NRF2-suppressed vascular calcification by regulating the antioxidant pathway in chronic kidney disease.

Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo differentiation and osteogenic transition, is a common complication of chronic kidney disease (CKD). Recent findings show that nuclear factor-erythroid-2-related factor 2 (NRF2) is an evolutionarily conserved antioxidant and beneficial in preventing vascular senescence and calcification. The roles of NRF2 in the initiation and progression of VC in CKD still need further investigation. CKD-associated VC model rats exhibited significant upregulation of NRF2, NAD(P)H: quinone oxidoreductase-1 (NQO1), osteogenic markers such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2) and osteopontin (OPN), and ß-catenin compared to CKD rats. Immunohistochemistry further verified these results. In addition, rat aortic VSMCs were isolated and subjected to four treatments: normal control, phosphorus-induced (Pi), Pi + NRF2 activator DMF, and Pi + NRF2 inhibitor ML385. The reactive oxygen species (ROS) generation, malondialdehyde (MDA) content, and calcium deposition of the four treatments were determined. The mRNA and protein expression levels of NRF2, NQO1, and haem oxygenase 1 (HO1) and the osteogenic markers ALP, Runx1, OPN, bone morphogenetic protein 2 (BMP2), and ß-catenin were quantified by RT-PCR and western blotting. VSMC apoptosis was calculated by flow cytometry. The in vitro results suggested that intracellular oxidative stress and calcification were closely associated with NRF2 activity and that the activation of NRF2 could significantly suppress osteogenic transition and apoptosis in VSMCs. Thus, this study indicated that the NRF2-related antioxidant pathway can positively respond to and protect against the initiation and progression of VC in CKD by reducing oxidative stress. This study may contribute insights facilitating the application of the NRF2 antioxidative system as a therapeutic treatment for vascular diseases such as CKD.

Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice.

BACKGROUND AND PURPOSE: Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)-reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. METHODS: SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation-reoxygenation (3 hours 0.5%-1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. RESULTS: SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post-oxygen-glucose deprivation-reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P=0.017) versus controls. CONCLUSIONS: Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.

R-sulforaphane modulates the expression profile of AhR, ERα, Nrf2, NQO1, and GSTP in human breast cell lines.

Our previous study showed remarkable differences in the effect of R-sulforaphane (R-SFN) on the expression of CYPs 19, 1A1, 1A2, and 1B1 in ER(+) MCF7, ER( -) MDA-MB-231, and non-tumorigenic immortalized MCF10A (8). This study aimed to evaluate the effect of R-SFN on phase II enzymes induction and expression of AhR, Nrf2, and ERα in the same breast cell lines. The results showed increased expression of GSTP as a result of treatment with R-SFN in breast cancer cells. An increased NQO1 transcript and protein levels were found in all breast cells, with the most significant increase in MCF7 cells. Similarly, the enhancement of Nrf2 expression was noticed in all tested cells. AhR gene transcript and protein were decreased in MCF7 cells. In MDA-MB-231, increased AhR mRNA was not confirmed at the protein level. No differences were found in the expression of ERα. Overall, the results of the present study extended our earlier suggestions on the possible interference of R-SFN with estrogens homeostasis in breast cancer cells differing in ERα status, as well as in non-tumorigenic immortalized breast epithelial cells. While some of R-SFN effects might be beneficial and useful in breast cancer prevention, the others, particularly GSTP induction, may lead to adverse effects.

Royal Jelly Protects against Epidermal Stress through Upregulation of the NQO1 Expression.

Royal jelly (RJ) is secreted by honeybees and has been used as an apitherapy to obtain healthy skin since ancient times. However, the mechanism of the protective effects of RJ against skin aging and skin diseases caused by skin stress and its components have not been clarified. In this study, we attempted to understand the effect of RJ on epidermal function and observed that NAD(P)H quinone dehydrogenase 1 (NQO1) is significantly induced by RJ in keratinocytes. The expression of NQO1 was also increased in the 3D epidermal skin model. NQO1 is involved in antioxidation and detoxification metabolism, and we found that RJ protects against the epidermal stress caused by UVB and menadione through the upregulation of NQO1. We identified 10-hydroxy-2-decenoic acid (10H2DA), a major fatty acid in RJ, as an active compound in this reaction as it induced the expression of NQO1 and protected the skin against oxidative stress. We demonstrated that the protective effect of RJ against epidermal stress is mediated through the upregulation of NQO1 by 10H2DA.

