NQO1 Gene Research Articles

CoenzymeQ10, Biochanin A and Phloretin Attenuate Cr(VI)-Induced Oxidative Stress and DNA Damage by Stimulating Nrf2/HO-1 Pathway in the Experimental Model.

Hexavalent chromium [Cr(VI)] has emerged as a prevailing environmental and occupational contaminant over the past few decades. However, the knowledge is sparse regarding Cr(VI)-induced neurological aberrations, and its remediation through natural bioactive compounds has not been fully explored. This study intended to probe the possible invigorative effects of nutraceuticals such as coenzyme Q10 (CoQ10), biochanin A (BCA), and phloretin (PHL) on Cr(VI) intoxicated Swiss albino mice with special emphasis on Nrf2/HO-1/NQO1 gene expressions. Mice received potassium dichromate (75ppm) through drinking water and were simultaneously co-treated intraperitoneally with CoQ10 (10mg/kg), BCA, and PHL (50mg/kg) each for 30-day treatment period. The statistics highlight the elevated levels of lipid peroxidation (LPO) and protein carbonyl content (PCC) with a concomitant reduction in the superoxide dismutase (SOD), glutathione-S-transferase (GST), reduced glutathione (GSH), total thiols (TT), catalase (CAT), and cholinesterase activities in the Cr(VI)-exposed mice. The collateral assessment of DNA fragmentation, DNA breakages, and induced histological alterations was in conformity with the above findings in conjugation with the dysregulation in the Nrf2 and associated downstream HO-1 and NQO1 gene expressions. Co-treatment with the selected natural compounds reversed the above-altered parameters significantly, thereby bringing cellular homeostasis in alleviating the Cr(VI)-induced conciliated impairments. Our study demonstrated that the combination of different bioactive compounds shields the brain better against Cr(VI)-induced neurotoxicity by revoking the oxidative stress-associated manifestations. These compounds may represent a new potential combination therapy due to their ability to modulate the cellular antioxidant responses by upregulating the Nrf2/HO-1/NQO1 signaling pathway against Cr(VI)-exposed population. HIGHLIGHTS: Cr(VI)-associated heavy metal exposure poses a significant threat to the environment, especially to living organisms. Cr(VI) exposure for 30 days resulted in the free radical's generation that caused neurotoxicity in the Swiss albino mice. Natural compounds such as coenzyme Q10, biochanin A, and phloretin counteracted the neurotoxic effect due to Cr(VI) exposure in scavenging of free radicals by enhancing Nrf2/HO-1/NQO1 gene expressions in maintaining the cellular homeostasis.

Human Placental Extract Delays In Vitro Cellular Senescence through the Activation of NRF2-Mediated Antioxidant Pathway.

Senescent cells accumulate in the organs of aged animals and exacerbate organ dysfunction, resulting in age-related diseases. Oxidative stress accelerates cellular senescence. Placental extract, used in the alleviation of menopausal symptoms and promotion of wound healing and liver regeneration, reportedly protects against oxidative stress. In this study, we investigated the effects of human placental extract (HPE) on cellular senescence in normal human dermal fibroblasts (NHDFs) under oxidative stress conditions. We demonstrated that HPE delays the onset of cellular senescence. Next-generation sequencing analysis revealed that under oxidative stress conditions, HPE treatment enhanced the expression of the antioxidant genes CYGB, APOE, NQO1, and PTGS1. Further, HPE treatment under oxidative stress conditions increased the protein level of nuclear factor-erythroid factor 2-related factor 2 (NRF2)—a vital molecule in the antioxidant pathway—via post-transcriptional and/or post-translational regulations. These findings indicate that HPE treatment in NHDFs, under chronic oxidative stress, delays cellular senescence by mitigating oxidative stress via upregulation of the NRF2-mediated antioxidant pathway, and HPE treatment could potentially ameliorate skin-aging-associated damage, in vivo.

