RESUMO
Polyphenols have attracted great interest as potent antioxidant compounds and nutraceuticals; however, their antioxidant properties represent a multifaceted phenomenon, including pro-oxidant effects under particular conditions and complex behavior when multiple polyphenols are simultaneously present. Moreover, their intracellular behavior cannot always be predicted from their ability to counteract the production of ROS in acellular assays. The present work aimed to study the direct intracellular redox activity of two polyphenols, resveratrol and quercetin, singly and in mixture in a cellular short-term bioassay under both basal and pro-oxidant conditions. The study was carried out by spectrofluorimetric assessment of the intracellular fluorescence of CM-H2DCFDA-charged HeLa cells under either basal conditions, due to the reactive species associated with the normal cellular oxidative metabolism, or pro-oxidant conditions induced by H2O2 exposure. Under basal conditions, the obtained results showed a significant antioxidant effect of quercetin and a weaker antioxidant effect of resveratrol when used singly, while antagonism of their effect was detected in their equimolar mixtures at all the concentrations used. Under exposure of the cells to H2O2, quercetin exhibited a dose-dependent intracellular antioxidant activity whereas resveratrol manifested a pro-oxidant intracellular activity, while their equimolar mixtures showed an intracellular interaction between the 2 polyphenols, with additive effects at 5 µM and synergic at 25 µM and 50 µM. Thus, the results clarified the direct intracellular antioxidant/pro-oxidant activity of quercetin and resveratrol alone and in their equimolar mixtures in the cell model HeLa cells and highlighted that the antioxidant properties of polyphenols in mixtures at the cellular level depend not only on the nature of the compounds themselves but also on the type of interactions in the cellular system, which in turn are influenced by the concentration and the oxidative status of the cell.
Assuntos
Quercetina , Estilbenos , Humanos , Resveratrol/farmacologia , Quercetina/farmacologia , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio , Células HeLa , Polifenóis/farmacologia , Oxirredução , Estilbenos/farmacologiaRESUMO
At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).
Assuntos
Antioxidantes/isolamento & purificação , Biologia Molecular/métodos , Estresse Oxidativo , Espécies Reativas de Oxigênio/isolamento & purificação , Antioxidantes/química , Fluoresceínas/química , Corantes Fluorescentes/química , Peróxido de Hidrogênio/química , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/químicaRESUMO
Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ∆sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H2O2, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ∆sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ∆sigCDE than in the control strain. These results indicate that ∆sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ∆sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ∆sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ∆sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ∆sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.
Assuntos
Estresse Oxidativo/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Synechocystis/metabolismo , Synechocystis/efeitos da radiação , Raios Ultravioleta , Carotenoides/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/efeitos da radiação , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos da radiação , Modelos Biológicos , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Processos Fotoquímicos/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Substâncias Protetoras/farmacologia , Superóxidos/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/crescimento & desenvolvimentoRESUMO
The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Mediadores da Inflamação/farmacologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Chaperonas Moleculares , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triptofano/análogos & derivados , Triptofano/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22(phox) expression are greatly increased in human prostate cancer tissues, and knockdown of p22(phox) by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22(phox) in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22(phox) resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22(phox) in tumor angiogenesis and tumor growth, and suggest that p22(phox) is a potential novel target for prostate cancer treatment.
Assuntos
Sistema de Sinalização das MAP Quinases , NADPH Oxidases/metabolismo , Neovascularização Patológica/enzimologia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/patologia , Neoplasias da Próstata/patologia , Ensaio Tumoral de Célula-Tronco , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aß fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aß42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aß42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Proteínas Amiloidogênicas/efeitos adversos , Carboxil e Carbamoil Transferases/efeitos adversos , Proteínas de Escherichia coli/efeitos adversos , Neuroblastoma/tratamento farmacológico , Neurônios/efeitos dos fármacos , Pré-Albumina/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Modelos Moleculares , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Conformação Proteica , Multimerização Proteica , RatosRESUMO
Anemia of inflammation or chronic disease is a highly prevalent form of anemia. The inflammatory cytokine interleukin-6 (IL-6) negatively correlates with hemoglobin concentration in many disease states. The IL-6-hepcidin antimicrobial peptide axis promotes iron-restricted anemia; however the full role of IL-6 in anemia of inflammation is not well-defined. We previously reported that chronic inflammation had a negative impact on maturation of erythroid progenitors in a mouse model. We hypothesized that IL-6 may be responsible for impaired erythropoiesis, independent of iron restriction. To test the hypothesis we utilized the human erythroleukemia TF-1 cell line to model erythroid maturation and exposed them to varying doses of IL-6 over six days. At 10 ng/ml, IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation. While IL-6 did not decrease the expression of genes associated with hemoglobin synthesis, we observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining. We also observed that IL-6 down regulated expression of the gene SLC4a1 which is expressed late in erythropoiesis. Those findings suggested that IL-6-dependent inhibition of hemoglobin synthesis might occur. We investigated the impact of IL-6 on mitochondria. IL-6 decreased the mitochondrial membrane potential at all treatment doses, and significantly decreased mitochondrial mass at the highest dose. Our studies indicate that IL-6 may impair mitochondrial function in maturing erythroid cells resulting in impaired hemoglobin production and erythroid maturation. Our findings may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and draw attention to the potential for new therapeutic targets that affect late erythroid development.
