Protective role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cisplatin-induced nephrotoxicity

Although cisplatin is widely used as an anti-cancer agent, its use is significantly limited because of its tendency to induce nephrotoxicity through poorly understood mechanisms. NAD(P)H:quinone oxidoreductase 1 (NQO1) is well known to regulate ROS generation. The purpose of this study was to investigate whether NQO1 modulates cisplatin-induced renal failure associated with NADPH oxidase (NOX)-derived ROS production in an animal model. NQO1^−/− mice were treated with cisplatin (18 mg/kg) and renal function, oxidative stress, and tubular apoptosis were assessed. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In accordance with these results, the cellular NADPH/NADP ratio and NOX activity were markedly increased in the kidneys of NQO1^−/− mice compared to NQO1^+/+ mice. In addition, activation of NQO1 by βL treatment significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis. This study demonstrates that NQO1 protects cells against renal failure induced by cisplatin, and that this effect is mediated by decreased NOX activity via cellular NADPH/NADP modulation. These results provide convincing evidence that NQO1 might be beneficial for ameliorating renal failure induced by cisplatin.     

NQO1 involves in the imine bond reduction of sanguinarine and recombinant adeno-associated virus mediated NQO1 overexpression decreases sanguinarine-induced cytotoxicity in rat BRL cells

Although sanguinarine (SANG) can be transformed to dihydrosanguinarine (DHSA) in human and animals, the enzyme involved in the imine bond reduction of SANG is still unknown. In this study, we found that rat NAD(P)H:quinone oxidoreductase 1 expressed by prokaryotic system can transform SANG to DHSA in an NADPH dependent manner. We also found out that there was more DHSA in rAAV-NQO1 infected than rAAV-CYP1A1 and rAAV-control infected BRL cells. SANG decreased rat BRL cell proliferation and augmented cell apoptosis in a time and dose dependent manner. However, the influence of DHSA to BRL cells is not significant difference than SANG. SANG-induced apoptosis was correlated with the up-regulation of Bax/Bcl2 ratio and the down-regulation of Bcl2. SANG can also dose dependently down regulate NQO1 expression, but CYP1A1 expression was a little up regulated. Since CYP1A1 involving in SANG oxidative reactions and NQO1 involving in the transform of SANG to DHSA, we hypothesized that up regulation of NQO1 could reduce SANG cytotoxicity and up regulation of CYP1A1 could increase SANG cytotoxitity. Our further study showed that recombinant adeno-associated virus (rAAV) mediated overexpression of NQO1 significantly increased cell proliferation and decreased Bax/Bcl2 ratio, apoptosis, and cytotoxicity, whereas rAAV mediated CYP1A1 overexpression had opposite effects. These data illustrated that NQO1 involved in the imine bond reduction of sanguinarine and this was a less toxic metabolizing pathway than CYP1A1-metabolizing pathway.     

Inhibitors of NQO1: identification of compounds more potent than dicoumarol without associated off-target effects

The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple “off-target” effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977–81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.     

Tetrachlorobenzoquinone induces acute liver injury,up-regulates HO-1 and NQO1 expression in mice model: The protective role of chlorogenic acid

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol (PCP). Although PCP has been investigated extensively, there are only a few reports describing the toxicity effect of TCBQ, and no report regarding TCBQ-induced liver injury in vivo. In the current study, we aimed to examine the acute hepatic toxicity of TCBQ in the mice model. Chlorogenic acid (CGA) exhibits promising antioxidant activity in the past studies, thus, the second aim of this study was to evaluate the protective effect of CGA on TCBQ-induced liver injury. Our results indicated TCBQ-intoxication caused marked liver cell necrosis and inflammation but not apoptosis, and this damage was alleviated by CGA treatment. Meantime, TCBQ-intoxication enhanced serum ALT, AST activities, TBIL content, hepatic oxidative stress and lipid peroxidation, decreased GSH content and inhibited the activities of antioxidant enzymes. Western blot and immunohistochemical analysis showed that TCBQ marked up-regulated HO-1 and NQO1 expression. On the other hand, pretreatment of CGA reduced TCBQ-induced liver damage remarkably. Taking together, these results revealed that TCBQ has strong hepatic toxic effect, and at least a part of this effect is initiated by free radical and relieved with CGA administration.     