Abstract P2045: Transcriptome Versus Proteomic Discrepancy Is Associated With Structural-functional Dys-synchrony In Reductive Stress Myocardium

Background: A real-time action of Nrf2 in response to a stress is vital to preserve the redox homeostasis, but its activation under unstressed settings tilt the redox balance towards the reductive arm, leading to reductive stress (RS). In this study, we identified that >50% mismatch between mRNA and protein levels in RS myocardium undergoing structural/functional dys-synchrony. Methods: NGS-RNA Seq (for RNA), Tandem Mass Tagging (TMT) Mass spectrometry (for proteins) and speckle tracking strain echocardiography (STE) using Vevo2100 system was performed in the cardiac-specific constitutively active Nrf2 transgenic (CaNrf2-TG/TG) and non-transgenic (NTG) mice (n=4/group) at ~6-7 months of age. Results: Comparison of NGS and TMT data in TG vs. NTG hearts showed surprising results. Out of 104 commonly found gene products in NGS and proteins at a FC≥1.5, the levels of 50 proteins are not consistent with their RNA expression. The proteins that are steadily expressed according to their respective mRNA levels are classified as “transcription-sync proteins (T-SP)” and the ones that do not match are termed as “transcription-nonsync proteins. While changes in Nrf2-driven antioxidant genes ( Nqo1, Gsr, Gsta1-4, and Gstm1 ) were grouped under “T-SP”, other transcripts including Atp5pb, Ces1d, Cmbl, Cox7a1, Cpt1b, Fah and Gstk1 did not quantitatively match with their translated products in TG hearts. Further, STE demonstrated an impaired wall motion (diastolic and systolic velocity), increased fractional shortening (20% vs 50%; p<0.0001), decreased global longitudinal strain (-10% vs -25%, p<0.0002), increased IVRT (6.46 vs 13.8 ms; p<0.0001) and IVCT (8.06 vs 12.5 ms; p<0.0005) in TG vs. NTG mice. Changes in the systolic and diastolic functions with structural abnormalities demonstrate cardiac dys-synchronicity in TG hearts with chronic RS exhibiting impaired transcriptome and proteome profiles. Conclusions: Our comparative analysis of RNA and protein in RS hearts demonstrated that 50% of the proteins do not match with the quantitative changes of their transcripts. We highlight that the RS could be a novel mechanism for disproportionate transcription vs translation, which leads to structural and functional dys-synchrony in the TG hearts.

CASC9 potentiates gemcitabine resistance in pancreatic cancer by reciprocally activating NRF2 and the NF-κB signaling pathway.

Gemcitabine resistance is a frequently occurring and intractable obstacle in pancreatic cancer treatment. However, the underlying mechanisms require further investigation. Adaptive regulation of oxidative stress and aberrant activation of the NF-κB signaling pathway are associated with resistance to chemotherapy. Here, we found that gemcitabine upregulated the expression of CASC9 in a dose-dependent manner, partially via induction of reactive oxygen species, whereas inhibition of CASC9 expression enhanced gemcitabine-induced oxidative stress and apoptosis in pancreatic cancer cells. Furthermore, suppression of CASC9 level inhibited the expression of NRF2 and the downstream genes NQO1 and HO-1, and vice versa, indicating that CASC9 forms a positive feedback loop with NRF2 signaling and modulates the level of oxidative stress. Silencing CASC9 attenuated NF-κB pathway activation in pancreatic cancer cells and synergistically enhanced the cytotoxic effect of gemcitabine chemotherapy in vivo. In conclusion, our findings suggest that CASC9 plays a key role in driving resistance to gemcitabine through a reciprocal loop with the NRF2-antioxidant signaling pathway and by activating NF-κB signaling. Our study reveals potential targets that can effectively reverse resistance to gemcitabine chemotherapy.

The effect of curcumin on NRF2/Keap1 signalling pathway in the epididymis of mouse experimental cryptorchidism.

Nrf2/Keap1 pathway, which prevents cellular damage against reactive oxygen species production, is disrupted in epididymis following cryptorchidism. In this study, we aimed to use curcumin (Cur) as an activator of Nrf2 to decrease the effects of disruption in this pathway caused by cryptorchidism. In this study, animals were randomly divided into following groups: control, sham-surgery, sham-vehicle, sham-Cur50, sham-Cur100 , cryptorchidism, cryptorchidism-vehicle, cryptorchidism-Cur50 and cryptorchidism-Cur100 . For cryptorchidism induction, the left testicle was removed from the scrotum and sutured to the abdominal wall. Two weeks after surgery, Cur was given orally to animals. After 1 month, sperm parameters and testis histopathology were analysed. The expression of Nrf2, NQO1, HO1, and Keap1 genes was evaluated by real-time polymerase chain reaction. Our data showed that Cur, especially at high doses, could improve sperm parameters and testis histopathology, which were damaged following cryptorchidism induction. The expression of HO1, NQO1, and Nrf2 genes, which had decreased in the cryptorchidism group, showed a significant increase after administration of Cur in a dose-dependent manner. Cur, by inducing the expression of genes involved in the Nrf2/Keap1 pathway, could reduce the adverse effects of cryptorchidism and might be used as adjuvant therapy for decreasing cryptorchidism complications before surgery.

