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Extracellular vesicles (EVs) are nanosized vesicles released from most cell types that play a key role in cell-to-cell communication by carrying DNA, non-coding RNAs, proteins and lipids out of cells. The composition of EVs depends on the cell or tissue of origin and changes according to their pathophysiological conditions, making EVs a potential circulating biomarker of disease. Additionally, the natural tropism of EVs for specific organs and cells has raised the interest in their use as delivery vehicles. In this review, we provide an overview of EV biogenesis, isolation and characterization. We also discuss EVs in the context of endothelial pathophysiology, summarizing the current knowledge about their role in cell communication in quiescent and activated endothelial cells. In the last part, we describe the potential use of EVs as delivery vehicles of bioactive compounds and the current strategies to load exogenous cargo and to functionalize EVs to drive them to a specific tissue.

Abstract: A sedentary lifestyle has evoked a high risk of cardiovascular (CV) disease, diabetes, and obesity, all of them with high morbimortality rates and with a common denominator, hypertension. Numerous pharmacological drugs have been used for the treatment of hypertension. However, the side effects associated with the use of existing pharmacological therapies have triggered a demand for plant-based medications. In this connection, the aim of this review was to provide an in-depth analysis of the use of plant-derived bioactives for the effective management of hypertension. Phyto-constituents from leaves, bark, stem, roots, seeds, and fruits of medicinal plants grown in our differ-ent regions of the globe have been highly searched. Among them, polyphenols (e.g., flavonoids as quercetin, anthocyanins as cyanidin, tannins as ellagic acid, stilbenes as resveratrol, lignans as honokiol and others as hydroxytyrosol or curcumin), organosulfur compounds (e.g. s-allyl cysteine and allicin), fatty acids (e.g. α-lipoic acid, DHA and oleic acid), alkaloids (e.g. berberine or tetran-drine) and some terpenes have been intensively investigated for the management of hypertension, with effective ability being stated in controlling high blood pressure and related health problems both in vivo and in vitro studies. Some of the activities presented by these bioactive compounds are reducing oxidative stress, renin-angiotensin system control, SIRT1 activation, regulating platelet aggregation and COX activity, anti-atherogenic effects, anti-inflammatory properties, vasorelaxa-tion and other results that translate into the prevention or control of hypertension. The knowledge of these bioactive compounds is important in developing countries where traditional medicine is the majority, but it can also give rise to new approaches in hypertension therapy.

Simple Summary In recent years, aspartate has been increasingly acknowledged as a critical player in the metabolism of cancer cells which use this metabolite for nucleotide and protein synthesis and for redox homeostasis. Most intracellular aspartate derives from the mitochondrial catabolism of glutamine. To date at least four mitochondrial transporters have been involved in this metabolic pathway. Their involvement appears to be cancer type-specific and dependent on glutamine availability. Targeting these mitochondrial transporters may represent a new attractive strategy to fight cancer. The aim of this review is to dissect the role of each of these transporters in relation to the type of cancer and the availability of nutrients in the tumoral microenvironment. Abstract Aspartate has a central role in cancer cell metabolism. Aspartate cytosolic availability is crucial for protein and nucleotide biosynthesis as well as for redox homeostasis. Since tumor cells display poor aspartate uptake from the external environment, most of the cellular pool of aspartate derives from mitochondrial catabolism of glutamine. At least four transporters are involved in this metabolic pathway: the glutamine (SLC1A5_var), the aspartate/glutamate (AGC), the aspartate/phosphate (uncoupling protein 2, UCP2), and the glutamate (GC) carriers, the last three belonging to the mitochondrial carrier family (MCF). The loss of one of these transporters causes a paucity of cytosolic aspartate and an arrest of cell proliferation in many different cancer types. The aim of this review is to clarify why different cancers have varying dependencies on metabolite transporters to support cytosolic glutamine-derived aspartate availability. Dissecting the precise metabolic routes that glutamine undergoes in specific tumor types is of upmost importance as it promises to unveil the best metabolic target for therapeutic intervention.

