RESUMO
BACKGROUND: IARC has classified the betel nut as a human environmental carcinogen. Previous studies have found that arecoline (AR) is the major alkaloid present in the saliva of betel quid chewers. Saliva contains a large content of AR which has been further shown to cause mutation of oral mucosa cells, resulting in oral cancer. Whereas, to date, there are only few studies reported the hepatotoxicity associated with arecoline and betel nut chewing. Therefore, the main purpose of this study was to determine the toxic effects of AR and its oxidative metabolite, arecoline N-oxide (ARNO), in normal liver cell lines. METHODS: The cytotoxic, genotoxic, and mutagenic effects were detected by crystal violet staining, alkaline comet assay, and Salmonella mutagenicity test, respectively. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2-DCFDA assay. RESULTS: Our results demonstrated that ARNO exerted higher cytotoxicity, DNA damage, and mutagenicity than its parent compound arecoline in liver cells. Antioxidants, such as N-acetylcysteine, Trolox, and penicillamine, strongly protected liver cells from ARNO-induced DNA damage and ROS production. Furthermore, co-treatment with Mito-TEMPO also effectively blocked ARNO-induced ROS production in liver cells. Besides antioxidants, co-treatment with 1-aminobenzotriazole and methimazole nearly completely suppressed ARNO-induced ROS production in liver cells. CONCLUSIONS: Our data suggest that arecoline ingested from the habit of chewing betel quid can be primarily oxidized to ARNO, thereby enhancing its toxicity through increased ROS production. Considering the excellent protective effects of both mitochondria-targeted antioxidant and CYP450 inhibitor on ARNO-induced ROS production in liver cells, mitochondria CYP450-mediated metabolism of ARNO may be a key mechanism. Collectively, our results provide novel cellular evidence for the positive connection between habitual betel quid chewing and the risk for liver damage.
Assuntos
Arecolina/análogos & derivados , Óxidos N-Cíclicos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Areca/química , Arecolina/toxicidade , Linhagem Celular , Cromanos/farmacologia , Dano ao DNA , Fígado/citologia , Mitocôndrias/metabolismo , Testes de Mutagenicidade , Estresse Oxidativo , Penicilamina/farmacologia , Ratos , Salmonella/efeitos dos fármacosRESUMO
Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKß, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKß proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKß proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKß:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKß protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKß or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and ß-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKß and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKß, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKß had powerful antitumor properties and that ZAKα could promote tumor growth.
Assuntos
Neoplasias/prevenção & controle , Proteínas Quinases/fisiologia , Processamento Alternativo , Linhagem Celular Tumoral , Proliferação de Células , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Doxorrubicina/farmacologia , Humanos , MAP Quinase Quinase Quinases , Neoplasias/patologia , Isoformas de Proteínas , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers. However, cisplatin can induce nephrotoxicity and neurotoxicity, limiting its dosage and usage. Galangin, a natural flavonol, has been found to exhibit anti-oxidant and anti-inflammatory effects in vivo. Here, we investigated the effects of galangin on cisplatin-induced acute kidney injury (AKI) and its molecular mechanisms in mice. Galangin administration reduced the cisplatin-induced oxidative stress by decreasing renal MDA and 3-NT formations. Galangin administration also increased renal anti-oxidative enzyme activities (SOD, GPx, and CAT) and GSH levels depleted by cisplatin. Furthermore, galangin administration inactivated stress-induced Nrf2 protein and its downstream products, HO-1 and GCLC. In terms of the inflammatory response, galangin administration reduced IκBα phosphorylation, NF-κB phosphorylation and nuclear translocation, and then inhibited cisplatin-induced secretions of pro-inflammatory TNF-α, IL-1ß and IL-6. In addition, cisplatin-induced ERK and p38 phosphorylations were inhibited by galangin administration. In terms of cell death, galangin administration reduced levels of p53, pro-apoptotic Bax and activated caspase-3 to inhibit the cisplatin-induced apoptosis. Galangin administration also reduced the expression levels of RIP1 and RIP3 to inhibit cisplatin-induced RIP1/RIP3-dependent necroptosis. Therefore, galangin administration significantly ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation, and cell death through inhibitions of ERK and NF-κB signaling pathways. Galangin might be a potential adjuvant for clinical cisplatin therapy.