Abstract 5681: Metabolic targeting of NRF2 potentiates the efficacy of TRAP1 inhibitor GTPP in colorectal cancer

Abstract Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial homolog of HSP90 chaperones. It plays an important role in protection against oxidative stress and apoptosis by regulating reactive oxidative species (ROS) and may also exert the cytoprotective activities by regulating mitochondrial permeability transition pore opening. Recent studies have suggested that TRAP1 may regulate metabolism and mitochondrial homeostasis in cancer. To further elucidate the mechanistic role of TRAP1 in regulating tumor cell survival, we used cellular models to investigate cellular effects and signaling pathways regulated by TRAP1 in several colon cancer cell lines. Inhibition of TRAP1 by a small molecule inhibitor, Gamitrinib-triphenylphosphonium (GTPP), in colon cancer cells led to ROS production, ER stress, reduction of cell viability and induction of cell death. Interestingly, we observed wide-range of responses to TRAP1 inhibition among these colon cancer cell lines. In GTPP sensitive cell lines (SW48), GTPP induced upregulation of GRP78, CHOP and cleavage form PARP proteins. In contract, in GTPP resistant cells (DLD1 and RKO), GTPP treatment highly induced activation of antioxidant gene NRF2, leading to better cell survival under oxidative environment. NRF2 target genes HO1 and NQO1 were upregulated in GTPP-treated DLD1 cells, making these cells more resistant to GTPP treatment. Using reporter assay, we further found that transcriptional activation of antioxidative responds element (ARE) was increased in GTPP-treated DLD1 and RKO cells (GTPP resistant), but not in SW48 cells (GTPP sensitive). Combination of NRF2 inhibitor ML385 and GTPP led to excessive ROS production and exacerbated GTPP-induced cell death in GTPP resistant cells. These new findings for the first-time link TRAP1 and NRF2 in a common pathway regulating ROS production in colon cancer. Taken together, dual targeting of TRAP1 and NRF2 may potentially overcome the tumor cell resistance through disruption of mitochondrial redox homeostasis. Citation Format: Hong-Yuan Tsai, Ru Chen, Noah Shroyer, Sheng Pan. Metabolic targeting of NRF2 potentiates the efficacy of TRAP1 inhibitor GTPP in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5681.

Abstract 1439: Meta-analysis of the NQO1 gene C609T polymorphism: Increased risk of colorectal cancer

Abstract Background/objective: Colorectal cancer (CRC) is aggressive and widespread (3rd among malignancies) in the United States. Recent studies suggest that metabolic pathways play an essential role in CRC. NQO1 is an enzyme that contributes to chemoprotection for CRC. Previous studies showed that the link between the NQO1 polymorphisms and CRC risk yielded conflicting results. The aim of this meta-analysis is to test the connection between NQO1 C609T polymorphisms and CRC risk. Methods: PubMed, Web of Science, and Google Scholar were used in bibliographic searches until July 17, 2021. For the NQO1 C609T analysis, a total of 18 studies (7568 CRC cases and 9003 controls) were considered. Data was gathered and subsequently calculated using the pooled odds ratio (OR) (95% confidence interval, CI). Results: The relation between NQO1 polymorphism and CRC risk (T vs. C: OR=1.17, 95% CI=1.07-1.29, p<0.001; TT + CT vs. CC: OR=1.19, 95% CI=1.06-1.34, p<0.001) was significant. Furthermore, stratified investigation based on ethnicity indicated significant association between NQO1 polymorphism and CRC risk in only Caucasian populations (T vs. C: OR=1.15, 95% CI=1.08-1.24, p<0.001; TT + CT vs. CC: OR=1.17, 95% CI=1.08-1.27, p<0.001). No proof of bias in publications was found for NQO1 polymorphism (Eggers p=0.929). Conclusion: This study indicates that the C609T polymorphism of NQO1 is linked with CRC risk in Caucasian populations. Citation Format: Chaithanya Ganji, Raasil R. Basha, Lakkakula V.K.S. Bhaskar, Bassel F. El-Rayes. Meta-analysis of the NQO1 gene C609T polymorphism: Increased risk of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1439.