Theaflavins alleviate sevoflurane-induced neurocytotoxicity via Nrf2 signaling pathway.

Aim: Sevoflurane could induce apoptosis of rat hippocampal neurons, while theaflavins (TFs) have antioxidant and anti-inflammatory properties. This study aims to explore whether TFs could alleviate sevoflurane-induced neuronal cell injury.Materials and methods: Cells were treated by concentration gradient of sevoflurane and TFs. Cell viability, level of reactive oxygen species (ROS) and apoptosis rate were determined by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Quantitative PCR (qPCR) and western blot were performed to determine mRNA and protein expressions.Results: TFs promoted viability of cells under the treatment of sevoflurane, while it suppressed apoptosis and down-regulated ROS level in a concentration-dependent manner. TFs could also down-regulate expression levels of caspase-3 and caspase-9 and cytosol and intranuclear nuclear factor E2-related factor 2 (Nrf2) in rat hippocampal nerve cells, while it up-regulated those of heme oxygenase 1 (HO-1), NADPH quinine oxidoreductase 1 (NQO1), glutamate cysteine ligase (GCL) and peroxiredoxin 1 (Prx1).Conclusions: Our study suggests that TFs exert protective effects on sevoflurane-induced neurocytotoxicity and therefore could be used as a potential drug for treatment of neuronal injury.

NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis.

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.

Phase 1 study of ARQ 761, a ß-lapachone analogue that promotes NQO1-mediated programmed cancer cell necrosis.

BACKGROUND: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase expressed in multiple tumour types. ARQ 761 is a ß-lapachone (ß-lap) analogue that exploits the unique elevation of NQO1 found in solid tumours to cause tumour-specific cell death. METHODS: We performed a 3+3 dose escalation study of 3 schedules (weekly, every other week, 2/3 weeks) of ARQ 761 in patients with refractory advanced solid tumours. Tumour tissue was analysed for NQO1 expression. After 20 patients were analysed, enrolment was restricted to patients with NQO1-high tumours (H-score ≥ 200). RESULTS: A total of 42 patients were treated. Median number of prior lines of therapy was 4. Maximum tolerated dose was 390 mg/m2 as a 2-h infusion every other week. Dose-limiting toxicity was anaemia. The most common treatment-related adverse events were anaemia (79%), fatigue (45%), hypoxia (33%), nausea (17%), and vomiting (17%). Transient grade 3 hypoxia, reflecting possible methemoglobinaemia, occurred in 26% of patients. Among 32 evaluable patients, best response was stable disease (n = 12); 6 patients had tumour shrinkage. There was a trend towards improved efficacy in NQO1-high tumours (P = 0.06). CONCLUSIONS: ARQ 761 has modest single-agent activity, which appears associated with tumour NQO1 expression. Principal toxicities include anaemia and possible methemoglobinaemia.

Inflammation, oxidative stress, and higher expression levels of Nrf2 and NQO1 proteins in the airways of women chronically exposed to biomass fuel smoke.

The study was carried out to examine whether chronic exposure to smoke during daily household cooking with biomass fuel (BMF) elicits changes in airway cytology and expressions of Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2]), Keap1 (Kelch-like erythroid-cell-derived protein with CNC homology [ECH]-associated protein 1), and NQO1 (NAD(P)H:quinone oxidoreductase 1) proteins in the airways. For this, 282 BMF-using women (median age 34 year) and 236 age-matched women who cooked with liquefied petroleum gas (LPG) were enrolled. Particulate matter with diameters of < 10 µm (PM10) and < 2.5 µm (PM2.5) were measured in indoor air with real-time laser photometer. Routine hematology, sputum cytology, Nrf2, Keap1, NQO1, and generation of reactive oxygen species (ROS) along with the levels of superoxide dismutase (SOD) and catalase were measured in both groups. PM10 and PM2.5 levels were significantly higher in BMF-using households compared to LPG. Compared with LPG users, BMF users had 32% more leukocytes in circulation and their sputa were 1.4-times more cellular with significant increase in absolute number of neutrophils, lymphocytes, eosinophils, and alveolar macrophages, suggesting airway inflammation. ROS generation was 1.5-times higher in blood neutrophils and 34% higher in sputum cells of BMF users while erythrocyte SOD was 31% lower and plasma catalase was relatively unchanged, suggesting oxidative stress. In BMF users, Keap1 expression was reduced, the percentage of AEC with nuclear expression of Nrf2 was two- to three-times more, and NQO1 level in sputum cell lysate was two-times higher than that of LPG users. In conclusion, cooking with BMF was associated with Nrf2 activation and elevated NQO1 protein level in the airways. The changes may be adaptive cellular response to counteract biomass smoke-elicited oxidative stress and inflammation-related tissue injury in the airways.

