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San Sebastián de los Reyes, Spain

Cufi S.,Catalan Institute of Nanoscience and Nanotechnology | Cufi S.,Girona Biomedical Research Institute | Vazquez-Martin A.,Catalan Institute of Nanoscience and Nanotechnology | Vazquez-Martin A.,Girona Biomedical Research Institute | And 15 more authors.
Cell Cycle | Year: 2012

We have tested the hypothesis that the antidiabetic biguanide metformin can be used to manipulate the threshold for stress-induced senescence (SIS), thus accelerating the onset of cancer-protective cellular senescence in response to oncogenic stimuli. Using senescence-prone murine embryonic fibroblasts (MEFs), we assessed whether metformin treatment modified the senescence phenotype that is activated in response to DNA damaging inducers. Metformin significantly enhanced the number of MEFs entering a senescent stage in response to doxorubicin, an anthracycline that induces cell senescence by activating DNA damage signaling pathways (e.g., ATM/ATR) in a reactive oxygen species (ROS)-dependent manner. Using WI-38 and BJ-1 human diploid fibroblasts (HDFs), we explored whether metformin supplementation throughout their entire replicative lifespan may promote the early appearance of the biomarkers of replicative senescence. Chronic metformin significantly reduced HDFs' lifespan by accelerating both the loss of replicative potential and the acquisition of replicative senescence-related biomarkers (e.g., enlarged and flattened cell shapes, loss of arrayed arrangement, accumulation of intracellular and extracellular debris and SA-β-gal-positive staining). Metformin functioned as a bona fide stressful agent, inducing monotonic, dose-dependent, SIS-like responses in BJ-1 HDFs, which are highly resistant to ROS-induced premature senescence. Metformin-induced SIS in BJ-1 fibroblasts was accompanied by the striking activation of several microRNAs belonging to the miR-200s family (miR-200a, miR-141 and miR429) and miR-205, thus mimicking a recently described ability of ROS to chemosensitize cancer cells by specifically upregulating anti-EMT (epithelial-to-mesenchymal transition) miR-200s. Because the unlimited proliferative potential of stem cells results from their metabolic refractoriness to SIS, we finally tested if metformin treatment could circumvent the stress (e.g., ROS)-resistant phenotype of induced pluripotent stem cells (iPSCs). Metformin treatment drastically reduced both the number and the size of iPSC colonies and notably diminished the staining of the pluripotency marker alkaline phosphatase. Our current findings, altogether, reveal for the first time that metformin can efficiently lower the threshold for SIS to generate an "stressed" cell phenotype that becomes pre-sensitized to oncogenic-like stimuli, including DNA damaging, proliferative and/or stemness inducers. © 2012 Landes Bioscience. Source

Menendez J.A.,Catalan Institute of Nanoscience and Nanotechnology | Menendez J.A.,Girona Biomedical Research Institute | Vellon L.,Cell Reprogramming Unit | Oliveras-Ferraros C.,Catalan Institute of Nanoscience and Nanotechnology | And 5 more authors.
Cell Cycle | Year: 2011

Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced pluripotent stem cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of mitochondrial function and in the accelerated onset of the glycolytic metabolism that is required to fuel reprogramming. By critically exploring how mTOR-regulated senescence, bioenergetic infrastructure and autophagy can actively drive the reprogramming of somatic cells to pluripotency, we define a metabolic roadmap that may be helpful for designing pharmacological and behavioral interventions to prevent or retard the dysfunction/exhaustion of aging stem cell populations. © 2011 Landes Bioscience. Source

Vazquez-Martin A.,University of Franca | Vazquez-Martin A.,Girona Biomedical Research Institute | Fernandez-Arroyo S.,University of Granada | Cufi S.,University of Franca | And 10 more authors.
Rejuvenation Research | Year: 2012

The epithelial-to-mesenchymal transition (EMT) genetic program is a molecular convergence point in the life-threatening progression of organ fibrosis and cancer toward organ failure and metastasis, respectively. Here, we employed the EMT process as a functional screen for testing crude natural extracts for accelerated drug development in fibrosis and cancer. Because extra virgin olive oil (EVOO) (i.e., the juice derived from the first cold pressing of the olives without any further refining process) naturally contains high levels of phenolic compounds associated with the health benefits derived from consuming an EVOO-rich Mediterranean diet, we have tested the ability of an EVOO-derived crude phenolic extract to regulate fibrogenic and oncogenic EMT in vitro. High-performance liquid chromatography (HPLC) coupled to time-of-flight (TOF) mass spectrometry assays revealed that the EVOO phenolic extract was mainly composed (∼70%) of two members of the secoiridoid family of complex polyphenols, namely oleuropein aglycone-the bitter principle of olives-and its derivative decarboxymethyl oleuropein aglycone. EVOO secoiridoids efficiently prevented loss of proteins associated with polarized epithelial phenotype (i.e., E-cadherin) as well as de novo synthesis of proteins associated with mesenchymal migratory morphology of transitioning cells (i.e., vimentin). The ability of EVOO to impede transforming growth factor-β (TGF-β)-induced disintegration of E-cadherin-mediated cell-cell contacts apparently occurred as a consequence of the ability of EVOO phenolics to prevent the upregulation of SMAD4-a critical mediator of TGF-β signaling-and of the SMAD transcriptional cofactor SNAIL2 (Slug)-a well-recognized epithelial repressor. Indeed, EVOO phenolics efficiently prevented crucial TGF-β-induced EMT transcriptional events, including upregulation of SNAI2, TCF4, VIM (Vimentin), FN (fibronectin), and SERPINE1 genes. While awaiting a better mechanistic understanding of how EVOO phenolics molecularly shut down the EMT differentiation process, it seems reasonable to suggest that nontoxic Oleaceae secoiridoids certainly merit to be considered for aging studies and, perhaps, for ulterior design of more pharmacologically active second-generation anti-EMT molecules. © 2012, Mary Ann Liebert, Inc. Source