Assuntos
Injúria Renal Aguda/prevenção & controle , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Mediadores da Inflamação/metabolismo , Rim/efeitos dos fármacos , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/patologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/metabolismo , Rim/enzimologia , Rim/patologia , Masculino , Malondialdeído/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/análise , Inibidor de Quinase Dependente de Ciclina p27/análise , Humanos , MAP Quinase Quinase QuinasesRESUMO
Smoking increases the risk of cardiovascular disorders and leads to damage caused by inflammation and oxidative stress. The actin cytoskeleton is a key player in the response to inflammatory stimuli and is an early target of cellular oxidative stress. The purpose of this study was to investigate the changes in actin cytoskeleton dynamics in human endothelial EA.hy926 cells exposed to cigarette smoke extract (CSE). Immunostaining revealed that CSE exposure resulted in modification of the actin cytoskeleton and led to cell rounding in a dose- and time-dependent manner. In addition, the intracellular calcium concentration was increased by treatment with CSE. Pretreatment with antioxidants (lipoic acid, glutathione, N-acetyl cysteine, aminoguanidine, α-tocopherol, and vitamin C) significantly attenuated the CSE-induced actin cytoskeleton reorganization and cell rounding. Calcium ion chelators (EGTA, BAPTA-AM AM) and a potent store-operated calcium channel inhibitor (MRS 1845) also reduced CSE-induced intracellular calcium changes and attenuated actin cytoskeleton reorganization and cell morphology change. Moreover, the CSE-induced intracellular calcium increase was suppressed by pretreatment with the inositol trisphosphate receptor (IP3R) inhibitor xestospongin C, the phospholipase C (PLC) inhibitor U-73122, and the protein kinase C (PKC) inhibitor GF109203X. These results suggest that reactive oxygen species production and intracellular calcium increase play an essential role in CSE-induced actin disorganization and cell rounding through a PLC-IP3-PKC signaling pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1293-1306, 2016.
Assuntos
Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Nicotiana/química , Fumaça , Acetilcisteína/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Antioxidantes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quelantes/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Estrenos/farmacologia , Glutationa/metabolismo , Humanos , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Maleimidas/farmacologia , Microscopia de Fluorescência , Nitrendipino/análogos & derivados , Nitrendipino/farmacologia , Oxazóis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Nicotiana/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Andrographolide, a bioactive diterpenoid, is identified in Andrographis paniculata. In this study, we investigated the pharmacokinetics and bioavailability of andrographolide in rats and studied whether andrographolide enhances antioxidant defense in a variety of tissues and protects against carbon tetrachloride-induced oxidative damage. After a single 50-mg/kg administration, the maximum plasma concentration of andrographolide was 1µM which peaked at 30min. The bioavailability of andrographolide was 1.19%. In a hepatoprotection study, rats were intragastrically dosed with 30 or 50mg/kg andrographolide for 5 consecutive days. The results showed that andrographolide up-regulated glutamate cysteine ligase (GCL) catalytic and modifier subunits, superoxide dismutase (SOD)-1, heme oxygenase (HO)-1, and glutathione (GSH) S-transferase (GST) Ya/Yb protein and mRNA expression in the liver, heart, and kidneys. The activity of SOD, GST, and GSH reductase was also increased in rats dosed with andrographolide (p<0.05). Immunoblot analysis and EMSA revealed that andrographolide increased nuclear Nrf2 contents and Nrf2 binding to DNA, respectively. After the 5-day andrographolide treatment, one group of animals was intraperitoneally injected with carbon tetrachloride (CCl4) at day 6. Andrographolide pretreatment suppressed CCl4-induced plasma aminotransferase activity and hepatic lipid peroxidation (p<0.05). These results suggest that andrographolide is quickly absorbed in the intestinal tract in rats with a bioavailability of 1.19%. Andrographolide protects against chemical-induced oxidative damage by up-regulating the gene transcription and activity of antioxidant enzymes in various tissues.