Abstract 717: Title: Modulation of local cholesterol biosynthesis and response to oxidative stress in high-risk postmenopausal breast tissue with licochalcone A from licorice

Abstract Background: The risk of breast cancer increases with age, despite a drastic decline in circulatory estrogen after menopause. Women at increased risk can reduce this by use of anti-endocrine agents, but often decline these drugs to their adverse effects. Therefore, alternative strategies with greater acceptability are needed. We have previously shown that licochalcone A (LicA) from licorice (Glycyrrhiza inflata) suppresses aromatase expression and activity, enhances the activity of detoxifying phase II metabolism enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. However, its effects on the breast tissue of high-risk women have not been studied. We hypothesize that LicA creates a tumor preventive environment in the breast by locally modulating cholesterol and steroid hormone biosynthesis, and antioxidant/anti-inflammatory response. Methods: We prepared microstructures from fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. We exposed these microstructures to DMSO (control) and LicA (5 µM) for 24 h. After total RNA sequencing, we examined differential gene expression between treated and control samples using the limma R package. Enrichment results were generated by analyzing the up-regulated and down-regulated gene sets using Enrichr. A gene ontology (GO) pathway analysis was performed. The enriched pathways with combined enrichment scores > 4 and FDR < 0.05 were considered statistically significant. We performed confirmatory qPCR experiments in samples obtained from 18 additional women. Results: We observed significant (P < 0.05) upregulation up to 8-fold of antioxidant genes NQO1, HMOX1, G6PD, SQSTM1, PGD, and GPX2. Significant upregulation of Nrf2, the transcription factor involved in the activation of antioxidant enzymes, was evident. In addition, we observed the significant (P < 0.05) downregulation, ranging from 4 to 32-fold of cholesterol biosynthesis and transport genes, MSMO1, FDPS, EBP, SQLE, MVD, MVK, IDI1, DHCR7, NSDHL, FDFT1, LSS, ABCA1, ABCC3, ABCG1, steroid hormone biosynthesis genes HSD17B7, STS, CYP1A1, and CYP1B1, as well as lipid metabolism genes ACAT2, ACYL, FADS1, FADS2, ELOV6, CYP51A1, and LDLR. Some of the genes are shared between these pathways. The significant upregulation of the regulators of these pathways, SREBF1 and SREBF2 provided further confirmation. Conclusion: Our data suggest that LicA can lead to a tumor-preventive breast microenvironment by reducing cholesterol and steroid hormones and lowering oxidative stress mediators. These observations along with the popularity of natural products, suggests that LicA might be a good candidate to be studied further as an alternative breast cancer prevention agent. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Susan Clare, Seema A. Khan. Title: Modulation of local cholesterol biosynthesis and response to oxidative stress in high-risk postmenopausal breast tissue with licochalcone A from licorice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 717.

Role of Nrf2 in bisphenol effects: a review study.

Bisphenols (BPs), the main endocrine-disrupting chemicals used in polycarbonate plastics, epoxy-phenol resins, and some other manufacturers, have been interestingly focused to find their toxic effects in recent years. Due to the strong relation between bisphenols and some crucial receptors such as ERs, AR, glucocorticoid receptor, THRs, ERRs, hPXR, AhR, and etcetera, the disrupting and oncogenic role of these chemicals on reproductive, respiratory, and circulatory systems and a broad group of body tissues have been investigated. BPs induce oxidant enzymes, exert antioxidant enzymes from body cells, and result in the expression of proinflammatory genes, leading to cell apoptosis and inflammation. To maintain the homeostasis of human body cells, Nrf2, the key regulator of oxidative stress (Ashrafizadeh et al., 2020a; Ashrafizadeh et al., 2020c; Boroumand et al.,2018), confronts BP-induced ROS and RNS through the activation of antioxidant enzymes such as SOD1/2, CAT, GSH, GPX, HO-1, and etcetera. Chemicals and drugs such as LUT, NAC, GEN, L-NMMA, Ph2Se2, and GE can regulate the interactions between BPs and Nrf2. Despite the vital role of controlled levels of Nrf2 as an anti-inflammatory and antiapoptotic element, the uncontrolled activity of this transcription factor could lead to cell proliferation and tumorigenesis through NQO1, SLC7a11, Gclm, HMOX1, NQO1 gene activation, and some other genes. To avoid the excessive activity of Nrf2, some protein complexes like CUL3-RBX1-Keap1 (as the primary regulator), β-TrCP, and WDR23 regulate Nrf2's function. It is necessary to note that BPA, as the most famous member, is further reviewed due to its resemblance to the bisphenol family to each other.

