Prognosis of hormone-dependent breast cancer seems to be influenced by KEAP1, NRF2 and GSTM1 genetic polymorphisms
Influence of Glutathione S-transferase Mu1 (GSTM1) has long been studied in breast cancer and GSTM1 null genotype was correlated with breast cancer risk. Nuclear factor-erythroid 2-related factor-2 (NRF2) is a transcription factor that forms a complex with Kelch-like ECH-associated protein-1 (KEAP1). Recent studies have demonstrated that expression of these proteins is deregulated in several malignancies. Thus, in the present study we aim to distinguish GSTM1 heterozygous from wild type genotype in breast cancer patients and evaluate the presence and clinical significance of NRF2 and KEAP1 polymorphisms, alone or in association, with breast cancer prognosis, in cases confirmed to have GSTM1-present genotype. Study population consisted in 52 patients with breast cancer. Genomic DNA was extracted, GSTM1 was genotyped through multiplex PCR and gene dose was evaluated through real-time PCR. All cases were sequenced, through Sanger sequencing, for specific regions of NRF2 and KEAP1. Genotyping and clinicopathological data were correlated and statistical analysis was performed. GSTM1 wild type was identified in 1 case and 26 cases were identified as heterozygous, these data were correlated with Human Epidermal growth factor Receptor 2 (HER2) status (p value = 0.017). We also verified that most cancers diagnosed at younger ages had the presence of KEAP1 and/or NRF2 polymorphisms. The association of GSTM1 heterozygous genotype with rs1048290 and rs35652124 seems to be associated with HER2 (p < 0.05). Our results suggest that GSTM1 * 1/0 genotype and the cumulative presence of at least one allele mutated in KEAP1 and/or NRF2 polymorphisms might be associated with worse prognosis for breast cancer patients.
Current Status and Challenges of NRF2 as a Potential Therapeutic Target for Diabetic Cardiomyopathy
Diabetic cardiomyopathy is one of the main causes of heart failure and death in patients with diabetes mellitus. Reactive oxygen species produced excessively in diabetes mellitus cause necrosis, apoptosis, ferroptosis, inflammation, and fibrosis of the myocardium as well as impair the cardiac structure and function. It is increasingly clear that oxidative stress is a principal cause of diabetic cardiomyopathy. The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2) activates the transcription of more than 200 genes in the human genome. Most of the proteins translated from these genes possess anti-oxidant, anti-inflammatory, anti-apoptotic, anti-ferroptotic, and anti-fibrotic actions. There is a growing body of evidence indicating that NRF2 and its target genes are crucial in preventing high glucose-induced oxidative damage in diabetic cardiomyopathy. Recently, many natural and synthetic activators of NRF2 are shown to possess promising therapeutic effects on diabetic cardiomyopathy in animal models of diabetic cardiomyopathy. Targeting NRF2 signaling by pharmacological entities is a potential approach to ameliorating diabetic cardiomyopathy. However, the persistent high expression of NRF2 in cancer tissues also protects the growth of cancer cells. This "dark side" of NRF2 increases the challenges of using NRF2 activators to treat diabetic cardiomyopathy. In addition, some NRF2 activators were found to have off-target effects. In this review, we summarize the current status and challenges of NRF2 as a potential therapeutic target for diabetic cardiomyopathy.
Ethanol-Mediated Stress Promotes Autophagic Survival and Aggressiveness of Colon Cancer Cells via Activation of Nrf2/HO-1 Pathway
Epidemiological studies suggest that chronic alcohol consumption is a lifestyle risk factor strongly associated with colorectal cancer development and progression. The aim of the present study was to examine the effect of ethanol (EtOH) on survival and progression of three different colon cancer cell lines (HCT116, HT29, and Caco-2). Our data showed that EtOH induces oxidative and endoplasmic reticulum (ER) stress, as demonstrated by reactive oxygen species (ROS) and ER stress markers Grp78, ATF6, PERK and, CHOP increase. Moreover, EtOH triggers an autophagic response which is accompanied by the upregulation of beclin, LC3-II, ATG7, and p62 proteins. The addition of the antioxidant N-acetylcysteine significantly prevents autophagy, suggesting that autophagy is triggered by oxidative stress as a prosurvival response. EtOH treatment also upregulates the antioxidant enzymes SOD, catalase, and heme oxygenase (HO-1) and promotes the nuclear translocation of both Nrf2 and HO-1. Interestingly, EtOH also upregulates the levels of matrix metalloproteases (MMP2 and MMP9) and VEGF. Nrf2 silencing or preventing HO-1 nuclear translocation by the protease inhibitor E64d abrogates the EtOH-induced increase in the antioxidant enzyme levels as well as the migration markers. Taken together, our results suggest that EtOH mediates both the activation of Nrf2 and HO-1 to sustain colon cancer cell survival, thus leading to the acquisition of a more aggressive phenotype.