Assuntos
Antioxidantes/farmacocinética , Tetracloreto de Carbono/toxicidade , Diterpenos/farmacocinética , Estresse Oxidativo/efeitos dos fármacos , Animais , Disponibilidade Biológica , Diterpenos/sangue , Masculino , Estresse Oxidativo/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Chrysin, apigenin, and luteolin are flavones that differ in their number of hydroxyl groups in the B ring. In this study, we investigated the protection by chrysin, apigenin, and luteolin against tert-butyl hydroperoxide (tBHP)-induced oxidative stress and the possible mechanisms involved in rat primary hepatocytes. Chrysin, apigenin, and luteolin dose-dependently up-regulated the protein expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase (GCL) catalytic (GCLC) and modifier subunit (GCLM) and increased the intracellular glutathione (GSH) content and the ratio of GSH to oxidized GSH. Among the flavones studied, chrysin showed the greatest induction of HO-1, GCLC, and GCLM protein expression and GSH content. Cellular reactive oxygen species production induced by tBHP was attenuated by pretreatment with chrysin, apigenin, and luteolin (P < .05), and this protection was reversed by the GCL inhibitor l-buthionine-S-sulfoximine and the HO-1 inhibitor zinc protoporphyrin. Chrysin, apigenin, and luteolin activated extracellular signal-regulated protein kinase 2 (ERK2), nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, nuclear Nrf2-antioxidant responsive element (ARE) binding activity, and ARE-dependent luciferase activity. Both ERK2 and Nrf2 siRNAs attenuated chrysin-induced HO-1, GCLC, and GCLM protein expression. Taken together, these results suggest that chrysin, apigenin, and luteolin inhibit tBHP-induced oxidative stress by up-regulating HO-1, GCLC, and GCLM gene transcription via the ERK2/Nrf2/ARE signaling pathways in rat primary hepatocytes.
Assuntos
Apigenina/farmacologia , Flavonoides/farmacologia , Glutamato-Cisteína Ligase/genética , Heme Oxigenase (Desciclizante)/genética , Hepatócitos/efeitos dos fármacos , Luteolina/farmacologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Apigenina/química , Células Cultivadas , Relação Dose-Resposta a Droga , Flavonoides/química , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hepatócitos/metabolismo , Luteolina/química , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Regulação para Cima/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologiaRESUMO
Butachlor is the most commonly used herbicide on paddy fields in Taiwan and throughout Southeast Asia. Since paddy fields provide habitat for pond breeding amphibians, we examined growth, development, time to metamorphosis, and survival of alpine cricket frog tadpoles (Fejervarya limnocharis) exposed to environmentally realistic concentrations of butachlor. We documented negative impacts of butachlor on survival, development, and time to metamorphosis, but not on tadpole growth. The 96 h LC(50) for tadpoles was 0.87 mg/l, much lower than the 4.8 mg/l recommended dosage for application to paddy fields. Even given the rapid breakdown of butachlor, tadpoles would be exposed to concentrations in excess of their 96 h LC(50) for an estimated 126 h. We also documented DNA damage (genotoxicity) in tadpoles exposed to butachlor at concentrations an order of magnitude less than the 4.8 mg/l recommended application rate. We did not find that butachlor depressed cholinesterase activity of tadpoles, unlike most organophosphorus insecticides. We conclude that butachlor is likely to have widespread negative impacts on amphibians occupying paddy fields with traditional herbicide application.
Assuntos
Acetanilidas/toxicidade , Herbicidas/toxicidade , Ranidae/fisiologia , Poluentes Químicos da Água/toxicidade , Acetilcolinesterase/metabolismo , Agricultura , Animais , Relação Dose-Resposta a Droga , Crescimento e Desenvolvimento/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Testes de Mutagenicidade , Mutagênicos/toxicidade , Sistema Nervoso/efeitos dos fármacos , Ranidae/crescimento & desenvolvimento , Ranidae/metabolismo , Taiwan , Testes de Toxicidade Aguda , Testes de Toxicidade CrônicaRESUMO
Epidemiologic studies have demonstrated that chronic arsenic exposure is associated with the incidence of chronic diseases. This association is partly related to the increase in reactive oxygen species (ROS) overload and protein oxidation that result from arsenic exposure. In this study, we intended to identify proteins susceptible to oxidative carbonylation by sodium arsenite and the impact of carbonylation on the function of these proteins in human umbilical vein endothelial cells (HUVECs). The 2,4-dinitrophenylhydrazine (DNPH) dot-blot assay revealed that arsenite (0-50 µM) dose-dependently increased protein carbonylation. Consistent with these findings, the cellular ROS level as measured by 2',7'-dichlorofluorescein diacetate (DCHF-DA) assay was increased in cells exposed to arsenite. By two-dimensional gel electrophoresis and matrix assist laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS), one glycolytic enzyme, enolase-α, two cytoskeleton proteins, fascin (F-actin associated protein) and vimentin, and two protein quality control proteins, HSC70 (heat-shock cognate protein 70), and PDIA3 (protein disulfide isomerase family A, member 3) were identified to be arsenic-sensitive carbonlyated proteins. Accompanied by carbonylation, enolase-α activity was dose-dependently decreased and the F-actin filament network was disturbed. Taken together, our results suggest that arsenite exposure results in the generation of carbonylated proteins, and the resultant changes in energy metabolism and in the cytoskeletal network may partly lead to cell damage.