Background: Increased levels of oxidative stress/cell inflammation contribute to colorectal cancer (CRC) onset. Nuclear factor-erythroid 2-related factor 2 (Nrf2) and its controlled growth factor erv1-like (Gfer) gene regulate redox‐sensitive and anti-inflammatory mechanisms, respectively, which can contribute to promoting cancer development. Aim: We evaluated Nrf2 and Gfer RNA expression and Nrf2 protein expression in colon mucosa in order to establish their possible involvement in the early stage of CRC. Methods: Forty subjects were enrolled after a histological evaluation of their colon biopsies. They included 20 subjects with a sporadic colorectal adenoma (SpCA group) and 20 without precancerous lesions (controls). Biopsy samples were processed for gene expression analysis and protein expression, using Real-time PCR and immunofluorescence confocal microscopy, respectively. Results: Nrf2 and Gfer mRNA expression were significantly reduced (p=0.007 and p

Background Glioma is the most common primary cancer in central nervous system, especially in brain. Paclitaxel (PTX) is a microtubule stabilizing agent with anticancer potential, but its clinical application to brain tumours is limited by drug resistance, side effects, and lower brain penetration. Purpose Herein we explored the in vitro effects, in glioma C6 cells, of the combination of PTX with curcumin, a natural compound with chemotherapeutic activity, in order to improve cytotoxic effects and overcome PTX limitations. Results Our data confirmed PTX antiproliferative activity that was improved by curcumin. These effects were confirmed by clonogenic assay and G0/G1 cell cycle arrest. PTX significantly promoted generation of intracellular reactive species (RS), while curcumin did not affect RS production; the combination of the two drugs resulted in a slight but significant increase in RS levels. Furthermore, we found a constitutive activation of NF-κB in C6 cell line that was inhibited by PTX and curcumin. Interestingly, combination of the drugs totally inhibited NF-κB nuclear translocation and reduced IκB phosphorylation. Our results also supported the involvement of p53-p21 axis in the anticancer effects of curcumin and PTX. The combination of the two drugs further increased p53 and p21 levels enhancing the antiproliferative effects. Furthermore, PTX plus curcumin most impressively activated caspase-3, effector of apoptosis pathways, and reduced the expression of the anti-apoptotic protein Bcl-2. Conclusion In conclusion, our findings demonstrated that combination of PTX and curcumin exerts a potentiated anti-glioma efficacy in vitro that may help in reducing dosage and/or minimizing side effects of cytotoxic therapy.

The novel Coronavirus, SARS-CoV-2 disease (COVID-19) was defined as a global pandemic and induced a severe public health crisis in 2020. Covid-19 viral infection targets the human respiratory system and, at present, no specific treatment has been identified even though certain drugs have been studied and considered apparently effective in viral progression by reducing the complications in the lung epithelium. Researchers and clinicians are still struggling to find a vaccine or a specific innovative therapeutic strategy to counter COVID-19 infection.Here we describe our study indicating that SARS-CoV-2 genome contains motif sequences in the 5´UTR leader sequence that can be selectively recognized by specific human non-coding RNAs (ncRNAs), such as micro and long non-coding RNAs (miRNAs and lncRNA). Notably, some of these ncRNAs have been already utilized as oligo-based drugs in pulmonary and virus-associated diseases. We identified three selective motifs at the 5´UTR leader sequence of SARS-CoV-2 that allow viral recognition and binding of a specific group of miRNAs, some of them characterized by “GU” seed alignments. Additionally, one seed motif within miRNAs has been found to be able to bind the 5’UTR leader sequence. Among miRNAs having thermodynamically stable binding site against leader sequence and that are able interacted with Spike transcript some are involved in pulmonary arterial hypertension and anti-viral response, i.e. miR-204, miR-3661, and miR-1343. Moreover, several miRNA candidates have been already validated in vivo and specific oligo sequence are indeed available for their inhibition or overexpression.Four lncRNAs (H19, Hotair, Fendrr, and LINC05) directly interact with spike transcript (mRNA) and viral genome.In conclusion, we suggest that specific miRNAs and lncRNAs can be potential candidates to design oligonucleotide-drugs to treat COVID-19 and that our study can provide candidate hypothesis to be eventually tested in further experimental studies.

