RESUMO
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.
Assuntos
Antioxidantes/metabolismo , Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Calcificação Vascular/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Modelos Animais de Doenças , Heme Oxigenase (Desciclizante)/biossíntese , Masculino , NAD(P)H Desidrogenase (Quinona)/biossíntese , Ratos , Ratos WistarRESUMO
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.
Assuntos
Infarto da Artéria Cerebral Média/tratamento farmacológico , Lipossomos/uso terapêutico , Nanopartículas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Antioxidantes/metabolismo , Linhagem Celular , Endotélio Vascular/patologia , Glucose/deficiência , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Humanos , Hipóxia , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologia , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/genéticaRESUMO
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.
Assuntos
Antioxidantes/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos/farmacologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Animais , Abelhas , Células Cultivadas , Epiderme/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/análise , Humanos , Queratinócitos/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pele/patologia , Regulação para CimaRESUMO
This study aimed to evaluate the cytotoxicity and genotoxicity from Inga laurina leaves extracts and fractions and obtain their chemical profile. The chemical profile of the crude extract from I. laurina leaves and its fractions was investigated through 1H NMR, RP-HPLC-PDA by co-injection with authentic standards and HPLC-MS. The quinone reductase induction as a biomarker for cancer chemoprevention was evaluated in murine hepatocellular carcinoma line, whereas the cytotoxicity was evaluated by sulforhodamine B assay (SRB) using HepG2 cell line and genotoxicity was evaluated by comet assay. The phytochemical analysis of the leaves crude extract and its fractions showed the presence of 2-hydroxyethyl-dodecanoate and the phenolic compounds: gallic acid, methyl gallate, p-coumaric acid, cinnamic acid, myricetin-3-O-(2â³-O-galoyl)-α-rhamnopyranoside, proanthocyanidin A-2 and myricetrin. All the fractions tested were not considered cytotoxic against the selected human cancer cell lines, they did not cause genotoxic in some concentrations damage and induced the enzyme quinone reductase.
Assuntos
Fabaceae/química , Mutagênicos/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , Dano ao DNA , Indução Enzimática/efeitos dos fármacos , Células Hep G2 , Humanos , Camundongos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder, caused by the selective death of dopaminergic neurons in the substantia nigra pars compacta. ß-caryophyllene (BCP) is a phytocannabinoid with several pharmacological properties, producing anti-inflammatory and antihypertensive effects. In addition, BCP protects dopaminergic neurons from neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), yet it remains unclear if this effect is due to its antioxidant activity. To assess whether this is the case, the effect of BCP on the expression and activity of NAD(P)H quinone oxidoreductase (NQO1) was evaluated in mice after the administration of MPTP. Male C57BL/6 J mice were divided into four groups, the first of which received saline solution i.p. in equivalent volume and served as a control group. The second group received MPTP. The second group received MPTP hydrochloride (5 mg/kg, i.p.) daily for seven consecutive days. The third group received BCP (10 mg/kg) for seven days, administered orally and finally, the fourth group received MPTP as described above and BCP for 7 days from the fourth day of MPTP administration. The results showed that BCP inhibits oxidative stress-induced cell death of dopaminergic neurons exposed to MPTP at the same time as it enhances the expression and enzymatic activity of NQO1. Also, the BCP treatment ameliorated motor dysfunction and protected the dopaminergic cells of the SNpc from damage induced by MPTP. Hence, BCP appears to achieve at least some of its antioxidant effects by augmenting NQO1 activity, which protects cells from MPTP toxicity. Accordingly, this phytocannabinoid may represent a promising pharmacological option to safeguard dopaminergic neurons and prevent the progression of PD.
