The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

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Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donortreated cells. Mutated Akt1/PKB␣ (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that Snitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS-and/or oxidative stress-involved insulin resistance. Nitric oxide (NO)1 is an endogenous cell signaling molecule involved in the regulation of many physiological functions and in the mediation of a variety of pathophysiological processes. NO and NO-related compounds function as both protective and cytotoxic, dependent on the cellular context and the nature of the NO group. The multifaceted actions of the NO group can be classified into two categories: 1) authentic NO-mediated, cGMPdependent, and 2) reactive nitrogen species-mediated, cGMPindependent actions. Nitrosative post-translational modifications, including protein S-nitrosylation and tyrosine nitration, are involved in the cGMP-independent actions. The cGMP-dependent actions play critical roles in a variety of physiological processes, including NO-mediated vasodilation. In contrast, cGMP-independent, nitrosative protein modifications are postulated to be involved in the pathological responses (1-4).Nitric-oxide synthases (NOSs) consist of three distinct genes, inducible nitric-oxide synthase (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). NO is generated by iNOS to a much greater extent, to over 1,000-fold, compared with that produced by the constitutive NOSs, eNOS and nNOS (2, 5). iNOS and nitrosative stress have been implicated in many human diseases, including insulin resistance (6, 7), atherosclerosis (8), inflammation, and neurodegenerative disorders (9). This is largely based on the evidence that iNOS deficiency results in significant amelioration of, or resistance to, these diseases. However, little is known about the molecular mechanisms by which iNOS causes and/or exacerbates these diseases. Furthe...

Objective-Cilostazol, a selective inhibitor of PDE3, has a protective effect on endothelium after ischemic vascular damage, through production of nitric oxide (NO). The purpose of the present study was to clarify the molecular mechanisms underlying the preventive effect of treatment with cilostazol on oxidative stress-induced premature senescence in human endothelial cells. Methods and Results-Prematurely

Abstract-Vascular calcification is clinically important in the development of cardiovascular disease. It is reported that hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) inhibited vascular calcification in several clinical trials. However, the mechanism is poorly understood. Recently, it has been suggested that apoptosis is one of the important processes regulating vascular smooth muscle cell (VSMC) calcification. In this study, we investigated the effect of statins on VSMC calcification by testing their effect on apoptosis, focusing in particular on regulation of the survival pathway mediated by growth arrest-specific gene 6 (Gas6), a member of the vitamin K-dependent protein family, and its receptor, Axl. In human aortic smooth muscle cells (HASMC), statins significantly inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner (reduced by 49% at 0.1 mol/L atorvastatin). The inhibitory effect of statins was mediated by preventing apoptosis, which was increased by Pi in a concentration-dependent manner, and not by inhibiting sodium-dependent phosphate cotransporter (NPC) activity, another mechanism regulating HASMC calcification. Furthermore, the antiapoptotic effect of statins was dependent on restoration of Gas6, whose expression was downregulated by Pi. Restoration of Gas6 mRNA by statins was mediated by mRNA stabilization, and not by an increase in transcriptional activity. Suppression of Gas6 using small interfering RNA and the Axl-extracellular domain abolished the preventive effect of statins on Pi-induced apoptosis and calcification. These data demonstrate that statins protected HASMC from Pi-induced calcification by inhibiting apoptosis via restoration of the Gas6-Axl pathway. Key Words: calcification Ⅲ statins Ⅲ apoptosis Ⅲ Gas6 Ⅲ Axl V ascular calcification, such as coronary and aortic calcification, is a significant feature of vascular pathology, because this lesion is associated with cardiovascular disease. 1,2 It has been recognized that statins exhibit various protective effects against atherosclerosis, including modification of endothelial function, 3 decreased inflammation, 4 and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration, 5 all of which cannot be accounted for by lipid reduction. One of the interesting pleiotropic effects of statins is the inhibition of vascular calcification. Results from clinical trials suggest an association of statin use with slowed progression of calcific aortic stenosis 6 -8 and coronary artery calcification. 9 Statins also inhibited calcification of atherosclerotic plaques in experimental hyperlipidemic animals. 10,11 On the other hand, some recent clinical trials were not able to find such an inhibitory effect. 12,13 To clarify these discrepancies, it is important to identify the detailed regulatory mechanism of vascular calcification and the target of effect of statins.Based on clinical findings, 14 inorganic phosphate (Pi) has been shown to be an important inducer of VSM...

