Alterations in mitochondrial activity and morphology have been demonstrated in human cells and tissues from individuals with Down syndrome (DS), as well as in DS mouse models. An impaired activity of the transcriptional coactivator PGC-1α/PPARGC1A due to the overexpression of chromosome 21 genes, such as NRIP1/RIP140, has emerged as an underlying cause of mitochondrial dysfunction in DS. We tested the hypothesis that the activation of the PGC-1α pathway might indeed reverse this mitochondrial dysfunction. To this end, we investigated the effects of metformin, a PGC-1α-activating drug, on mitochondrial morphology and function in DS foetal fibroblasts. Metformin induced both the expression of PGC-1α and an augmentation of its activity, as demonstrated by the increased expression of target genes, strongly promoting mitochondrial biogenesis. Furthermore, metformin enhanced oxygen consumption, ATP production, and overall mitochondrial activity. Most interestingly, this treatment reversed the fragmentation of mitochondria observed in DS and induced the formation of a mitochondrial network with a branched and elongated tubular morphology. Concomitantly, cristae remodelling occurred and the alterations observed by electron microscopy were significantly reduced. We finally demonstrated that the expression of genes of the fission/fusion machinery, namely OPA1 and MFN2, was reduced in trisomic cells and increased by metformin treatment. These results indicate that metformin promotes the formation of a mitochondrial network and corrects the mitochondrial dysfunction in DS cells. We speculate that alterations in the mitochondrial dynamics can be relevant in the pathogenesis of DS and that metformin can efficiently counteract these alterations, thus exerting protective effects against DS-associated pathologies.
Objective. Stimuli activating vascular smooth muscle cell death can constrain the neointimal response to arterial damage and prevent vascular thickening. Conversely, endothelial cell death increases endothelial dysfunction and thrombosis risk. We investigated the combined effect of atorvastatin and TNF-α on vascular cell death. Keywords: Our study suggests that atorvastatin enhances TNF-α-induced cell death in vascular smooth muscle- but not endothelial – cells; by a cell-context-dependent mechanism, involving PKCβ inhibition
Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.
Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.