Assuntos
Antioxidantes/uso terapêutico , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/prevenção & controle , NAD(P)H Desidrogenase (Quinona)/biossíntese , Sesquiterpenos Policíclicos/uso terapêutico , Animais , Antioxidantes/farmacologia , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Sesquiterpenos Policíclicos/farmacologia , Distribuição AleatóriaRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/biossíntese , Glutationa S-Transferase pi/biossíntese , Isotiocianatos/farmacologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Receptores de Hidrocarboneto Arílico/biossíntese , Sulfóxidos/farmacologia , Anticarcinógenos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Humanos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Biflavonoides/farmacologia , Catequina/farmacologia , Hipocampo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/fisiologia , Neurônios/metabolismo , Síndromes Neurotóxicas/prevenção & controle , Sevoflurano/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3/biossíntese , Caspase 9/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Glutamato-Cisteína Ligase/biossíntese , Heme Oxigenase-1/biossíntese , Hipocampo/metabolismo , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Neurônios/fisiologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Peroxirredoxinas/biossíntese , Cultura Primária de Células , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
One million females are diagnosed worldwide every year with breast cancer, and the mortality rate of these patients remains high. Several treatments, including surgery, are available for breast cancer. ßLapachone (ßLap), a natural quinone compound, has been developed for cancer treatment due to its strong cytotoxic effect through its action on NAD(P)H:quinone oxidoreductase 1 (NQO1)dependent activity. However, the mechanism in regards to how ßLap induces cytotoxicity in breast cancer cells is still elusive. In the present study, we showed that ßLap induced apoptotic cell death via activation of protein kinase A (PKA) in NQO1overexpressing MDAMB231 human breast cancer cells. This PKAdependent cell death was observed solely in NQO1overexpressing 231 cells via the high production of reactive oxygen species (ROS). Cell survival of antioxidant [Nacetylcysteine (NAC)]treated NQO1overexpressing 231 cells was significantly recovered, and NQO1negative 231 cells did not respond to ßLap. Antiapoptotic proteins such as Bcl2 and BclxL were decreased, while proapoptotic proteins, including cytochrome c, activation of caspase3, and cleavage of PARP were increased after ßLap treatment of NQO1overexpressing 231 cells. Furthermore, PKA activators, forskolin or dibutyrylcAMP, an analog of cAMP, aggravated the ßLapinduced apoptotic cell death by decreasing antiapoptotic proteins and further activating proapoptotic proteins in NQO1positive 231 cells. Treatment with a PKA inhibiter, H89, significantly increased cell viability even in NQO1overexpressing cells treated with ßLap. These data showed that ßLap activated PKA via ROS accumulation, subsequently leading to apoptotic cell death in NQO1positive breast cancer cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NAD(P)H Desidrogenase (Quinona)/biossíntese , Naftoquinonas/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant, which has a wide range of toxic effects on organisms. This study investigated the cytotoxic effects on human hepatocytes (L02â¯cells) after treated with 0, 5, 10, 20, and 40⯵M of TBBPA. Results showed that TBBPA significantly increased intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and the ratio of oxidized/reduced glutathione (GSSG/GSH) dose-dependently. TBBPA also decreased the cell mitochondrial membrane potential (MMP), caused the release of cytochrome C (Cyt C) to cytoplasm and promoted the expression of caspase-9 and caspase-3, and finally increased the level of apoptosis. The ROS inhibitor N-acetyl-L-cysteine (NAC) relieved the oxidative stress responses, and prevented the decrease of MMP and increase of apoptosis. In addition, TBBPA promoted the expression of antioxidant genes related to Nrf2, such as quinone oxidoreductase 1 (NQO1), catalase (CAT), and heme oxygenase 1 (HO-1). Oxidative stress initiated by TBBPA, activated mitochondrial apoptosis and Nrf2 pathway, and increased the degree of apoptosis in L02â¯cells.
Assuntos
Apoptose/efeitos dos fármacos , Retardadores de Chama/toxicidade , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Bifenil Polibromatos/toxicidade , Acetilcisteína/farmacologia , Caspase 3/biossíntese , Caspase 9/biossíntese , Catalase/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Hepatócitos/efeitos dos fármacos , Humanos , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Secondary brain injury imposes great effects on the prognosis of patients following traumatic brain injury (TBI). Accumulating evidence suggests that nuclear factor erythroid 2-related factor 2 (Nrf2) could play a neuroprotective role in experimental TBI models by regulating the expression of numerous antioxidant, anti-inflammatory, and neuroprotective proteins. However, whether Nrf2 is activated in patients following TBI is still unknown. In this study, human brain tissues were obtained during surgery from patients suffering from TBI. The purpose of this study was to investigate the expression of Nrf2 and Nrf2-regulated gene products, NAD(P)H quinine oxidoreductase 1, and glutathione S-transferase in human injured brain tissue after TBI. Our results revealed that the nuclear level of Nrf2 was significantly increased in injured brain tissues, whereas the cytoplasmic level of Nrf2 was markedly decreased. In addition, the expression of NAD(P)H quinine oxidoreductase 1 and glutathione S-transferase was significantly upregulated. Nrf2 may be activated and confer neuroprotection against secondary brain injury following TBI. Therefore, Nrf2 could serve as a promising molecular target for the treatment of TBI.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Fator 2 Relacionado a NF-E2/biossíntese , Adulto , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/biossíntese , Transportadores de Ânions Orgânicos/biossínteseRESUMO
The elevated expression of NQO1 in many human solid tumors along with its ability to activate quinone-based anticancer agents makes it an excellent target for enzyme-directed drug development. NQO1 plays an important role in melanogenesis and given its correlation with a poor patient outcome we propose this enzyme as an intriguing target for molecular-based therapeutic regimen against melanoma. Unfortunately, the natural product ß-Lapachone (ß-Lap), whose antitumor activity is based on NQO1, reported dose-limiting toxicity which hampered its pre-clinical and clinical use. Therefore, new effective and safe therapeutic NQO1-bioactivatable agents for melanoma treatment are desirable. Regarding NQO1, we demonstrated that halogenated ß-Lap derivative named PFB is an excellent substrate and effective tumor-selective anticancer compound. In addition, PFB resulted more attractive than the parent ß-Lap for treating metastatic-derived melanoma cells. In this context, it would be interesting to design strategies to induce NQO1 activity in cancer cells as a promising combinatorial approach with bioreductive drugs. In this sense, we had reported that photodynamic therapy (PDT) significantly upregulated NQO1 expression. Based on this event, here we demonstrated that the cytotoxic regimen consisting of PFB plus PDT improved synergistic therapeutic combination on melanoma cells. In conclusion, our contribution provides a strong rationale for using therapies that associate photo- and chemotherapy to effectively treat melanoma with modular NQO1 status.