Metformin prevention of doxorubicin resistance in MCF-7 and MDA-MB-231 involves oxidative stress generation and modulation of cell adaptation genes
Metformin was shown to sensitize multidrug resistant breast cancer cells; however, the mechanisms involved in this capacity need to be clarified. We investigated oxidative stress and inflammatory-related pathways during the induction of doxorubicin resistance in MCF-7 and MDA-MB-231 human breast cancer cells (DOX-res group), and evaluated metformin-induced cellular responses that resulted in the prevention of doxorubicin resistance (Met-DOX group). Microarray analysis demonstrated that DOX-res changed the expression of genes involved in oxidative stress (OS) and the TGF- β1 pathway. The DOX-res group presented increased thiols and reduced lipoperoxidation, increased levels of nitric oxide, nuclear NF-kB and Nrf2, and reduced nuclear p53 labelling. Analysis of the TGF-β1 signaling pathway by RT-PCR array showed that DOX-res developed adaptive responses, such as resistance against apoptosis and OS. Metformin treatment modified gene expression related to OS and the IFN-α signaling pathway. The Met-DOX group was more sensitive to DOX-induced OS, presented lower levels of nitric oxide, nuclear NF-kB and Nrf2, and increased nuclear p53. Analysis of the IFN-α signaling pathway showed that Met-DOX presented more sensitivity to apoptosis and OS. Our findings indicate that metformin is a promising tool in the prevention of chemoresistance in patients with breast cancer submitted to doxorubicin-based treatments.
Three novel genetic variants in NRF2 signaling pathway genes are associated with pancreatic cancer risk
Pancreatic cancer (PanC) is one of the most lethal solid malignancies, and metastatic PanC is often present at the time of diagnosis. Although several high- and low-penetrance genes have been implicated in PanC, their roles in carcinogenesis remain only partially elucidated. Because the nuclear factor erythroid2-related factor2 (NRF2) signaling pathway is involved in human cancers, we hypothesize that genetic variants in NRF2 pathway genes are associated with PanC risk. To test this hypothesis, we assessed associations between 31,583 common SNPs in 164 NRF2-related genes and PanC risk using three published genome-wide association study (GWAS) datasets, which included 8,474 cases and 6,944 controls of European descent. We also performed the expression quantitative trait loci (eQTL) analysis to assess the genotype-phenotype correlation of the identified significant SNPs using publicly available data in the 1000 Genomes Project. We found that three novel SNPs (i.e., rs3124761, rs17458086 and rs1630747) were significantly associated with PanC risk (P = 5.17×10 , 5.61×10 and 5.52×10 , respectively). A combined analysis using the number of unfavorable genotypes (NUGs) of these three SNPs suggested that carriers of 2-3 NUGs had an increased risk of PanC (P < 0.0001), compared with those carrying 0-1 NUGs. Furthermore, the eQTL analysis revealed that both rs3124761 T and the rs17458086 C alleles were associated with increased mRNA expression levels of SLC2A6 and SLC2A13, respectively (P < 0.05). In conclusion, genetic variants in NRF2 pathway genes could play a role in susceptibility to PanC, and further functional exploration of the underlying molecular mechanisms is warranted. This article is protected by copyright. All rights reserved.