Assuntos
Arsenitos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Carbonilação Proteica , Proteínas/química , Compostos de Sódio/farmacologia , Eletroforese em Gel Bidimensional , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Docosahexaenoic acid (DHA) is known to regulate autophagy in cancer cells. We explored whether oxidative stress-induced growth inhibitor 1 (OSGIN1) is involved in the regulation of autophagy by DHA in breast cancer cells and the possible mechanisms involved. DHA upregulated the levels of OSGIN1, LC3-II and SQSTM1/p62. By contrast, DHA dose-dependently decreased the levels of mTOR and p-mTORS2448 expression. Using GFP/RFP-LC3 fluorescence staining, we showed that cells treated with DHA showed a dose-dependent response in autophagic signals. OSGIN1 Overexpression mimicked DHA treatment in that LC3-II and GFP/RFP-LC3 signals as well as the expression of p-AMPKαT172 and p-RaptorS792 were significantly increased, whereas mTOR, p-mTORS2448, and p-ULK1S757 expression were decreased. With knockdown of OSGIN1 expression, these outcomes were reversed. Moreover, OSGIN1 overexpression transiently elevated the accumulation of OSGIN1 and reactive oxygen species (ROS) in the mitochondrial fraction and subsequently increased p-AMPKαT172 and p-RaptorS792 expression. Upon pretreatment with Mito-TEMPO, a scavenger of mitochondrial ROS, these outcomes were reversed. Taken together, these results suggest that DHA can transiently elevate the generation of ROS in mitochondria and promote autophagosome formation through activation of the p-AMPKαT172/p-Raptor S792 and inactivation of the p-mTORS2448/p-ULK1Ser757 signaling pathways, and these effects depend on OSGIN1 protein in MCF-7 cells.
Assuntos
Adenilato Quinase/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ativação Enzimática , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Mutant p53 (mutp53) commonly loses its DNA binding affinity to p53 response elements (p53REs) and fails to induce apoptosis fully. However, the p53 mutation does not predict chemoresistance in all subtypes of breast cancers, and the critical determinants remain to be identified. In this study, mutp53 was found to mediate chemotherapy-induced long intergenic noncoding RNA-p21 (lincRNA-p21) expression by targeting the G-quadruplex structure rather than the p53RE on its promoter to promote chemosensitivity. However, estrogen receptor alpha (ERα) suppressed mutp53-mediated lincRNA-p21 expression by hijacking mutp53 to upregulate damaged DNA binding protein 2 (DDB2) transcription for subsequent DNA repair and chemoresistance. Levels of lincRNA-p21 positively correlated with the clinical responses of breast cancer patients to neoadjuvant chemotherapy and had an inverse correlation with the ER status and DDB2 level. In contrast, the carboplatin-induced DDB2 expression was higher in ER-positive breast tumor tissues. These results demonstrated that ER status determines the oncogenic function of mutp53 in chemoresistance by switching its target gene preference from lincRNA-p21 to DDB2 and suggest that induction of lincRNA-p21 and targeting DDB2 would be effective strategies to increase the chemosensitivity of mutp53 breast cancer patients.
