632 research outputs found

    Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation [version 3]

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    Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells

    The pharmacological regulation of cellular mitophagy

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    Small molecules are pharmacological tools of considerable value for dissecting complex biological processes and identifying potential therapeutic interventions. Recently, the cellular quality-control process of mitophagy has attracted considerable research interest; however, the limited availability of suitable chemical probes has restricted our understanding of the molecular mechanisms involved. Current approaches to initiate mitophagy include acute dissipation of the mitochondrial membrane potential (ΔΨm) by mitochondrial uncouplers (for example, FCCP/CCCP) and the use of antimycin A and oligomycin to impair respiration. Both approaches impair mitochondrial homeostasis and therefore limit the scope for dissection of subtle, bioenergy-related regulatory phenomena. Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation. We have summarized the current status of mitophagy modulators and analyzed the available chemical tools, commenting on their advantages, limitations and current applications

    Interactomic and Pharmacological Insights on Human Sirt-1

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    Sirt-1 is defined as a nuclear protein involved in the molecular mechanisms of inflammation and neurodegeneration through the de-acetylation of many different substrates even if experimental data in mouse suggest both its cytoplasmatic presence and nucleo-cytoplasmic shuttling upon oxidative stress. Since the experimental structure of human Sirt-1 has not yet been reported, we have modeled its 3D structure, highlighted that it is composed by four different structural regions: N-terminal region, allosteric site, catalytic core and C-terminal region, and underlined that the two terminal regions have high intrinsic disorder propensity and numerous putative phosphorylation sites. Many different papers report experimental studies related to its functional activators because Sirt-1 is implicated in various diseases and cancers. The aim of this article is (i) to present interactomic studies based human Sirt-1 to understand its most important functional relationships in the light of the gene–protein interactions that control major metabolic pathways and (ii) to show by docking studies how this protein binds some activator molecules in order to evidence structural determinants, physico-chemical features and those residues involved in the formation of complexes

    Elucidating the role of EP300/CBP mediated FOXO3 acetylation in tumorigenesis and cancer drug resistance

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    Forkhead Box O3 (FOXO3) is a tumour suppressor whose functional activity is predominantly regulated by post-translational modifications (PTMs). Of all PTMs, the best-studied PTM of FOXO proteins is phosphorylation, however, acetylation by EP300 or the CREB-binding protein (CBP) has been increasingly recognised as a novel axis regulating FOXO proteins. Herein, using the BT474 breast cancer cells and its lapatinib resistant derivative BT474 LapR cells, I observed that higher FOXO3 and acetylated Ac-FOXO3 levels correlate with lapatinib sensitivity. Subsequent ectopic expression of EP300, as well as CBP, led to an increase in acetylated-FOXO3 in sensitive, but not in resistant BT474 cells. Moreover, lapatinib sensitive BT474 cells exhibited enhanced lapatinib cytotoxicity upon over-expression of wild-type but not acetylation-deficient EP300 or CBP as revealed by drug sensitivity assays. Besides, using SIRT1/6 specific siRNAs and chemical inhibitor, I also found that SIRT1 and SIRT6 play a part in fine-tuning FOXO3 acetylation and lapatinib sensitivity. Notably, we next explored that not only SIRT1/6 but also SIRT2 plays a significant role in regulating FOXO3 acetylation and lapatinib sensitivity of BT474 and nasopharyngeal cancers. Taken together, these results suggest the involvement of acetyltransferases EP300/CBP and nuclear sirtuins in regulating FOXO3 acetylation and lapatinib sensitivity of breast cancers. Despite having a tumour-suppressive role, some reports suggested the oncogenic role of EP300 in the development and progression of cancers. To uncover this, I used Hs578T cells which have E-cadherin promoter hypermethylated. Interestingly, overexpression of EP300 led to the up-regulation of mesenchymal and stemness markers, increases in cell migration, invasion, anchorage-independent growth, and drug resistance. Moreover, genome-wide expression profiling identified Aldo-keto reductases AKR1C1-3 as positive modulators of cancer stemness and drug resistance, since their pharmacological inhibition with flufenamic acid restored both doxorubicin and paclitaxel sensitivity and reduced mammosphere formation. Thus, EP300 functions as an oncogene via the up-regulation of AKR1Cs in cells where the E-cadherin promoter is hypermethylated. This important finding offers the rationale of using current AKR1Cs inhibitors in the treatment of triple-negative breast cancers.Open Acces

    Phytochemicals and Cancer Chemoprevention: Epigenetic Friends or Foe?

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    Cancer, as one of the non-communicable diseases, remains one of the leading causes of death around the world. Since immune cells that infiltrate tumors engage in an extensive and dynamic crosstalk with cancer cells, inflammatory responses play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis. Inflammation also affects immune surveillance and therapy sensitivity. Recent successes of therapeutic interventions in cancer and inflammatory diseases using epigenetic modifiers such as histone deacetylase inhibitors and inhibitors of DNA methylation suggest that epigenetic reprogramming plays an important role in the aetiology of these diseases. Epigenetic changes in DNA methylation patterns at CpG sites (epimutations) or corrupt chromatin states of tumor promoting genes and noncoding RNAs, recently emerged as major governing factors in tumor progression and cancer drug sensitivity. Epigenetic defects (epimutations) are thought to be more easily reversible (when compared with genetic defects) and, as such, have inspired efforts to identify novel compounds that correct epimutations or prevent disease progression. Given the fact that epigenetic modifications occur early in carcinogenesis and represent potentially initiating events in cancer development, they have been identified as promising new targets for chemoprevention strategies. Numerous clinical, epidemiological and laboratory studies have identified various promising nutritional anti-inflammatory compounds as chemopreventive agents, which affect carcinogenic epigenetic marks in the body and the host immune system, and protect against aggressive cancer malignancies. This has recently launched reexploration of chemopreventive phytochemicals for identification of epigenetic targets which allow epigenetic (re)programming of cancer stem cells, prevent metastasis or sensitize for drug sensitivity. This review will discuss mechanisms of epigenome plasticity by cancer-inflammation and chemopreventive phytochemicals