Effects of 17β-Estradiol on Colorectal Cancer Development after Azoxymethane/Dextran Sulfate Sodium Treatment of Ovariectomized Mice
Estrogen is known to have a protective effect in colorectal cancer (CRC) development. Previously, we reported the anti-inflammatory and antitumorigenic effects of 17β-estradiol (E2) in azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated male mice. The aim of this study was to investigate whether ovariectomy in a female AOM/DSS mouse model increases colorectal tumorigenesis and whether tumorigenesis is reduced by estrogen supplementation after ovariectomy. Clinical symptoms and histological severity of colitis and the levels of inflammatory mediators were evaluated in the colon of AOM/DSS-treated ovariectomized (OVX) mice. The levels of E2, myeloperoxidase (MPO), and NF-κB-dependent cytokines (interleukin (IL)-1β and IL-6) were measured by ELISA. Furthermore, quantitative real-time (qRT) PCR and Western blot analysis were performed. Ovariectomy did not aggravate AOM/DSS-induced colitis at 2 weeks. At weeks 10 and 16, ovariectomy significantly increased tumor number and incidence rate in only the proximal colon after AOM/DSS treatment (F_AOM/DSS vs OVX_AOM/DSS), and these increases were significantly reduced by E2 supplementation (OVX_AOM/DSS vs OVX_AOM/DSS/E2). However, ovariectomy did not affect CRC development in the distal colon (F_AOM/DSS vs OVX_AOM/DSS). At week 2, E2 administration to AOM/DSS-treated OVX mice attenuated the histological severity of colitis by decreasing the protein and/or mRNA levels of estrogen receptor alpha (ERα) and NF-κB-related mediators (i.e., COX-2, TNF-α, and IL-6) and by enhancing estrogen receptor beta (ERβ) and nuclear Nrf2 protein expression and the mRNA expression of related antioxidant enzyme genes (i.e., HO-1, GCLC, GCLM, and NQO1). Endogenous estrogen in females protects against the development of proximal colon cancer, and exogenous E2 replacement in OVX female mice showed protective effects against AOM/DSS-induced colitis and carcinogenesis. The mechanism could involve modulating ERs-, NF-κB- and Nrf2-mediated pathways.
Luteolin Shifts Oxaliplatin-Induced Cell Cycle Arrest at G₀/G₁ to Apoptosis in HCT116 Human Colorectal Carcinoma Cells
Certain antioxidative flavonoids are known to activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates cellular antioxidants and detoxifying response and is reportedly highly activated in many types of cancers. Few studies on the potential undesired effects of flavonoid intake during chemotherapy have been conducted, yet Nrf2 activators could favor cancer cell survival by attenuating chemotherapeutic efficiency. This study aimed to examine if luteolin, an Nrf2 activator, hinders chemotherapeutic activity of oxaliplatin, a potent anticancer agent for colorectal cancer, in HCT116 cells. Luteolin treatment strongly increased the transcriptional activity of the antioxidant response element in HCT116 cells and induced the protein expression of heme oxygenase-1, which were indicative of its Nrf2-inducing potential. Intriguingly, 25 μM luteolin reduced cell viability through apoptotic induction, which was intensified in p53-expressing cells while 1 μM oxaliplatin caused cell cycle arrest at G₀/G₁-phase via the p53/p21-dependent mechanism. Moreover, luteolin treatment was found to reduce oxaliplatin-treated p53-null cell viability and colony counts further, thereby demonstrating an additional effect of luteolin in the killing of human colorectal tumor HCT116 cells not expressing functional p53 protein. The findings suggest that luteolin can induce p53-mediated apoptosis regardless of oxaliplatin treatment and may eliminate oxaliplatin-resistant p53-null colorectal cells.
Luteolin promotes apoptotic cell death via upregulation of Nrf2 expression by DNA demethylase and the interaction of Nrf2 with p53 in human colon cancer cells
Luteolin, a dietary flavone, modulates various signaling pathways involved in carcinogenesis. In this study, we investigated the molecular mechanism that underlies the apoptotic effects of luteolin mediated by DNA demethylation of the nuclear factor erythroid 2-related factor 2 (Nrf2) promoter and the interaction of Nrf2 and p53, a tumor suppressor, in human colon cancer cells. Luteolin increased the expression of apoptosis-related proteins and antioxidant enzymes. In DNA methylation, luteolin inhibited the expression of DNA methyltransferases, a transcription repressor, and increased the expression and activity of ten-eleven translocation (TET) DNA demethylases, a transcription activator. Methyl-specific polymerase chain reaction and bisulfite genomic sequencing indicated that luteolin decreased the methylation of the Nrf2 promoter region, which corresponded to the increased mRNA expression of Nrf2. In addition, luteolin increased TET1 binding to the Nrf2 promoter, as determined using a chromatin immunoprecipitation (ChIP) assay. TET1 knockdown decreased the percentages of luteolin-treated cells in sub-G phase and cells with fragmented nuclei. Furthermore, complex formation between p53 and Nrf2 was involved in the apoptotic effects of luteolin. These results provide insight into the mechanism that underlies the anticancer effects of luteolin on colon cancer, which involve the upregulation of Nrf2 and its interaction with the tumor suppressor.
