Metformin has been proposed to operate as an agonist of SIRT1, a nicotinamide adenine dinucleotide (NAD
)-dependent deacetylase that mimics most of the metabolic responses to calorie restriction. ...Herein, we present an
analysis focusing on the molecular docking and dynamic simulation of the putative interactions between metformin and SIRT1. Using eight different crystal structures of human SIRT1 protein, our computational approach was able to delineate the putative binding modes of metformin to several pockets inside and outside the central deacetylase catalytic domain. First, metformin was predicted to interact with the very same allosteric site occupied by resveratrol and other sirtuin-activating compounds (STATCs) at the amino-terminal activation domain of SIRT1. Second, metformin was predicted to interact with the NAD
binding site in a manner slightly different to that of SIRT1 inhibitors containing an indole ring. Third, metformin was predicted to interact with the C-terminal regulatory segment of SIRT1 bound to the NAD
hydrolysis product ADP-ribose, a "C-pocket"-related mechanism that appears to be essential for mechanism-based activation of SIRT1. Enzymatic assays confirmed that the net biochemical effect of metformin and other biguanides such as a phenformin was to improve the catalytic efficiency of SIRT1 operating in conditions of low NAD
. Forthcoming studies should confirm the mechanistic relevance of our computational insights into how the putative binding modes of metformin to SIRT1 could explain its ability to operate as a direct SIRT1-activating compound. These findings might have important implications for understanding how metformin might confer health benefits
maintenance of SIRT1 activity during the aging process when NAD
levels decline.
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.
Transforming Growth Factor-b (TGFb) is a major driving force of the Epithelial-to-Mesenchymal (EMT) genetic program, which becomes overactive in the pathophysiology of many age-related human ...diseases. TGFb-driven EMT is sufficient to generate migrating cancer stem cells by directly linking the acquisition of cellular motility with the maintenance of tumor-initiating (stemness) capacity. Chronic diseases exhibiting excessive fibrosis can be caused by repeated and sustained infliction of TGFb-driven EMT, which increases collagen and extracellular matrix synthesis. Pharmacological prevention and/or reversal of TGFb-induced EMT may therefore have important clinical applications in the management of cancer metastasis as well as in the prevention and/or treatment of end-state organ failures. Earlier studies from our group have revealed that clinically-relevant concentrations of the biguanide derivative metformin, the most widely used oral agent to lower blood glucose concentration in patients with type 2 diabetes and metabolic syndrome, notably decreased both the self-renewal and the proliferation of trastuzumab-refractory breast cancer stem cell populations. Given that: a.) tumor-initiating cancer stem cells display a significant enrichment in the expression of basal/mesenchymal or myoepithelial markers, including an increased secretion of TGFb; b.) metformin treatment impedes the ontogeny of generating the stem cell phenotype by transcriptionally repressing key drivers of the EMT genetic program (e.g. ZEB1, TWIST1, SNAIL2 Slug, TGFbs), we recently hypothesized that prevention of TGFb-induced EMT might represent a common molecular mechanism underlying the anti-cancer stem cells and anti-fibrotic actions of metformin. Remarkably, metformin exposure not only impedes TGFb-promoted loss of the epithelial marker E-cadherin in MCF-7 breast cancer cells but it prevents further TGF-induced cell scattering and accumulation of the mesenchymal marker vimentin in Madin-Darby canine kidney (MDCK) cells. We now propose that metformin, by weakening the ability of TGFb signaling to fully induce mesenchymal cell states in a variety of pathological processes including fibrosis (e.g. chronic renal disease, non-alcoholic steatohepatitis, heart failure or sclerosis) and malignant progression (and likely by reducing TGFb-regulated inflammation and immune responses -inflamm-aging-), molecularly behaves as a bona fide anti-aging modality.
Autophagy in stem cells Guan, Jun-Lin; Simon, Anna Katharina; Prescott, Mark ...
