Epidermal growth factor protects against myocardial ischaemia reperfusion injury through activating Nrf2 signalling pathway

Alleviating the oxidant stress associated with myocardial ischaemia reperfusion has been demonstrated as a potential therapeutic approach to limit ischaemia reperfusion (I/R)-induced cardiac damage. It is reported that EGFR/erbB2 signalling is an important cardiac survival pathway in cardiac function and activation of EGFR has a cardiovascular effect in global ischaemia. Epidermal growth factor (EGF), a typical EGFR ligand, was considered to have a significant role in activating EGFR. However, no evidence has been published whether exogenous EGF has protective effects on myocardial ischaemia reperfusion. This study aims to investigate the effects of EGF in I/R-induced heart injury and to demonstrate its mechanisms. H9c2 cells challenged with H₂O₂ were used for in vitro biological activity and mechanistic studies. The malondialdehyde (MDA) and Superoxide Dismutase (SOD) levels in H9c2 cells were determined, and the cell viability was assessed by MTT assay. Myocardial I/R mouse administrated with or without EGF were used for in vivo studies. Pretreatment of H9c2 cells with EGF activated Nrf2 signalling pathway, attenuated H₂O₂-increased MDA and H₂O₂-reduced SOD level, followed by the inhibition of H₂O₂-induced cell death. In in vivo animal models of myocardial I/R, administration of EGF reduced infarct size and myocardial apoptosis. These data support that EGF decreases oxidative stress and attenuates myocardial ischaemia reperfusion injury via activating Nrf2.     

Methylglyoxal induces cell death through endoplasmic reticulum stress‐associated ROS production and mitochondrial dysfunction

Diabetic retinopathy (DR) and age‐related macular degeneration (AMD) are two important leading causes of acquired blindness in developed countries. As accumulation of advanced glycation end products (AGEs) in retinal pigment epithelial (RPE) cells plays an important role in both DR and AMD, and the methylglyoxal (MGO) within the AGEs exerts irreversible effects on protein structure and function, it is crucial to understand the underlying mechanism of MGO‐induced RPE cell death. Using ARPE‐19 as the cell model, this study revealed that MGO induces RPE cell death through a caspase‐independent manner, which relying on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, intracellular calcium elevation and endoplasmic reticulum (ER) stress response. Suppression of ROS generation can reverse the MGO‐induced ROS production, MMP loss, intracellular calcium increase and cell death. Moreover, store‐operated calcium channel inhibitors MRS1845 and YM‐58483, but not the inositol 1,4,5‐trisphosphate (IP3) receptor inhibitor xestospongin C, can block MGO‐induced ROS production, MMP loss and sustained intracellular calcium increase in ARPE‐19 cells. Lastly, inhibition of ER stress by salubrinal and 4‐PBA can reduce the MGO‐induced intracellular events and cell death. Therefore, our data indicate that MGO can decrease RPE cell viability, resulting from the ER stress‐dependent intracellular ROS production, MMP loss and increased intracellular calcium increase. As MGO is one of the components of drusen in AMD and is the AGEs adduct in DR, this study could provide a valuable insight into the molecular pathogenesis and therapeutic intervention of AMD and DR.     

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.     

Igalan induces detoxifying enzymes mediated by the Nrf2 pathway in HepG2 cells

Igalan is one of the sesquiterpene lactones found in Inula helenium L., which is used as the traditional medicine to treat inflammatory diseases. However, the pharmacological effects of igalan have not been characterized. In this study, we isolated igalan from I. helenium L. and evaluated the effects of igalan on signaling pathways and expression of target genes in HepG2 cells. Igalan activated the nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway by increasing the inactive form of GSK3β, the phosphorylated form of AKT, and the nuclear accumulation of Nrf2. Thus, target genes of Nrf2 such as HO‐1 and NQO1 increased in HepG2 cells. Moreover, igalan inhibited the tumor necrosis factor‐α (TNF‐α)‐induced nuclear factor‐κB activation and suppressed the expression of its target genes, including TNF‐α, interleukin (IL)‐6, and IL‐8 in HepG2 cells. Our results indicate the potential of igalan as an activator of cellular defense mechanisms and a detoxifying agent.     

Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions.     

Role of oxidative stress and caspase 3 in CD47-mediated neuronal cell death

CD47 or integrin-associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin-1 or the specific CD47-activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time-dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways.     

Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation,ROS generation and mitochondrial dysfunction

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.     

Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines

Recent evidence suggests that reactive oxygen species (ROS) play an important role in the pathogenesis of various illnesses, and the ROS and antioxidant enzymes are highly associated with cell differentiation and diseases. In this study, we tested the hypothesis that specific antioxidant enzymes are differentially expressed in hepatocellular carcinoma (HCC) cell lines with various degrees of differentiation. We compared the expression of several antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GRx), and glutathione peroxidase (GPx) in five HCC cell lines with well (Hep G2 and Hep 3B) or poor (HA22T/VGH, HA55T/VGH, and SK-Hep-1) differentiation. Our results showed that both well-differentiated HCC cell lines expressed extremely higher CAT and GRx enzyme activities than all three poorly differentiated ones. Moreover, the protein and mRNA levels of CAT were much higher in two well-differentiated HCC cell lines than in all three poorly differentiated ones. Both well-differentiated HCC cell lines also showed a higher protein or mRNA expression of Cu/ZnSOD and MnSOD than three poorly differentiated ones. Our results demonstrate that specific antioxidant enzymes (especially, CAT and GRx) are differentially expressed in HCC cell lines with well or poor differentiation. These findings suggest that CAT and GRx are two potential differentiation markers for HCC.     

Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and oxidant-dependent apoptosis in Xenopus laevis oocytes

Ceramide regulates many cellular processes, including cell growth, differentiation, and apoptosis. Although the effects of exogenous bacterial neutral sphingomyelinase (SMase) in Xenopus laevis oocytes have been investigated, its microinjection into oocytes has not been reported previously. Thus, we compared the incubation versus microinjection of the neutral Bacillus cereus sphingomyelinase (bSMase) to examine whether the topology of ceramide generation determines its effects on the fate of oocytes. In agreement with previous findings, incubation of mature stage VI oocytes with bSMase increased ceramide levels in oocyte extracts over time, causing the germinal vesicle breakdown indicative of maturation, without evidence of cytotoxicity. In contrast, bSMase microinjection, which increased ceramide levels in a time- and dose-dependent manner, resulted in oocyte apoptosis characterized by reactive oxygen species (ROS) generation, reduced glutathione (GSH) depletion in cytosol and mitochondria, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Microinjection of acidic SMase from human placenta recapitulated the apoptotic effects of bSMase microinjection. Preincubation of oocytes with GSH-ethyl ester before bSMase microinjection prevented ROS generation and mitochondrial downstream events, thus protecting oocytes from bSMase-induced death. These findings show a divergent action of bSMase-induced ceramide on oocyte maturation or apoptosis depending on the intracellular site where ceramide is generated.     

Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.