Calorie restriction and dietary restriction mimetics: a strategy for improving healthy aging and longevity.

Improvements in health care have increased human life expectancy in recent decades, and the elderly population is thus increasing in most developed countries. Unfortunately this still means increased years of poor health or disability. Since it is not yet possible to modify our genetic background, the best anti-aging strategy is currently to intervene on environmental factors, aiming to reduce the incidence of risk factors of poor health. Calorie restriction (CR) with adequate nutrition is the only non-genetic, and the most consistent non-pharmacological intervention that extends lifespan in model organisms from yeast to mammals, and protects against the deterioration of biological functions, delaying or reducing the risk of many age-related diseases. The biological mechanisms of CR's beneficial effects include modifications in energy metabolism, oxidative stress, insulin sensitivity, inflammation, autophagy, neuroendocrine function and induction of hormesis/xenohormesis response. The molecular signalling pathways mediating the anti-aging effect of CR include sirtuins, peroxisome proliferator activated receptor G coactivator-1α, AMP-activated protein kinase, insulin/insulin growth factor-1, and target of rapamycin, which form a pretty interacting network. However, most people would not comply with such a rigorous dietary program; research is thus increasingly aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic molecules/ treatments without lowering food intake, particularly in mid- to late-life periods. Likely candidates act on the same signalling pathways as CR, and include resveratrol and other polyphenols, rapamycin, 2-deoxy-D-glucose and other glycolytic inhibitors, insulin pathway and AMP-activated protein kinase activators, autophagy stimulators, alpha-lipoic acid, and other antioxidants.

Phenolic compounds present in Sardinian wine extracts protect against the production of inflammatory cytokines induced by oxysterols in CaCo-2 human enterocyte-like cells.

Cholesterol auto-oxidation products, namely oxysterols, are widely present in cholesterol-rich foods. They are thought to potentially interfere with homeostasis of the human digestive tract, playing a role in intestinal mucosal damage. This report concerns the marked up-regulation in differentiated CaCo-2 colonic epithelial cells of two key inflammatory interleukins, IL-6 and IL-8, caused by a mixture of oxysterols representative of a high cholesterol diet. This strong pro-inflammatory effect appeared to be dependent on the net imbalance of red-ox equilibrium with the production of excessive levels of reactive oxygen species through the colonic NADPH-oxidase NOX1 activation. Induction of NOX1 was markedly while not fully inhibited by CaCo-2 cell pre-incubation with phenolic extracts obtained from well-selected wines from typical grape varieties grown in Sardinia. Oxysterol-dependent NOX1 activation, as well as interleukin synthesis, were completely prevented by Cannonau red wine extract that contains an abundant phenolic fraction, in particular phenolic acids and flavonoids. Conversely, cell pre-treatment with Vermentino white wine extract with smaller phenolic fraction showed only a partial NOX1 down-regulation and was ineffective in interleukin synthesis induced by dietary oxysterols. It is thus likely that the effects of Sardinian wine extracts against intestinal inflammation induced by dietary oxysterols are mainly due to their high phenolic content: low doses of phenolics would be responsible only for direct scavenging oxysterol-dependent ROS production. Besides this direct activity, an excess of phenolic compounds detectable in red wine, may exert an additional indirect action by blocking oxysterol-related NOX1 induction, thus totally preventing the pro-oxidant and pro-inflammatory events triggered by dietary oxysterols.

Autophagic degradation contributes to muscle wasting in cancer cachexia.

Muscle protein wasting in cancer cachexia is a critical problem. The underlying mechanisms are still unclear, although the ubiquitin-proteasome system has been involved in the degradation of bulk myofibrillar proteins. The present work has been aimed to investigate whether autophagic degradation also plays a role in the onset of muscle depletion in cancer-bearing animals and in glucocorticoid-induced atrophy and sarcopenia of aging. The results show that autophagy is induced in muscle in three different models of cancer cachexia and in glucocorticoid-treated mice. In contrast, autophagic degradation in the muscle of sarcopenic animals is impaired but can be reactivated by calorie restriction. These results further demonstrate that different mechanisms are involved in pathologic muscle wasting and that autophagy, either excessive or defective, contributes to the complicated network that leads to muscle atrophy. In this regard, particularly intriguing is the observation that in cancer hosts and tumor necrosis factor α-treated C2C12 myotubes, insulin can only partially blunt autophagy induction. This finding suggests that autophagy is triggered through mechanisms that cannot be circumvented by using classic upstream modulators, prompting us to identify more effective approaches to target this proteolytic system.

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line.

