Targeting Endothelial SIRT1 for the Prevention of Arterial Aging

Cardiovascular diseases are the leading cause of morbidity and mortality in the elderly population all over the world. Arterial aging is the earliest manifestation and a key risk factor for age-induced cardiovascular abnormalities. The longevity regulator Sirtuin 1 (SIRT1) is abundantly expressed in the endothelium of the arteries and elicits potent protective functions against arterial aging. Targeting endothelial SIRT1 represents a promising approach for the prevention and treatment of cardiovascular diseases. This chapter provides an overview of SIRT1’s regulation and function in endothelial cells and discusses the potential applications of targeting endothelial SIRT1 for arterial aging-related cardiovascular diseases

Human Sirt-1: Molecular Modeling and Structure-Function Relationships of an Unordered Protein

BACKGROUND: Sirt-1 is a NAD+-dependent nuclear deacetylase of 747 residues that in mammals is involved in various important metabolic pathways, such as glucose metabolism and insulin secretion, and often works on many different metabolic substrates as a multifunctional protein. Sirt-1 down-regulates p53 activity, rising lifespan, and cell survival; it also deacetylases peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and its coactivator 1 alpha (PGC-1alpha), promoting lipid mobilization, positively regulating insulin secretion, and increasing mitochondrial dimension and number. Therefore, it has been implicated in diseases such as diabetes and the metabolic syndrome and, also, in the mechanisms of longevity induced by calorie restriction. Its whole structure is not yet experimentally determined and the structural features of its allosteric site are unknown, and no information is known about the structural changes determined by the binding of its allosteric effectors. METHODOLOGY: In this study, we modelled the whole three-dimensional structure of Sirt-1 and that of its endogenous activator, the nuclear protein AROS. Moreover, we modelled the Sirt-1/AROS complex in order to study the structural basis of its activation and regulation. CONCLUSIONS: Amazingly, the structural data show that Sirt-1 is an unordered protein with a globular core and two large unordered structural regions at both termini, which play an important role in the protein-protein interaction. Moreover, we have found on Sirt-1 a conserved pharmacophore pocket of which we have discussed the implication

Estudio de inhibidores de Sirtuinas como futuros agentes antitumorales

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de .Biología Fecha de lectura: 15-01-201

Targeting FOXO3a and Sirtuins in breast cancer

SIRT proteins play an important role in the survival and drug resistance of tumour cells, especially during chemotherapy. In this study, we investigated the potency, specificity and cellular targets of three recently identified SIRT inhibitors, Sirtinol, Salermide and EX527. Our results identify the specificity and cellular targets of these novel inhibitors, and suggest that SIRT inhibitors require combined targeting of both SIRT1 and SIRT2 to induce p53 acetylation and cell death in breast cancer cells. Assessing the role of sirtuins in chemoresistance, cisplatin resistant cell lines were developed and characterized. Cisplatin resistant cells (CisR) were found to have higher levels of SIRT1, repressing the activation of FOXO3a. MCF-7 cells overexpressing SIRT1 were also shown to be protected against cisplatin treatment, highlighting its role in resistance. Sirtuins have many cellular targets, including alpha-tubulin, PARP, ku70 and FOXO3a. Because of the similarities between the regulation of p53 and FOXO3a, we decided to elucidate the effect of sirtinol treatment on FOXO3a. Sirtinol treatment was shown to stabilize FOXO3a, similar to its effect on p53, but at lower concentrations. We have shown previously that FOXO3a is an important downstream mediator of the cytotoxicity of receptor tyrosine kinase targeted therapies. To examine the conjecture whether sirtuin inhibitors could increase the proapoptotic potency of lapatinib through stabilization of FOXO3a, lapatinib was treated alone and in combination with sirtinol and EX527. Sirtinol was found to synergise with lapatinib treatment (in cells containing mutant p53) and this was dependent on the presence of FOXO proteins, highlighting the use of sirtuin inhibitors in increasing the efficacy of therapies that indirectly target FOXO3a

