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The important functions of the endothelium and the relationship between cardiovascular risk factors and endothelial dysfunction suggest the primary role of this tissue as a target for dietary strategies aimed at the prevention from related diseases. Cereals are key component of a healthy and balanced diet, and the presence of non-specific lipid-transfer protein 2 (nsLTP2) in wheat represents an added value to contribute to maintain the functionality of the vascular endothelium and consequently of the cardiovascular system. Indeed, nsLTP2 downregulates the expression of the main cell adhesion molecules induced by a pro-inflammatory cytokine and, meanwhile, upregulates heme oxigenase-1, exerting a cytoprotective/anti-inflammatory activity. Therefore, nsLTP2 might represent a food-derived tool to protect the vascular system against several pathological conditions.

Numerous studies support the protective role of bioactive peptides against cardiovascular diseases. Cereals represent the primary source of carbohydrates, but they also contain substantial amounts of proteins, therefore representing a potential dietary source of bioactive peptides with nutraceutical activities. The analysis of wheat extracts purified by chromatographic techniques by means of HPLC‐UV/nanoLC‐nanoESI‐QTOF allowed the identification of a signal of about 7 kDa which, following data base searches, was ascribed to a nonspecific lipid‐transfer protein (nsLTP) type 2 from Triticum aestivum(sequence coverage of 92%). For the first time nsLTP2 biological activities have been investigated. In particular, in experiments with human umbilical vein endothelial cells (HUVEC), nsLTP2 displayed antioxidant and cytoprotective activities, being able to significantly decrease reactive oxygen species (ROS) levels and to reduce lactate dehydrogenase (LDH) release, generated following oxidative (hydrogen peroxide) and inflammatory (tumor necrosis factor α, interleukin‐1β, and lipopolysaccharide) stimulation. The obtained promising results suggest potential protective role of nsLTP2 in vascular diseases prevention. nsLTP 2 peptide is resistant to proteases throughout the gastrointestinal tract and exerts antioxidant and cytoprotective activities. These characteristics could be exploited in vascular diseases prevention.

Sulforaphane, a biologically active isothiocyanate compound extracted from cruciferous vegetables, has been shown to exert cytotoxic effects on many human cancer cells, including leukemia. However, the exact molecular mechanisms behind the action of sulforaphane in hematological malignancies are still unclear. Like other cancer cells, leukemia cells produce high level of reactive oxygen species; in particular, hydrogen peroxide derived from Nox family is involved in various redox signal transduction pathways, promoting cell proliferation and survival. Recent evidence show that many tumour cell types express elevated level of aquaporin isoforms, and we previously demonstrated that aquaporin-8 acts as H2O2 transport facilitator across the plasma membrane of B1647 cells, a model of acute myeloid human leukemia. Thus, the control of AQP8-mediated H2O2 transport could be a novel strategy to regulate cell signalling and survival. To this purpose, we evaluated whether sulforaphane could somehow affect aquaporin-8-mediated H2O2 transport and/or Nox-mediated H2O2 production in B1647 cell line. Results indicated that sulforaphane inhibited both aquaporin-8 and Nox2 expression, thus decreasing B1647 cells viability. Moreover, the data obtained by coimmunoprecipitation technique demonstrated that these two proteins are linked to each other; thus, sulforaphane has an important role in modulating the downstream events triggered by the axis Nox2-aquaporin-8. Cell treatment with sulforaphane also reduced the expression of peroxiredoxin-1, which is increased in almost all acute myeloid leukemia subtypes. Interestingly, sulforaphane concentrations able to trigger these effects are achievable by dietary intake of cruciferous vegetables, confirming the importance of the beneficial effect of a diet rich in bioactive compounds.