Copper induces oxidative stress and apoptosis of hippocampal neuron via pCREB/BDNF/ and Nrf2/HO-1/NQO1 pathway

Disordered copper metabolism has been suggested to occur to several neurological conditions, including Alzheimer's disease and Parkinson's disease. However, the underlying mechanism was still unclear. This might link to copper-induced hippocampal neuronal apoptosis and decrease in neurons viability. Our vitro experiment showed copper exposure induced oxidative stress and promoted apoptosis of HT22 murine hippocampal neuronal cell. Mechanistically, we found copper, on the one hand, prevented phosphorylation of cAMP response element binding protein (CREB) to decrease expression its downstream target protein Brain-derived neurotrophic factor (BDNF), and to decrease mitochondrial membrane potential and Bcl-2/Bax ratio; on the other hand, copper-induced reactive oxygen species (ROS), promoted lipid peroxidation, reduced antioxidant enzyme activity of GSH-Px. Copper-induced oxidative damage further decreased the phosphorylation of CREB, decreased expression of Bcl-2, enhanced expression of Bax, and accelerated the dissociation of keap1-Nrf2 complex, promoted the nuclear translocation of Nrf2, stimulate the expression of antioxidant molecules HO-1 and NQO1. In conclusion, we found copper inhibited pCREB/BDNF signaling pathway by prevent CREB from phosphorylation, further found that oxidative damage not only inhibited neuroprotective signaling pathways and induced apoptosis, but activated antioxidant protection signals Nrf2/HO-1/NQO1 signaling pathway.     

Pharmacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells

NAD(P)H quinone oxidoreductase (NQO1) has multiple functions in the cell including an ability to act as a detoxifying enzyme and as a protein chaperone. The latter property is particularly important in oncology as one of the client proteins of NQO1 is p53. The inhibitor, dicoumarol, is classically used to probe the biological properties of NQO1, but interpretation of enzyme function is compromised by the multiple “off-target” effects of this agent. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of recombinant human NQO1 have been identified (Nolan et al., J Med Chem 2009;52:7142-56) The purpose of the work reported here is to demonstrate the functional activity of these agents for inhibiting NQO1 in cells. To do this, advantage was taken of the NQO1-mediated toxicity of the chemotherapeutic drug EO9 (Apaziquone). The toxicity of this drug is substantially reduced when the function of NQO1 is inhibited and many of the coumarin-based compounds are more efficient than dicoumarol for inhibiting EO9 toxicity. The ability to do this appears to be related to their capacity to inhibit NQO1 in cell free systems. In conclusion, agents have been identified that may be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.     

Identification of NQO1 and ferrochelatase as interaction partners for neuroprotective N‐{[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐arylamides

Affinity chromatography was used to identify potential cellular targets that are responsible for neuroprotective activity of N‐{[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐arylamides. Active and inactive representatives of N‐{[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐arylamides bearing an extended linker were synthesized and immobilized on an agarose‐based matrix. This was followed by the identification of specifically bound proteins isolated out of the whole rat brain extract. Inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) was identified as candidates for cellular targets.     

Pre-clinical pharmacokinetics of the acridine antitumour candidate AC04 and its 1-oxo-metabolite plasma profile

1. This work aimed to investigate plasma pharmacokinetics and tissue distribution of a new acridine derivative 5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione (AC04) and its 1-oxo-AC04 metabolite disposition in Wistar rats.

2. After a single AC04 1.5?mg/kg intravenous (i.v.) bolus dose, blood samples were taken up to 120?h. Plasma samples were deproteinization, and AC04 and metabolite were quantified by validated liquid chromatography in tandem with mass spectrometry method. Protein binding was determined by ultrafiltration. AC04 tissue disposition was evaluated after i.v. bolus dose.

3. Individual AC04 concentration–time profiles were best fitted by a two-compartment model showing CL_tot of 3.4?±?3.4?L/h/kg, Vd_SS of 137.9?±?91.4?L/kg, AUC_0–∞ of 788?±?483 ng·h/mL and a t_1/2 of 45.5?±?31.5?h. Protein binding was 98.1?±?1.6%. AC04 showed higher penetration into the lung, spleen and liver, with AUC_0–96 of 798,443, 263,211 and 303,722 ng·h/mL, respectively. The 1-oxo-AC04 metabolite represented 10% of AC04 plasma concentration, showing a t_1/2 of 23.2?±?10.4?h.

4. These results suggest that, despite the small free plasma fraction, AC04 penetrates extensively reaching high concentrations in most tissues residing for a long time, which is important for its activity on solid tumours. All results combined indicate that AC04 is potentially a good antitumour candidate.

     

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities,mRNA and protein levels in rats

Abstract

Context: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes.

Objective: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver.

Materials and methods: Male Wistar Albino rats were injected with EH at a dose of 37.5?mg/kg i.p. daily for 9?d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively.

Results: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54?±?1.21 and 4.48?±?1.67?nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537?±?0.011 and 0.109?±?0.01?nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069?±?0.015 to 0.107?±?0.026?nmol/min/mg and from 163.34?±?92 to 588.3?±?14?nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment.

Discussion and conclusion: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.     