Assuntos
Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/radioterapia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Naftoquinonas/uso terapêutico , Fotoquimioterapia/métodos , Radiossensibilizantes/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Naftoquinonas/farmacologia , Radiossensibilizantes/farmacologiaRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/análise , NAD(P)H Desidrogenase (Quinona)/biossíntese , Naftoquinonas/uso terapêutico , Necrose/induzido quimicamente , Neoplasias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Naftoquinonas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10nM), oltipraz (100µM) and simvastatin (1µM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.
Assuntos
Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1B1/biossíntese , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Receptores de Hidrocarboneto Arílico/biossíntese , Sinvastatina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos/biossíntese , Dibenzodioxinas Policloradas/farmacologia , Pirazinas/farmacologia , Tionas , TiofenosRESUMO
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.
Assuntos
Vesículas Extracelulares/enzimologia , Glutaminase/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Quinona Redutases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Vesículas Extracelulares/metabolismo , Glutaminase/biossíntese , Glutamina/metabolismo , Humanos , Immunoblotting , Masculino , Modelos Moleculares , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/metabolismo , Metástase Neoplásica , Estresse Oxidativo , Neoplasias da Próstata/metabolismo , Quinona Redutases/biossíntese , Quinona Redutases/químicaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Exposição Ocupacional/efeitos adversos , Estresse Oxidativo , Mucosa Respiratória/metabolismo , Lesão por Inalação de Fumaça/metabolismo , Fumaça/efeitos adversos , Adulto , Feminino , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mucosa Respiratória/patologia , Lesão por Inalação de Fumaça/patologiaRESUMO
Purpose: We previously demonstrated the anti-inflammatory and antioxidant effects of exenatide. We now hypothesized that exenatide also increases the plasma concentration of interleukin-1 receptor antagonist (IL-1RA), an endogenous anti-inflammatory protein, and modulates the nuclear factor erythroid 2ârelated factorâKelchlike ECH-associated protein 1âantioxidant response element (Nrf-2âKeap-1âARE) system to induce key antioxidant enzymes to suppress inflammatory and oxidative stress. Methods: Twenty-four patients with obesity and type 2 diabetes receiving combined oral and insulin therapy were randomly assigned to receive either exenatide 10 µg or placebo twice a day for 12 weeks. Results: Exenatide increased IL-1RA concentration by 61% (from 318 ± 53 to 456 ± 88 pg/mL; P < 0.05). Exenatide treatment also suppressed Keap-1 protein (P < 0.05) and increased messenger RNA expression of NQO-1, glutathione S-transferase PI, heme oxygenase-1, and p21 and increased NAD(P)H dehydrogenase [quinone] 1 protein (P < 0.05) in mononuclear cells. Conclusions: Because IL-1RA protects, maintains, and stimulates ß-cell function in humans and Nrf-2âKeap-1âARE protects ß cells in animals with experimental diabetes, these actions of exenatide may contribute to a potential protective effect on ß cells in diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/sangue , Obesidade/tratamento farmacológico , Peptídeos/farmacologia , Peçonhas/farmacologia , Elementos de Resposta Antioxidante/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Quimioterapia Combinada , Indução Enzimática/efeitos dos fármacos , Exenatida , Feminino , Glutationa S-Transferase pi/biossíntese , Heme Oxigenase-1/biossíntese , Humanos , Insulina/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Obesidade/sangue , Obesidade/complicaçõesRESUMO
Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market.