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage. However, the treatment of colon cancer cells with a diet-compatible mixture of oxysterols does not elicit the same responses than individual components added to the cells at the same concentrations at which they are present in the mixture. Sixty µM oxysterol mixture showed a slight pro-apoptotic effect on human colon cancer CaCo-2 cell line, evaluated in terms of caspase-3 and caspase-7 activation; conversely, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and 5α,6α-epoxycholesterol were identified to be able to induce a significant pro-apoptotic effect if added to cell culture singly; 7ß-hydroxycholesterol had stronger action than other compounds. The enhanced production of reactive oxygen species through up-regulation of the colonic NADPH-oxidase isoform NOX1 appeared to be the key event in oxysterol-induced apoptosis in these colon cancer cells. As regards pro-inflammatory effects of oxysterols, IL-8 and MCP-1 were evaluated for their chemotactic activity. Only MCP-1 production was significantly induced by 7ß-hydroxycholesterol, as well as by cholesterol and oxysterol mixture. However, oxysterol-induced inflammation appeared to be NOX1-independent, suggesting a secondary role of this enzyme in inducing inflammation in colon cancer cells. A selective cell death induced by specific oxysterols against colon cancer cells, mainly exploiting their ability to activate NOX1 in generating oxidative reactions, might represent a promising field of investigation in colorectal cancer, and might bring new insights on strategies in anticancer therapy.

Plaque oxysterols induce unbalanced up-regulation of matrix metalloproteinase-9 in macrophagic cells through redox-sensitive signaling pathways: Implications regarding the vulnerability of atherosclerotic lesions.

An imbalance in the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) contributes to atherosclerotic plaque destabilization and rupture. Here we determined whether oxysterols accumulating in advanced atherosclerotic lesions play a role in plaque destabilization. In human promonocytic U937 cells, we investigated the effects of an oxysterol mixture of composition similar to that in advanced human carotid plaques on the expression and synthesis of MMP-9 and its endogenous inhibitors TIMP-1 and TIMP-2. A marked increment of MMP-9 gene expression, but not of its inhibitors, was observed by real-time RT-PCR; MMP-9 gelatinolytic activity was also found increased by gel zymography. Consistently, a net increment of MMP-9 protein level was also observed by immunoblotting. Using antioxidants or specific inhibitors or siRNAs, we demonstrated that the oxysterol mixture induces MMP-9 expression through: (i) overproduction of reactive oxygen species, probably by NADPH-oxidase and mitochondria; (ii) up-regulation of mitogen-activated protein kinase signaling pathways via protein kinase C; and (iii) up-regulation of activator protein-1- and nuclear factor-κB-DNA binding. These results suggest, for the first time, that oxysterols accumulating in advanced atherosclerotic lesions significantly contribute to plaque vulnerability by promoting MMP-9/TIMP-1/2 imbalance in phagocytic cells.

Pro-oxidant and proapoptotic effects of cholesterol oxidation products on human colonic epithelial cells: a potential mechanism of inflammatory bowel disease progression.

With the aim of investigating whether cholesterol oxidation products could contribute to the pathogenesis of the intestinal epithelial barrier dysfunction that occurs in human inflammatory bowel disease (IBD), differentiated versus undifferentiated CaCo-2 cells, an accepted model for human intestinal epithelial cells, were challenged with a dietary-representative mixture of oxysterols. Only differentiated colonic cells were susceptible to the proapoptotic action of the oxysterol mixture, checked both by enzymatic and by morphological methods, mainly because of a very low AKT phosphorylation pathway compared to the undifferentiated counterparts. Enhanced production of reactive oxygen species by a colonic NADPH oxidase hyperactivation seemed to represent the key event in oxysterol-induced up-regulation of the mitochondrial pathway of programmed death of differentiated CaCo-2 cells. These in vitro findings point to the pro-oxidant and cytotoxic potential of cholesterol oxidation products, of both dietary and endogenous origin, as an important mechanism of induction and/or worsening of the functional impairment of enteric mucosa that characterizes IBD.

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture.

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa.

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.

Early involvement of ROS overproduction in apoptosis induced by 7-ketocholesterol.

Cholesterol oxidation products are increasingly considered as much more bioactive than the parent compound in the multifactor and multistep process that characterizes atherosclerosis. In particular, 7-ketocholesterol has been reported to induce oxidative stress as well as a marked pro-apoptotic effect in vascular cells including macrophages. With the aim to investigate a possible pathogenic correlation between the two events, cultivated murine macrophages were challenged with a concentration of 7-ketocholesterol actually detectable in human vasculature. Conclusive proof was obtained of a primary role of NADPH-oxidase in the overproduction of reactive oxygen species within cells treated with the oxysterol. In addition, such oxidative burst occurred very early after cell intoxication and it was definitely demonstrated as able to lead cells to apoptotic death. In fact, two metabolic inhibitors of NADPH-oxidase and the antioxidant epicatechin very well counteracted 7-ketocholesterol-induced apoptosis by preventing the oxysterol pro-oxidant action.