Cancer cells are characterized by an increase in spatially localized reactive oxygen species (ROS) production and by an altered redox environment compared to normal cells. Consequently, signalling pathways that promote cell proliferation, survival, angiogenesis and metastasis are hyper-activated. In particular, hydrogen peroxide (H2O2) derived from NOX family is involved in various redox signal transduction pathways and the aquaporin 8 (AQP8) has been identified as a H2O2 transport facilitator across the plasma membrane. Recent evidence demonstrated that many tumor cell types express elevated level of aquaporin isoforms and highlighted a positive correlation between histological tumor grade and the AQP expression. Sulforaphane (SFN), an isothiocyanate compound present in abundance in cruciferous vegetables, has been found to induce therapeutic effects against a wide array of malignancies in both experimental and epidemiological studies . Therefore, this study aimed at the evaluation of the potential effect of SFN on the modulation of AQP8, NOX2 and p-VEGFR-2 expression in B1647 cell line, a model of acute myeloid leukemia. In fact, we previously demonstrated that AQP8 funnels NOX-derived H2O2, triggered by endogenously generated VEGF, which, in turn, provokes VEGFR-2 phosphorylation and the consequent modulation of many cellular activities, resulting in cell survival and proliferation. As peroxiredoxin (Prx1) represents a member of the so called thiol-based antioxidant system that acts as redox switches to modulate redox signalling pathways, the effect of SFN on Prx1 expression was also evaluated. Unravelling the role of AQP8 in cancer redox signalling, and the effect exerted by SFN on its modulation, can offer new potential target for anti-cancer therapy, suggesting the importance of dietary co-treatment.

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

Cereals and legumes are key components of a healthy and balanced diet. Accordingly, many national nutritional guidelines emphasize their health promoting properties by placing them at the base of nutritional food pyramids. This concept is further validated by the observed correlation between a lower risk and occurrence of chronic diseases and the adherence to dietary patterns, like the Mediterranean diet, in which cereal grains, legumes and derived products represent a staple food. In the search for a dietary approach to control/prevent chronic degenerative diseases, protein derived bioactive peptides may represent one such source of health-enhancing components. These peptides may already be present in foods as natural components or may derive from hydrolysis by chemical or enzymatic treatments (digestion, hydrolysis or fermentation). Many reports are present in the literature regarding the bioactivity of peptides in vitro and a wide range of activities has been described, including antimicrobial properties, blood pressure-lowering (ACE inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption/bioavailability, cyto- or immunomodulatory effects, and opioid-like activities. However it is difficult to translate these observed effects to human. In fact, the active peptide may be degraded during digestion, or may not be absorbed or reach the target tissues at a concentration necessary to exert its function. This review will focus on bioactive peptides identified in cereals and legumes, from an agronomical and biochemical point of view, including considerations about requirements for the design of appropriate clinical trials necessary for the assessment of their nutraceutical effect in vivo.

Background and aims Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipid-lowering and anti-inflammatory activity. However, its role in regulating LOX1-mediated signaling is still unknown. The aim of this study was to investigate the effect of BBR on oxLDL- and TNFα-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) and to compare it with that of lovastatin (LOVA). Methods and results Cytotoxicity was determined by lactate dehydrogenase assay. Antioxidant capacity was measured with chemiluminescent and fluorescent method and intracellular ROS levels through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively. HUVECs exposure to oxLDL (30 μg/ml) or TNFα (10 ng/ml) for 24 h led to a significant increase in LOX1 expression, effect abrogated by BBR (5 μM) and LOVA (5 μM). BBR but not LOVA treatment abolished the TNFα-induced cytotoxicity and restored the activation of Akt signaling. In spite of a low direct antioxidant capacity, both compounds reduced intracellular ROS levels generated by treatment of TNFα but only BBR inhibited NOX2 expression, MAPK/Erk1/2 signaling and subsequent NF-κB target genes VCAM and ICAM expression, induced by TNFα. Conclusions These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFα - induced LOX1 expression and oxidative stress, key events that lead to NOX, MAPK/Erk1/2 and NF-κB activation linked to endothelial dysfunction. Chemical compounds studied in this article Berberine (PubChem CID: 2353); Lovastatin (PubChem CID: 53232).