The term &ldquo, normobaric oxygen paradox&rdquo, (NOP), describes the response to the return to normoxia after a hyperoxic event, sensed by tissues as oxygen shortage, and resulting in up-regulation of the Hypoxia-inducible factor 1&alpha, (HIF-1&alpha, ) transcription factor activity. The molecular characteristics of this response have not been yet fully characterized. Herein, we report the activation time trend of oxygen-sensitive transcription factors in human peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects after one hour of exposure to mild (MH), high (HH) and very high (VHH) hyperoxia, corresponding to 30%, 100%, 140% O2, respectively. Our observations confirm that MH is perceived as a hypoxic stress, characterized by the activation of HIF-1&alpha, and Nuclear factor (erythroid-derived 2)-like 2 (NRF2), but not Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB). Conversely, HH is associated to a progressive loss of NOP response and to an increase in oxidative stress leading to NRF2 and NF-kB activation, accompanied by the synthesis of glutathione (GSH). After VHH, HIF-1&alpha, activation is totally absent and oxidative stress response, accompanied by NF-kB activation, is prevalent. Intracellular GSH and Matrix metallopeptidase 9 (MMP-9) plasma levels parallel the transcription factors activation pattern and remain elevated throughout the observation time. In conclusion, our study confirms that, in vivo, the return to normoxia after MH is sensed as a hypoxic trigger characterized by HIF-1&alpha, activation. On the contrary, HH and VHH induce a shift toward an oxidative stress response, characterized by NRF2 and NF-kB activation in the first 24 h post exposure.

Exosome-like nanoparticles (ELNs) are attracting interest as important vehicles of intercellular communication, both in prokaryotes and eukaryotes. Recently, dietary nanoparticles similar to mammalian exosomes have attracted attention for these features. In particular they appear to be relevant in the modulation of several cellular processes as well as candidate carriers of bioactive molecules (proteins, lipids, and nucleic acids, including miRNAs) with therapeutic value. Herein, we investigated the cellular uptake of blueberry-derived ELNs (B-ELNs) by a human stabilized endothelial cell line (EA.hy926) and the ability of B-ELNs to modulate the expression of inflammatory genes as the response of tumor necrosis factor-&alpha, (TNF-&alpha, ). Our results indicate that 1) EA.hy926 cells internalize B-ELNs in a dose-dependent manner, 2) pretreatment with B-ELNs counters TNF-&alpha, induced reactive oxygen species (ROS) generation and loss of cell viability and modulates the differential expression of 29 genes (fold change &gt, 1.5) induced by TNF-&alpha, compared to control, 3) pathway analysis reveals their involvement in a total of 340 canonical pathways, 121 KEGG pathways, and 121 GO Biological processes, and 4) the intersection between differentially expressed (DE) genes and miRNAs contained in B-ELNs unveils a set of candidate target genes, such as prostaglandin I2 synthase (PTGIS), mitogen-activated protein kinase 14 (MAPK14), and phosphodiesterase 7A (PDE7A), for ELNs-contained cargo. In conclusion, our study indicates that B-ELNs can be considered candidate therapeutic carriers of bioactive compounds potentially able to protect vascular system against various stressors.

Pharmacological treatment of colorectal carcinoma currently proceeds through the administration of a combination of different chemotherapeutic agents. In the case of rectal carcinoma, radiation therapy also represents a therapeutic strategy. In an attempt at translating much-needed new targeted therapy to the clinics, p38 mitogen activated protein kinase (MAPK) inhibitors have been tested in clinical trials involving colorectal carcinoma patients, especially in combination with chemotherapy; however, despite the high expectations raised by a clear involvement of the p38 MAPK pathway in the response to therapeutic treatments, poor results have been obtained so far. In this work, we review recent insights into the exact role of the p38 MAPK pathway in response to currently available therapies for colorectal carcinoma, depicting an intricate scenario in which the p38 MAPK node presents many opportunities, as well as many challenges, for its perspective exploitation for clinical purposes.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour. UCP2 is shown in yeast and mammalian cells to transport aspartate out of mitochondria, thus enabling KRAS-mutated pancreatic ductal adenocarcinoma cells to perform glutaminolysis to support cancer growth.

The antioxidants role in cell response regulation attracted great interest in the last decades and it is undergoing to a profound reconsideration. The mere concept of "biological antioxidant" has been frequently misconceived or misused, possibly leading to the misinterpretation of some experimental observation. Organosulfur compounds in general and α-lipoic acid, a dithiol molecule, can be considered a typical example of the kind. Reduced α-lipoic acid, dehydrolipoic acid has been in fact originally considered a bona fide, reducing, electron donor molecule. A more recent approach, according to stoichiometric and thermodynamic evidences, lead to a reinterpretation of the biochemical role of "antioxidants". The electrophilic nature of oxidized nucleophilic molecules, including α-lipoic acid, renders more plausible a mechanism based on the ability to activate Nrf2/EpRE mediated hormetic response. In this study, we demonstrate that nmolar concentrations of oxidized α-lipoic acid, but not dehydrolipoic acid, protect human umbilical primary endothelial cells (HUVEC) from TNF-α induced dysfunction, inhibit NF-κB activation and block apoptosis following the activation of Nrf2 transcription factor. Our observations corroborate the concept that the major, if not the unique, mechanism by which α-lipoic acid can non-enzymatically exert its reducing activity is related to the electrophilic nature of the oxidized form.