Modulation of NAD(P)H:Quinone oxidoreductase (NQO1) activity mediated by 5-arylamino-2-methyl-4,7-dioxobenzothiazoles and their cytotoxic potential

Synthesized 5-arylamino-2-methyl-4,7-dioxobenzothiazoles 3a-3o were evaluated for modulation of NAD(P)H: quinone oxidoreductase (NQO1) activity with the cytosolic fractions derived from cultured human lung cancer cells and their cytotoxicity in cultured several human solid cancer cell lines. The 4,7-dioxobenzothiazoles affected the reduction potential by NQO1 activity and showed a potent cytotoxic activity against human cancer cell lines. The tested compounds 3a, 3b, 3g, 3h, 3n and 3o were considered as more potent cytotoxic agents, and comparable modulators of NQO1 activity.     

Effects of 6-arylamino-5,8-quinolinediones and 6-chloro-7-ary-lamino-5,8-isoquinolinediones on NAD(P)H: quinone oxidoreductase (NQO1) activity and their cytotoxic potential

Synthesized 6-arylamino-5,8-quinolinediones 4a-4j and 6-chloro-7-arylamino-5,8-isoquinolinediones 5a-5g were evaluated for effects on NAD(P)H: quinone oxidoreductase (NQO1) activity with the cytosolic fractions derived from cultured human lung cancer cells and their cytotoxicity in cultured several human solid cancer cell lines. The 5,8-quinolinediones 4 and 5,8-isoquinolinediones 5 affected the reduction potential by NQO1 activity and showed a potent cytotoxic activity against human cancer cell lines. The tested compounds 4a, 5c, 5f, and 5g were considered as more potent cytotoxic agents. The compounds 4d, 5b, 5c, 5e and 5g were comparable modulators of NQO1 activity.     

A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by stable transfection. The level of NQO1 over-expression ranged from 14 to 29 times the NQO1 activity in the wild-type CHO cells. This panel of cell lines, allowed investigation of the protective role of NQO1 in quinone cytotoxicity. It could be demonstrated that menadione toxicity was significantly reduced in all NQO1-transfected CHO clones compared to the wild-type cells, but the clones did not show differences in their level of protection against menadione. This observation pointed at a critical threshold concentration of NQO1 above which a further increase does not provide further protection against quinone cytotoxicity. Additional studies in which the NQO1 activity was inhibited by dicoumarol showed that only dicoumarol concentrations of about five times the EC(50) for NQO1 inhibition were able to reduce NQO1 levels below the apparent threshold, making the cells more sensitive. The level of this threshold was estimated to be in the range of base line NQO1 activities observed in several tissues and species. Thus, the results of the present study indicate that beneficial effects of NQO1 induction by, for example, cruciferous vegetables might be absent or present depending on the NQO1 activity threshold for optimal protection and the basal level of NQO1 expression in the tissue and species of interest.     

Antimelanoma activity of the redox dye DCPIP (2,6-dichlorophenolindophenol) is antagonized by NQO1

Altered redox homeostasis involved in the control of cancer cell survival and proliferative signaling represents a chemical vulnerability that can be targeted by prooxidant redox intervention. Here, we demonstrate that the redox dye 2,6-dichlorophenolindophenol (DCPIP) may serve as a prooxidant chemotherapeutic targeting human melanoma cells in vitro and in vivo. DCPIP-apoptogenicity observed in the human melanoma cell lines A375 and G361 was inversely correlated with NAD(P)H:quinone oxidoreductase (NQO1) expression levels. In A375 cells displaying low NQO1 activity, DCPIP induced apoptosis with procaspase-3 and PARP cleavage, whereas G361 cells expressing high levels of enzymatically active NQO1 were resistant to DCPIP-cytotoxicity. Genetic (siRNA) or pharmacological (dicoumarol) antagonism of NQO1 strongly sensitized G361 cells to DCPIP apoptogenic activity. DCPIP-cytotoxicity was associated with the induction of oxidative stress and rapid depletion of glutathione in A375 and NQO1-modulated G361 cells. Expression array analysis revealed a DCPIP-induced stress response in A375 cells with massive upregulation of genes encoding Hsp70B’ (HSPA6), Hsp70 (HSPA1A), heme oxygenase-1 (HMOX1), and early growth response protein 1 (EGR1) further confirmed by immunodetection. Systemic administration of DCPIP displayed significant antimelanoma activity in the A375 murine xenograft model. These findings suggest feasibility of targeting tumors that display low NQO1 enzymatic activity using DCPIP.     