Identification of Nrf2/STAT3 axis in induction of apoptosis through sub-G cell cycle arrest mechanism in HT-29 colon cancer cells
We investigated the role of stattic as an adjuvant molecule to increase the cytotoxicity of 5-fluorouracil (5-FU) through specific inhibition of molecular targets, signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-29 colon cancer cells. Cytotoxicity and apoptotic effects were investigated by methylthiazolyldiphenyl-tetrazolium bromide assay and flow cytometry analysis, respectively. Real-time polymerase chain reaction was applied to assess the messenger RNA (mRNA) level of STAT3, Nrf2, and apoptotic genes including Bax, Bcl-xl, and Bcl-2. The antitumor effect of 5-FU in combination with stattic induced synergistic effect in HT-29 cells with combination indexes (CIs) 0.49. Flow cytometric results related to apoptotic confirmed that there was up to 40% increase in the population of apoptotic cells in HT-29 colon cancer cells incubated with 5-FU and stattic compared with control groups. Our data from gene expression determined a substantial diminish in the mRNA levels of the Nrf2 and antiapoptotic gene Bcl-2 along with a noticeable increase in the level of the proapoptotic Bax in HT-29 colon cells that underwent cotreatment with 5-FU and stattic (P < 0.05). Moreover, the results exhibited that stattic can be used as adjuvant chemotherapy besides the 5-FU. This therapeutic approach in colon cancer could mediate 5-FU chemoresistance via modulating therapeutic targets (ie, STAT3 and Nrf2 pathways) and decreased 5-FU-related adverse effects.
Triptolide induces oxidative damage in NRK-52E cells through facilitating Nrf2 degradation by ubiquitination via the GSK-3β/Fyn pathway
Triptolide (TP) isolated from Tripterygium wilfordii Hook F. (TWHF) shows extensive anti-inflammation, immunosuppression and anti-tumor properties. However, its therapeutic potential is limited by its severe side effects, especially the nephrotoxicity. This study intended to explore the role of the GSK-3β/Fyn pathway in TP-induced oxidative damage and the potential mechanism of Nrf2 protein downregulation. Our data showed that TP induced oxidative stress and cell damage in the rat renal tubular epithelial cell line NRK-52E cells by activation of GSK-3β and nuclear translocation of Fyn, which resulted in decreased Nrf2 nuclear translocation. Moreover, TP significantly induced Nrf2 degradation by ubiquitination, which was blocked by the proteasome inhibitor MG132. In addition, cotreatment with a typical GSK-3β inhibitor, lithium chloride, promoted the nuclear translocation of Nrf2 and decreased the nuclear translocation of Fyn, which led to reduced cell damage, LDH leakage, glutathione depletion and cell apoptosis. Collectively, our results indicated that TP induced oxidative damage in NRK-52E cells by facilitating Nrf2 degradation by ubiquitination via the GSK-3β/Fyn pathway.
Nrf2 in cancers: A double-edged sword
The Nrf2/Keap1 pathway is an important signaling cascade responsible for the resistance of oxidative damage induced by exogenous chemicals. It maintains the redox homeostasis, exerts anti-inflammation and anticancer activity by regulating its multiple downstream cytoprotective genes, thereby plays a vital role in cell survival. Interestingly, in recent years, accumulating evidence suggests that Nrf2 has a contradictory role in cancers. Aberrant activation of Nrf2 is associated with poor prognosis. The constitutive activation of Nrf2 in various cancers induces pro-survival genes and promotes cancer cell proliferation by metabolic reprogramming, repression of cancer cell apoptosis, and enhancement of self-renewal capacity of cancer stem cells. More importantly, Nrf2 is proved to contribute to the chemoresistance and radioresistance of cancer cells as well as inflammation-induced carcinogenesis. A number of Nrf2 inhibitors discovered for cancer treatment were reviewed in this report. These provide a new strategy that targeting Nrf2 could be a promising therapeutic approach against cancer. This review aims to summarize the dual effects of Nrf2 in cancer, revealing its function both in cancer prevention and inhibition, to further discover novel anticancer treatment.
