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1.
Eur J Med Chem ; 189: 111981, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978780

RESUMO

Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumor. Pharmacological treatment of this malignance is limited by the selective permeability of the blood-brain barrier (BBB) and relies on a single drug, temozolomide (TMZ), thus making the discovery of new compounds challenging and urgent. Therefore, aiming to discover new anti-glioma drugs, we developed robust machine learning models for predicting anti-glioma activity and BBB penetration ability of new compounds. Using these models, we prioritized 41 compounds from our in-house library of compounds, for further in vitro testing against three glioma cell lines and astrocytes. Subsequently, the most potent and selective compounds were resynthesized and tested in vivo using an orthotopic glioma model. This approach revealed two lead candidates, 4m and 4n, which efficiently decreased malignant glioma development in mice, probably by inhibiting thioredoxin reductase activity, as shown by our enzymological assays. Moreover, these two compounds did not promote body weight reduction, death of animals, or altered hematological and toxicological markers, making then good candidates for lead optimization as anti-glioma drug candidates.

2.
Mol Cell Biochem ; 462(1-2): 11-23, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31446616

RESUMO

BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-ß and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.


Assuntos
Proteína BRCA1/metabolismo , Polaridade Celular , Inflamação/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/patologia , NF-kappa B/metabolismo , Animais , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Dano ao DNA , Inflamação/metabolismo , Lipopolissacarídeos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Camundongos , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Ratos
3.
Int J Biol Macromol ; 134: 660-672, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31054301

RESUMO

The antitumor activity of DVL, a lectin purified from Dioclea violacea seeds, on the U87 human glioma cell line was evaluated and compared with Canavalia ensiformis lectin (ConA). Treatment with DVL (10-100 µg/mL; 24-96 h) induced alterations in cell morphology, decreased cell numbers and clonogenic survival in a time- and concentration-dependent manner. DVL caused significant decreases in cell viability and impaired cell migration. Mechanistically, DVL treatment (12 h) disrupted mitochondrial electrochemical gradient, without ROS accumulation or caspase activation. In the absence of apoptosis, DVL (30-100 µg/mL), instead, induced autophagy, as detected by acridine orange staining and cleavage of LC3I. Inhibition of autophagy with 3-Methyladenine (3-MA) and Chloroquine partially abrogated DVL, but not ConA, cytotoxicity. The modulation of signaling pathways that orchestrate autophagic and cell survival processes were analyzed. DVL (30-100 µg/mL) decreased Akt, mTORC1 and ERK1/2 phosphorylation and augmented JNK(p54) and p38MAPK phosphorylation. DVL was more potent than ConA for most parameters analyzed. Even though both lectins showed cytotoxicity to glioma cells, they spared primary astrocyte cultures. The results suggest a selective antiglioma activity of DVL by inhibiting U87 glioma cell migration and proliferation and inducing cell death, partially associated with autophagy, and likely involving Akt and mTORC1 dephosphorylation.


Assuntos
Autofagia/efeitos dos fármacos , Dioclea/química , Lectinas de Plantas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
J Nutr Biochem ; 67: 190-200, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30951973

RESUMO

Sexual hormone deficiency has been associated with metabolic changes, oxidative stress and subclinical inflammation in postmenopausal women. Hormone replacement therapies are effective in many instances, even though some patients either do not respond or are not eligible. The aim of this study was to evaluate the impact of short- (15 days) versus long-term (60 days) sexual hormone depletion and whether antioxidant supplementation with N-acetylcysteine (NAC) and alpha-lipoic acid (LA) improves oxidative stress, metabolic, and inflammatory parameters in ovariectomized (OVX) rats. Short-term OVX rapidly depleted circulating estrogen, causing uterine atrophy and body weight gain without affecting oxidative damage, inflammatory and lipid metabolism markers. In contrast, long-term OVX augmented oxidative damage in serum and peripheral tissues as well as increased serum total cholesterol, TNF-α and IL6 levels. Triglycerides, glucose and HDL cholesterol were not altered. Long-term OVX-induced oxidative stress was associated with depletion of GSH and total non-enzymatic antioxidants as well as decreased activity of Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), but not Superoxide Dismutase (SOD) and Catalase (CAT). NAC and LA supplementation prevented GSH and total non-enzymatic antioxidants depletion as well as restored GPx and GR activities, TNF-α, IL6 and cholesterol in OVX rats. NAC and LA effects appear to be independent on NRF2 activation and estrogen-like activity, since NAC/LA did not promote NRF2 activation and were not able to emulate estrogen effects in OVX rats and estrogen-receptor-positive cells. The herein presented data suggest that NAC and LA may improve some deleterious effects of sexual hormone depletion via estrogen-independent mechanisms.