HX-1171, a Novel Nrf2 Activator, Induces NQO1 and HMOX1 Expression.

HX-1171 (1-O-hexyl-2,3,5-trimethylhydroquinone) is a novel synthesized vitamin E derivative, which reportedly has positive effects on various diseases and conditions, such as liver fibrosis, hepatic cirrhosis, and cancer. In this study, we analyzed the transcriptional activity induced by HX-1171. Results from reverse transcription polymerase chain reaction and promoter assays reveal that HX-1171 increased the expression of NQO1 and HMOX1, encoding antioxidant-related enzymes, in A549 human lung epithelial cells. The activity of nuclear factor-E2-related factor (Nrf2), a key transcriptional factor for antioxidative enzymes, was examined in HX-1171-treated cells. Confocal microscopy and Western blotting showed that HX-1171 effectively induced the nuclear translocation and transcriptional activity of Nrf2. We conclude that HX-1171, a novel Nrf2 activator, may be a promising therapeutic agent for oxidative stress-induced diseases. J. Cell. Biochem. 118: 3372-3380, 2017. © 2017 Wiley Periodicals, Inc.

Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study.

STUDY HYPOTHESIS: Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent. STUDY FINDING: DC altered cell proliferation, decreased viability and increased apoptosis in Vero and MCF-7 cell lines but did not show any toxic effect on mouse ovarian tissues and developing oocytes in vitro and in vivo. WHAT IS KNOWN ALREADY: DC suppresses cell proliferation and increases apoptosis in various cancer cells such as breast, urogenital and melanoma. DC has also been reported to alter the anticancer effects of several chemotherapeutics, including cisplatin, gemcitabine and doxorubicin in prostate, liver and uroepithelial cancer cells, respectively. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Vero (African green monkey kidney epithelial cells) and MCF-7 (human cancerous breast epithelial cells) cell lines and mouse granulosa cells isolated from 21-day-old female BALB/c mice (n = 21) were used to assess the effects of DC (10, 50, 100 and 200 µm) for 24 and 48 h on cell proliferation, viability and apoptotic cell death. In vivo experiments were performed with a single i.p. injection of 32 mg/kg DC in 21-day-old female BALB/c mice (n = 12). Following 48 h, animals were sacrificed by cervical dislocation and histological sections of isolated ovaries were evaluated for apoptosis. Viability assays were based on the trypan blue dye exclusion method and an automated cell counter device was used. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and Annexin-V immunofluorescence were assessed by 3D confocal microscopy to address apoptotic cell death. We also assessed whether DC inhibits cell proliferation and viability through NQO1 [NAD(P)H Quinone Oxidoreductase 1], an intracellular inhibitor of reactive oxygen species (ROS). The meiotic spindle and chromosomes were studied in mouse oocytes by α-ß-tubulin and 7-aminoactinomycine D (7-AAD) immunostaining in vitro and in vivo. MAIN RESULTS AND THE ROLE OF CHANCE: DC does not block oocyte maturation and no significant alteration was noted in meiotic spindle or chromosome morphology in metaphase-II (M-II) stage oocytes following DC treatment in vitro or in vivo. In contrast, exposure to DC for 24 h suppressed cell proliferation (P = 0.026 at 200 µm), decreased viability (P = 0.002 at 200 µm) and increased apoptosis (P = 0.048 at 100 µm) in Vero and MCF-7 cell lines, compared with controls. These changes were not related to intracellular NQO1 levels. Mouse granulosa cells were unaffected by 50 or 100 µm DC treatment for 24 and 48 h in vitro. DC treatment in vivo did not alter the number of primordial follicles or the ratio of apoptosis in primordial, primary and secondary follicles, as well as in antral follicles, compared with the controls. LIMITATIONS, REASONS FOR CAUTION: DC was tested for ovarian toxicity only in isolated mouse oocytes/ovaries and healthy BALB/c mice. No cancer formation was used as an in vivo test model. The possibility that DC may potentiate ovarian toxicity when combined with traditional chemotherapeutic agents, such as mitomycin-C, cisplatin, gemcitabine and doxorubicin, must be taken into account, as DC is known to alter their effects in some cancer cells. WIDER IMPLICATIONS OF THE FINDINGS: The present study evaluated, for the first time, the effect of DC on ovarian tissue. The results suggested that DC is not toxic to ovarian tissues and developing oocytes; therefore, DC should be assessed further as a potential anticancer agent when female fertility preservation is a concern. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work includes data from dissertation thesis entitled 'Effects of dicoumarol on mitotic and meiotic cells as an anticancer agent' by DA, 2014 and was partly supported by The National Scientific and Technological Research Council of Turkey (SBAG-109S415) to AC, OC and SO. The authors confirm that this article content presents no conflicts of interest.

