Luis Goya

Cocoa flavonoids up-regulate antioxidant enzyme activity via the ERK1/2 pathway to protect against oxidative stress-induced apoptosis in HepG2 cells

Oxidative stress is widely recognized as an important mediator of apoptosis in liver cells and plays a pivotal role in the pathogenesis of several diseases. Cocoa flavonoids have shown a powerful antioxidant activity providing protection against oxidation and helping prevent oxidative stress-related diseases. However, the molecular mechanisms responsible for this protection are not fully understood. Thus, in this study we investigated the protective effect of a cocoa polyphenolic extract (CPE) against tert-butyl hydroperoxide (t-BOOH)-induced apoptosis and the molecular mechanisms involved in this process. Incubation of HepG2 cells with t-BOOH induced apoptosis as evidenced by caspase-3 activation. This effect was accompanied by increased reactive oxygen species formation and by transient activation of the extracellular regulated kinases (ERKs) as well as sustained activation of the c-Jun N-terminal kinases (JNKs). On the contrary, pretreatment of HepG2 cells with CPE prevented apoptosis through the reduction of reactive oxygen species generation and the modulation of the apoptotic pathways activated by t-BOOH. CPE treatment also activated survival signaling proteins, such as protein kinase B (AKT) and ERKs, and increased the activities of two antioxidant enzymes, glutathione peroxidase (GPx) and glutathione reductase (GR). ERKs implication on GPx and GR induction and the protective effect of CPE against t-BOOH-induced oxidative stress and apoptosis were confirmed through experiments with selective inhibitors. These findings suggest that CPE is an effective inductor of GPx and GR activities via ERK activation and that this up-regulation seems to be required to attenuate t-BOOH-induced injury.

Quercetin modulates Nrf2 and glutathione-related defenses in HepG2 cells: Involvement of p38

Dietary flavonoid quercetin has been suggested as a cancer chemopreventive agent, but the mechanisms of action remain unclear. This study investigated the influence of quercetin on p38-MAPK and the potential regulation of the nuclear transcription factor erythroid-2p45-related factor (Nrf2) and the cellular antioxidant/detoxifying defense system related to glutathione (GSH) by p38 in HepG2 cells. Incubation of HepG2 cells with quercetin at a range of concentrations (5-50μM) for 4 or 18h induced a differential effect on the modulation of p38 and Nrf2 in HepG2 cells, 50μM quercetin showed the highest activation of p38 at 4h of treatment and values of p38 similar to those of control cells after 18 h of incubation, together with the inhibition of Nrf2 at both incubation times. Quercetin (50μM) induced a time-dependent activation of p38, which was in concert with a transient stimulation of Nrf2 to provoke its inhibition afterward. Quercetin also increased GSH content, mRNA levels of glutamylcysteine-synthetase (GCS) and expression and/or activity of glutathione-peroxidase, glutathione-reductase and GCS after 4h of incubation, and glutathione-S-transferase after 18h of exposure. Further studies with the p38 specific inhibitor SB203580 showed that the p38 blockage restored the inhibited Nrf2 transcription factor and the enzymatic expression and activity of antioxidant/detoxificant enzymes after 4h exposure. In conclusion, p38-MAPK is involved in the mechanisms of the cell response to quercetin through the modulation of Nrf2 and glutathione-related enzymes in HepG2 cells.

Protection of Human HepG2 Cells against Oxidative Stress by Cocoa Phenolic Extract

Cocoa is a rich source of flavanols and procyanidin oligomers with antioxidative properties, providing protection against oxidation and nitration. The present study investigated the potential protective effect of a polyphenolic extract from cocoa on cell viability and antioxidant defenses of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Pretreatment of cells with 0.05-50 microg/mL of cocoa polyphenolic extract (CPE) for 2 or 20 h completely prevented cell damage and enhanced activity of antioxidant enzymes induced by a treatment with t-BOOH. Moreover, lower levels of GSH caused by t-BOOH in HepG2 cells were partly recovered by a pretreatment with CPE. Increased reactive oxygen species (ROS) induced by t-BOOH was dose-dependently prevented when cells were pretreated for 2 or 20 h with CPE. These results show that treatment of HepG2 in culture with CPE (within the physiological range of concentrations) confers a significant protection against oxidation to the cells.