Assuntos
Eritropoese/efeitos dos fármacos , Interleucina-6/farmacologia , Leucemia Eritroblástica Aguda/etiologia , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Eritropoese/genética , Humanos , Imunofenotipagem , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2'-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1-2h) incubations with 0-10µM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences.
Assuntos
Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , DNA-Formamidopirimidina Glicosilase/genética , Compostos de Metilmercúrio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Compostos de Metilmercúrio/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco , Regulação para CimaRESUMO
A crucial issue in male infertility work-up is to have reliable methods to detect oxidative stress in native semen samples. Here, we explored flow cytometric detection of Reactive Oxygen Species (ROS) in viable spermatozoa using native semen samples. To this aim, we challenged three fluorescent probes: CM-H2DCFDA, CellROX Green and MitoSOX Red. After excluding all non-sperm cells, each probe was coupled to a suitable stain to eliminate also semen apoptotic bodies and non-viable spermatozoa: Merocyanine 540 (M540) for CM-H2DCFDA and CellROX Green, and LIVE/DEAD Fixable Green Dead Cell Stain (LD-G) for MitoSOX Red. We found that CM-H2DCFDA was confined in the sperm midpiece, whereas CellROX Green and MitoSOX Red were localized in the head of spermatozoa. Treatment with H2O2 highly increased MitoSOX Red fluorescence (36.20 ± 5.24 vs 18.02 ± 2.25, %, p < 0.01), but not, or only slightly, the labelling with CMH2DCFDA (2.57 ± 1.70 vs 2.77 ± 1.43, p > 0.05) and CellROX Green (5.34 ± 3.18 vs 3.76 ± 2.04, p < 0.05), respectively. Menadione treatment highly increased CellROX Green (10.13 ± 5.85 vs 3.82 ± 2.70, p < 0.01) and MitoSOX Red (69.20 ± 27.14 vs 21.18 ± 7.96, %, p < 0.05), but not CM-H2DCFDA fluorescence (8.30 ± 11.56 vs 7.30 ± 9.19, p > 0.05). Further, only MitoSOX Red was able to detect spontaneous ROS generation during in vitro sperm incubation. We also detected DNA fragmentation by Comet and SCD Assay after sorting MitoSOX Red positive and negative sperm viable fractions. Results indicated that MitoSOX labelling in viable spermatozoa was strictly associated to sperm DNA fragmentation. In conclusion, MitoSOX Red/LD-G appears to be a promising method to detect oxidative stress in human semen for male infertility work-up.
Assuntos
Espécies Reativas de Oxigênio/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Adulto , Fragmentação do DNA , Citometria de Fluxo , Fluoresceínas , Corantes Fluorescentes , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Organofosforados , Estresse Oxidativo , FenantridinasRESUMO
Introduction: Neomenthol, a cyclic monoterpenoid, is a stereoisomer of menthol present in the essential oil of Mentha spp. It is used in food as a flavoring agent, in cosmetics and medicines because of its cooling effects. However, neomenthol has not been much explored for its anticancer potential. Additionally, targeting hyaluronidase, Cathepsin-D, and ODC by phytochemicals is amongst the efficient approach for cancer prevention and/or treatment. Objectives: To investigate the molecular and cell target-based antiproliferative potential of neomenthol on human cancer (A431, PC-3, K562, A549, FaDu, MDA-MB-231, COLO-205, MCF-7, and WRL-68) and normal (HEK-293) cell lines. Methods: The potency of neomenthol was evaluated on human cancer and normal cell line using SRB, NRU and MTT assays. The molecular target based study of neomenthol was carried out in cell-free and cell-based test systems. Further, the potency of neomenthol was confirmed by quantitative real-time PCR analysis and molecular docking studies. The in vivo anticancer potential of neomenthol was performed on mice EAC model and the toxicity examination was accomplished through in silico, ex vivo and in vivo approaches. Results: Neomenthol exhibits a promising activity (IC50 17.3 ± 6.49 µM) against human epidermoid carcinoma (A431) cells by arresting the G2/M phase and increasing the number of sub-diploid cells. It significantly inhibits hyaluronidase activity (IC50 12.81 ± 0.01 µM) and affects the tubulin polymerization. The expression analysis and molecular docking studies support the in vitro molecular and cell target based results. Neomenthol prevents EAC tumor formation by 58.84% and inhibits hyaluronidase activity up to 10% at 75 mg/kg bw, i.p. dose. The oral dose of 1000 mg/kg bw was found safe in acute oral toxicity studies. Conclusion: Neomenthol delayed the growth of skin carcinoma cells by inhibiting the tubulin polymerization and hyaluronidase activity, which are responsible for tumor growth, metastasis, and angiogenesis.