Investigation of C609T polymorphism in the NQO1 gene in patients diagnosed with colorectal cancer in the Azerbaijani population

Investigation of C609T polymorphism in the NQO1 gene in patients diagnosed with colorectal cancer in the Azerbaijani population

In vitro comparison of various antioxidants and flavonoids from Rooibos as beta cell protectants against lipotoxicity and oxidative stress-induced cell death.

Oxidative stress and lipotoxicity effects on pancreatic β cells play a major role in the pathogenesis of type 2 diabetes (T2D). Flavonoids and antioxidants are under study for their cytoprotective effects and antidiabetic potential. In this study, we aimed to compare the protective effect of the Rooibos components aspalathin, isoorientin, 3-hydroxyphloretin (3-OH) and green Rooibos extract (GRT) itself, and exendin-4 and N-acetylcysteine (NAC) as reference molecules, against lipotoxicity and oxidative stress. The insulin-producing β cell line INS1E was exposed to hydrogen peroxide or streptozotocin (STZ) to induce oxidative stress, and palmitate to induce lipotoxicity. Cell viability was assessed by a MTS cell viability assay. Antioxidant response and antiapoptotic gene expression was performed by qRT-PCR. Glucose transporter 2 (GLUT 2) transporter inhibition was assessed through 2-NBDG uptake. GRT and the flavonoids aspalathin and 3-hydroxyphloretin offered significant protection against oxidative stress and lipotoxicity. GRT downregulated expression of pro-apoptotic genes Txnip and Ddit3. The flavonoids aspalathin and 3-hydroxyphloretin also downregulated these genes and in addition upregulated expression of antioxidant response genes Hmox1, Nqo1 and Sod1. Isoorientin gave no cytoprotection. Cytoprotection by Rooibos components was significantly higher than by NAC or exendin-4. Rooibos components strongly protect INS1E β cells against diabetogenic stress. Cytoprotection was associated with the upregulation of antioxidant response genes of the NRF2/KEAP1 pathway or suppression of the TXN system. The Rooibos molecules offered better protection against these insults than exendin-4 and NAC, making them interesting candidates as β cell cytoprotectants for therapeutic or nutraceutical applications.

RNA-Seq Analysis of Protection against Chronic Alcohol Liver Injury by Rosa roxburghii Fruit Juice (Cili) in Mice

Rosa roxburghii Tratt. fruit juice (Cili) is used as a medicinal and edible resource in China due to its antioxidant and hypolipidemic potentials. The efficacy of Cili in protecting alcohol-induced liver injury and its underlying mechanism was investigated. C57BL/6J mice received a Lieber-DeCarli liquid diet containing alcohol to produce liver injury. After the mice were adapted gradually to 5% alcohol, Cili (4 mL and 8 mL/kg/day for 4 weeks) were gavaged for treatment. The serum enzyme activities, triglyceride levels, histopathology and Oil-red O staining were examined. The RNA-Seq and qPCR analyses were performed to determine the protection mechanisms. Cili decreased serum and liver triglyceride levels in mice receiving alcohol. Hepatocyte degeneration and steatosis were improved by Cili. The RNA-Seq analyses showed Cili brought the alcohol-induced aberrant gene pattern towards normal. The qPCR analysis verified that over-activation of CAR and PXR (Cyp2a4, Cyp2b10 and Abcc4) was attenuated by Cili. Cili alleviated overexpression of oxidative stress responsive genes (Hmox1, Gsta1, Gstm3, Nqo1, Gclc, Vldlr, and Cdkn1a), and rescued alcohol-downregulated metabolism genes (Angptl8, Slc10a2, Ces3b, Serpina12, C6, and Selenbp2). Overall, Cili was effective against chronic alcohol liver injury, and the mechanisms were associated with decreased oxidative stress, improved lipid metabolism through modulating nuclear receptor CAR-, PXR-and Nrf2-mediated pathways.