Autophagy,
06/2013, Volume:
9, Issue:
6
Journal Article
Peer reviewed
Open access
Autophagy is a highly conserved cellular process by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. As a major intracellular degradation and ...recycling pathway, autophagy is crucial for maintaining cellular homeostasis as well as remodeling during normal development, and dysfunctions in autophagy have been associated with a variety of pathologies including cancer, inflammatory bowel disease and neurodegenerative disease. Stem cells are unique in their ability to self-renew and differentiate into various cells in the body, which are important in development, tissue renewal and a range of disease processes. Therefore, it is predicted that autophagy would be crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells given their relatively long life in the organisms. In contrast to the extensive body of knowledge available for somatic cells, the role of autophagy in the maintenance and function of stem cells is only beginning to be revealed as a result of recent studies. Here we provide a comprehensive review of the current understanding of the mechanisms and regulation of autophagy in embryonic stem cells, several tissue stem cells (particularly hematopoietic stem cells), as well as a number of cancer stem cells. We discuss how recent studies of different knockout mice models have defined the roles of various autophagy genes and related pathways in the regulation of the maintenance, expansion and differentiation of various stem cells. We also highlight the many unanswered questions that will help to drive further research at the intersection of autophagy and stem cell biology in the near future.
The expression and activity of Fatty Acid Synthase (FASN; the sole enzyme capable of the reductive de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine ...dinucleotide phosphate -NADPH-) is extremely low in nearly all nonmalignant adult tissues, whereas it is significantly up-regulated or activated in many cancer types, thus creating the potential for a large therapeutic index. Since the pioneering observation that inhibition of FASN activity by the mycotoxin cerulenin preferentially kills cancer cells and retards the growth of tumors in xenografts models, numerous in vitro and in vivo studies have confirmed the potential of FASN as a target for antineoplastic intervention. Other FASN inhibitors such as the cerulenin derivative C75, the beta-lactone orlistat, the green tea polyphenol epigallocatechin-3-gallate (EGCG) and other naturally occurring flavonoids (i.e., luteolin, quercetin, and kaempferol), as well as the antibiotic triclosan, have been identified and have been shown to limit cancer cell growth by inducing apoptotic cell death. Though the exact mode of action of these FASN inhibitors is under discussion, it has been revealed that depletion of end-product fatty acids, toxic intracellular accumulation of supra-physiological concentrations of the FASN substrate malonyl-CoA and/or limited membrane synthesis and/or functioning by altered production of phospholipids partitioning into detergent-resistant membrane microdomains (lipid raft-aggregates), can explain, at least in part, the cytostatic, cytotoxic as well as the apoptotic effects occurring upon pharmacological inhibition of FASN activity in cancer cells. Moreover, several cancer-associated molecular features including nonfunctioning p53, overexpression of the Her-2/neu (erbB-2) oncogene, and hyperactivation of the PI-3'K down-stream effector protein kinase B (AKT), appear to determine an exacerbated sensitivity to FASN inhibition-induced cancer cell death. Although few of these inhibitors are expected to be "exclusively" selective for FASN, the potential of FASN as a target for antineoplastic intervention has eventually been confirmed by RNA interference (RNAi)-knockdown of FASN. Certainly, future studies should definitely elucidate the ultimate biochemical link between FASN inhibition and cancer cell death. Although the combination of FASN structural complexity and until recently the lack of X-ray crystallography data of mammalian FASN created a significant challenge in the exploitation of FASN as a valuable target for drug development, it is hoped that the improvement in the selectivity and potency of forthcoming novel FASN-targeted small molecule inhibitors by taking advantage, for instance, of the recent 4.5 A resolution X-ray crystallographic map of mammalian FASN, will direct the foundation of a new family of chemotherapeutic agents in cancer history.
Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without ...compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds 1, 2 and 5 were cytotoxic in the low micromolar range against a panel of molecularly diverse human cancer cell lines. Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.
The acquisition of and departure from stemness in cancer tissues might not only be hardwired by genetic controllers, but also by the pivotal regulatory role of the cellular metabotype, which may act ...as a "starter dough" for cancer stemness traits. We have coined the term metabostemness to refer to the metabolic parameters causally controlling or functionally substituting the epitranscriptional orchestration of the genetic reprograming that redirects normal and tumor cells toward less-differentiated cancer stem cell (CSC) cellular states. Certain metabotypic alterations might operate as pivotal molecular events rendering a cell of origin susceptible to epigenetic rewiring required for the acquisition of aberrant stemness and, concurrently, of refractoriness to differentiation. The metabostemness attribute can remove, diminish, or modify the nature of molecular barriers present in Waddington's epigenetic landscapes, thus allowing differentiated cells to more easily (re)-enter into CSC cellular macrostates. Activation of the metabostemness trait can poise cells with chromatin states competent for rapid dedifferentiation while concomitantly setting the idoneous metabolic stage for later reprograming stimuli to finish the journey from non-cancerous into tumor-initiating cells. Because only a few permitted metabotypes will be compatible with the operational properties owned by CSC cellular states, the metabostemness property provides a new framework through which to pharmacologically resolve the apparently impossible problem of discovering drugs aimed to target the molecular biology of the cancer stemness itself. The metabostemness cancer hallmark generates a shifting oncology theory that should guide a new era of metabolo-epigenetic cancer precision medicine.
