RESUMO
Epidemiological and experimental studies have demonstrated that combined exposure to the pesticides paraquat (PQ) and maneb (MB) increases the risk of developing Parkinson's disease. However, the mechanisms mediating the toxicity induced by combined exposure to these pesticides are not well understood. The aim of this study was to investigate the mechanism(s) of neurotoxicity induced by exposure to the pesticides PQ and MB isolated or in association (PQ + MB) in SH-SY5Y neuroblastoma cells. PQ + MB exposure for 24 and 48 h decreased cell viability and disrupted cell membrane integrity. In addition, PQ + MB exposure for 12 h decreased the mitochondrial membrane potential. PQ alone increased reactive oxygen species (ROS) and superoxide anion generation and decreased the activity of mitochondrial complexes I and II at 12 h of exposure. MB alone increased ROS generation and depleted intracellular glutathione (GSH) within 6 h of exposure. In contrast, MB exposure for 12 h increased the GSH levels, the glutamate cysteine ligase (GCL, the rate-limiting enzyme in the GSH synthesis pathway) activity, and increased nuclear Nrf2 staining. Pretreatment with buthionine sulfoximine (BSO, a GCL inhibitor) abolished the MB-mediated GSH increase, indicating that MB increases GSH synthesis by upregulating GCL, probably by the activation of the Nrf2/ARE pathway. BSO pretreatment, which did not modify cell viability per se, rendered cells more sensitive to MB-induced toxicity. In contrast, treatment with the antioxidant N-acetylcysteine protected cells from MB-induced toxicity. These findings show that the combined exposure of SH-SY5Y cells to PQ and MB induced a cytotoxic effect higher than that observed when cells were subjected to individual exposures. Such a higher effect seems to be related to additive toxic events resulting from PQ and MB exposures. Thus, our study contributes to a better understanding of the toxicity of PQ and MB in combined exposures.
Assuntos
Sobrevivência Celular , Maneb , Neuroblastoma , Paraquat , Espécies Reativas de Oxigênio , Paraquat/toxicidade , Humanos , Maneb/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Glutationa/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Linhagem Celular Tumoral , Fator 2 Relacionado a NF-E2/metabolismo , Butionina Sulfoximina/farmacologiaRESUMO
Buthionine sulfoximine (BSO) is a synthetic amino acid that blocks the biosynthesis of reduced glutathione (GSH), an endogenous antioxidant cellular component present in tumor cells. GSH levels have been associated with tumor cell resistance to chemotherapeutic drugs and platinum compounds. Consequently, by depleting GSH, BSO enhances the cytotoxicity of chemotherapeutic agents in drug-resistant tumors. Therefore, the aim of this study was to conduct a systematic review with meta-analysis of preclinical studies utilizing BSO in cancer treatments. The systematic search was carried out using the following databases: PubMed, Web of Science, Scopus, and EMBASE up until March 20, 2023, in order to collect preclinical studies that evaluated BSO, alone or in association, as a strategy for antineoplastic therapy. One hundred nine investigations were found to assess the cytotoxic potential of BSO alone or in combination with other compounds. Twenty-one of these met the criteria for performing the meta-analysis. The evidence gathered indicated that BSO alone exhibits cytotoxic activity. However, this compound is generally used in combination with other antineoplastic strategies, mainly chemotherapy ones, to improve cytotoxicity to carcinogenic cells and treatment efficacy. Finally, this review provides important considerations regarding BSO use in cancer treatment conditions, which might optimize future studies as a potential adjuvant antineoplastic therapeutic tool.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Butionina Sulfoximina/farmacologia , Butionina Sulfoximina/uso terapêutico , Metionina Sulfoximina/uso terapêutico , Metionina Sulfoximina/toxicidade , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Two ternary copper(II) complexes with 2,2'-biquinoline (BQ) and with sulfonamides: sulfamethazine (SMT) or sulfaquinoxaline (SDQ) whose formulae are Cu(SMT)(BQ)Cl and Cu(SDQ)(BQ)Cl·CH3OH, in what follows SMTCu and SDQCu, respectively, induced oxidative stress by increasing ROS level from 1.0 µM and the reduction potential of the couple GSSG/GSH2. The co-treatment with L-buthionine sulfoximine (BSO), which inhibits the production of GSH, enhanced the effect of copper complexes on tumor cell viability and on oxidative damage. Both complexes generated DNA strand breaks given by-at least partially-the oxidation of pyrimidine bases, which caused the arrest of the cell cycle in the G2/M phase. These phenomena triggered processes of apoptosis proven by activation of caspase 3 and externalization of phosphatidylserine and loss of cell integrity from 1.0 µM. The combination with BSO induced a marked increase in the apoptotic population. On the other hand, an improved cell proliferation effect was observed when combining SDQCu with a radiation dose of 2 Gy from 1.0 µM or with 6 Gy from 1.5 µM. Finally, studies in multicellular spheroids demonstrated that even though copper(II) complexes did not inhibit cell invasion in collagen gels up to 48 h of treatment at the higher concentrations, multicellular resistance outperformed several drugs currently used in cancer treatment. Overall, our results reveal an antitumor effect of both complexes in monolayer and multicellular spheroids and an improvement with the addition of BSO. However, only SDQCu was the best adjuvant of ionizing radiation treatment.
