Neri Mercatelli

Regulation of the p27Kip1 tumor suppressor by miR‐221 and miR‐222 promotes cancer cell proliferation

MicroRNAs (miRNAs) are potent post‐transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR‐221 and miR‐222 (miR‐221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR‐221&222 to maintain low p27Kip1 levels and continuous proliferation. Interestingly, high levels of miR‐221&222 appear in glioblastomas and correlate with low levels of p27Kip1 protein. Thus, deregulated expression of miR‐221&222 promotes cancerous growth by inhibiting the expression of p27Kip1.

miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27Kip1. We recognize two target sites for the microRNAs in the 3′ untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G1 to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27Kip1 in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27Kip1, and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27Kip1 down-regulation.

The Inhibition of the Highly Expressed Mir-221 and Mir-222 Impairs the Growth of Prostate Carcinoma Xenografts in Mice

Background MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity. Methodology/Principal Findings Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression. Conclusions/Significance These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.

NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells.

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription.

Role of exercise-induced reactive oxygen species in the modulation of heat shock protein response

Abstract The multiple roles that have been associated with heat shock proteins (HSPs), inside and outside cells are remarkable. HSPs have been found to play a fundamental role in multiple stress conditions and to offer protection from subsequent insults. Exercise, because of the physiological stresses associated with it, is one of the main stimuli associated with a robust increase of different HSPs in several tissues. Given the combination of physiological stresses induced by exercise, and the ‘cross-talk’ that occurs between signaling pathways in different tissues, it is likely that exercise induces the HSP expression through a combination of ‘stressors’, among which reactive oxygen species (ROS) could play a major role. Indeed, although an imbalance between ROS production and antioxidant levels results in oxidative stress, causing damage to lipids, proteins, and nucleic acids with a possible activation of the programed cell death pathway, at moderate concentrations ROS play an important role as regulatory mediators in signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and re-establish redox homeostasis. The aim of this review is to provide a critical update on the role of exercise-induced ROS in the modulation of the HSPs response, focusing on experimental results from animal and human studies where the link between redox homeostasis and HSPs’ expression in different tissues has been addressed.

Platelet-rich plasma and skeletal muscle healing: a molecular analysis of the early phases of the regeneration process in an experimental animal model.

Platelet-rich plasma (PRP) has received increasing interest in applied medicine, being widely used in clinical practice with the aim of stimulating tissue healing. Despite the reported clinical success, there is still a lack of knowledge when considering the biological mechanisms at the base of the activity of PRP during the process of muscle healing. The aim of the present study was to verify whether the local delivery of PRP modulates specific molecular events involved in the early stages of the muscle regeneration process. The right flexor sublimis muscle of anesthetized Wistar rats was mechanically injured and either treated with PRP or received no treatment. At day 2 and 5 after surgery, the animals were sacrificed and the muscle samples evaluated at molecular levels. PRP treatment increased significantly the mRNA level of the pro-inflammatory cytokines IL-1β, and TGF-β1. This phenomenon induced an increased expression at mRNA and/or protein levels of several myogenic regulatory factors such as MyoD1, Myf5 and Pax7, as well as the muscular isoform of insulin-like growth factor1 (IGF-1Eb). No effect was detected with respect to VEGF-A expression. In addition, PRP application modulated the expression of miR-133a together with its known target serum response factor (SRF); increased the phosphorylation of αB-cristallin, with a significant improvement in several apoptotic parameters (NF-κB-p65 and caspase 3), indexes of augmented cell survival. The results of the present study indicates that the effect of PRP in skeletal muscle injury repair is due both to the modulation of the molecular mediators of the inflammatory and myogenic pathways, and to the control of secondary pathways such as those regulated by myomiRNAs and heat shock proteins, which contribute to proper and effective tissue regeneration.

Exercise-induced ROS in heat shock proteins response.