Molecular mechanisms of calorie restriction's protection against age-related sclerosis.

The current knowledge on the molecular mechanisms of the protective effect of calorie restriction (CR) against age-related fibrosclerosis is tentatively reviewed with specific reference to the role of oxidative stress in aging. The effects of oxidative stress are often mediated by its own final products. Of these, 4-hydroxy-2,3-nonenal (HNE) induces the expression and synthesis of transforming growth factor beta1 (TGFbeta1) and activates nuclear binding of transcription factor activator protein 1 (AP-1) thus stimulating fibrogenesis. Several studies have shown that, as well as extending mean and maximum life span in a variety of species, CR delays the onset and slows the progression of a variety of age-associated diseases, including diabetes, cardiovascular diseases and neoplasia. However, the anti-aging mechanisms of CR are still not clearly understood. Of the numerous hypotheses put forward, one that still remains popular is protection against the age-associated increase of oxidative stress and consequent cell damage. CR protects the rat aorta from the age-related increase of both oxidative damage and fibrosis; as regards the possible mechanism/s of CR's protection against fibrosclerosis, it is conceivable that, by decreasing oxidative stress, CR reduces HNE levels and consequently TGFbeta1 expression and collagen deposition, likely by down-regulating the activation of Jun-N terminal kinase and of AP-1. Through the modulation of reactive oxygen species and oxidative stress CR may also attenuate the age-associated increase in the inflammatory milieu, thus preserving vascular functional integrity by suppressing the age-associated increase in inflammatory enzyme activities and prostanoids.

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells.

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.

Role of 4-hydroxy-2,3-nonenal in the pathogenesis of fibrosis.

Transient activation of fibroblasts or fibroblast-like cells to proliferate and to produce elevated quantities of extracellular matrix is essential to fibrosis. This activation is regulated by several cytokines produced by various inflammation-associated cells. Among these, transforming growth factor beta1 (TGFbeta1) is considered of major importance. Many studies have shown that lipid peroxidation play a key role in the initiation and progression of fibrosis in different organs. In fact, 4-hydroxy-2,3-nonenal (HNE), the major aldehydic product of lipid peroxidation, is able to induce TGFbeta1 expression and synthesis, and activation of activator protein-1 (AP-1) transcription factor. In this study, using the murine macrophage line J774-A1, we show that these effects are strictly related to the chemical structure of HNE, since neither 2-nonenal nor nonanal are biologically active to the same extent. Moreover, we demonstrate that HNE can indeed contribute to the onset of fibrosis by stimulating AP-1 binding to DNA and consequently inducing TGFbeta1 expression, since thiol-group reagents, such as N-ethylmaleimide and 4-(chloro-mercuri)-benzenesulfonic acid, that down-modulate HNE entrance and localisation inside the cell, prevent both phenomena. The possibility to control fibrogenic cytokine levels by means of antioxidant or dietetic treatments opens new potential pharmacological and nutritional horizons in the treatment of many chronic diseases characterised by excessive fibrosis.

4-hydroxynonenal and TGF-beta1 concur in inducing antiproliferative effects on the CaCo-2 human colon adenocarcinoma cell line.

4-Hydroxynonenal (HNE) has been demonstrated to exert its antiproliferative effect by up-regulating the c-Jun-N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family (MAPKs). Transforming growth factor-beta1 (TGF-beta1) is the major negative regulatory factor in controlling cell proliferation, and Smads are its intracellular transducers. Recent data on human colon adenocarcinoma has shown a low HNE content paralleled by a marked alteration of TGF-beta1 levels within the tumor mass. The two events appear related because of the demonstrated marked ability of HNE to up-regulate expression and synthesis of TGF-beta1; the combined decreases of HNE and TGF-beta1 found in cancer cells provide a favorable condition for neoplastic progression. Furthermore, HNE is likely able to interact with the cytokine to enhance apoptosis and increase intracellular reactive oxygen species (ROS) formation in the CaCo-2 colon carcinoma cell line. The probable mechanism whereby HNE and TGF-beta1 interact to induce apoptosis is through cross-talk between the main signaling pathways of the two molecules (JNK and Smads), and the observed ROS production might only contribute to amplifying the apoptotic pathways. The network between the two signaling pathways here involved is now under investigation.

Oxysterol mixtures prevent proapoptotic effects of 7-ketocholesterol in macrophages: implications for proatherogenic gene modulation.

Oxysterols are common components of oxidized low-density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7-ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7-ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase-9 activation, and eventually caspase-3 activation. Subsequent experiments showed that when 7-ketocholesterol was administered to cells together with another oxysterol, namely 7betaOH-cholesterol, the strong proapoptotic effect of 7-ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7-ketocholesterol, when administered alone, to strongly up-regulate the steady-state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up-regulation of proapoptotic factor, p21, after treatment with 7-ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.