Complete Disaggregation of MCF-7-derived Breast Tumour Spheroids with Very Low Concentrations of α-Mangostin Loaded in CD44 Thioaptamer-tagged Nanoparticles.

Background: 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.

Effects of α-Mangostin on Viability, Growth and Cohesion of Multicellular Spheroids Derived from Human Breast Cancer Cell Lines.

Background: α-Mangostin (αMG) is extracted from Garcinia mangostana Linn and exerts antiproliferative activities. Although several researches on αMG 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 αMG 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 αMG 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 αMG 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 αMG. By contrast, αMG 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 αMG 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 αMG 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 αMG 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.

Autologous 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.

Antiapoptotic and antiautophagic effects of eicosapentaenoic acid in cardiac myoblasts exposed to palmitic acid.

Apoptosis is a programmed cell death that plays a critical role in cell homeostasis. In particular, apoptosis in cardiomyocytes is involved in several cardiovascular diseases including heart failure. Recently autophagy has emerged as an important modulator of programmed cell death pathway. Recent evidence indicates that saturated fatty acids induce cell death through apoptosis and this effect is specific for palmitate. On the other hand, n-3 polyunsaturated fatty acids (PUFAs) have been implicated in the protection against cardiovascular diseases, cardiac ischemic damage and myocardial dysfunction. In the present study we show that n-3 PUFA eicosapentaenoic acid (EPA) treatment to culture medium of H9c2 rat cardiomyoblasts protects cells against palmitate-induced apoptosis, as well as counteracts palmitate-mediated increase of autophagy. Further investigation is required to establish whether the antiautophagic effect of EPA may be involved in its cytoprotective outcome and to explore the underlying biochemical mechanisms through which palmitate and EPA control the fate of cardiac cells.