Oxygen is a fundamental element for the life of a large number of living organisms allowing an efficient energetic utilization of substrates. Organisms relying on oxygen evolved complex structures for oxygen delivery and biochemical machineries dealing with its safe utilization and the ability to overcome the potentially harmful consequences of changes in oxygen availability. On fact, cells composing complex Eukaryotic organisms are set to live within an optimum narrow range of oxygen, quite specific for each cell type. Minute modifications of oxygen availability, either positive or negative, induce the expression of specific genes, the major actors of this responses being the transcription factors HIF and Nrf2 that control the attempt to cope with low oxygen (hypoxia) or to either high oxygen or to an oxygen "overflow," respectively. This review describes the interaction between these two transcription factors and their interaction with the transcription factor NF-κB acting as a pivotal determinant of final cell response. © 2018 BioFactors, 44(3):207-218, 2018.

Increased plasma levels of free fatty acids, including palmitic acid (PA), cause insulin resistance in endothelium characterized by a decreased synthesis of insulin-mediated vasodilator nitric oxide (NO), and by an increased production of the vasoconstrictor protein, endothelin-1. Several in vitro and in vivo studies suggest that anthocyanins, natural phenols commonly present in food and vegetables from Mediterranean Diet, exert significant cardiovascular health-promoting activities. These effects are possibly mediated by a positive regulation of the transcription factor Nrf2 and activation of cellular antioxidant and cytoprotective genes. The present study examined, at a molecular level, the effects of cyanidin-3-O-glucoside (C3G), a widely distributed anthocyanin, on PA-induced endothelial dysfunction and insulin resistance in human umbilical vein endothelial cells (HUVECs). Our results indicate that C3G pretreatment effectively reverses the effects of PA on PI3K/Akt axis, and restores eNOS expression and NO release, altered by PA. We observed that these effects were exerted by changes on the phosphorylation of IRS-1 on specific serine and tyrosine residues modulated by PA through the modulation of JNK and IKK activity. Furthermore, silencing Nrf2 transcripts demonstrated that the protective effects of C3G are directly related to the activation of Nrf2.

Background Breast cancer is worldwide commonly found malignancy in women and effective treatment is regarded as a huge clinical challenge even in the presence of several options. Extensive literature is available that demonstrating polyphenols, the richly introduce phytopharmaceuticals as anticancer agents. Among these polyphenols, resveratrol, silibinin, quercetin, genistein, curcumin reported to have an awesome potential against breast cancer. However, till now no comprehensive survey found about the anticarcinogenic properties of luteolin against breast cancer. Scope and approach This review targeted the available literature on luteolin in the treatment of breast cancer, effects in combination with other anticancer drugs with possible mechanisms. Key findings and conclusion An outstanding therapeutic potential of luteolin in the treatment of breast cancer has been recorded not just as a chemopreventive and chemotherapeutic agent yet complemented by its synergistic effects with other anticancer therapies such as cyclophosphamide, doxorubicin, and NSAID such as celecoxib, and possible underlying mechanisms. Ideally, this review will open new dimensions for luteolin as an effective and safe therapeutic agent in diminishing breast cancer.

