NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Modulation of NRF2 signaling pathway by nuclear receptors: Implications for cancer

Nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also known as Nfe2l2) plays a critical role in regulating cellular defense against electrophilic and oxidative stress by activating the expression of an array of antioxidant response element-dependent genes. On one hand, NRF2 activators have been used in clinical trials for cancer prevention and the treatment of diseases associated with oxidative stress; on the other hand, constitutive activation of NRF2 in many types of tumors contributes to the survival and growth of cancer cells, as well as resistance to anticancer therapy. In this review, we provide an overview of the NRF2 signaling pathway and discuss its role in carcinogenesis. We also introduce the inhibition of NRF2 by nuclear receptors. Further, we address the biological significance of regulation of the NRF2 signaling pathway by nuclear receptors in health and disease. Finally, we discuss the possible impact of NRF2 inhibition by nuclear receptors on cancer therapy.     

Down-Regulation of ID2-AS1 Alleviates the Neuronal Injury Induced by 1-Methy1-4-Phenylpyridinium in Human Neuroblastoma Cell Line SH-SY5Y Cells Through Regulating miR-199a-5p/IFNAR1/JAK2/STAT1 Axis

We aimed to illustrate the roles and molecular mechanisms of ID2-AS1 in parkinson’s disease (PD). Methods: qRT-PCR detected the expression of ID2-AS1. CCK-8, LDH release assays the effect of ID2-AS1 knockdown on PD cells. Flow cytometry and Western Blot were used to detect the effect of ID2-AS1 inhibition on PD cell apoptosis. ELISA analysis showed that ID2-AS1 inhibition can reduce the inflammation of PD cells. ROS activity assay showed that inhibiting ID2-AS1 attenuated the oxidative stress induced by 1-methy1-4-phenylpyridinium (MPP+). RNA binding protein immunoprecipitation assay showed that ID2-AS1 is mainly located in the cytoplasm. The luciferase reporter assay is used to verify the interaction. In our study, ID2-AS1 was concentration-dependently and time-dependently up-regulated in MPP+?-treated human neuroblastoma cell line SH-SY5Y. ID2-AS1 knockdown enhanced cell proliferation and decreased cell death in PD cells. Knockdown of ID2-AS1 attenuates MPP+?-induced cytotoxicity in SH-SY5Y cells. ID2-AS1 is a sponge of miR-199a-5p. IFNAR1 is a target of miR-199a-5p. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+?triggered neuronal injury. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+?-triggered JAK2/STAT1 activation. Overall, down-regulation of ID2-AS1 alleviated the neuronal injury in PD through regulating miR-199a-5p/IFNAR1/JAK2/STAT1 axis.

     

Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.     

Fibronectin-1 modulated by the long noncoding RNA OIP5-AS1/miR-200b-3p axis contributes to doxorubicin resistance of osteosarcoma cells

Chemoresistance has been an obstacle in the further improvement of 5-year survival rates of osteosarcoma (OS) patients, but the underlying mechanism of chemo-resistance remains unclear. A comprehensive analysis of mRNAs and noncoding RNAs related to OS chemo-resistance could help solve this problem. In the current study, we first identified that fibronectin-1 (FN1), screened by microarray analysis in three paired chemo-resistant and chemo-sensitive OS cell lines, was significantly upregulated in the chemo-resistant OS cell lines and tissues and was related to unfavourable prognosis. Further functional assays revealed that FN1 inhibition greatly increased the sensitivity of OS cells to doxorubicin in vitro and in vivo, whereas FN1 overexpression had the opposite effect. Moreover, mechanistic investigation demonstrated, by a series of assays that included luciferase reporter gene, RNA immunoprecipitation, RNA pull-down and rescue assays, that FN1 expression was regulated by the oncogenic long noncoding RNA (lncRNA) OIP5-AS1 through sponging miR-200b-3p. Thus, these results indicated the role and potential application of the lncRNA OIP5-AS1/miR-200b-3p/FN1 regulatory pathway as a promising target in treatment of OS chemo-resistance.     

Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis

Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy or apoptosis. The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between NRF2/KEAP1 signaling and regulation of apoptosis and autophagy is not well understood. In this hypothesis article we discuss how KEAP1 protein and its direct interactants (such as PGAM5, prothymosin α, FAC1 (BPTF), and p62) provide a molecular foundation for a possible cross-talk between NRF2/KEAP1, apoptosis, and autophagy pathways. We present a hypothesis for how NRF2/KEAP1 may interfere with the cellular apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner-PGAM5. Based on very recent experimental evidence, new hypotheses for a cross-talk between NF-κB and the NRF2/KEAP1 pathway in the context of autophagy-related "molecular hub" protein p62 are also presented. The roles of KEAP1 molecular binding partners in apoptosis regulation during carcinogenesis and in neurodegenerative diseases are also discussed.     

The protective effect of sulforaphane against oxidative stress in granulosa cells of patients with polycystic ovary syndrome (PCOS) through activation of AMPK/AKT/NRF2 signaling pathway

Increased production of reactive oxygen species (ROS) in granulosa cells (GCs) causes oxidative stress (OS) and plays a role in pathogenesis of polycystic ovary syndrome (PCOS). Sulforaphane (SFN) has received a great deal of attention as potent antioxidant because of its ability to induce expression of antioxidant enzymes through nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Therefore, the present study was done to investigate the protective effect of SFN against OS in granulosa-lutein cells (GLCs) of patients with PCOS through activation of AMP-activated protein kinase (AMPK)/AKT/NRF2 signaling pathway. GLCs were isolated from patients with PCOS and healthy fertile women, as control group, during egg retrieval procedure. Level of intracellular ROS and apoptosis was determined in the isolated cells. For investigating the protective effect of SFN against ROS production and apoptosis in GLCs, the cells were cultured for 24 h in the presence or absence of SFN. Finally, expression of AMPK, AKT, and NRF2 proteins and genes was evaluated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The results indicated the increased ROS and apoptosis levels in GLCs isolated from patients with PCOS compared to the control group. Addition of SFN to culture medium of GLCs of patients with PCOS reduced intracellular ROS and apoptosis levels, and increased expression of AMPK, AKT, and NRF2 proteins and genes. Our findings demonstrated the protective effect of SFN against OS by lowering level of ROS and apoptosis possibly through activation of AMPK, AKT, and NRF2 proteins and genes expression.     

Long noncoding RNA SOX21-AS1 regulates the progression of triple-negative breast cancer through regulation of miR-520a-5p/ORMDL3 axis

Recently, long noncoding RNAs (lncRNAs) have been reported as a new kind of controllers about cancer processes in biology. In spite of the dysregulation of lncRNAs in various kinds of cancers, only a little of the information was effective on the expression configuration and inner effects of lncRNAs in triple-negative breast cancer (TNBC). This study valued the expression of lncRNA SOX21-AS1 and the biological role it played in TNBC. In our research, SOX21-AS1 had a high expression in TNBC cell lines. The functional experiments showed that knockdown of SOX21-AS1 obviously restrained cell proliferation, migration, invasion, and epithelial-mesenchymal transition process and promoted cell apoptosis. Mechanistically, SOX21-AS1 was found to bind with miR-520a-5p. Besides, ORMDL3 was identified as a downstream target of miR-520a-5p, and the suppressed ORMDL3 expression induced by silenced SOX21-AS1 could be restored by miR-520a-5p inhibition. Further, data from rescue assays revealed that SOX21-AS1 could regulate the malignancy of TNBC via miR-520a-5p/ORMDL3 axis. All in all, we identified that SOX21-AS1 regulated the cellular process of TNBC cells via antagonizing miR-520a-5p availability to upregulate ORMDL3 expression.     

DNA damage and oxidative stress in human cells infected by Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.