none 13 no Objectives: Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins also inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipid-lowering and anti-inflammatory activity. However, its role in regulating LOX1- mediated signalling is still unknown. The aim of this study was to compare the effects of BBR and lovastatin (LOVA) on oxLDL- and TNFa-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs). Methods: Cytotoxicity was determined by LDH assay. Antioxidant capacity was measured with chemiluminescent and ORAC method and intracellular ROS levels in HUVECs through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively. Results: HUVECs exposure to oxLDL (30mg/ml) or TNFa (10ng/ml) for 24h led to a significant increase in LOX1 expression, effect abrogated by BBR (5mM) and LOVA (1mM). BBR but not LOVA treatment abolished the TNFa induced cytotoxicity and restored the activation of Akt signalling. Although both compounds reduced intracellular ROS levels induced by H2O2 (100mM) or TNFa, only BBR inhibited NOX2 expression, MAPK/Erk signalling and subsequent NF-kB e related genes VCAM and ICAM expression, induced by TNFa. Conclusions: These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFa - induced LOX1 expression and oxidative stress, key events that lead to NOX2, MAPK/Erk and NF-kB activation linked to endothelial dysfunction. Cicero, A; Caliceti, C; Rizzo, P; Ferrari, R; Fortini, F; Aquila, G; Shehu, J; Leoncini, E; Zambonin, L; Rizzo, B; Guardigli, M; Roda, A; Hrelia, S Cicero, A; Caliceti, C; Rizzo, P; Ferrari, R; Fortini, F; Aquila, G; Shehu, J; Leoncini, E; Zambonin, L; Rizzo, B; Guardigli, M; Roda, A; Hrelia, S

The isothiocyanate sulforaphane (SF), abundant in Cruciferous vegetables, is known to induce antioxidant/detoxification enzymes in many cancer cell lines, but studies focused on its cytoprotective action in nontransformed cells are just at the beginning. Since we previously demonstrated that SF elicits cardioprotection through an indirect antioxidative mechanism, the aim of this study was to analyze the signaling pathways through which SF exerts its protective effects. Using cultured rat cardiomyocytes, we investigated the ability of SF to activate Akt/protein kinase B (PKB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways, which are implicated in cardiac cell survival, and to increase the phosphorylation of Nuclear factor E2-related factor 2 (Nrf2) and its binding to the antioxidant response element. By means of specific inhibitors, we demonstrated that the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway represents a mechanism through which SF influences both expression and activity of glutathione reductase, glutathione-S-transferase, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase-1, analyzed by western immunoblotting and spectrophotometric assay, respectively, and modulates Nrf2 binding and phosphorylation resulting in a cytoprotective action against oxidative damage. Results of this study confirm the importance of phase II enzymes modulation as cytoprotective mechanism and support the nutritional assumption of Cruciferous vegetables as source of nutraceutical cardioprotective agents.

Oxidative stress plays a major role in the pathophysiology of cardiac disorders, but the experimental data on the protective effects of exogenous antioxidants are controversial. A promising cardioprotective strategy may be through the induction of the endogenous antioxidants and phase II enzymes by chemical inducers. Sulforaphane is an isothiocyanate derived from cruciferous vegetables, and it has gained attention mainly as a potential chemopreventive agent in part through the induction of detoxifying enzymes. Accordingly, this study was undertaken to investigate the time-dependent induction of gene transcription, protein expression, and enzyme activity of antioxidant and phase II enzymes [glutathione reductase, glutathione-S-transferase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase-1, thioredoxin reductase] by sulforaphane in cultured rat neonatal cardiomyocytes. The potential cardioprotective action of sulforaphane was confirmed by the decrease in intracellular reactive oxygen species production, the increase in cell viability, and the decrease in DNA fragmentation after long-term treatment accompanied by the induction of antioxidants and phase II enzymes in cardiomyocytes.

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism.

Apha-1-adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha-1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved.