MO454: Increased Senescent Cell Burden and Alterations in NRF2 Pathway Drive Uraemic Vascular Calcification in Humans

Abstract BACKGROUND AND AIMS Vascular calcification is a clinical sequelae of chronic kidney disease (CKD) that is associated with high cardiovascular-related mortality in end-stage kidney failure (ESKF) patients. Increased senescent cell burden and dysregulation of the NRF2 pathway, the master regulator of antioxidant genes, have been suggested to play an important role in the onset of multiple non-communicable burden-of-lifestyle diseases, including but not limited to diabetes, obesity and CKD. Thus, we investigated if senescence and NRF2 pathways may serve as drivers of uraemia-induced vascular calcification in human arteries. METHOD ESKF patients without epigastric artery calcification and coronary artery calcification (CAC) (n = 8) were matched with patients with severely calcified epigastric arteries and the presence of CAC (n = 8). In all subjects, samples of epigastric arteries were obtained during living donor kidney transplantation surgery. Gene (qPCR) and protein expression (immunohistochemistry) of cellular senescence markers (p16, p21), NRF2 pathway candidates (NRF2, NQO1, CAT, SOD1) and calcification markers (RUNX2, ALPL) were assessed. In addition, four groups of aortic vascular smooth muscle cells (VSMCs) were treated as follows: i) control media; ii) osteogenic media (Pi = 2.5mM); iii) osteogenic media + pooled serum from non-ESKD subjects; or iv) osteogenic media + pooled uraemic serum from ESKF patients that had severely calcified epigastric arteries (n = 8). Calcification in cell culture experiments was assessed using BoneTag Optical Probe and normalized for protein content, while gene expression analysis for a selection of calcification, senescence and NRF2-related markers, and SA-beta-GAL staining, were performed (n = 4 each). RESULTS At the gene expression level, severely calcified epigastric arteries as assessed by von Kossa staining (Figure 1a) had preserved ALPL expression but significantly increased RUNX2 expression (P < 0.01; Figure 1b) when compared with non-calcified vessels. Gene expression of p16, but not p21, was increased in the calcified compared with non-calcified group (P < 0.001, Figure 1c), while both p16 and p21 protein expressions were elevated in the media layer of calcified vessels (Figure 1c). NRF2 and downstream target gene expressions remained similar between the two groups, however, NRF2 protein expression was increased in the non-calcified group. In cell culture studies, uraemic serum-induced VSMC calcification was increased when compared to non-uraemic serum and osteogenic controls (P < 0.0001, Figure 2a). This was further accompanied by increased ALPL gene expression (P < 0.0001, Figure 2b) and reduced NRF2, SOD1 and NQO1 gene expression (all P < 0.01, Figure 2c). SA-beta-gal staining and p16/p21 gene expression analysis revealed that uraemic serum-induced calcification was not associated with increased senescent cell burden. CONCLUSION Established calcification in ESKF patients is associated with increased senescent cell burden, but preserved NRF2 pathway markers, in epigastric arteries. In contrast, induced calcification was accompanied by the diminished activity of the NRF2 pathway, but not senescence pattern, using an in vitro approach in VSMCs. Our translational data indicate that established and induced calcification may reflect different pathophysiological processes, supporting data from a parallel study using 5/6 rats (Laget, Hobson et al., unpublished data). Future studies should consider the timing of initiation of NRF2 agonist/senolytic treatment when targeting calcification in ESKF patients.

Accelerated evolution of Vkorc1 in desert rodent species reveals genetic preadaptation to anticoagulant rodenticides.

Some rodent species living in arid areas show elevated physiological tolerance to anti-vitamin K rodenticides (AVKs), which seems to be due to some unknown selective pressures that rodents may experience in desert habitats. Genes involved in the ϒ-carboxylation of blood coagulation, including vitamin K epoxide reductase complex, subunit 1 (Vkorc1), ϒ-glutamyl-carboxylase (Ggcx) and NAD(P)H quinone one dehydrogenase (Nqo1) are associated with anticoagulant resistance, or some levels of elevated tolerance, in rodents. To detect whether the DNA sequences of the three genes are also under natural selection in the desert rodent species, we analyzed the Vkorc1, Ggcx and Nqo1 genes of the desert rodents and compared them with other rodent species. We found an accelerated evolutionary rate in Vkorc1 of desert rodents, especially in Mus spretus, Nannospalax galili and Psammomys obesus. By contrast, signals of positive selection were absent for Ggcx and Nqo1 in all species. Mapping the amino acid variations on the VKORC1 protein three-dimensional model suggested most interspecific amino acid variations occur on the outer surface of the VKORC1 pocket, whereas most intraspecific amino acid changes and known AVK resistance mutations occurred on the inner surface and endoplasmic reticulum luminal loop regions. Some desert-species-specific amino acid variations were found on the positions where known resistance mutations occurred, indicating these variations might be related to the elevated physical tolerance to AVKs in desert rodents. The evolution of Vkorc1 has been accelerated in some desert rodent species, indicating genetic preadaptation to anticoagulant rodenticides. Positive selection and relaxed selection have been detected in Psammomys obesus and Nannospalax galili, indicating the two rodent species might also show tolerance to AVKs, which needs further verification. © 2022 Society of Chemical Industry.

