatment modulates SASP and SASP-related microRNAs in endothelial cells and in circulating angiogenic cells

Endothelial cell senescence is characterized by acquisition of senescence-associated secretory phenotype (SASP), able to promote inflammaging and cancer progression. Emerging evidence suggest that preventing SASP development could help to slow the rate of aging and the progression of age-related diseases, including cancer. Aim of this study was to evaluate whether and how adalimumab, a monoclonal antibody directed against tumor necrosis factor-\u3b1 (TNF-\u3b1), a major SASP component, can prevent the SASP. A three-pronged approach has been adopted to assess the if adalimumab is able to: i) modulate a panel of classic and novel senescence- and SASP-associated markers (interleukin [IL]-6, senescence associated-\u3b2-galactosidase, p16/Ink4a, plasminogen activator inhibitor 1, endothelial nitric oxide synthase, miR-146a-5p/Irak1 and miR-126-3p/Spred1) in human umbilical vein endothelial cells (HUVECs); ii) reduce the paracrine effects of senescent HUVECs' secretome on MCF-7 breast cancer cells, through wound healing and mammosphere assay; and iii) exert significant decrease of miR-146a-5p and increase of miR-126-3p in circulating angiogenic cells (CACs) from psoriasis patients receiving adalimumab in monotherapy. TNF-\u3b1 blockade associated with adalimumab induced significant reduction in released IL-6 and significant increase in eNOS and miR-126-3p expression levels in long-term HUVEC cultures. A significant reduction in miR-146a-5p expression levels both in long-term HUVEC cultures and in CACs isolated from psoriasis patients was also evident. Interestingly, conditioned medium from senescent HUVECs treated with adalimumab was less consistent than medium from untreated cells in inducing migration- and mammosphere- promoting effects on MCF-7 cells. Our findings suggest that adalimumab can induce epigenetic modifications in cells undergoing senescence, thus contributing to the attenuation of SASP tumor-promoting effects

Intervisionauti

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