Cells have evolved multiple and sophisticated stress response mechanisms aiming to prevent macromolecular (including proteins, lipids, and nucleic acids) damage and to maintain or re-establish cellular homeostasis. Heat shock proteins (HSPs) are among the most highly conserved, ubiquitous, and abundant proteins in all organisms. Originally discovered more than 50 years ago through heat shock stress, they display multiple, remarkable roles inside and outside cells under a variety of stresses, including also oxidative stress and radiation, recognizing unfolded or misfolded proteins and facilitating their restructuring. Exercise consists in a combination of physiological stresses, such as metabolic disturbances, changes in circulating levels of hormones, increased temperature, induction of mild to severe inflammatory state, increased production of reactive oxygen and nitrogen species (ROS and RNS). As a consequence, exercise is one of the main stimuli associated with a robust increase in different HSPs in several tissues, which appears to be also fundamental in facilitating the cellular remodeling processes related to the training regime. Among all factors involved in the exercise-related modulation of HSPs level, the ROS production in the contracting muscle or in other tissues represents one of the most attracting, but still under discussion, mechanism. Following exhaustive or damaging muscle exercise, major oxidative damage to proteins and lipids is likely involved in HSP expression, together with mechanically induced damage to muscle proteins and the inflammatory response occurring several days into the recovery period. Instead, the transient and reversible oxidation of proteins by physiological concentrations of ROS seems to be involved in the activation of stress response following non-damaging muscle exercise. This review aims to provide a critical update on the role of HSPs response in exercise-induced adaptation or damage in humans, focusing on experimental results where the link between redox homeostasis and HSPs expression by exercise has been addressed. Further, with the support of in vivo and in vitro studies, we discuss the putative molecular mechanisms underlying the ROS-mediated modulation of HSP expression and/or activity during exercise.

αB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts

Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin.

Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training.

Reactive oxygen and nitrogen species are important signal molecules for adaptations to training. Due to the antioxidant properties of vitamin C and E, supplementation has been shown to blunt adaptations to endurance training. In this study, we investigated the effects of vitamin C and E supplementation and endurance training on adaptations in endogenous antioxidants and heat shock proteins (HSP). Thirty seven males and females were randomly assigned to receive Vitamin C and E (C + E; C: 1000 mg, E: 235 mg daily) or placebo (PLA), and underwent endurance training for 11 weeks. After 5 weeks, a subgroup conducted a high intensity interval session to investigate acute stress responses. Muscle and blood samples were obtained to investigate changes in proteins and mRNA related to the antioxidant and HSP system. The acute response to the interval session revealed no effects of C + E supplementation on NFκB activation. However, higher stress responses to exercise in C + E group was indicated by larger translocation of HSPs and a more pronounced gene expression compared to PLA. Eleven weeks of endurance training decreased muscle GPx1, HSP27 and αB‐crystallin, while mnSOD, HSP70 and GSH remained unchanged, with no influence of supplementation. Plasma GSH increased in both groups, while uric acid decreased in the C + E group only. Our results showed that C + E did not affect long‐term training adaptations in the antioxidant‐ and HSP systems. However, the greater stress responses to exercise in the C + E group might indicate that long‐term adaptations occurs through different mechanisms in the two groups.

Physical activity in the prevention of human diseases: role of epigenetic modifications

Epigenetic modification refers to heritable changes in gene function that cannot be explained by alterations in the DNA sequence. The current literature clearly demonstrates that the epigenetic response is highly dynamic and influenced by different biological and environmental factors such as aging, nutrient availability and physical exercise. As such, it is well accepted that physical activity and exercise can modulate gene expression through epigenetic alternations although the type and duration of exercise eliciting specific epigenetic effects that can result in health benefits and prevent chronic diseases remains to be determined. This review highlights the most significant findings from epigenetic studies involving physical activity/exercise interventions known to benefit chronic diseases such as metabolic syndrome, diabetes, cancer, cardiovascular and neurodegenerative diseases.