The Unusual Cosubstrate Specificity of NQO2: Conservation Throughout the Amniotes and Implications for Cellular Function.

Human Quinone Reductase 2 (NQO2) is a pharmacological target and has appeared in numerous screening efforts as an off-target interactor with kinase-targeted drugs. However the cellular functions of NQO2 are not known. To gain insight into the potential cellular functions of NQO2, we have carried out a detailed evolutionary analysis. One of the most striking characteristics of NQO2 is that it uses conventional dihydronicotinamide cosubstrates, NADH and NADPH, extremely inefficiently, raising questions about an enzymatic function in cells. To characterize the ability of NQO2 to serve as an enzyme, the NQO2 gene was disrupted in HCT116 cells. These NQO2 knockouts along with the parental cells were used to demonstrate that cellular NQO2 is unable to catalyze the activation of the DNA cross-linking reagent, CB1954, without the addition of exogenous dihydronicotinamide riboside (NRH). To find whether the unusual cosubstrate specificity of NQO2 has been conserved in the amniotes, recombinant NQO2 from a reptile, Alligator mississippiensis, and a bird, Anas platyrhynchos, were cloned, purified, and their catalytic activity characterized. Like the mammalian enzymes, the reptile and bird NQO2 were efficient catalysts with the small and synthetic cosubstrate N-benzyl-1,4-dihydronicotinamide but were inefficient in their use of NADH and NADPH. Therefore, the unusual cosubstrate preference of NQO2 appears to be conserved throughout the amniotes; however, we found that NQO2 is not well-conserved in the amphibians. A phylogenetic analysis indicates that NQO1 and NQO2 diverged at the time, approximately 450 MYA, when tetrapods were beginning to evolve.

COPD lung studies of Nrf2 expression and the effects of Nrf2 activators

BackgroundNrf2 regulates cellular antioxidant defence in lung cells, including epithelial cells and alveolar macrophages (AM). The Nrf2/Keap-1 pathway can be modulated by activators with different modes of action; electrophilic compounds and protein–protein interaction (PPI) inhibitors.We assessed Nrf2 and Keap-1 protein and gene levels in COPD compared to controls and the effect of Nrf2 activators on COPD AM.MethodsLung resected tissue from non-smokers, smokers and COPD patients were analysed for epithelial and AM expression of Nrf2 and Keap-1 by imunoshistochemistry and by qPCR in isolated AM. AM were cultured with Nrf2 activators CDDO, C4X_6665, GSK7, MMF and Sulforaphane. Expression of Nrf2 target genes NQO1, HMOX1 SOD1 and TXNRD1 and NQO1 activity were assessed.ResultsNrf2 and Keap-1 expression was not altered in the epithelium or AM of COPD patients compared to controls. NQO1 activity was downregulated, while NQO1, HMOX1, SOD1 and TXNRD1 gene expression increased in COPD patients. All Nrf2 activators increased NQO1 activity, and NQO1, HMOX1, SOD1 and TXNRD1 expression in AMs from both COPD and smokers. The potency of C4X_6665 on NQO1 activity and regulation of Nrf2 target gene expression was higher than other compounds.ConclusionThere is evidence of dysregulation of the Nrf2 signalling pathway in AM from COPD patients. The higher potency of the novel PPI Nrf2 compound C4X_6665 for inducing antioxidant activity and gene expression compared to electrophilic and other PPI Nrf2 activators highlights the therapeutic potential of this compound to address Nrf2 pathway dysregulation in COPD AM.

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Review on NAD(P)H dehydrogenase quinone 1 (NQO1) pathway.

