1,721,197 research outputs found
Doxorubicin and α-Mangostin oppositely affect luminal breast cancer cell stemness evaluated by a new retinaldehyde-dependent ALDH assay in MCF-7 tumor spheroids
Full text linkAccording to cancer stem cell theory, only a limited number of self-renewing and cloning cells are responsible for tumor relapse after a period of remittance. The aim of the present study was to investigate the effects of Doxorubicin and α-Mangostin, two antiproliferative drugs, on both tumor bulk and stem cells in multicellular tumor spheroids originated from the luminal MCF-7 breast cancer cell line. A new and original fluorimetric assay was used to selectively measure the activity of the retinaldehyde-dependent isoenzymes of aldehyde dehydrogenase (RALDH), which are markers of a subpopulation of breast cancer stem cells. The administration of 5 μg/ml (12.2 μM) α-Mangostin for 48 h provoked: i) a marked disaggregation of the spheroids, leading to a doubling of their volume (p < 0.01), ii) a 40 % decrease in cell viability (p < 0.01), evaluated by the acid phosphatase assay, and iii) a reduction by more than 90 % of RALDH activity. By contrast, Doxorubicin given for 48 h in the range of 0.1–40 μM did not significantly reduce cell viability and caused only a modest modification of the spheroid morphology. Moreover, 40 μM Doxorubicin increased RALDH activity 2.5-fold compared to the untreated sample. When the two drugs were administered together using 5 μg/ml α-Mangostin, the IC50 of Doxorubicin referred to cell viability decreased six-fold and the RALDH activity was further reduced. In conclusion, the combined administration of Doxorubicin and α-Mangostin provoked a significant cytotoxicity and a remarkable inhibition of RALDH activity in MCF-7 tumor spheroids, suggesting that these drugs could be effective in reducing cell stemness in luminal breast cancer
Cancer stem cells and mesenchymal stem cells in the hypoxic tumor niche: Two different targets for one only drug
No full textPutative cancer stem cells (CSCs) reside in a hypoxic microenvironment where mesenchymal stem cells (MSCs) are also present. In this niche MSCs seem to promote the generation of CSCs and sustain tumor progression. Therefore, it may assume clinical relevance to produce a drug which kills not only CSCs but also MSCs. We hypothesized that bifunctional nanoparticles, loaded with a HIF-1 alpha inhibitor and conjugated with an aptamer targeting a common receptor of CSCs and MSCs, may fulfill this strategy. The nanoparticle should ensure that: (1) the conveyed drug is less susceptible to degradation, (2) the common receptor of CSCs and MSCs is recognized by a superselective aptamer, and (3) receptor-mediated internalization is the main process to enter target cells. Small RNA or DNA aptamers represent an advantage over antibodies because do not cause immune reactions, are better internalized into the target cell, are more resistant to degradation, their cost of production are lower, and the purity of the oligonucleotide ligand is extremely elevated. Concerning the drugs to be delivered, we suggest to employ those exerting an anti-HIF-1 alpha activity because they should be harmful for hypoxic CSCs and MCSs in their tumor niche but provide very limited toxicity, or even none, to well-oxygenated normal cells. Corresponding experimental approaches to perform pre-clinical studies and verify this hypothesis are also addressed
Nitric oxide regulates multiple functions and fate of adult progenitor and stem cells
No full textNitric oxide is an endogenous gas which exerts autocrine/paracrine actions by different signaling pathways and/or direct interactions with intracellular compounds and structures. Several processes are regulated by nitric oxide in stem cells including self-renewal, viability, migration, proliferation, and differentiation. The modulation of cell functions depends on its concentrations because opposite effects can be observed when low and high levels of nitric oxide are compared. Here, the responses to nitric oxide of adult stem/progenitor cells which are often used in regenerative medicine, including mesenchymal stem cells, hematopoietic stem cells, neural stem cells, endothelial progenitor cells, satellite cells, and fibro-adipogenic precursor cells, are reviewed. Therapeutic strategies which employ drugs releasing nitric oxide or modulating nitric oxide intracellular pathways are suggested to perform new ex vivo preconditioning or in vivo treatments suitable for stem/progenitor cell therapy and tissue engineering applications
Complete disaggregation of MCF-7-derived breast tumour spheroids with very low concentrations of α-mangostin loaded in CD44 thioaptamer-tagged nanoparticles