Antioxidant effects of sulforaphane in human HepG2 cells and immortalised hepatocytes
Sulforaphane (SFN) has shown anti-cancer effects in cellular and animal studies but its effectiveness has been limited in human studies. Here, the effects of SFN were measured in both human hepatocytes (HHL5) and hepatoma (HepG2) cells. Results showed that SFN inhibited cell viability and induced DNA strand breaks at high doses (≥20 μM). It also activated the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and increased intracellular glutathione (GSH) levels at 24 h. Pre-treatment with a low dose SFN (≤5 μM) protected against hydrogen peroxide (HO)-induced cell damage. High doses of SFN were more toxic towards HHL5 compared to HepG2 cells; the difference is likely due to the disparity in the responses of Nrf2-driven enzymes and -GSH levels between the two cell lines. In addition, HepG2 cells hijacked the cytoprotective effect of SFN over a wider dose range (1.25-20 μM) compared to HHL5. Manipulation of levels of GSH and Nrf2 in HepG2 cells confirmed that both molecules mediate the protective effects of SFN against HO. The non-specific nature of SFN in the regulation of cell death and survival could present undesirable risks, i.e. be more toxic to normal cells, and cause chemo-resistance in tumor cells. These issues should be addressed in the context for cancer prevention and treatment before large scale clinical trials are undertaken.
Carnosol, a dietary diterpene from rosemary () activates Nrf2 leading to sestrin 2 induction in colon cells
Rosemary is abundant with phytochemicals and has recently been approved as an antioxidant food preservative in the European Union. The safety of rosemary is well established, however, the benefits on gastrointestinal health are less known. Our overall hypothesis is that the phytochemicals in rosemary including carnosol have the potential to promote gastrointestinal health by activation of the antioxidant sestrin-2 when consumed in our diet.
Low expression of pentraxin 3 and nuclear factor-like 2 implying a relatively longer overall survival time in gliomas
Pentraxin 3 (PTX3) and nuclear factor-like 2 (Nrf2) are known to induce tumor progression in certain malignancies but act as tumor suppressors in other human neoplasms. In this study, we not only tested the association between PTX3 expression and the World Health Organization (WHO) tumor grading system but also evaluated overall patient survival under variable expression of PTX3 and Nrf2 in primary brain tumors (PBTs). Immunohistochemistry (IHC) was performed for PTX3 and Nrf2 in 10 nonneoplastic brain tissues and 197 PBTs. IHC scores were calculated as the degree of cytoplasmic and nuclear PTX3 and Nrf2 staining intensity multiplied by the percentage of positively stained tissue area. The correlation between PTX3 and Nrf2 IHC scores and tumor grades as well as overall survival time was analyzed by Pearson product-moment correlation and Kaplan-Meier estimate. According to our results, PTX3 IHC scores showed a positive correlation with the WHO grades of gliomas and meningiomas. In addition, we also observed that higher PTX3 expression was associated with poor prognosis in gliomas but not in meningiomas. The concordance between PTX3 and Nrf2 immunohistochemistry (IHC) scores was analyzed using linear regression analysis. When compared to groups with high IHC scores for either one or both biomarkers, gliomas with low expression of both PTX3 and Nrf2 showed significantly better prognosis. In conclusion, we demonstrated that high PTX3 expression implied aggressive tumor behavior and shorter survival time in glioma patients. In addition, our results also showed that gliomas with low PTX3 and Nrf2 immunohistochemical expression could imply a longer overall survival time. Therefore, the combination of lower PTX3 and Nrf2 immunohistochemical expression may be important in offering a better prognosis in gliomas, although the detailed mechanism is yet to be elucidated.
Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome
The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.