Oncometabolites are defined as small-molecule components (or enantiomers) of normal metabolism whose accumulation causes signaling dysregulation to establish a milieu that initiates carcinogenesis. ...In a similar manner, we propose the term "gerometabolites" to refer to small-molecule components of normal metabolism whose depletion causes signaling dysregulation to establish a milieu that drives aging. In an investigation of the pathogenic activities of the currently recognized oncometabolites R(-)-2-hydroxyglutarate (2-HG), fumarate, and succinate, which accumulate due to mutations in isocitrate dehydrogenases (IDH), fumarate hydratase (FH), and succinate dehydrogenase (SDH), respectively, we illustrate the fact that metabolic pseudohypoxia, the accumulation of hypoxia-inducible factor (HIFα) under normoxic conditions, and the subsequent Warburg-like reprogramming that shifts glucose metabolism from the oxidative pathway to aerobic glycolysis are the same mechanisms through which the decline of the "gerometabolite" nicotinamide adenine dinucleotide (NAD)
+
reversibly disrupts nuclear-mitochondrial communication and contributes to the decline in mitochondrial function with age. From an evolutionary perspective, it is reasonable to view NAD
+
-driven mitochondrial homeostasis as a conserved response to changes in energy supplies and oxygen levels. Similarly, the natural ability of 2-HG to significantly alter epigenetics might reflect an evolutionarily ancient role of certain metabolites to signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency. However, when chronically altered, these responses become conserved causes of aging and cancer. Because HIFα-driven pseudohypoxia might drive the overproduction of 2-HG, the intriguing possibility exists that the decline of gerometabolites such as NAD
+
could promote the chronic accumulation of oncometabolites in normal cells during aging. If the sole activation of a Warburg-like metabolic reprogramming in normal tissues might be able to significantly increase the endogenous production of bona fide etiological determinants in cancer, such as oncometabolites, this undesirable trade-off between mitochondrial dysfunction and activation of oncometabolites production might then pave the way for the epigenetic initiation of carcinogenesis in a strictly metabolic-dependent manner. Perhaps it is time to definitely adopt the view that aging and aging diseases including cancer are governed by a pivotal regulatory role of metabolic reprogramming in cell fate decisions.
The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We ...here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.
Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.
Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.
The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.
The metabolic features of cancer stem (CS) cells and the effects of specific nutrients or metabolites on CS cells remain mostly unexplored. A preliminary study to delineate the nutritional phenome of ...CS cells exploited the landmark observation that upon experimental induction into an epithelial-to-mesenchymal (EMT) transition, the proportion of CS-like cells drastically increases within a breast cancer cell population. EMT-induced CS-like cells (HMLERshEcad) and isogenic parental cells (HMLERshCntrol) were simultaneously screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput metabolic phenotyping platform comprising >350 wells that were pre-loaded with different carbohydrates/starches, alcohols, fatty acids, ketones, carboxylic acids, amino acids, and bi-amino acids. The generation of "phenetic maps" of the carbon and nitrogen utilization patterns revealed that the acquisition of a CS-like cellular state provided an enhanced ability to utilize additional catabolic fuels, especially under starvation conditions. Crucially, the acquisition of cancer stemness activated a metabolic infrastructure that enabled the vectorial transfer of high-energy nutrients such as glycolysis end products (pyruvate, lactate) and bona fide ketone bodies (β-hydroxybutyrate) from the extracellular microenvironment to support mitochondrial energy production in CS-like cells. Metabolic reprogramming may thus constitute an efficient adaptive strategy through which CS-like cells would rapidly obtain an advantage in hostile conditions such as nutrient starvation following the inhibition of tumor angiogenesis. By understanding how specific nutrients could bioenergetically boost EMT-CS-like phenotypes, "smart foods" or systemic "metabolic nichotherapies" may be tailored to specific nutritional CSC phenomes, whereas high-resolution heavy isotope-labeled nutrient tracking may be developed to monitor the spatiotemporal distribution and functionality of CS-like cells in real time.
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