Assuntos
Neoplasias Cutâneas , Tubulina (Proteína) , Animais , Proliferação de Células , Células HEK293 , Humanos , Hialuronoglucosaminidase , Camundongos , Simulação de Acoplamento Molecular , Polimerização , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O2 â¢-) and hydroxyl radical (â¢OH) and non-radical oxygen species such as hydrogen peroxide (H2O2), singlet oxygen (O2). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics ( Ray et al., 2012 ). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer's disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.
RESUMO
Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2,7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).
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A Bax-dependent increase of reactive oxygen species (ROS) and other reactive species (RS) occurs after withdrawing NGF from mouse sympathetic neurons in cell culture. Possible mechanisms underlying the increased ROS/RS are leakage of electrons from the mitochondrial electron transport chain secondary to caspase cleavage of respiratory complexes or leakage secondary to depletion of cytochrome c from the chain. We previously demonstrated that deletion of Bax or caspase 3 from these cells reduces ROS/RS production to near baseline levels indicating a central role for both Bax and caspase 3 in generating the ROS/RS. Here we depleted cytochrome c to a similar level in neurons from wild type and bax hemizygous or knockout mice by NGF withdrawal or treatment with H2O2. Death was prevented with a caspase inhibitor that caused a partial reduction of ROS/RS levels but did not completely prevent the ROS/RS increase. ROS/RS was highest in bax wild-type cells, lowest in bax knockout cells, and at an intermediate level in the bax hemizygous cells. These and our previous findings indicate that Bax and caspase 3 are necessary for the increased ROS/RS after withdrawing NGF from these cells and that little or none of the increased ROS/RS are secondary to a depletion of cytochrome c from the electron transport chain.
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The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.
Assuntos
Fibroblastos/metabolismo , Lamina Tipo A/metabolismo , Mitocôndrias/metabolismo , Lâmina Nuclear/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/antagonistas & inibidores , Lamina Tipo A/genética , Potencial da Membrana Mitocondrial , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/patologia , Lâmina Nuclear/química , Estresse Oxidativo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/agonistas , Transdução de Sinais , Fatores de TempoRESUMO
Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 µg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 µg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/imunologia , Caspases/imunologia , Capeamento Imunológico/efeitos dos fármacos , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Apoptose/imunologia , Linfoma de Burkitt/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Microdomínios da Membrana/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologiaRESUMO
This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 µM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 µM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.
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Excessive acetaminophen (APAP) use is one of the most common causes of acute liver failure. Various types of cell death in the damaged liver are linked to APAP-induced hepatotoxicity, and, of these, necrotic cell death of hepatocytes has been shown to be involved in disease pathogenesis. Until recently, necrosis was commonly considered to be a random and unregulated form of cell death; however, recent studies have identified a previously unknown form of programmed necrosis called receptor-interacting protein kinase (RIPK)-dependent necrosis (or necroptosis), which is controlled by the kinases RIPK1 and RIPK3. Although RIPK-dependent necrosis has been implicated in a variety of disease states, including atherosclerosis, myocardial organ damage, stroke, ischemia-reperfusion injury, pancreatitis, and inflammatory bowel disease. However its involvement in APAP-induced hepatocyte necrosis remains elusive. Here, we showed that RIPK1 phosphorylation, which is a hallmark of RIPK-dependent necrosis, was induced by APAP, and the expression pattern of RIPK1 and RIPK3 in the liver overlapped with that of CYP2E1, whose activity around the central vein area has been demonstrated to be critical for the development of APAP-induced hepatic injury. Moreover, a RIPK1 inhibitor ameliorated APAP-induced hepatotoxicity in an animal model, which was underscored by significant suppression of the release of hepatic enzymes and cytokine expression levels. RIPK1 inhibition decreased reactive oxygen species levels produced in APAP-injured hepatocytes, whereas CYP2E1 expression and the depletion rate of total glutathione were unaffected. Of note, RIPK1 inhibition also conferred resistance to oxidative stress in hepatocytes. These data collectively demonstrated a RIPK-dependent necrotic mechanism operates in the APAP-injured liver and inhibition of this pathway may be beneficial for APAP-induced fulminant hepatic failure.