    Identification of Four Potential Epigenetic Modulators from the NCI Structural Diversity Library Using a Cell-Based Assay

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    Epigenetic pathways help control the expression of genes. In cancer and other diseases, aberrant silencing or overexpression of genes, such as those that control cell growth, can greatly contribute to pathogenesis. Access to these genes by the transcriptional machinery is largely mediated by chemical modifications of DNA or histones, which are controlled by epigenetic enzymes, making these enzymes attractive targets for drug discovery. Here we describe the characterization of a locus derepression assay, a fluorescence-based mammalian cellular system which was used to screen the NCI structural diversity library for novel epigenetic modulators using an automated imaging platform. Four structurally unique compounds were uncovered that, when further investigated, showed distinct activities. These compounds block the viability of lung cancer and melanoma cells, prevent cell cycle progression, and/or inhibit histone deacetylase activity, altering levels of cellular histone acetylation

    Role of Sirtuin-1 in the pathogenesis of hypertension in spontaneously hypertensive rats : molecular mechanisms

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    Il a été démontré que la sirtuine 1 (Sirt-1), une histone désacétylase de classe III, est surexprimée dans le coeur des rats spontanément hypertendus (SHR). Nous avons récemment montré que les cellules musculaires lisses vasculaires (CMLV) des SHR présentent une expression accrue de Sirt-1 par rapport aux rats Wistar Kyoto (WKY) de même âge qui contribue à l’augmentation de la régulation de la protéine Giα impliquée dans la pathogenèse de l'hypertension. La présente étude a été effectuée pour étudier le rôle de l'augmentation de l'expression de la Sirt-1 dans la pathogenèse de l'hypertension chez les SHR et pour explorer les mécanismes moléculaires impliqués dans cette réponse. Dans cette étude, un inhibiteur sélectif de la Sirt-1, EX-527 (5 mg/kg de poids corporel), a été injecté par voie intrapéritonéale chez des rats SHR adultes de 8 semaines et des rats WKY de même âge, deux fois par semaine pendant 3 semaines. La pression artérielle (PA) et la fréquence cardiaque ont été mesurées deux fois par semaine par la méthode non invasive du brassard autour de la queue. Le traitement avec l’inhibiteur spécifique de la Sirt-1, l'EX-527, a atténué les augmentations de PA (de 76 mmHg) et de fréquence cardiaque chez les rats SHR. La surexpression de Sirt-1 et des protéines Giα dans le coeur, les CMLV et l'aorte a été atténuée au niveau des contrôles par l'inhibiteur de la Sirt-1. L'inhibition de la Sirt-1 a également atténué les niveaux accrus des anions superoxydes, l’activité de la NADPH oxydase et la surexpression des sous-unités de la NADPH oxydase ; les protéines Nox2, Nox4 et P47phox dans les CMLV isolées des SHR traités par l’EX-527. De plus, les niveaux réduits du monoxyde d'azote synthase endothélial (eNOS) et du monoxyde d'azote (NO) et les niveaux accrus de la peroxynitrite (ONOO-) dans les CMLV des SHR ont également été rétablis à des niveaux contrôles par l'inhibiteur de la Sirt-1. Ces résultats suggèrent que l'inhibition de la surexpression de la Sirt-1, en diminuant les niveaux accrus des protéines Giα et du stress nitro-oxydant, atténue la PA élevée chez les rats SHR. Il est donc possible de suggérer que les inhibiteurs de la Sirt-1 puissent être utilisés comme des agents thérapeutiques dans le traitement des complications cardiovasculaires associées à l'hypertension.Sirtuin-1 (Sirt-1), class III histone deacetylase, has been shown to be overexpressed in hearts from spontaneously hypertensive rats (SHR). We recently showed that vascular smooth muscle cells (VSMC) from SHR exhibit enhanced expression of Sirt-1 as compared to age-matched Wistar Kyoto (WKY) rats, which contributes to the upregulation of Giα protein implicated in the pathogenesis of hypertension. The present study was undertaken to investigate the role of upregulated Sirt-1 expression in the pathogenesis of hypertension in SHR and to explore the underlying molecular mechanisms involved in this response. For this study, a selective inhibitor of Sirt-1, EX-527 (5mg/kg of body weight), was injected intraperitoneally into 8-week-old adult SHR and age-matched WKY rats twice per week for 3 weeks. The blood pressure (BP) and heart rate was measured twice a week by the CODA™ non-invasive tail cuff method. Treatment of SHR with Sirt-1-specific inhibitor, EX-527, attenuated high BP by 76 mmHg and inhibited the augmented heart rate. The overexpression of Sirt-1 and Giα proteins in heart, VSMC and aorta was attenuated to the control levels by Sirt-1 inhibitor. Inhibition of Sirt-1 also attenuated the enhanced levels of superoxide anion, NADPH oxidase activity and the overexpression of NADPH oxidase subunits; Nox2, Nox4 and P47phox proteins in VSMC isolated from EX-527-treated SHR. Furthermore, the decreased levels of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) and increased levels of peroxynitrite (ONOO-) in VSMC from SHR were also restored to control levels by Sirt-1 inhibitor. These results suggest that the inhibition of overexpression of Sirt-1 through decreasing the enhanced levels of Giα proteins and nitro-oxidative stress attenuates the high BP in SHR. It may thus be suggested that inhibitors of Sirt-1 may have the potential to be used as therapeutic agents in the treatment of cardiovascular complications associated with hypertension
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