选择性PARP-1抑制剂的研究进展

聚腺苷二磷酸核糖聚合酶[Poly(ADP-ribose) polymerase,PARP]作为一种参与DNA修复的关键酶,已成为抗肿瘤药物开发的重要靶点。PARP抑制剂通过抑制DNA修复及协同致死作用可有效杀死肿瘤细胞,已被广泛应用于多种肿瘤的治疗。但大部分已被批准的PARP抑制剂因在抑制PARP-1的同时对PARP-2也有抑制作用而产生不良反应。因此,提高PARP-1抑制剂的选择性是解决这一问题的重要策略。本文综述已报道的选择性PARP-1抑制剂的研究进展,包括研究方法、构效关系、药理作用等。     

Role of NAD(P)H:quinone oxidoreductase 1 in clofibrate-mediated hepatoprotection from acetaminophen

Mice pretreated with the peroxisome proliferator clofibrate (CFB) are resistant to acetaminophen (APAP) hepatotoxicity. Whereas the mechanism of protection is not entirely known, CFB decreases protein adducts formed by the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant properties that is responsible for the reduction of cellular quinones. We hypothesized that CFB increases NQO1 activity, which in turn enhances the conversion of NAPQI back to the parent APAP. This could explain the decreases in APAP covalent binding and glutathione depletion produced by CFB without affecting APAP bioactivation to NAPQI. Administration of CFB (500 mg/kg, i.p.) to male CD-1 mice for 5 or 10 days increased NQO1 protein and activity levels. To evaluate the capacity of NQO1 to reduce NAPQI back to APAP, we utilized a microsomal activating system. Cytochrome P450 enzymes present in microsomes bioactivate APAP to NAPQI, which binds the electrophile trapping agent, N-acetyl cysteine (NAC). We analyzed the formation of APAP–NAC metabolite in the presence of human recombinant NQO1. Results indicate that NQO1 is capable of reducing NAPQI. The capacity of NQO1 to amelioriate APAP toxicity was then evaluated in primary hepatocytes. Primary hepatocytes isolated from mice dosed with CFB are resistant to APAP toxicity. These hepatocytes were also exposed to ES936, a high affinity, and irreversible inhibitor of NQO1 in the presence of APAP. Concentrations of ES936 that resulted in over 94% inhibition of NQO1 activity did not increase the susceptibility of hepatocytes from CFB treated mice to APAP. Whereas NQO1 is mechanistically capable of reducing NAPQI, CFB-mediated hepatoprotection does not appear to be dependent upon enhanced expression of NQO1.     

白细胞介素-1β和黄芪对气管上皮细胞转化生长因子-β_1表达的影响

目的检测经白细胞介素(IL)-1β、IL-1β和黄芪共同干预后兔气道上皮细胞转化生长因子-β1(TGF-β1)蛋白的表达水平,探讨IL-1β对气管上皮细胞TGF-β1表达的影响以及黄芪在哮喘气管重塑中的防治作用。方法体外培养兔气管上皮细胞,①实验组加入终浓度为1ng/ml的IL-1β,于不同时间点收集培养上清液及贴有细胞的盖玻片;②实验组分别加入不同浓度的IL-1β;③各组均加入终浓度为10ng/ml的IL-1β,同时实验组分别加入不同浓度的黄芪和地塞米松。②与③均于24h后收集培养上清液及贴有细胞的盖玻片。采用免疫细胞化学染色和双抗体夹心酶联免疫吸附试验(ELISA)测定TGF-β1蛋白的表达。结果①经IL-1β1ng/ml处理后TGF-β1的表达在24h点[上皮细胞吸光度值(0.613±0.022),上清液含量(701±32)pg/ml]明显高于其余各时间点(P分别<0.05和0.01)。②与对照组[(0.138±0.009),(216±28)pg/ml]比较,IL-1β 0.1ng组[(0.156±0.003),(267±12)pg/ml]、1ng组[(0.614±0.020),(710±32)pg/ml]、10ng组[(0.917±0.050),(940±34)pg/ml]TGF-β1表达均增高,差异有统计学意义(P分别<0.05和0.01);经直线相关分析培养上清液中TGF-β1的含量与所加IL-1β的浓度呈正相关(r=0.906,P<0.01)。③与IL-1β组[(0.904±0.047),(935±32)pg/ml]比较,黄芪50mg组[(0.397±0.020),(398±52)pg/ml]、黄芪200mg组[(0.144±0.005),(258±45)pg/ml]、黄芪500mg组[(0.401±0.005),(414±22)pg/ml]和地塞米松组[(0.155±0.003),(247±44)pg/ml]TGF-β1表达水平均降低,差异有统计学意义(P分别<0.05和0.01)。结论IL-1β可促进气管上皮细胞TGF-β1的蛋白表达,黄芪对这一过程有抑制作用,早期应用黄芪可能延缓气管重塑的形成与发展。     

Nerolidol inhibits the LOX-1 / IL-1β signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea

PurposeNerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis.MethodsThe minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1β (IL-1β). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot.ResultsNerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1β were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1β levels in A. fumigatus stimulated HCECs.ConclusionNerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1β signaling.