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.

Since in the aged heart an increased basal production of reactive oxygen species (ROS) has been demonstrated, and the resistance to ROS attack could be ameliorated by antioxidant supplementation, we verified the protective effect of selenium, as sodium selenite (SS) or seleno methionine (SM), in cultured rat cardiomyocytes aged in vitro. In normoxia, glutathione peroxidase (GPx) activity and total antioxidant activity were higher in old than in young cardiomyocytes, suggesting the existence of a compensatory increase of antioxidant defenses. When aged cells were submitted to hypoxia/reoxygenation, GPx activity was not modified; while total antioxidant activity decreased, conjugated diene level increased. Selenium supplementation, particularly as SM, was able to increase GPx, and consequently total antioxidant activity, and to decrease conjugated diene production. The observed ability of selenium supplementation to protect aged cardiomyocytes from hypoxia/reoxygenation damage underlines the importance of an optimal selenium dietary intake, particularly in the elderly.

Lunasin is a peptide whose anticancer properties are widely reported. Originally isolated from soybean seeds, lunasin was also found in cereal (wheat, rye, barley and Triticale), Solanum and amaranthus seeds. However, it was recently reported that searches of transcript and DNA sequence databases for wheat and other cereals failed to identify sequences with similarity to those encoding the lunasin peptide in soy. In order to clarify the presence or absence of lunasin in wheat varieties, a broad investigation based on chemical (LC-ESI-MS) and molecular (PCR) analyses was conducted. Both approaches pointed out the absence of lunasin in the investigated wheat genotypes; in particular no compounds with a molecular weight similar to that of lunasin standard and no lunasin-related sequences were found in the analysed wheat samples. These findings confirm the hypothesis, reported in recent researches, that lunasin is not a wheat-derived peptide.

Summary Background and aims Peroxidation of membrane lipids, altering cell integrity and function, plays an important part in the onset and development of cardiac damage following ischemia and reperfusion. Cells maintain their membrane lipid homeostasis by substituting peroxidized lipids with new polyunsaturated fatty acids. The microsomal enzymatic system converting essential fatty acids to highly unsaturated fatty acids (HUFAs) contributes to this repairing mechanism. The membrane of the endoplasmic reticulum could be one of the potential targets of free radicals generated in ischemia/reperfusion, thus causing a reduced efficacy of the system required for HUFA biosynthesis. To verify this hypothesis, and the consequent modification in fatty acid composition, we exposed cultured rat cardiomyocytes to different periods of hypoxia (H), eventually followed by reoxygenation (R). Furthermore, the effectiveness of antioxidants like α-tocopherol and a green tea extract in counteracting H/R damage towards HUFA biosynthesis was tested. Methods and results Linoleic (LA) and α-linolenic acid (ALA) conversion was measured by pre-labelling cells with [1- 14 C]LA or [1- 14 C]ALA for 1 h; total lipid fatty acid composition was determined by gas chromatographic analysis. H profoundly affected HUFA biosynthesis, and this effect was much more evident on LA than on ALA. Conversion of both substrates was partially restored during R due to the readmission of the final acceptor of the desaturating complex. Fatty acid composition data were in agreement with the modifications observed in essential fatty acid conversion. Antioxidant protection appeared to be related to the duration of H, and to be more effective during H than during R. Conclusion This study points out the importance of possessing good antioxidant defenses not only after, but mainly prior to the onset of H.

Extracts fromStevia rebaudianaBertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently,Steviaextracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies onSteviaand steviol glycosidesin vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercialSteviaextracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin andSteviaextracts increased the phosphorylation of PI3K and Akt. Furthermore,Steviaextracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis thatSteviaextracts could mimic insulin effects modulating PI3K/Akt pathway.

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems. Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases., This work was supported by grants from the Italian Ministry of Economic Development (Project “Made in Italy-over 50”), Emilia Romagna region, Italy (Project “BioPane”, CUP J31J09000430002) and the Italian Ministry of Agriculture (Project “Pane della salute” MiPAF-OIGA, CUP 31J09000370001) and by Fondazione del Monte di Bologna e Ravenna (Italy).