Hydroxytyrosol induces antioxidant/detoxificant enzymes and Nrf2 translocation via extracellular regulated kinases and phosphatidylinositol‐3‐kinase/protein kinase B pathways in HepG2 cells

Hydroxytyrosol (HTy) is a natural polyphenol abundant in olive oil, which possesses multiple biological actions. Particularly, HTy has cytoprotective activity against oxidative-stress-induced cell damage, but the underlying mechanisms of action remain unclear. Here, we have investigated the molecular mechanism involved in the protection exerted by HTy on tert-butyl hydroperoxide-induced damage in human HepG2 liver cells. Treatment of HepG2 cells with HTy increased the expression and the activity of glutathione-related enzymes such as glutathione peroxidase, glutathione reductase and glutathione S-transferase. HTy also induced the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Moreover, two important signalling proteins involved in Nrf2 translocation, the protein kinase B and the extracellular regulated kinases, were also activated by HTy. Further studies with specific inhibitors confirmed that both molecular pathways are critical for the nuclear translocation of Nrf2, the increased enzyme expression and activity and the beneficial effect against oxidative stress induced by HTy. In conclusion, together with the inherent radical scavenging activity of HTy, our results provide an additional mechanism of action to prevent oxidative stress damage through the modulation of signalling pathways involved in antioxidant/detoxifying enzymes regulation.

Epicatechin induces NF-κB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) via phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) signalling in HepG2 cells

The dietary flavonoid epicatechin has been reported to exhibit a wide range of biological activities. The objective of the present study was to investigate the time-dependent regulation by epicatechin on the activity of the main transcription factors (NF-kappaB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor (Nrf2)) related to antioxidant defence and survival and proliferation pathways in HepG2 cells. Treatment of cells with 10 microm-epicatechin induced the NF-kappaB pathway in a time-dependent manner characterised by increased levels of IkappaB kinase (IKK) and phosphorylated inhibitor of kappaB subunit-alpha (p-IkappaBalpha) and proteolytic degradation of IkappaB, which was consistent with an up-regulation of the NF-kappaB-binding activity. Time-dependent activation of the AP-1 pathway, in concert with enhanced c-Jun nuclear levels and induction of Nrf2 translocation and phosphorylation were also demonstrated. Additionally, epicatechin-induced NF-kappaB and Nrf2 were connected to reactive oxygen species intracellular levels and to the activation of cell survival and proliferation pathways, being phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) associated to Nrf2 modulation and ERK to NF-kappaB induction. These data suggest that the epicatechin-induced survival effect occurs by the induction of redox-sensitive transcription factors through a tight regulation of survival and proliferation pathways.

Cocoa flavonoids attenuate high glucose-induced insulin signalling blockade and modulate glucose uptake and production in human HepG2 cells.

Insulin resistance is the primary characteristic of type 2 diabetes. Cocoa and its main flavanol, (-)-epicatechin (EC), display some antidiabetic effects, but the mechanisms for their preventive activities related to glucose metabolism and insulin signalling in the liver remain largely unknown. In the present work, the preventive effect of EC and a cocoa polyphenolic extract (CPE) on insulin signalling and on both glucose production and uptake are studied in insulin-responsive human HepG2 cells treated with high glucose. Pre-treatment of cells with EC or CPE reverted decreased tyrosine-phosphorylated and total levels of IR, IRS-1 and -2 triggered by high glucose. EC and CPE pre-treatment also prevented the inactivation of the PI3K/AKT pathway and AMPK, as well as the diminution of GLUT-2 levels induced by high glucose. Furthermore, pre-treatment of cells with EC and CPE avoided the increase in PEPCK levels and the diminished glucose uptake provoked by high glucose, returning enhanced levels of glucose production and decreased glycogen content to control values. These findings suggest that EC and CPE improved insulin sensitivity of HepG2 treated with high glucose, preventing or delaying a potential hepatic dysfunction through the attenuation of the insulin signalling blockade and the modulation of glucose uptake and production.