Full text linkBackground: α-Mangostin (αMG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free αMG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, αMG 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 αMG 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 μg/ml final concentrations of αMG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment. Results: The NPs without thioaptamer and conveying 0.1 μg/ml αMG 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 μg/ml αMG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 – 72 h. By contrast, only concentrations over 1.0 μg/ml of free αMG 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 αMG-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 αMG-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 αMG 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
Autologous adipose tissue transplantation: regenerative potential of lipoaspirates obtained by different processing systems
No full textEffects od alpha-mangostin on cell growth, viability and metabolic activity evaluated in a hypoxic model of breast cancer spheroids
No full textIntroduction. 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: “Distruzione selettiva di cellule sta-minali tumorali ipossiche mediante uso di nanoparticelle bifunzionali”
Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes
No full textHigh tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure. Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications
Effects of alpha-mangostin on viability, growth and cohesion of multicellular spheroids derived from human breast cancer cell lines
Full text linkBackground: alpha-Mangostin (a-MG) is extracted from Garcinia mangostana Linn and exerts antiproliferative activities. Although several researches on a-MG were performed using cell monolayers, the in vitro pharmacological effects on 3D cancer models have never been investigated. Aim of the present study was to find new anticancer properties of a-MG by evaluating the changes that this compound provokes in multicellular tumour spheroids (MCTSs). Methods: MCTSs were generated from MDA-MB-231 and MCF-7 breast tumour cell lines and then treated with 0.1-30 μg/ml a-MG for 24 and 48 h. MCTS size, density, and cell migration were determined by software elaboration of phase contrast images captured by a digital camera. Cell viability was evaluated by resazurin and acid phosphatase assays, while cell apoptosis was assessed by a fluorescent assay of caspase activity. The distribution of living cells inside MCTSs was shown by live/dead fluorescence staining. Results: A dose-dependent decrease in cell viability was obtained by treating MDA-MB-231 spheroids with a-MG for 48 h (IC50 = 0.70-1.25 μg/ml). A significant reduction in spheroid volume, paralleled by its increased compactness, was observed only at concentration of 30 μg/ml, but not with lower doses of a-MG. By contrast, a-MG in the range of 5-15 μg/ml increased the size of MCTSs due to a parallel reduction in cell aggregation. The same window of concentrations was also able to stimulate cell apoptosis in a dose-dependent manner. Bimodal volumetric effects were also obtained by treating the spheroids generated from the MCF-7 cells with 0.1·30 μg/ml a-MG for 48 h. Finally, doses higher than 5 μg/ml caused a progressive impairment in cell migration from the edge of MDA-MB-231 MCTSs. Conclusion: After exposure at doses of a-MG just above IC50, MDA-MB-231 spheroids showed a significant reduction in cell adhesion that did not stimulate cell migration but, on the contrary, blunted cell motility. These findings suggest a novel anticancer feature of a-MG that could be taken into consideration to improve conventional drug penetration into the tumour bulk
Lull pgm system: A suitable technique to improve the regenerative potential of autologous fat grafting
No full textAutologous fat grafting and methods of purification of harvested tissue have become one of the most current themes in regenerative medicine. The aim of this study was to evaluate the in vitro regenerative potential of abdomen lipoaspirates subjected to a combined washing-decantation purifying procedure, the Lull pgm System (Lull). Blood cells and stromal-vascular fraction (SVF) cells contained in the aspirates were investigated and compared with those obtained through more conventional fat-processing methods, that is, the decantation and Coleman's centrifugation techniques. The lowest number of erythrocytes, which are proinflammatory cells, was observed in the Lull samples, corresponding to about 50% of those isolated by decantation and centrifugation. The highest amount of SVF cells were isolated from the Lull samples whose number of colony forming units, representative of the amount of adipose-derived stem cells (ADSCs), was about fourfold and sixfold higher than in the decantation and centrifugation samples, respectively. Adipocyte and osteoblast commitment of SVF cells obtained from all the three procedures also confirmed that the subpopulation of ADSCs was actively represented in the processed aspirates. Moreover, the growth rate of the SVF cells was more accentuated in the samples obtained from decantation and Lull than centrifugation. In conclusion, Lull seems to be the best processing technique for adipose tissue graft with respect to decantation and centrifugation, because it clears more efficiently the fat from proinflammatory blood cells and provides the greatest number of proliferating SFV cells and ADSCs
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