An investigation of roles for SIRT1 and dietary polyphenols in modulating the ageing process through DNA methylation

Dietary restriction (DR) can increase lifespan across evolutionarily distinct species, from yeast to rodents. The NAD+-dependent (class III) histone deacetylase SIRT1 in mammals, and its ortholog in other species, may play a major role in this response, but may affect ‘healthspan’ (number of years of good health), rather than lifespan per se. Ageing is accompanied by changes in genome methylation, which may be causal in the ageing process. Since histones are one of the many substrates that are deacetylated by SIRT1, we hypothesised that epigenetic effects of SIRT1 activity – and specifically effects on DNA methylation, which is associated closely with histone acetylation – mediate some of the beneficial effects of DR that contribute to increased healthspan. We also propose that dietary polyphenols may act at the cellular level in a similar way. To test this hypothesis, we first investigated effects of altering SIRT1 expression, by overexpression of a transgene or siRNA-mediated knockdown, on global DNA methylation (methylation of the LINE-1 element) in the human intestinal cell line, Caco-2. We also measured effects on global DNA methylation of dietary isoflavones and resveratrol, under control conditions as well as conditions of SIRT1 overexpression. Measured effects on global DNA methylation revealed possible complex interactions between SIRT1 and these dietary polyphenols but were dependent on the assay used to measure methylation at the LINE-1 element (COBRA or pyrosequencing), so were not considered to be robust observations. We investigated factors that may affect SIRT1 expression – specifically we examined SIRT1 promoter activity in response to polyphenols, effects of promoter methylation and effects of age. Treatment of Caco-2 cells with dietary polyphenols had no effect on the SIRT1 promoter in a promoter-reporter construct. In contrast, methylation of the SIRT1 promoter reduced reporter gene expression in this model. Age did not appear to change the levels of SIRT1 protein expressed in mouse intestinal tissue when comparing young and older mice. An in silico analysis was carried out to investigate if overlaps between groups of genes compiled from published and publically-available data found to i) associate with SIRT1, ii) show altered expression in response to DR, and iii) show altered methylation with ageing were greater than expected by chance, which would support the hypothesis, and provided targets to investigate possible site-specific effects of SIRT1 on DNA methylation. Ten genes were found to fit into the ‘three way’ overlap, which was statistically greater than expected by chance. Pyrosequencing assays could be optimised for only 8 of these 10 genes, so we focused further investigations on this sub-set. Significant effects of SIRT1 overexpression and/or knockdown on methylation were observed on at least one CpG site in the promoters of six of these genes (CDC7, EIF5, IRX3, KLF3, PTPRG, TBX3) and expression at the mRNA level of all of the eight genes (also PCYT1A and SLC39A4) was affected significantly. To gain a more comprehensive view of the extent to which DNA methylation at specific loci may be affected by SIRT1 expression levels microarray-based analysis of the methylation pattern across the genome in Caco-2 cells was carried out under conditions where SIRT1 was overexpressed or where expression was reduced by siRNA. In parallel, we measured the response to expressing SIRT1 at different levels at the level of the transcriptome. Overlaps that were statistically greater than expected by chance between these data and the lists of genes compiled from published and publically-available data used for the in silico analysis were found to exist between the complied list of genes reported to respond to dietary restriction and the set of genes we found to show altered expression in response to changing the level of SIRT1 expression and the set of genes we found to be differentially-methylated in response to reducing the level of SIRT1 expression. This observation is in broad support of our overarching hypothesis. The findings of this study indicate that effects of SIRT1 on methylation of specific genes may correspond with altered expression under conditions of dietary restriction. The data reveal a large number of gene targets for which causal links between these modifications roles in modulating the ageing process could be investigated.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Agents for Hepatocellular Carcinoma: Synthesis and Mode of Action

Ph.DDOCTOR OF PHILOSOPH