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules and occurs in a biphasic pattern: an early phase after 1–2 h and a late phase after 12–24 h. While it is widely accepted that reactive oxygen species are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H2O2-induced cardioprotection in rat neonatal cardiomyocytes. We focused on antioxidant and phase II enzymes and their modulation by protein kinase signaling pathways and nuclear-factor-E2-related factor-1 (Nrf1) and Nrf2. H2O2preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H2O2preconditioning counteracted oxidative stress-induced apoptosis by decreasing caspase-3 activity, increasing Bcl2 expression and selectively increasing the expression and activity of antioxidant and phase II enzymes through Nrf1 and Nrf2 translocation to the nucleus. The downregulation of Nrf1 and Nrf2 by small interfering RNA reduced the expression level of phase II enzymes. Specific inhibitors of phosphatidylinositol 3-kinase/Akt and p38 MAPK activation partially reduced the cardioprotection elicited by H2O2preconditioning and the induction and activity of phase II enzymes. These findings demonstrate, for the first time, a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes triggered by H2O2preconditioning.

Sulforaphane (SF), one of the most important isothiocyanates in the human diet, present in cruciferous vegetables, is known to have chemopreventive activities in different tissues. No data are available on its effects in the prevention of skeletal muscle damage. In this study, we investigated the potential protective effects of SF treatment on muscle damage and oxidative stress induced by an acute bout of exhaustive exercise in rats. Male Wistar rats were treated with SF (25 mg/kg body wt ip) for 3 days before undergoing an acute exhaustive exercise protocol in a treadmill (+7% slope and 24 m/min). Acute exercise resulted in a significant increase in plasma lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities. It also resulted in a significant increase in thiobarbituric acid-reactive substances, in a significant decrease in tissue total antioxidant capacity, and in a significant decrease in NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in vastus lateralis muscle. SF treatment significantly increased muscle NQO1, glutathione- S-transferase, and glutathione reductase expression and activity, with no effect on glutathione peroxidase and thioredoxin reductase. The observed SF-induced upregulation of phase II enzymes was accompanied by a significant increase in nuclear erythroid 2 p45-related factor 2 expression and correlated with a significant increase in total antioxidant capacity and a decrease in plasma LDH and CPK activities. Our data demonstrate that SF acts as an indirect antioxidant in skeletal muscle and could play a critical role in the modulation of the muscle redox environment, leading to the prevention of exhaustive exercise-induced muscle damage.

Ten benzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Several compounds showed a significant antiproliferative activity on K562 cells, although to a different extent, whereas compound 1i showed a highly significant activity on SW620 cells, comparable to that of doxorubicin. Both the substituents in the quinone ring and the position of the nitrogen atom in the pyridine moiety play a crucial role for the biological activity.

The possibility of enhancing glutathione peroxidase (GPx) activity and cytosolic total antioxidant activity (TAA) in normoxia and hypoxia/reoxygenation (H/R) by the supplementation of different concentrations of sodium selenite (SS) or selenomethionine (SM) was investigated in cultured rat cardiomyocytes. To assess the entity of oxidative stress due to H/R, levels of conjugated dienes containing lipids were determined. In normoxia, GPx activity and TAA increased in parallel with the increase in SS and SM supplementation. H/R did not influence GPx activity but lowered TAA; both SS and SM supplementations were effective in increasing GPx activity, the most effective concentration being 1 microM. At this SS and SM concentration, TAA returned to a normoxic value. Conjugated diene production, increased by H/R, was reduced by SS and SM supplementation, the 1 microM concentration appearing to be the most effective one. According to these data Se supplementation represents another possibility to counteract oxidative damage in the myocardium.

Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.