The protective effect of melatonin on smoke-induced vascular injury in rats and humans: a randomized controlled trial.

Smoking is one of the most harmful lifestyles in the world. Very few studies have investigated the effects of melatonin in smoke-induced vascular injury. This study was designed to investigate whether melatonin could protect rats and humans from smoke-induced vascular injury. 32 male rats and a double-blind randomized controlled trial (RCT) containing 63 participants formed the subjects of this study. In rats, 10 mg/kg of melatonin was intraperitoneally injected. Blood samples and abdominal artery were harvested two weeks later. Melatonin decreased the expression of platelet endothelial cell adhesion molecule-1 (CD31), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) compared with the smoke exposed group (P < 0.05), whereas endothelial nitric oxide synthase (eNOS), nuclear erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone oxidoreductase 1 (NQO-1), catalytic glutamate cysteine ligase (GCLC) and heme oxygenase-1 (HO-1) recovered markedly (P < 0.05). In humans, 3 mg/day of melatonin was taken orally by the participants. Blood samples were drawn at baseline and after two weeks of treatment. Compared with the oral placebo group, melatonin decreased the concentration of fibrinogen (Fbg) (P = 0.04) and free fatty acids (FFA) (P = 0.04) in smokers, along with the decreased expression of ICAM-1, VCAM-1 and ET-1 (P = 0.004, P = 0.001, P < 0.0001, respectively). In contrast, Nrf2 and HO-1 expression were markedly increased (P = 0.0001, P = 0.0049, respectively) after smokers took melatonin orally. In summary, our present data suggest that melatonin could ameliorate smoke-induced vascular injury.

NAD(P)H: quinone oxidoreductase 1 inducer activity of novel 4-aminoquinazoline derivatives.

Fourteen novel 4-aminoquinazoline derivatives 2-15 were designed and synthesized. The structure of the newly synthesized compounds was established on the basis of elemental analyses, IR, (1)H-NMR, (13)C-NMR, and mass spectral data. The compounds were evaluated for their potential cytoprotective activity in murine Hepa1c1c7 cells. All of the synthesized compounds showed concentration-dependent ability to induce the cytoprotective enzyme NAD(P)H: quinone oxidoreductase (NQO1) with potencies in the low- to sub-micromolar range. This approach offers an encouraging framework which may lead to the discovery of potent cytoprotective agents.

Synthesis, molecular modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity of novel 2-phenylquinazolin-4-amine derivatives.

Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This work presents the design and synthesis of novel 2-phenylquinazolin-4-amine derivatives (2-12) and evaluation of their NAD(P)H: quinone oxidoreductase 1 (NQO1) inducer activity in murine cells. Also, molecular docking of all the new compounds was performed to assess their ability to inhibit Keap1-Nrf2 protein-protein interaction through occupying the Keap1-Nrf2-binding domain which biologically leads to a consequent Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds have the ability to interact with Keap1; however compound 7, the most active compound in this study, showed more interactions with key amino acids.

Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway.

Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL), one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone1) (NQO1). However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and improved the activities of superoxide dismutase (SOD) and catalase (CAT), resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents.

CONTEXT: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery. OBJECTIVE: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity. MATERIALS AND METHODS: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells. RESULTS: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC50 values ranging between 0.18 and 17 µg/mL) and five inhibited iNOS (IC50 values ranging between 0.32 and 12.9 µg/mL). In the aromatase assay, only two isolates mediated inhibition (IC50 values 12.2 and 10.5 µg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5 µg/mL), and five extracts inhibited the growth of MCF-7 cells (IC50 values ranging from 0.56 to 17.5 µg/mL). Six active cultures were derived from woody parts of the plant material. CONCLUSION: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.