Hyperglycemia contributes to dysregulate endothelial function associated with diabetes, leading to initiation and propagation of vascular complications and dysfunction. Caffeic acid (CA), a dietary hydroxycinnamic acid abundant in coffee, has been reported to exert antidiabetic effects in rat models. Herein, we investigated the molecular effects of physiological concentrations of CA (10 nM) against endothelial dysfunction induced by high glucose (HG) in human endothelial cells (HUVECs). HUVECs were exposed to HG 25 mM, to mimic diabetic condition, in presence of CA. Intracellular redox status (reduced glutathione, superoxide dismutase (SOD) and total antioxidant activity levels), and NF-κB pathway were examined. We also evaluated the involvement of NF-E2-related factor 2 (Nrf2)/electrophile responsive element (EpRE) pathway. Our data show that CA inhibits HG-induced nuclear translocation of NF-κB and the downstream expression of endothelial adhesion molecule 1 and restores antioxidant levels by upregulating Nrf2/EpRE pathway. Our data suggest that CA can suppress several aspects of HG-induced endothelial dysfunction through the modulation of intracellular redox status controlled by the transcription factor Nrf2. These findings highlight that low physiological concentration of CA achievable specifically upon food consumption are able to prevent endothelial dysfunction associated with inflammation and oxidative stress induced by high concentration of glucose. © 2016 BioFactors, 43(1):54-62, 2017.

Intestinal epithelium represents a protective physical barrier and actively contributes to the mucosal immune system. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF-κB signaling and inflammatory pathways in endothelial cells, efficiently triggers extravasation of neutrophils from the vasculature, therefore contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF-κB activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Many papers reported that anthocyanins, a group of compounds belonging to flavonoids, possess anti-inflammatory effects and modulate NF-κB activity. In this study, by using a coculture in vitro system, we aimed to evaluate the effects of TNF-α-stimulated intestinal cells on endothelial cells activation, as well as the protective effects of cyanidin-3-glucoside (C3G). In this model, TNF-α induced nuclear translocation of NF-κB and TNF-α and IL-8 gene expression in Caco-2 cells, whereas C3G pretreatment dose-dependently reduced these effects. Furthermore, TNF-α-stimulated Caco-2 cells induced endothelial cells activation with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF-κB levels in HUVECs, which were inhibited by C3G. We demonstrated that selective inhibition of the NF-κB pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. Thus, anthocyanins could contribute to the management of chronic gut inflammatory diseases.

Hyperglycemia contributes to dysregulate endothelial function associated with diabetes, leading to initiation and propagation of vascular complications and dysfunction. Caffeic acid (CA), a dietary hydroxycinnamic acid abundant in coffee, has been reported to exert antidiabetic effects in rat models. Herein, we investigated the molecular effects of physiological concentrations of CA (10 nM) against endothelial dysfunction induced by high glucose (HG) in human endothelial cells (HUVECs). HUVECs were exposed to HG 25 mM, to mimic diabetic condition, in presence of CA. Intracellular redox status (reduced glutathione, superoxide dismutase (SOD) and total antioxidant activity levels), and NF-κB pathway were examined. We also evaluated the involvement of NF-E2-related factor 2 (Nrf2)/electrophile responsive element (EpRE) pathway. Our data show that CA inhibits HG-induced nuclear translocation of NF-κB and the downstream expression of endothelial adhesion molecule 1 and restores antioxidant levels by upregulating Nrf2/EpRE pathway. Our data suggest that CA can suppress several aspects of HG-induced endothelial dysfunction through the modulation of intracellular redox status controlled by the transcription factor Nrf2. These findings highlight that low physiological concentration of CA achievable specifically upon food consumption are able to prevent endothelial dysfunction associated with inflammation and oxidative stress induced by high concentration of glucose. © 2016 BioFactors, 43(1):54-62, 2017.

The term hypoxia refers to conditions characterized by a relative restriction of oxygen supply. It is usually associated to a paradoxical overproduction of reactive oxygen species (ROS) and to the activation of several transcription factors, including HIF-1α, which in turn trigger angiogenic and apoptotic response. In this study we have investigated the mechanisms by which the anthocyanin cyanidin-3-O-glucoside (C3G) modulates hypoxia induced response in human endothelial cells (HUVECs). In fact, hypoxia induces an increase of ROS generation in HUVECs paralleled by a loss of antioxidant cellular capacity. According to the observed increase of HO-1 mRNA expression, pretreatment of C3G to HUVEC reduces the entity of oxidative stress thanks to the activation of cellular antioxidant response. C3G also attenuates HIF-1α protein accumulation conditions supporting the hypothesis of a major role of oxidative stress in the presence of low oxygen. Furthermore, the increased expression of angiogenesis and apoptosis markers (MMP-2 and caspase-3) due to HIF-1α activation by hypoxia is reduced in C3G pretreated cells. Overall, our data suggest that the modulation of intracellular redox status induced by C3G may be an important protective mechanism against endothelial damage in hypoxic conditions.