RESUMO
Cigarette smoke is a mixture of chemicals that cause direct or indirect oxidative stress in different cell lines. We investigated the effect of nonfractionated cigarette smoke extract (CSE) on protein carbonylation in human THP-1 cells. Cells were exposed to various concentrations (2.5-20%) of CSE for 30 min, and protein carbonylation was assessed by use of the sensitive 2,4-dinitrophenylhydrazine immuno-dot blot assay. CSE-induced protein carbonylation exhibited a dose-response relation with CSE concentrations. However, with prolonged exposure to CSE, significant decrements were observed when compared with the 30 min exposure. Cotreatment of THP-1 cells with antioxidants (N-acetyl-cysteine, S-allyl-cysteine, and alpha-tocopherol) and copper(II) ion chelators (d-penicillamine) during CSE exposure significantly reduced protein carbonylation, whereas cotreatment with antioxidants (vitamin C and trolox) and a metal chelator (EDTA), iron chelator (1,10-phenanthroline), or copper(I) chelator (neocuprin) did not decrease CSE-induced protein carbonylation in THP-1 cells. These results suggest that protein carbonylation is induced by CSE in THP-1 cells via a copper(II)-catalyzed reaction and not an iron-catalyzed reaction. Furthermore, the copper(II) ions involved in this CSE-induced protein carbonylation are derived from the intracellular pool, not via uptake from the extracellular medium. We speculate that natural copper(II) chelators may prevent some of the health problems caused by cigarette smoking, including lung disease, renal failure, and diabetes.
Assuntos
Nicotiana/toxicidade , Carbonilação Proteica , Fumaça , Antioxidantes/farmacologia , Catálise , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Imunoensaio , Penicilamina/química , Penicilamina/farmacologia , Nicotiana/químicaRESUMO
The present study was designed to investigate the pathways involved in the effect of betel nut arecoline on cell viability in 3T3-L1 preadipocytes. Arecoline, but not arecaidine or guvacine, inhibited preadipocyte viability in a concentration- and time-dependent manner. Arecoline arrested preadipocyte growth in the G2/M phase of the cell cycle; decreased the total levels of cyclin-dependent kinase 1 (CDK1), p21, and p27 proteins; increased p53 and cyclin B1 protein levels; and had no effect on CDK2 protein levels. These results suggested that arecoline selectively affected a particular CDK subfamily. Arecoline inhibited AMP-activated protein kinase (AMPK) activity; conversely, the AMPK activator, AICAR, blocked the arecoline-induced inhibition of cell viability. Pre-treatment with the antioxidant, N-acetylcysteine, prevented the actions of arecoline on cell viability, G2/M growth arrest, reactive oxygen species (ROS) production, and the levels of CDK1, p21, p27, p53, cyclin B1, and phospho-AMPK proteins. These AMPK- and ROS-dependent effects of arecoline on preadipocyte growth may be related to the mechanism underlying the modulatory effect of arecoline on body weight.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Arecolina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteínas de Ciclo Celular/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , CamundongosRESUMO
Polyglutamine (polyQ)-expanded mutant ataxin-3 protein, which is prone to misfolding and aggregation, leads to cerebellar neurotoxicity in spinocerebellar ataxia type 3 (SCA3), an inherited PolyQ neurodegenerative disease. Although the exact mechanism is unknown, the pathogenic effects of mutant ataxin-3 are associated with dysregulation of transcription, protein degradation, mitochondrial function, apoptosis, and antioxidant potency. In the present study we explored the protective role and possible mechanism of caffeic acid (CA) and resveratrol (Res) in cells and Drosophila expressing mutant ataxin-3. Treatment with CA and Res increased the levels of antioxidant and autophagy protein expression with consequently corrected levels of reactive oxygen species, mitochondrial membrane potential, mutant ataxin-3, and the aggregation of mutant ataxin-3 in SK-N-SH-MJD78 cells. Moreover, in SK-N-SH-MJD78 cells, CA and Res enhanced the transcriptional activity of nuclear factor erythroid-derived-2-like 2 (Nrf2), a master transcription factor that upregulates the expression of antioxidant defense genes and the autophagy gene p62. CA and Res improved survival and motor performance in SCA3 Drosophila. Additionally, the above-mentioned protective effects of CA were also observed in CA-supplemented SCA3 Drosophila. Notably, blockade of the Nrf2 pathway by use of small interfering RNA annulled the health effects of CA and Res on SCA3, which affirmed the importance of the increase in Nrf2 activation by CA and Res. Additional studies are need to dissect the protective role of CA and Res in modulating neurodegenerative progression in SCA3 and other polyQ diseases.
