John T. Pinto

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrols properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

Resveratrol induces mitochondrial biogenesis in endothelial cells

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells.

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted H(2)O(2)-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular H(2)O(2) levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Anti-oxidative and anti-inflammatory vasoprotective effects of caloric restriction in aging: Role of circulating factors and SIRT1

Endothelial dysfunction, oxidative stress and inflammation are associated with vascular aging and promote the development of cardiovascular disease. Caloric restriction (CR) mitigates conditions associated with aging, but its effects on vascular dysfunction during aging remain poorly defined. To determine whether CR exerts vasoprotective effects in aging, aortas of ad libitum (AL) fed young and aged and CR-aged F344 rats were compared. Aging in AL-rats was associated with impaired acetylcholine-induced relaxation, vascular oxidative stress and increased NF-kappaB activity. Lifelong CR significantly improved endothelial function, attenuated vascular ROS production, inhibited NF-kappaB activity and down-regulated inflammatory genes. To elucidate the role of circulating factors in mediation of the vasoprotective effects of CR, we determined whether sera obtained from CR animals can confer anti-oxidant and anti-inflammatory effects in cultured coronary arterial endothelial cells (CAECs), mimicking the effects of CR. In CAECs cultured in the presence of AL serum TNFalpha elicited oxidative stress, NF-kappaB activation and inflammatory gene expression. By contrast, treatment of CAECs with CR serum attenuated TNFalpha-induced ROS generation and prevented NF-kappaB activation and induction of inflammatory genes. siRNA knockdown of SIRT1 mitigated the anti-oxidant and anti-inflammatory effects of CR serum. CR exerts anti-oxidant and anti-inflammatory vascular effects, which are likely mediated by circulating factors, in part, via a SIRT1-dependent pathway.

Age-Associated Vascular Oxidative Stress, Nrf2 Dysfunction, and NF-κB Activation in the Nonhuman Primate Macaca mulatta

Aging promotes oxidative stress in vascular endothelial and smooth muscle cells, which contribute to the development of cardiovascular diseases. NF-E2-related factor 2 (Nrf2) is a transcription factor, which is activated by reactive oxygen species in the vasculature of young animals, leading to adaptive upregulation of numerous reactive oxygen species detoxifying and antioxidant genes. The present study was designed to elucidate age-associated changes in the homeostatic role of Nrf2-driven free radical detoxification mechanisms in the vasculature of nonhuman primates. We found that carotid arteries of aged rhesus macaques (Macaca mulatta, age: ≥20 years) exhibit significant oxidative stress (as indicated by the increased 8-iso-PGF2α and 4-HNE content and decreased glutathione and ascorbate levels) as compared with vessels of young macaques (age:~10 years) that is associated with activation of the redox-sensitive proinflammatory transcription factor, nuclear factor-kappaB. However, age-related oxidative stress does not activate Nrf2 and does not induce Nrf2 target genes (NQO1, GCLC, and HMOX1). In cultured vascular smooth muscle cells (VSMCs) derived from young M mulatta, treatment with H(2)O(2) and high glucose significantly increases transcriptional activity of Nrf2 and upregulates the expression of Nrf2 target genes. In contrast, in cultured vascular smooth muscle cells cells derived from aged macaques, H(2)O(2)- and high glucose-induced Nrf2 activity and Nrf2-driven gene expression are blunted. High glucose-induced H(2)O(2) production was significantly increased in aged vascular smooth muscle cells compared with that in vascular smooth muscle cells from young M mulatta. Taken together, aging is associated with Nrf2 dysfunction in M mulatta arteries, which likely exacerbates age-related cellular oxidative stress, promoting nuclear factor-kappaB activation and vascular inflammation in aging.

Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

Recent studies demonstrate that age-related dysfunction of NF-E2-related factor-2 (Nrf2)-driven pathways impairs cellular redox homeostasis, exacerbating age-related cellular oxidative stress and increasing sensitivity of aged vessels to oxidative stress-induced cellular damage. Circulating levels of insulin-like growth factor (IGF)-1 decline during aging, which significantly increases the risk for cardiovascular diseases in humans. To test the hypothesis that adult-onset IGF-1 deficiency impairs Nrf2-driven pathways in the vasculature, we utilized a novel mouse model with a liver-specific adeno-associated viral knockdown of the Igf1 gene using Cre-lox technology (Igf1(f/f) + MUP-iCre-AAV8), which exhibits a significant decrease in circulating IGF-1 levels (~50%). In the aortas of IGF-1-deficient mice, there was a trend for decreased expression of Nrf2 and the Nrf2 target genes GCLC, NQO1 and HMOX1. In cultured aorta segments of IGF-1-deficient mice treated with oxidative stressors (high glucose, oxidized low-density lipoprotein, and H(2)O(2)), induction of Nrf2-driven genes was significantly attenuated as compared with control vessels, which was associated with an exacerbation of endothelial dysfunction, increased oxidative stress, and apoptosis, mimicking the aging phenotype. In conclusion, endocrine IGF-1 deficiency is associated with dysregulation of Nrf2-dependent antioxidant responses in the vasculature, which likely promotes an adverse vascular phenotype under pathophysiological conditions associated with oxidative stress (eg, diabetes mellitus, hypertension) and results in accelerated vascular impairments in aging.