Procyanidin B2 induces Nrf2 translocation and glutathione S-transferase P1 expression via ERKs and p38-MAPK pathways and protect human colonic cells against oxidative stress

PurposeProcyanidin B2 (PB2) is a naturally occurring flavonoid widely found in cocoa, red wine and grape juice. Recent studies have suggested that PB2 could protect against oxidative stress- and chemical-induced injury in colonic cells by modulating the endogenous cellular defence. However, the precise mechanism for this protection is not fully understood. Herein, we examined the effect of PB2 on the expression of one of the major antioxidant/detoxificant enzymes related to intestinal protection, the glutathione S-transferase P1 (GSTP1), and the molecular mechanisms involved.MethodsHuman colonic Caco-2 cells were treated with PB2 at different times and enzymatic activity, and mRNA and protein levels of GSTP1 were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation states of specific proteins central to intracellular signalling cascades were also investigated.ResultsPB2 induced the expression and activity of GSTP1 and the nuclear translocation of Nrf2. Interestingly, two important signalling proteins involved in Nrf2 translocation, the extracellular signal-regulated protein kinases (ERKs) and the p38 mitogen-activated protein kinase (MAPK) were also activated. Further experiments with specific inhibitors of both pathways confirmed their critical role in the beneficial effects induced by PB2.ConclusionsThe present results show that PB2 protects against oxidative injury in colonic cells and up-regulate the expression of GSTP1 via a mechanism that involves ERK and p38 MAPK activation and Nrf2 translocation. These results provide a molecular basis for the potential contribution of PB2 in the prevention of oxidative stress-related intestinal injury and gut pathologies.

Potential for preventive effects of cocoa and cocoa polyphenols in cancer.

Prevention of cancer through the diet is receiving increasing interest, and cocoa because of its polyphenolic compounds has become an important potential chemopreventive and therapeutic natural agent. Cocoa and its main polyphenols have been reported to interfere at the initiation, promotion and progression of cancer. Cocoa flavonoids have been demonstrated to influence several important biological functions in vitro and in vivo by their free radical scavenging ability or through the regulation of signal transduction pathways to stimulate apoptosis and to inhibit inflammation, cellular proliferation, apoptosis, angiogenesis and metastasis. Nevertheless, these molecular mechanisms of action are not completely characterized and many features remain to be elucidated. The aim of this review is to provide insights into the molecular basis of the potential chemopreventive activity of cocoa and its polyphenolic components by summarizing cell culture and animal models studies, as well as interventional and epidemiological studies on humans.

Cocoa flavonoid epicatechin protects pancreatic beta cell viability and function against oxidative stress

SCOPE Diabetes mellitus is associated with reductions in glutathione, supporting the critical role of oxidative stress in its pathogenesis. Antioxidant food components such as flavonoids have a protective role against oxidative stress-induced degenerative and age-related diseases. Flavonoids such as epicatechin (EC) constitute an important part of the human diet; they can be found in green tea, grapes, and cocoa and possess multiple biological activities. This study investigates the chemo-protective effect of EC against oxidative stress induced by tert-butylhydroperoxide (t-BOOH) on Ins-1E pancreatic beta cells. METHODS AND RESULTS Cell viability, oxidative status, phosphorylated Jun kinase (p-JNK) expression, and insulin secretion were evaluated. Ins-1E cells treatment with 5-20 μM EC for 20 h evoked no cell damage and enhanced antioxidant enzymes and insulin secretion. Addition of 50 μM t-BOOH for 2 h induced reactive oxygen species, p-JNK, and carbonyl groups and decreased GSH and insulin secretion. Pretreatment of cells with EC prevented the t-BOOH-induced reactive oxygen species, carbonyl groups, p-JNK expression and cell death, and recovered insulin secretion. CONCLUSION Ins-1E cells treated with EC showed a remarkable recovery of cell viability and insulin secretion damaged by t-BOOH, indicating that integrity of secreting and surviving machineries in the EC-treated cells was notably protected against the oxidative insult.

Quercetin modulates NF-κB and AP-1/JNK pathways to induce cell death in human hepatoma cells

Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. The aim of this study was to investigate the regulatory effect of quercetin (50 μ M) on two main transcription factors (NF-κ B and AP-1) related to survival/proliferation pathways in a human hepatoma cell line (HepG2) over time. Quercetin induced a significant time-dependent inactivation of the NF-κ B pathway consistent with a downregulation of the NF-κ B binding activity (from 15 min onward). These features were in concert with a time-dependent activation (starting at 15 min and maintained up to 18 h) of the AP-1/JNK pathway, which played an important role in the control of the cell death induced by the flavonoid and contributed to the regulation of survival/proliferation (AKT, ERK) and death (caspase-3, p38, unbalance of Bcl-2 proapoptotic and antiapoptotic proteins) signals. These data suggest that NF-κ B and AP-1 play a main role in the tight regulation of survival/proliferation pathways exerted by quercetin and that the sustained JNK/AP-1 activation and inhibition of NF-κ B provoked by the flavonoid induced HepG2 death.