Assuntos
Ataxina-3/genética , Ácidos Cafeicos/uso terapêutico , Doença de Machado-Joseph/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Resveratrol/uso terapêutico , Animais , Antioxidantes/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Vias Eferentes/efeitos dos fármacos , Humanos , Doença de Machado-Joseph/genética , Mutação/genética , Fator 2 Relacionado a NF-E2/genética , Agregação Patológica de Proteínas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Transgenes/genética , Regulação para CimaRESUMO
Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains unclear. Our previous study showed that arsenite significantly induces oxidative DNA adducts and DNA-protein cross-links in several mammalian cell lines. In the present study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the possible target in the genomic DNA of human lymphoblastoid cells that were exposed to sodium arsenite. Treatment with both 10 and 80 microM arsenite for 4h induced significant changes in RAPD profiles compared with the control pattern. Two 10-mer RAPD primers (D11 and F1) produced the most distinguishable banding profiles between arsenite-treated and control genomic DNA. The sequencing of four arsenite-sensitive RAPD bands showed that the RB1CC1 and PACE4 genes might be the DNA targets of sodium arsenite treatment. We propose that arsenite may induce sequence- or gene-specific damage and then change the RAPD profile in human lymphoblastoid cells. The results of our study also show that RAPD combined with other techniques is a good tool for detecting alterations in genomic DNA and for the direct screening of new molecular markers related to arsenite-induced carcinogenesis.
Assuntos
Arsenitos/toxicidade , Carcinógenos/toxicidade , Dano ao DNA , DNA/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Linfócitos/efeitos dos fármacos , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Compostos de Sódio/toxicidade , Proteínas Relacionadas à Autofagia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Transformada , DNA/metabolismo , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Humanos , Linfócitos/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a polyglutamine (polyQ) repeat in the protein ataxin-3 which is involved in susceptibility to mild oxidative stress induced neuronal death. Here we show that caffeic acid (CA) and resveratrol (Res) decreased reactive oxygen species (ROS), mutant ataxin-3 and apoptosis and increased autophagy in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells containing mutant ataxin-3. Furthermore, CA and Res improved survival and locomotor activity and decreased mutant ataxin-3 and ROS levels in tBH-treated SCA3 Drosophila. CA and Res also altered p53 and nuclear factor-κB (NF-κB) activation and expression in tBH-treated cell and fly models of SCA3, respectively. Blockade of NF-κB activation annulled the protective effects of CA and Res on apoptosis, ROS, and p53 activation in tBH-treated SK-N-SH-MJD78 cells, which suggests the importance of restoring NF-κB activity by CA and Res. Our findings suggest that CA and Res may be useful in the management of oxidative stress induced neuronal apoptosis in SCA3.
Assuntos
Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Doença de Machado-Joseph/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila , Humanos , Doença de Machado-Joseph/tratamento farmacológico , Doença de Machado-Joseph/etiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Peroxynitrite is a major oxidizing and nitrating biological agent formed at sites of inflammation. Peroxynitrite can cause DNA damage and is thought to contribute to inflammation-related carcinogenesis. This study describes a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct determination of peroxynitrite-derived 8-nitroguanine (8-nitroGua) in DNA hydrolysates. This method exhibited a sensitive detection limit of 3 fmol and inter- and intraday imprecision of <10% and was applied to systemically examine the formation and stability of peroxynitrite-derived 8-nitroGua in different DNA substrates under various conditions. The 8-nitroGua formation was maximal at pH 8. The formation rate of 8-nitroGua in different DNA substrates decreased in the order of monodeoxynucleoside>single-stranded DNA>double-stranded DNA. A stability test revealed that the half-life for the depurination of 8-nitroGua from DNA was short and affected by both the temperature and DNA structure. When present in monodeoxynucleoside, the half-life of 8-nitroGua was estimated to be ~6min at 25°C and 2.3h at ~0°C. In single-stranded DNA, the half-life varied from 1.6h at 37°C to 533h at -20°C, whereas the half-life increased from 2.4h at 37°C to 1115h at -20°C in double-stranded DNA. We demonstrated that the measurement of 8-nitroGua in isolated DNA is not practicable because 8-nitroGua is unstable and lost during DNA extraction from cell. Therefore, we suggest that directly detecting cellular 8-nitroGua following nuclear membrane lysis is an alternative measure of the nitrative damage of nucleic acids, accounting for both DNA and RNA lesions within cells.