Chronic intestinal inflammatory disorders, such as Inflammatory Bowel Diseases (IBDs), are characterized by excessive release of proinflammatory mediators, intestinal barrier dysfunction and excessive activation of NF-kB cascade. Previous studies shown that TNF-α plays a central role in intestinal inflammation of IBDs and supported beneficial effects of flavonoids against chronic inflammatory diseases. In this study, we employed an in vitro model of acute intestinal inflammation using intestinal Caco-2 cells exposed to TNF-α. The protective effects of cyanidin-3-glucoside (C3G), an anthocyanin widely distributed in mediterranean diet, were then evaluated. Caco-2 cells exposure to TNF-α activated NF-kB proinflammatory pathway and induced IL6 and COX-2 expression. Cells pretreatment for 24h with C3G (20-40μM) prevented TNF-α-induced changes, and improved intracellular redox status. Our results demonstrated that C3G, also without any kind of stimulus, increased the translocation of the transcription factor Nrf2 into the nucleus so activating antioxidant and detoxifying genes. In conclusion, C3G exhibited protective effects through the inhibition of NF-kB signalling in Caco-2 cells and these beneficial effects appear to be due to its ability to activate cellular protective responses modulated by Nrf2. These data suggest that anthocyanins could contribute, as complementary or preventive approaches, to the management of chronic inflammatory diseases.

Acetaminophen (N-acetyl-p-aminophenol, APAP) overdosage can produce fatal centrilobular hepatic necrosis in humans. The present study attempted to investigate the protective effect of an anthocyanin-rich extract from bilberries and blackcurrants (AE) against APAP-induced acute hepatic damage in rats. Treatment with AE normalised blood activities of glutamate oxaloacetate and glutamate pyruvate transaminase and prevented APAP-induced plasmatic and tissutal alterations in biomarkers of oxidative stress, probably due to various bioproperties of the components of the extract.

Colorectal cancer is the fourth most common type of cancer worldwide, and adenocarcinoma cells that form the majority of colorectal tumors are markedly resistant to antineoplastic agents. Epidemiological studies have demonstrated that consumption of fruits and vegetables that are rich in polyphenols, is linked to reduced risk of colorectal cancer. In the present study, the effect of a standardized anthocyanin (ACN)‑rich extract on proliferation, apoptosis and cell cycle in the Caco-2 human colorectal cancer cell line was evaluated by trypan blue and clonogenic assays and western blot analysis of cleaved caspase‑3 and p21Waf/Cif1. The results of the current study demonstrated that the ACN extract markedly decreased Caco‑2 cell proliferation, induced apoptosis by activating caspase‑3 cleavage, and upregulated cyclin‑dependent kinase inhibitor 1 (p21Waf/Cif1) expression in a dose dependent manner. Furthermore, ACN extract was able to produce a dose‑dependent increase of intracellular reactive oxygen species (ROS) in Caco‑2 cells, together with a light increase of the cell total antioxidant status. In conclusion, the present study demonstrated that a standardized berry anthocyanin rich extract inhibited proliferation of Caco‑2 cells by promoting ROS accumulation, inducing caspase‑3 activation, and upregulating the expression of p21Waf/Cif1.

Free fatty acids (FFA), commonly elevated in diabetes and obesity, have been shown to impair endothelial functions and cause oxidative stress, inflammation, and insulin resistance. Anthocyanins represent one of the most important and interesting classes of flavonoids and seem to play a role in preventing cardiovascular diseases. Herein, we investigated the in vitro protective effects of cyanidin-3-O-glucoside (C3G) on cell signaling pathways in human umbilical vein endothelial cells (HUVECs) exposed to palmitic acid (PA), the most prevalent saturated FFA in circulation. Our data reported a significant augmentation of free radicals and oxidative stress in HUVECs exposed to PA for 3h, while C3G pretreatment improved intracellular redox status altered by FFA. Moreover, C3G significantly inhibited NF-κB proinflammatory pathway and adhesion molecules induced by PA, and these effects were attributed to the activation of Nrf2/EpRE pathway. In fact, C3G induced Nrf2 nuclear localization and activation of cellular antioxidant and cytoprotective genes at baseline and after PA exposure in endothelial cells. Our data confirm the hypothesis that natural Nrf2 inducers, such as C3G, might be a potential therapeutic strategy to protect vascular system against various stressors preventing several pathological conditions.