Some thiazolylbenzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Compounds 2d and 2e show a very good activity on K562 cells, whereas compounds 2a and 2b are active on SW620 cells. The importance of the methoxy group on the quinone moiety is confirmed and the function at 4-position of the thiazole ring plays a determining role for the activity.

The recognition that oxidative stress plays a major role in the pathophysiology of cardiac disorders has led to extensive investigation of the protective effects of exogenous antioxidants. Another strategy may be through induction of the endogenous antioxidants/phase 2 enzymes. Accordingly, this study was undertaken to investigate the inducibility of endogenous antioxidants/phase 2 enzymes (reduced glutathione (GSH), GSH peroxidase (GPx), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1)) by the isothiocyanate sulforaphane (SF) in cultured rat cardiomyocytes. Due to the role of the nuclear factors E2-related factor 1 (nrf1) and 2 (nrf2) in inducing phase 2 enzymes, we also investigated the role of SF in the translocation of these factors from cytosol to the nucleus. Enzymatic activities were determined spectrophotometrically, GSH by the fluorescent indicator monochlorobimane, reactive oxygen species (ROS) formation spectrofluorimetrically by 2′,7′-dichlorofluorescein diacetate, cell viability by MTT assay, nrf1 and nrf2 translocation to nucleus by laser confocal microscopy. GPx activity was not affected by SF, but GR, GST and NQO1 activities and GSH levels increased with a time-dependent pattern following SF supplementation. As a consequence SF supplementation led to decreased ROS levels and increased cell viability following oxidative stress. Laser confocal microscopy revealed the translocation of both nrf1 and nrf2 to the nucleus. In conclusion, SF acts as an indirect antioxidant leading to cardioprotection against oxidative stress.

This study investigated potential cardiotoxicity as exerted by Erbicin-derived-immunoagents (EDIAs), novel human anti-ErbB2 immunoagents engineered by fusion of a human anti-ErbB2 scFv, Erbicin, with either a human RNase or the Fc region of a human IgG1. EDIAs are strongly cytotoxic on ErbB2-positive cells in vitro and in vivo and bind to an epitope different from that of Herceptin, a humanized anti-ErbB2 mAb effective in the therapy of breast carcinoma, but cardiotoxic in a high percentage of cases. Toxicity and apoptosis were tested in vitro by 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT), DNA fragmentation, and immunoblotting analyses. Echocardiography was measured in mice after treatment with each immunoagent. Cardiac fibrosis and detection of apoptosis were examined by Sirius red staining of collagen and TUNEL assay, respectively. EDIAs were found in vitro to have no adverse effects on cardiac cells for which Herceptin is severely toxic. In vivo studies on a mouse model showed that the EDIAs did not alter cardiac function, whereas Herceptin and doxorubicin, used as positive controls, significantly reduced the fractional shortening parameter. Cardiac fibrosis and apoptosis were not significantly affected in mice treated with EDIAs. Thus, EDIAs could fulfill the therapeutic need of patients ineligible for Herceptin treatment due to cardiac dysfunction.

The maintenance of the endothelial function, challenged by oxidative stress and inflammation, is a fundamental target of preventive dietary strategies. The aim of this study was to investigate the protective activities of a wheat peptide belonging to the family of the "non-specific lipid transfer protein type 2" (nsLTP2). Results showed that HUVECs pretreatment with nsLTP2 significantly decreased both oxidative stress and cell damage induced by inflammation, and significantly counteracted the increased expression of adhesion molecules (VCAM-1, ICAM-1, E-selectin). Moreover, nsLTP2 increased the expression of HO-1, similarly to statins. As HO-1 has been shown to ameliorate inflammation in part through its ability to inhibit expression of endothelial adhesion molecules, experiments were also conducted using two HO-1 inhibitors, showing that the induced expression of HO-1 contributes to the anti-inflammatory activity exerted by nsLTP2. Taken together, obtained results suggest a nutraceutical role played by nsLTP2 in maintaining vascular health. This work was supported by BACCHUS project (FP7 European Commission Grant Agreement 312090)