Objective-Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have pleiotropic vascular protective effects besides cholesterol lowering. Recently, experimental and clinical studies have indicated that senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. Therefore, the present study was performed to determine whether statins would reduce endothelial senescence and to clarify the molecular mechanisms underlying the antisenescent property of statins. Methods and Results-Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide (H 2 O 2 ), as judged by senescence-associated ␤-galactosidase assay and cell morphological appearance. Atorvastatin, pravastatin, and pitavastatin inhibited the oxidative stress induced-endothelial senescence. These statins phosphorylated Akt at Ser473 and subsequently led to increased expression of endothelial nitric oxide synthase (eNOS), SIRT1, and catalase. Treatment with LY294002 or Akt short interfering RNA decreased the eNOS activation, SIRT1 expression, and antisenescent property of atorvastatin. Moreover, in streptozotocin-diabetic mice, administration of pitavastatin increased eNOS, SIRT1, and catalase expression and decreased endothelial senescence, but levels remained unaltered in Sirt1 knockout mice. Key Words: endothelium Ⅲ nitric oxide synthase Ⅲ SIRT1 Ⅲ senescence Ⅲ statin T he 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, are effective in lowering the plasma concentration of low-density lipoprotein cholesterol and are widely used in patients with hypercholesterolemia. Recently, experimental and clinical evidence has indicated that the pleiotropic effects of statins involve improvement or restoration of endothelial function, enhanced activity of endothelial nitric oxide synthase (eNOS), and decreased oxidative stress. 1 Oxidative stress is implicated in the pathogenesis of cardiovascular diseases, such as atherosclerosis. 2 Excessive production of reactive oxygen species inflicts damage on endothelial cells and leads to the onset of endothelial senescence. Senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. 3 Histological study of human atherosclerotic lesions has demonstrated the existence of endothelial cells that exhibit the morphological features of senescence. 4 Assmus et al have shown that statins reduce senescence and increase proliferation of endothelial progenitor cells. 5 In Saccharomyces cerevisiae, the silent information regulator 2 (Sir2) family of genes governs budding exhaustion and replicative life span. 6,7 Sir2 has been identified as an NAD ϩ -dependent histone deacetylase and is responsible for maintenance of chromatin silencing and genome stability. 8 Sir2 genes are conserved during evolution, and 7 homologs of sirtuins (Sirt1 to Sirt7) have been cloned in mammals. Mammalian sirtuin 1 (Sirt1), the closest homolog of Sir2, regulates the cell cycle, senescence, apoptosis, and metabolism by interacting with a number of mo...

SIRT1 plays an essential role in preventing hyperphosphatemia-induced arterial calcification via inhibition of osteoblastic transdifferentiation. In addition, Pi-induced SMC calcification may be associated with both premature and replicative cellular senescence.

Sir2 (silent information regulator-2), an NAD -dependent histone deacetylase, is highly conserved in organisms ranging from archaea to humans. Yeast Sir2 is responsible for silencing at repeated DNA sequences in mating-type loci, telomeres and rDNA, and plays critical roles in DNA repair, stress resistance and longevity. The phenomenon of human aging is known to be a critical cardiovascular risk factor. Senescence of endothelial cells has been proposed to be involved in vascular dysfunction and atherogenesis. Recent studies have demonstrated that mammalian Sirt1 NAD -dependent protein deacetylase, the closest homologue of Sir2, regulates vascular angiogenesis, homeostasis and senescence. This review focuses on SIRT1 as a potential therapeutic target against atherosclerosis.J Atheroscler Thromb, 2010; 17:431-435.