RESUMO
Past work has shown that exposure to gamma rays and protons elicit a persistent oxidative stress in rodent and human neural stem cells (hNSCs). We have now adapted these studies to more realistic exposure scenarios in space, using lower doses and dose rates of these radiation modalities, to further elucidate the role of radiation-induced oxidative stress in these cells. Rodent neural stem and precursor cells grown as neurospheres and human neural stem cells grown as monolayers were subjected to acute and multi-dosing paradigms at differing dose rates and analyzed for changes in reactive oxygen species (ROS), reactive nitrogen species (RNS), nitric oxide and superoxide for 2 days after irradiation. While acute exposures led to significant changes in both cell types, hNSCs in particular, exhibited marked and significant elevations in radiation-induced oxidative stress. Elevated oxidative stress was more significant in hNSCs as opposed to their rodent counterparts, and hNSCs were significantly more sensitive to low dose exposures in terms of survival. Combinations of protons and γ-rays delivered as lower priming or higher challenge doses elicited radioadaptive changes that were associated with improved survival, but in general, only under conditions where the levels of reactive species were suppressed compared to cells irradiated acutely. Protective radioadaptive effects on survival were eliminated in the presence of the antioxidant N-acetylcysteine, suggesting further that radiation-induced oxidative stress could activate pro-survival signaling pathways that were sensitive to redox state. Data corroborates much of our past work and shows that low dose and dose rate exposures elicit significant changes in oxidative stress that have functional consequences on survival.
Assuntos
Raios gama , Células-Tronco Neurais/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Fótons , Acetilcisteína/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
It is known that flavonoids possess, among others, antioxidant and antitumoral properties that depend on their molecular structure. The central objective if this study was to investigate the potential antioxidant and antiproliferative properties of the flavonol morin and its new oxovanadium(IV) complex (VOmor) that was synthesized in order to modify the morin chemical structure. Two osteoblast (UMR106 and MC3T3E1), two breast tumor (T47D and SKBR3) and breast epithelial cell lines in culture were used for the antitumoral determinations. Additionally, a comparative study of their antioxidant capacities using different radicals (DPPH, ABTS(+), OH, O2(-), ROO) was performed. Selected mechanisms of action were studied using the breast cancer cell lines. Results obtained show that morin and its complex behaved as good antioxidant agents for some of the radicals and that the complexation improved the behavior with respect to OH and O2(-) radicals being morin more effective as ROO scavenger. A considerable variation in sensitivity was observed in the breast cancer cells but non-specificity was found for the treatment of osteosarcoma. Moreover, the compounds did not affect the normal proliferation of the breast epithelial mammal cells. The mechanistic studies demonstrated that the complex did not generate reactive oxygen species in the cells (confirming the in vitro studies) and did not produce any damage of DNA. The plasmatic membrane was observed to be damaged only in the SKBR3 cell line. In contrast, the perturbation of the mitochondrial membrane potential and the activation of caspase 3/7 for the breast tumor cells revealed an apoptotic cell death process. All these results collectively suggested that VOmor complex could serve as promising pharmacologically active substance against breast cancer treatment.
Assuntos
Antineoplásicos/química , Antioxidantes/química , Complexos de Coordenação/química , Flavonoides/química , Vanádio/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Camundongos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
ß-Adrenergic receptor stimulation plays an important role in cardiomyocyte stress responses, which may result in apoptosis and cardiovascular degeneration. We previously demonstrated that toxicity of the ß-adrenergic agonist isoproterenol on H9c2 cardiomyoblasts depends on the stage of cell differentiation. We now investigate ß-adrenergic receptor downstream signaling pathways and stress responses that explain the impact of muscle cell differentiation on hyper-ß-adrenergic stimulation-induced cytotoxicity. When incubated with isoproterenol, differentiated H9c2 muscle cells have increased cytosolic calcium, cyclic-adenosine monophosphate content and oxidative stress, as well as mitochondrial depolarization, increased superoxide anion, loss of subunits from the mitochondrial respiratory chain, decreased Bcl-xL content, increased p53 and phosphorylated-p66Shc as well as activated caspase-3. Undifferentiated H9c2 cells incubated with isoproterenol showed increased Bcl-xL protein and increased superoxide dismutase 2 which may act as protective mechanisms. We conclude that the differentiation of H9c2 is associated with differential regulation of stress responses, which impact the toxicity of several agents, namely those acting through ß-adrenergic receptors and resulting in mitochondrial disruption in differentiated cells only.