Allyl mercaptan, a garlic-derived organosulfur compound, inhibits histone deacetylase and enhances Sp3 binding on the P21WAF1 promoter

Histone deacetylase (HDAC) inhibitors have the potential to derepress epigenetically silenced genes in cancer cells, leading to cell cycle arrest and apoptosis. In the present study, we screened several garlic-derived small organosulfur compounds for their ability to inhibit HDAC activity in vitro. Among the organosulfur compounds examined, allyl mercaptan (AM) was the most potent HDAC inhibitor. Molecular modeling, structure activity and enzyme kinetics studies with purified human HDAC8 provided evidence for a competitive mechanism (K(i) = 24 microM AM). In AM-treated human colon cancer cells, HDAC inhibition was accompanied by a rapid and sustained accumulation of acetylated histones in total cellular chromatin. Chromatin immunoprecipitation assays confirmed the presence of hyperacetylated histone H3 on the P21WAF1 gene promoter within 4 h of AM exposure, and there was increased binding of the transcription factor Sp3. At a later time, 24 h after AM treatment, there was enhanced binding of p53 in the distal enhancer region of the P21WAF1 gene promoter. These findings suggest a primary role for Sp3 in driving P21 gene expression after HDAC inhibition by AM, followed by the subsequent recruitment of p53. Induction of p21Waf1 protein expression was detected at time points between 3 and 72 h after AM treatment and coincided with growth arrest in G(1) of the cell cycle. The results are discussed in the context of other anticarcinogenic mechanisms ascribed to garlic organosulfur compounds and the metabolic conversion of such compounds to potential HDAC inhibitors in situ.

Effects of a series of organosulfur compounds on mitotic arrest and induction of apoptosis in colon cancer cells

We previously reported that the garlic-derived compound S-allylmercaptocysteine (SAMC) causes growth inhibition, mitotic arrest, and induction of apoptosis in SW480 human colon cancer cells by inducing microtubule depolymerization and c-Jun NH2 terminus kinase-1 activation. In the present study, we compared the aforementioned effects of SAMC to those of a series of garlic-derived and other organosulfur compounds. Among the 10 compounds tested, only SAMC, diallyl disulfide (DADS), and S-trityl-L-cysteine (trityl-cys) cause significant inhibition of cell growth with IC50 values of 150, 56, and 0.9 μmol/L, respectively. These three compounds also induce G2-M cell cycle arrest and apoptosis. Further studies reveal that, like SAMC, the garlic-derived compound DADS exerts antiproliferative effects by binding directly to tubulin and disrupting the microtubule assembly, thus arresting cells in mitosis and triggering mitochondria-mediated signaling pathways that lead to apoptosis. However, the synthetic compound trityl-cys exerts its effect on M-phase arrest and growth inhibition by mechanisms that involve spindle impairment but do not involve disruption of microtubule structure or dynamics. Furthermore, trityl-cys does not induce marked loss of mitochondrial membrane potential or release of cytochrome c, but it does induce caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Structure-function analysis suggests that both the allyl and the disulfide moieties are important features for the antiproliferative effects of SAMC and DADS. These findings may be useful in the identification, synthesis, and development of organosulfur compounds that have anticancer activity.

Inhibition of caspase-3 activity and activation by protein glutathionylation

Protein glutathionylation is a post-translational modification that may account for a broad mechanism of redox signaling. The caspase family of cysteine proteases represents a potential target for regulation by glutathionylation. To examine this, caspase proteins, derived from HL-60 cells after activation with actinomycin D, were incubated with GSSG. Total protein glutathionylation was enhanced and caspase-3 activity was inhibited in a dose- and time-dependent manner by GSSG. Caspase inhibition was reversible by thiol-specific reducing reagents. Proteolytic activation of caspases was also affected, as the activation of procaspase-3 and procaspase-9 in HL-60 cell extracts induced by cytochrome c and dATP was inhibited by pre-incubation with GSSG. When biotin-labeled GSSG was incubated with recombinant caspase-3, biotin label was found associated with both p12 and p17 subunits of active caspase-3 by non-reducing SDS-PAGE. Caspase-3 glutathionylation was confirmed by matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis of GSSG-treated recombinant caspase-3. Specific sites of glutathionylation were identified as Cys(135) of the p17 protein (the active site) and Cys(45) of the p12 protein. These results indicate that glutathionylation of caspase can occur at physiologically relevant concentrations of GSSG and results in the inhibition of caspase activation and activity.

Antiproliferative Effects of Allium Derivatives from Garlic

There is increasing evidence that allium derivatives from garlic have significant antiproliferative actions on human cancers. Both hormone-responsive and hormone-unresponsive cells lines respond to these derivatives. The effects shown by allium derivatives include induction of apoptosis, regulation of cell cycle progression and modification of pathways of signal transduction. Allium derivatives appear to regulate nuclear factors involved in immune function and inflammation, as well as in cellular proliferation. Our own studies indicate that allium derivatives inhibit proliferation of the human prostate cancer cell line (LNCaP) and the human breast cancer cell line (MCF-7). Further research is required to clarify the mechanisms of inhibition of cellular proliferation by allium derivatives and to explore their potential application to cancer prevention and control.