Menendez J.A.,Catalan Institute of Nanoscience and Nanotechnology | Menendez J.A.,Girona Biomedical Research Institute | Cufi S.,Catalan Institute of Nanoscience and Nanotechnology | Cufi S.,Girona Biomedical Research Institute | And 7 more authors.
Aging | Year: 2011

By activating the ataxia telangiectasia mutated (ATM)-mediated DNA Damage Response (DDR), the AMPK agonist metformin might sensitize cells against further damage, thus mimicking the precancerous stimulus that induces an intrinsic barrier against carcinogenesis. Herein, we present the new hypothesis that metformin might function as a tissue sweeper of pre-malignant cells before they gain stem cell/tumor initiating properties. Because enhanced glycolysis (the Warburg effect) plays a causal role in the gain of stem-like properties of tumor-initiating cells by protecting them from the pro-senescent effects of mitochondrial respiration-induced oxidative stress, metformin's ability to disrupt the glycolytic metabotype may generate a cellular phenotype that is metabolically protected against immortalization. The bioenergetic crisis imposed by metformin, which may involve enhanced mitochondrial biogenesis and oxidative stress, can lower the threshold for cellular senescence by pre-activating an ATM-dependent pseudo-DDR. This allows an accelerated onset of cellular senescence in response to additional oncogenic stresses. By pushing cancer cells to use oxidative phosphorylation instead of glycolysis, metformin can rescue cell surface major histocompatibility complex class I (MHC-I) expression that is downregulated by oncogenic transformation, a crucial adaptation of tumor cells to avoid the adaptive immune response by cytotoxic T-lymphocytes (CTLs). Aside from restoration of tumor immunosurveillance at the cell-autonomous level, metformin can activate a senescence-associated secretory phenotype (SASP) to reinforce senescence growth arrest, which might trigger an immune-mediated clearance of the senescent cells in a non-cell-autonomous manner. By diminishing the probability of escape from the senescence anti-tumor barrier, the net effect of metformin should be a significant decrease in the accumulation of dysfunctional, pre-malignant cells in tissues, including those with the ability to initiate tumors. As life-long or late-life removal of senescent cells has been shown to prevent or delay the onset or progression of age-related disorders, the tissue sweeper function of metformin may inhibit the malignant/metastatic progression of premalignant/senescent tumor cells and increase the human lifespan. © Menendez et al. Source

Cufi S.,Catalan Institute of Oncology Girona ICO Girona | Cufi S.,Girona Biomedical Research Institute IDIBGI | Vazquez-Martin A.,Catalan Institute of Oncology Girona ICO Girona | Vazquez-Martin A.,Girona Biomedical Research Institute IDIBGI | And 6 more authors.
Cell Cycle | Year: 2011

The molecular mechanisms used by breast cancer stem cells (BCSCs) to survive and/or maintain their undifferentiated CD44 +CD24 -/low mesenchymal-like antigenic state remain largely unexplored. Autophagy, a key homeostatic process of cytoplasmic degradation and recycling evolved to respond to stress conditions, might be causally fundamental in the biology of BCSCs. Stable and specific knockdown of autophagy-regulatory genes by lentiviral-delivered small hairpin (sh) RNA drastically decreased the number of JIMT-1 epithelial BC cells bearing CD44 +CD24 -/low cell-surface antigens from ∼75% in parental and control (-) shRNA-transduced cells to 26% and 7% in ATG8/LC3 shRNA- and ATG12 shRNA-transduced cells, respectively. Autophagy inhibition notably enhanced transcriptional activation of CD24 gene, potentiating the epithelial-like phenotype of CD44 +CD24 + cells vs. the mesenchymal CD44 +CD24 -/low progeny. EMT-focused Real Time RT-PCR profiling revealed that genetic ablation of autophagy transcriptionally repressed the gene coding for the mesenchymal filament vimentin (VIM). shRNA-driven silencing of the ATG12 gene and disabling the final step in the autophagy pathway by the antimalarial drug chloroquine both prevented TGFβ1-induced accumulation of vimentin in JIMT-1 cells. Knockdown of autophagy-specific genes was sufficient also to increase by up to 11-times the number of CD24 + cells in MDA-MB-231 cells, a BC model of mesenchymal origin that is virtually composed of CD44 +CD24 -/low cells. Chloroquine treatment augmented the number of CD24 + cells and concomitantly reduced constitutive overexpression of vimentin in MDA-MB-231 cells. This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of BCSCs. © 2011 Landes Bioscience. Source

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