NQO1 protein expression predicts poor prognosis of non-small cell lung cancers.

BACKGROUND: High-level expression of NAD(P)H: quinoneoxidoreductase 1 (NQO1) has been correlated with many types of human cancers, suggesting that NQO1 plays important roles in tumor occurrence and progression. This study attempted to explore the role of NQO1 in tumor progression and prognostic evaluation of non-small cell lung cancer (NSCLC). METHODS: Total 164 tissue samples, including 150 NSCLC paired with the adjacent non-tumor tissues and 14 normal lung tissues, were picked-up for immunohistochemical (IHC) staining of the NQO1 protein, and immunofluorescence (IF) staining was also performed to detect the subcellular localization of the NQO1 protein in A549 human lung cancer cells. The correlation between NQO1 expression and clinicopathological characteristics were evaluated by Chi-square test and Fisher's exact tests. The disease-free survival (DFS) and overall survival (OS) rates of NSCLC patients were calculated by the Kaplan-Meier method, and univariate and multivariate analyses were performed using the Cox proportional hazards regression model. RESULTS: The NQO1 protein showed a mainly cytoplasmic staining pattern in lung cancer cells, including adenocarcinoma and squamous cell carcinoma (SCC). Both positive rate and strongly positive rate of NQO1 protein expression were significantly higher in NSCLC (59.3% and 28.0%) than that in adjacent non tumor (8.0% and 1.3%) and normal lung tissues (0%). The positive rate of NQO1 was related with clinical stage and lymph node metastasis, and the strongly positive rate of NQO1 protein was significantly correlated with tumor size, poor differentiation, advanced clinical stage and lymph node metastasis in NSCLC. Additionally, survival analyses showed that the patients with NQO1 positive expression had lower OS rates compared with those with NQO1 negative expression in the groups of T1-2, T3-4, without LN metastasis and stage I-II of NSCLC, respectively; however, in the groups of patients with LN metastasis or III-IV stages, OS rate was not correlated with NQO1 expression status. Moreover, multivariate analysis suggested that NQO1 emerged as a significant independent prognostic factor along with tumor size, differentiation, lymph node metastasis and clinical stage in patients with NSCLC. CONCLUSIONS: NQO1 is upregulated in NSCLC, and it may be a useful poor prognostic biomarker and a potential therapeutic target for patients with NSCLC.

Neuroprotective efficacy of naringin on 3-nitropropionic acid-induced mitochondrial dysfunction through the modulation of Nrf2 signaling pathway in PC12 cells.

Oxidative stress and mitochondrial dysfunction are implicated in neuronal apoptosis associated with Huntington's disease. Naringin is the flavanone present in grapefruit and related citrus species possess diverse pharmacological and therapeutic properties including antioxidant, anti-apoptotic, and neuroprotective properties. The aim of this study was to investigate the protective effect of naringin on 3-nitropropionic acid (3-NP)-induced neurotoxicity in pheochromocytoma cells (PC12) cells and to explore its mechanism of action. Naringin protects PC12 cells from 3-NP neurotoxicity, as evaluated the by cell viability assays. The lactate dehydrogenase release was decreased upon naringin treatment in 3-NP-induced PC12 cells. Naringin treatment enhances the antioxidant defense by increasing the activities of enzymatic antioxidants and the level of reduced glutathione. The increase in levels of reactive oxygen species and lipid peroxidation induced by 3-NP were significantly decreased by naringin. PC12 cells induced with 3-NP showed decrease in the mitochondrial membrane potential and mitochondrial respiratory complex enzymes, succinate dehydrogenase and cytochrome c oxidase activities, and it was significantly altered to near normal upon naringin treatment. Naringin reduced the 3-NP-induced apoptosis through the modulation in expressions of B-cell lymphoma 2 and Bcl-2-associated X protein. Further, naringin enhances the nuclear translocation of Nrf2 and induces the NAD(P)H: quinone oxidoreductase-1 and Heme oxygenase-1 expressions through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. Taken together, the above findings suggest that naringin augments cellular antioxidant defense capacity and reduces the 3-NP-induced neurotoxicity in PC12 cells through the PI-3K/Akt-dependent Nrf2 activation in PC12 cells.

Induction of complementary function reductase enzymes in colon cancer cells by dithiole-3-thione versus sodium selenite.