NQO1 is an enzyme present in humans which is encoded by NQO1 gene. It is a protective antioxidant agent, versatile cytoprotective agent and regulates the oxidative stresses of chromatin binding proteins for DNA damage in cancer cells. The oxidization of cellular pyridine nucleotides causes structural alterations to NQO1 and changes in its capacity to binding of proteins. A strategy based on NQO1 to have protective effect against cancer was developed by organic components to enhance NQO1 expression. The quinone derivative compounds like mitomycin C, RH1, E09 (Apaziquone) and β-lapachone causes cell death by NQO1 reduction of two electrons. It was also known to be overexpressed in various tumor cells of breast, lung, cervix, pancreas and colon when it was compared with normal cells in humans. The mechanism of NQO1 by the reduction of FAD by NADPH to form FADH2 is by two ways to inhibit cancer cell development such as suppression of carcinogenic metabolic activation and prevention of carcinogen formation. The NQO1 exhibit suppression of chemical-mediated carcinogenesis by various properties of NQO1 which includes, detoxification of quinone scavenger of superoxide anion radical, antioxidant enzyme, protein stabilizer. This review outlines the NQO1 structure, mechanism of action to inhibit the cancer cell, functions of NQO1 against oxidative stress, drugs acting on NQO1 pathways, clinical significance.

Anti-Inflammatory Activity of Monosubstituted Xestoquinone Analogues from the Marine Sponge Neopetrosia compacta.

Chronic inflammation is recognized as a contributor to multiple chronic diseases, such as cancer, cardiovascular, and autoimmune disorders. Here, a natural products-initiated discovery of anti-inflammatory agents from marine sponges was undertaken. From the screening of 231 crude extracts, a total of 30 extracts showed anti-inflammatory activity with no direct cytotoxic effects at 50 μg/mL on RAW 264.7 (ATCC®TIB-71™) murine macrophage cells stimulated with 1 μg/mL lipopolysaccharide (LPS). Bioactivity-guided purification of the anti-inflammatory extract from the sponge Neopetrosia compacta led to the isolation of xestoquinone (1), adociaquinone B (2), adociaquinone A (3), 14-hydroxymethylxestoquinone (4), 15-hydroxymethylxestoquinone (5), and an inseparable 2:1 mixture of 14-methoxyxestoquinone and 15-methoxyxestoquinone (6). Compounds 1–6 caused a concentration-dependent reduction of nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells, with 4–6 having low micromolar IC50 and acceptable selectivity index. Gene expression analysis using qRT-PCR showed that 1, 5, and 6 downregulated Il1b and Nos2 expression by 2.1- to 14.8-fold relative to the solvent control at 10 μM. Xestoquinone (1) and monosubstituted analogues (4–6), but not the disubstituted adociaquinones (2 and 3), caused Nrf2 activation in a luciferase reporter MCF7 stable cells. Compounds 5 and 6 caused a modest increase in Nqo1 gene expression at 10 μM. The anti-inflammatory activity of xestoquinone (1) and monosubstituted analogues (4–6) may, in part, be mediated by Nrf2 activation, leading to attenuation of inflammatory mediators such as IL-1β and NOS2.

Arsenic trioxide inhibits the functions of lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal interstitial lung disease. Currently, no treatment can block or reverse the development of lung fibrosis in patients suffering from IPF. Recent studies indicate that arsenic trioxide (ATO), a safe, effective anti-cancer pro-oxidant drug, prevents the differentiation of normal human lung fibroblasts (NHLFs) in vitro and reduces experimental pulmonary fibrosis in vivo. In this context, we investigated the anti-fibrotic effects of ATO on the main fibrosis functions of human lung fibroblasts (HLFs) isolated from patients with IPF.IPF and non-IPF (control) HLFs were incubated with 0.01–1 μM ATO and stimulated with pro-fibrotic factors (PDGF-BB or TGF-β1). We measured their rates of proliferation, migration and differentiation and the cell stress response triggered by ATO.ATO did not affect cell viability but strongly inhibited the proliferation and migration of PDGF-BB-stimulated IPF and control HLFs. ATO also prevented myofibroblastic differentiation, as assessed by the expression of α-smooth muscle actin (α-SMA) and collagen-1, and the phosphorylation of SMAD2/3 in TGF-β1-stimulated HLFs. These antifibrotic effects were associated with increased expression of the transcription factor NRF2 and its target genes NQO1 and HMOX1. Genetic silencing of NRF2 inhibited the ATO-induced cell stress response but did not prevent the ATO-dependent inhibition of α-SMA expression in TGF-β1-stimulated HLFs.The results demonstrate that ATO, at concentrations similar to exposure in blood plasma of ATO-treated cancer patients, counteracted pro-fibrotic activities of HLFs from IPF patients. We propose to consider ATO for clinical exploration to define the therapeutic potential in patients with IPF.