Assuntos
DNA/química , Células Endoteliais/química , Guanina/análogos & derivados , Ácido Peroxinitroso/química , Animais , Células CHO , Bovinos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cricetulus , DNA/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Guanina/análise , Guanina/química , Guanina/metabolismo , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Cinética , Limite de Detecção , Variações Dependentes do Observador , Ácido Peroxinitroso/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKCδ and STAT3α activation and GSK3ß phosphorylation; up-regulated Wnt-1, ß-catenin, and STAT3α expression; and increased the nuclear translocation of ß-catenin and STAT3α. Rottlerin, a PKCδ inhibitor, abrogated the increases in STAT3α activation and ß-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3α DNA binding activity and ß-catenin expression. Knockdown of ß-catenin attenuated TPA-induced fascin-1 and STAT3α expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, ß-catenin, and STAT3α expression as well. TPA also induced Wnt-1 expression and secretion, and blocking Wnt-1 signaling abrogated ß-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKCδ and STAT3α activation, GSK3ß phosphorylation, and Wnt-1, ß-catenin, STAT3α, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKCδ and Wnt-1 pathways, which lead to STAT3α activation, GSK3ß inactivation, and ß-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKCδ and Wnt-1 signaling.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Movimento Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Proteínas dos Microfilamentos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Humanos , Proteína Quinase C-delta/metabolismo , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Arsenic is recognized to be a nonmutagenic carcinogen because it induces DNA damage only at very high concentrations. However, many more DNA strand breaks could be detected by digesting the DNA of arsenite-treated cells with endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K. By doing so, arsenite could be shown to induce DNA damage in human cells within a pathologically meaningful concentration range. Oxidized guanine products were detected in all arsenite-treated human cells examined. DNA-protein cross-links were also detected in arsenite-treated NB4 and HL60 cells. In human umbilical vein endothelial cells, the induction of oxidized guanine products by arsenite was sensitive to inhibitors of nitric oxide (NO) synthase but not to oxidant modulators, whereas the opposite result was obtained in vascular smooth muscle cells. On the other hand, the arsenite-induced oxidized guanine products and DNA-protein cross-links in NB4 and HL60 cells were sensitive to modulators of calcium, NO synthase, oxidant, and myeloperoxidase. Therefore, although oxidized guanine products were detected in all the human cells treated with arsenite, the pathways could be different in different cell types. Because the sensitivity and the mechanism of arsenic intoxication are cell specific, it is important that target tissues and target cells are used for investigations. It is also important that pathologically or pharmacologically meaningful concentrations of arsenic are used. This is because in most cases we are dealing with the chronic effect rather than acute toxicity.
Assuntos
Arsenitos/toxicidade , Adutos de DNA , DNA/química , Teratogênicos/toxicidade , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Guanina/química , Humanos , Ácido Hipocloroso/química , Óxido Nítrico Sintase/farmacologia , Estresse Oxidativo , Ligação Proteica , Espécies Reativas de Oxigênio/efeitos adversosRESUMO
BACKGROUND: Because lymphocytes from fragile X patients have been reported as hypersensitive to bleomycin-induced chromatid breaks and because the number of trinucleotide repeats in families with fragile X syndrome has a propensity to expand, we have investigated the possibility that fragile X cells may be hypersensitive to DNA damage and have a lower capacity for DNA repair. METHODS: Lymphocytes from normal and fragile X syndrome donors were immortalized by Epstein-Barr virus transformation. Characteristics of fragile X syndrome including the folate-sensitive fragile site on chromosome Xq27.3, length of CGG repeat expansion, and FMRP expression in Epstein-Barr virus-transformed lymphoblastoid cell lines were analyzed by standard cytogenetic methods, Southern blot, and Western blot, respectively. Analysis of DNA damage and repair induced by hydrogen peroxide, bleomycin, ethyl methanesulfonate, 4-nitroquinoline-N-oxide, etoposide, and mitomycin C was carried out by single-cell gel electrophoresis assay (known as comet assay). RESULTS: Lymphoblastoid cell lines from fragile X donors had a folate-sensitive fragile site on chromosome Xq27.3, no or low FMRP expression, and expansion of the CGG repeat. Results of comet assay showed that fragile X cells were not more sensitive to mutagen-induced DNA strand breaks and did not have lower DNA repair capacity in comparison with normal cells. Furthermore, one fragile X cell line showed hyposensitivity to DNA strand breaks induced by hydrogen peroxide, bleomycin, and ethyl methansulfonate. CONCLUSIONS: The results of this study do not support the notion that CGG trinucleotide expansion in fragile X syndrome is caused by permanent deficiency in DNA repair.