Assuntos
Anticarcinógenos/metabolismo , Brassica/genética , Brassica/metabolismo , Glucosinolatos/genética , Glucosinolatos/metabolismo , Anticarcinógenos/análise , Brassica/química , Suplementos Nutricionais/análise , Topos Floridos/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Glucose/análogos & derivados , Glucose/análise , Glucose/genética , Glucose/metabolismo , Glucosinolatos/análise , Humanos , Hidrólise , Imidoésteres/análise , Imidoésteres/metabolismo , Metaboloma , Técnicas Analíticas Microfluídicas , NAD(P)H Desidrogenase (Quinona)/biossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/biossíntese , Raízes de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Distribuição TecidualRESUMO
Dihydroartemisinin (DHA) has been shown to inhibit the viability of various cancer cells. Previous studies have revealed that the mechanisms involved in the inhibitory effects of DHA are based on theactivation of p53 and the mitochondrial-related cell death pathway. However, the exact association between upstream signaling and the activation of cell death pathway remains unclear. In this study, we found that DHA treatment induced the upregulation of caveolin 1 (Cav1) and mitochondrial carrier homolog 2 (MTCH2) in HeLa cells, and this was associated with the DHA-induced inhibition of cell viability and DHA-induced apoptosis. Additionally, the overexpression of Cav1 and MTCH2 in HeLa cells enhanced the inhibitory effects of DHA on cell viability. Moreover, we also found that the upregulation of Cav1 contributed to the DHA-mediated p53 activation and the downregulation of the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), which have been reported to contribute to the activation of the cell death pathway. Of note, we also found that DHA induced the nuclear translocation and accumulation of both Cav1 and p53, indicating a novel potential mechanism, namely the regulation of p53 activation by Cav1. On the whole, our study identified Cav1 and MTCH2 as the molecular targets of DHA and revealed a new link between the upstream Cav1/MTCH2 upregulation and the downstream activation of the cell death pathway involved in the DHA-mediated inhibition of cell viability.
Assuntos
Artemisininas/farmacologia , Caveolina 1/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/biossíntese , NAD(P)H Desidrogenase (Quinona)/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Neoplasias do Colo do Útero/patologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Waltheria indica L. is traditionally used in several countries against inflammatory related diseases and cancer, mainly as a decoction of the aerial parts. AIM OF THE STUDY: The transcription factor NF-κB is known to induce tumor promotion and progression and is considered a major player in inflammation-driven cancers. Therefore, inhibitors of this pathway possess cancer chemopreventive and chemotherapeutic activities. This study aimed first to confirm the use of Waltheria indica as a traditional anti-inflammatory remedy by assessing the NF-κB inhibitory activity and then to identify the major bioactive compounds. The isolated compounds were also tested for their QR inducing property, a complementary strategy in cancer chemoprevention able to target tumor initiation. Finally, the relevance of in vitro results was examined by investigating the occurrence of the active compounds in traditional preparations. MATERIALS AND METHODS: Compounds were isolated from the dichloromethane extract of the aerial parts using flash chromatography and semi-preparative HPLC. NF-κB inhibitory activity of pure compounds from Waltheria indica was assessed using a luciferase reporter assay in HEK293 cells. Their QR inducing activity was also assessed in Hepa1c1c7 cells. RESULTS: Twenty-nine compounds, of which 5 are new, were obtained from the dichloromethane extract and tested for their cancer chemoprevention activity. Eleven compounds inhibited NF-κB and/or induced QR in the low to mid µM range. Chrysosplenol E (20) was active in both tests. Two of the most potent NF-κB inhibitors, waltherione A (4) and waltherione C (5), as well as 20 were found in the traditional decoction, in which 4 and 5 were major compounds. CONCLUSION: The presence of potent NF-κB inhibitors and QR inducing compounds in the decoction of the aerial parts of Waltheria indica supports its traditional use in inflammatory-related diseases and cancer chemoprevention.
Assuntos
Anti-Inflamatórios/farmacologia , Anticarcinógenos/farmacologia , Malvaceae/química , Extratos Vegetais/farmacologia , Anti-Inflamatórios/isolamento & purificação , Anticarcinógenos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Indução Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Medicina Tradicional/métodos , NAD(P)H Desidrogenase (Quinona)/biossíntese , NF-kappa B/metabolismo , Componentes Aéreos da PlantaRESUMO
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.