Assuntos
Cobre , Neoplasias Pulmonares , Apoptose , Butionina Sulfoximina/farmacologia , Cobre/química , Cobre/farmacologia , Glutationa/metabolismo , Humanos , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Quinolinas , Radiação Ionizante , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: Colon cancer is one of the most important causes of death in the entire world. New pharmacological strategies are always needed, especially in resistant variants of this pathology. We have previously reported that drugs such as menadione (MEN), D, L-buthionine-S,R-sulfoximine or calcitriol, used in combination, enhanced cell sensibility of breast and colon tumour models, due to their ability to modify the oxidative status of the cells. Melatonin (MEL), a hormone regulating circadian rhythms, has anti-oxidant and anti-apoptotic properties at low concentrations, while at high doses, it has been shown to inhibit cancer cell growth. OBJECTIVE: The objective of this study is to determine the antitumoral action of the combination MEN and MEL on colon cancer cells. METHODS: Caco-2 cells were employed to evaluate the effects of both compounds, used alone or combined, on cellular growth/morphology, oxidative and nitrosative stress, and cell migration. RESULTS: MEN plus MEL dramatically reduced cell proliferation in a time and dose-dependent manner. The antiproliferative effects began at 48 h. At the same time, the combination modified the content of superoxide anion, induced the formation of reactive nitrogen species and enhanced catalase activity. Cell migration process was delayed. Also, changes in nuclear morphology consistent with cell death were observed. CONCLUSION: The enhanced effect of simultaneous use of MEN and MEL on Caco-2 cells suggests that this combined action may have therapeutic potential as an adjuvant on intestinal cancer acting in different oncogenic pathways.
Assuntos
Neoplasias do Colo , Melatonina , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Butionina Sulfoximina/farmacologia , Células CACO-2 , Neoplasias do Colo/tratamento farmacológico , Humanos , Melatonina/farmacologia , Estresse Oxidativo , Vitamina K 3/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Cellular damage related to oxidative stress (OS) is implicated in periodontal diseases (PD). Melatonin (MEL) has multiple functions, and it has been described as a potential treatment for PD. We aim at evaluating the protective effects of MEL on an in vitro model of cellular damage triggered by glutamate (GLUT) and DL-buthionine sulfoximine (BSO), on gingival cells (GCs) in culture. MATERIAL AND METHODS: A primary culture of GCs from Wistar rats was developed in order to test the protective property of MEL; BSO and GLUT were administered alone as well as in combination with MEL. The viability and apoptosis were measured with MTT assay and TUNEL, respectively, and the concentration of superoxide anion ( O 2 - ) was measured with the NBT method. RESULTS: The combination of BSO and GLUT treatment resulted in a decreased viability of GCs. This was evidenced by the increase in both the production of superoxide anion and apoptosis. After MEL administration, the oxidant and pro-apoptotic effects of BSO and GLUT were totally counteracted. CONCLUSIONS: These findings demonstrated that MEL has an effective protective role on GCs subjected to cellular damage in a model of OS and cytotoxicity triggered by BSO and GLUT. Consequently, MEL could be used as a therapeutic agent in PD which begin with a significative loss of GCs.
Assuntos
Melatonina , Animais , Butionina Sulfoximina/toxicidade , Ácido Glutâmico , Glutationa , Melatonina/farmacologia , Ratos , Ratos WistarRESUMO
Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10 mM) plus 6-OHDA (200 µM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5 mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.
Assuntos
Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Antioxidantes/metabolismo , Butionina Sulfoximina/metabolismo , Morte Celular/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase , Humanos , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Glutationa Peroxidase GPX1RESUMO
Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.
Assuntos
Antioxidantes/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa/análogos & derivados , Proteínas de Protozoários/genética , Espermidina/análogos & derivados , Tiorredoxinas/genética , Trypanosoma cruzi/efeitos dos fármacos , Amida Sintases/genética , Amida Sintases/metabolismo , Butionina Sulfoximina/farmacologia , Linhagem Celular , Combinação de Medicamentos , Resistência a Medicamentos/genética , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Peróxido de Hidrogênio/farmacologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Nitroimidazóis/farmacologia , Oxirredução , Estresse Oxidativo , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Espermidina/antagonistas & inibidores , Espermidina/biossíntese , Tiorredoxinas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
Dendritic cells (DCs) are one of the principal host cells of the obligate intracellular parasite Leishmania that can survive and reproduce within cells due to the ability to regulate different cellular events, including apoptosis. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. We have previously reported that Leishmania mexicana promastigotes and amastigotes inhibit camptothecin-induced apoptosis of monocyte-derived dendritic cells (moDCs) through the downregulation of p38 and JNK phosphorylation. The upregulation of glutathione (GSH), the most important regulator of reactive oxygen species (ROS) concentration, has proven to protect cells from apoptosis through the inhibition of JNK1. Another mechanism employed by cells for the protection of apoptosis is the expression of anti-apoptotic proteins of the Bcl-2 family. The aim of this study was to determine if GSH, ROS, and Bcl-xL participate in the inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes. GSH quantification assays showed that camptothecin and BSO (an inhibitor of glutathione synthesis) strongly decreased intracellular GSH concentration in moDC, while infection with L. mexicana promastigotes had no effect in the level of GSH. On the other hand, infection with L. mexicana promastigotes of BSO- and camptothecin-treated moDC diminished the concentration of ROS and induced the expression of the anti-apoptotic protein Bcl-xL. Our findings suggest that inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes is preferentially regulated by the expression of anti-apoptotic proteins of the Bcl-2 family rather than by the redox status of the cell.
Assuntos
Apoptose/fisiologia , Células Dendríticas/fisiologia , Células Dendríticas/parasitologia , Glutationa/metabolismo , Leishmania mexicana/imunologia , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Camptotecina/farmacologia , Células Cultivadas , Regulação para Baixo , Humanos , FosforilaçãoRESUMO
BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.
Assuntos
Resistência a Medicamentos , Leishmaniose Visceral/parasitologia , Antimônio/farmacologia , Butionina Sulfoximina , Testes de Sensibilidade Parasitária , Inibidores EnzimáticosRESUMO
BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.