5.
Biochem Pharmacol ; 163: 440-450, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30878553

RESUMO

Glioblastoma multiforme is the most aggressive type of primary brain tumor associated with few therapeutic opportunities and poor prognosis. In this study, we evaluated the efficacy of combining temozolomide (TMZ) with suberoylanilide hydroxamic acid (SAHA) - a specific histone deacetylases inhibitor - in glioma models in vitro and in vivo. In glioma cell lines, combined TMZ/SAHA promoted more cytotoxicity, G2/M arrest and apoptosis than either drugs alone. G2/M arrest was detected as soon as 24 h post drug exposure and preceded apoptosis, which occurred from 72 h treatment. TMZ and SAHA, alone or combined, also stimulated autophagy as evaluated by means of acridine orange staining and immunodetection of LC3I-II conversion and p62/SQSTM1 degradation. Time-course of autophagy accompanied G2/M arrest and preceded apoptosis, and blockage of late steps of autophagy with chloroquine (CQ) augmented SAHA/TMZ toxicity leading to apoptosis. In orthotopic gliomas in vivo, combined SAHA/TMZ showed better antitumor efficacy than either drugs alone, and adding CQ to the regimen improved antiglioma effects of SAHA and TMZ monotherapies without further benefit on combined SAHA/TMZ. In summary, the herein presented data suggest that autophagy acts as a protective response that impairs efficacy of SAHA and TMZ. Inhibiting autophagy termination with CQ may offer means to improve antitumor effects of SAHA and TMZ in gliomas and possibly other cancers.


Assuntos
Cloroquina/uso terapêutico , Temozolomida/uso terapêutico , Vorinostat/uso terapêutico , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/administração & dosagem , Quimioterapia Combinada , Técnicas de Silenciamento de Genes , Humanos , Ratos , Ratos Wistar , Temozolomida/administração & dosagem , Vorinostat/administração & dosagem
6.
Toxicol In Vitro ; 51: 23-33, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29730415

RESUMO

Achyrocline satureioides, popularly known as "marcela", is a medicinal plant found in South America. This plant is rich in flavonoids, which have been reported to exert numerous biological activities. The aim of this study was to purify, identify and evaluate the mechanisms underlining anticancer activity of A. satureioides flavonoids in glioma cell lines (U87, U251 and C6) as well as their comparative toxicity in normal brain cells (primary astrocytes, neurons and organotypic hippocampal cultures). The main flavonoids present in A. satureioides are luteolin, quercetin, 3-O-methyl-quercetin and achyrobichalcone, the later a very unique metabolite present in this plant. Isolated flavonoids as well as A. satureioides extracts reduced proliferation and clonogenic survival, and induced apoptosis of glioma cell lines. In addition, A. satureioides flavonoids potentiated the cytotoxic effect and apoptosis induction by the glioma chemotherapeutic temozolomide (TMZ). Importantly, A. satureioides flavonoids were less cytotoxic to astrocytes, neuron:astrocytes co-cultures and hippocampal cultures if compared to gliomas. Investigation of 10 cancer-related pathways showed a reduced activation of MYC and the Map kinases ERK and JNK by A. satureioides flavonoid-enriched extract, an effect not observed when individual flavonoids were evaluated. Altogether, the herein presented results show that A. satureioides extract possesses a combination of flavonoids, some unique for this plant, which have synergistic anticancer activity and potential for further studies in vivo.