Cellular induction of reductase enzymes can alter the susceptibility of cells toward drugs and chemicals. In this study, we compared the capacity of a single dose of sodium selenite and 3H-1,2-dithiole-3-thione (D3T) to influence the drug-relevant reducing capacity of HT29 cells over time, and defined the protein-specific contribution to this activity on the basis of selected reaction monitoring mass spectrometry. Thioredoxin reductase 1 (TrxR1) protein levels and activity were inducible up to 2.2-fold by selenium. In contrast, selenium had only a minor influence on prostaglandin reductase 1 (PTGR1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) activity and protein levels. D3T, a strong Nrf2 inducer, induced all the reductases and additionally increased the cytotoxicity of hydroxymethylacylfulvene, a bioreductive DNA-alkylating drug. The data and experimental approaches allow one to define induction potency for reductase enzymes PTGR1, TrxR1, and NQO1 in HT29 cells and link these to changes in drug cytotoxicity.

Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminophen-induced hepatotoxicity in mice?

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the expression and activity of cytoprotective genes during periods of oxidative stress. It has previously been shown that some Nrf2 genes are more highly expressed in livers of female than male mice. This could explain previously reported sex-related differences in susceptibility to acetaminophen (APAP) hepatotoxicity in mice, where females show greater resistance to APAP hepatotoxicity. Here, we examined, for the first time, differences in mRNA and protein expression for Nrf2 and a battery of Nrf2-dependent genes in naïve wild-type (WT) and overnight-fasted WT and Nrf2-null male and female mice following APAP treatment. Alanine aminotransferase (ALT) activity was measured as an indicator of hepatotoxicity. Hepatic mRNA and protein levels were measured by quantitative polymerase chain reaction and western blotting, respectively. Contrary to expectations, basal Nrf2 mRNA and protein expression were significantly lower in livers of naïve female than male mice. Although mRNA and/or protein expression of quinone oxidoreductase 1 and multidrug resistance-associated protein 4 was more pronounced in livers of female than male mice under some of the conditions examined, no higher global expression of Nrf2-dependent genes was detected in female mice. Furthermore, ALT activity was significantly elevated in overnight-fasted WT and Nrf2-null male mice following APAP treatment, but no increases in ALT were observed in either genotype of female mice. These results indicate that factors other than Nrf2 are responsible for the lower susceptibility of female mice to APAP hepatotoxicity.

NQO1 overexpression is associated with poor prognosis in squamous cell carcinoma of the uterine cervix.

BACKGROUND: NQO1 (NAD(P)H: quinone oxidoreductase-1), located on chromosome 16q22, functions primarily to protect normal cells from oxidant stress and electrophilic attack. Recent studies have revealed that NQO1 is expressed at a high level in most human solid tumors including those of the colon, breast, pancreas, ovaries and thyroid, and it has also been detected following the induction of cell cycle progression and proliferation of melanoma cells. In this study, we aimed to investigate the clinicopathological significance of upregulated NQO1 protein expression in squamous cell carcinomas (SCCs) of the uterine cervix. METHODS: The localization of the NQO1 protein was determined in the SiHa cervical squamous cancer cell line using immunofluorescence (IF) staining, and immunohistochemical (IHC) staining performed on paraffin-embedded cervical SCC specimens from 177 patients. For comparison, 94 cervical intraepithelial neoplasia (CIN) and 25 normal cervical epithelia samples were also included. QRT-PCR was performed on RNA from fresh tissues to detect NQO1 mRNA expression levels, and HPV infection status was genotyped using oligonucleotide microarray. Disease-free survival (DFS) and 5-year overall survival (OS) rates for all cervical SCC patients were calculated using the Kaplan-Meier method, and univariate and multivariate analysis was performed using the Cox proportional hazards regression model. RESULTS: The NQO1 protein showed a mainly cytoplasmic staining pattern in cervical cancer cells, and only three cases of cervical SCC showed a nuclear staining pattern. The strongly positive rate of NQO1 protein expression was significantly higher in cervical SCCs and CINs than in normal cervical epithelia. High-level NQO1 expression was closely associated with poor differentiation, late-stage, lymph node metastasis and high-risk for HPV infection. Additionally, high-level NQO1 expression was associated with lower DFS and 5-year OS rates, particularly for patients with early-stage cervical SCCs. Furthermore, Cox analysis revealed that NQO1 expression emerged as a significant independent hazard factor for DFS rate in patients with cervical SCC. CONCLUSIONS: NQO1 overexpression might be an independent biomarker for prognostic evaluation of cervical SCCs.