Assuntos
Achyrocline , Antineoplásicos/farmacologia , Flavonoides/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Flores , Glioma/tratamento farmacológico , Glioma/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Masculino , Neurônios/efeitos dos fármacos , Ratos Wistar
7.
Cancer Lett ; 425: 101-115, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29608984

RESUMO

Molecular targeted compounds are emerging as a strategy to improve classical chemotherapy. Herein, we describe that using low dose of the multikinase inhibitor sorafenib improves cyclophosphamide antitumor activity by inhibiting angiogenesis, metastasis and promoting tumor healing in MDA-MB231 xenografts and the 4T1-12B syngeneic breast cancer metastasis model. Mechanistic studies in MDA-MB231 cells revealed that alkylation upregulates inflammatory genes/proteins such as COX-2, IL8, CXCL2 and MMP1 in a MEK1/2-ERK1/2-dependent manner. These proteins enrich the secretome of cancer cells, stimulating cell invasion and angiogenesis via autocrine and paracrine mechanisms. Sorafenib inhibits MEK1/2-ERK1/2 pathway thereby decreasing inflammatory genes and mitigating cell invasion and angiogenesis at basal and alkylation-induced conditions whereas NRF2 and ER stress pathways involved in alkylation survival are not affected. In non-invasive/non-angiogenic breast cancer cells (SKBR3 and MCF7), alkylation did not elicit inflammatory responses with the only sorafenib effect being ERK1/2-independent ROS-dependent cytotoxicity when using higher drug concentrations. In summary, our data show that alkylating agents may elicit inflammatory responses that seems to contribute to malignant progression in specific breast cancer cells. Identifying and targeting drivers of this phenotype may offer opportunities to optimize combined drug regimens between classical chemotherapeutics and targeted agents.


Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Sorafenibe/administração & dosagem , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclofosfamida/farmacologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Comput Biol Chem ; 72: 62-76, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29414098

RESUMO

The genes of the NFκB pathway are involved in the control of a plethora of biological processes ranking from inhibition of apoptosis to metastasis in cancer. It has been described that Gliobastoma multiforme (GBM) patients carry aberrant NFκB activation, but the molecular mechanisms are not completely understood. Here, we present a NFκB pathway analysis in tumor specimens of GBM compared to non-neoplasic brain tissues, based on the different kind of cycles found among genes of a gene co-expression network constructed using quantized data obtained from the microarrays. A cycle is a closed walk with all vertices distinct (except the first and last). Thanks to this way of finding relations among genes, a more robust interpretation of gene correlations is possible, because the cycles are associated with feedback mechanisms that are very common in biological networks. In GBM samples, we could conclude that the stoichiometric relationship between genes involved in NFκB pathway regulation is unbalanced. This can be measured and explained by the identification of a cycle. This conclusion helps to understand more about the biology of this type of tumor.


Assuntos
Retroalimentação Fisiológica , Glioblastoma/genética , NF-kappa B/metabolismo , Transdução de Sinais/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Curadoria de Dados , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Glioblastoma/fisiopatologia , Humanos , Análise em Microsséries , NF-kappa B/genética , Transdução de Sinais/fisiologia
9.
Cell Physiol Biochem ; 42(6): 2507-2522, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28848092

RESUMO

BACKGROUND/AIMS: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. METHODS: Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. RESULTS: Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. CONCLUSIONS: Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Células A549 , Citocinas/genética , Citocinas/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/farmacologia , Humanos , Immunoblotting , Imunoprecipitação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Células U937
10.
Mol Neurobiol ; 54(9): 6903-6916, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-27771902

RESUMO

Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), ß-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, ß-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.


Assuntos
Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Neurônios/citologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
Cancer Lett ; 390: 176-187, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007636

RESUMO

The tumor microenvironment is being increasingly recognized as a key factor in cancer aggressiveness. In this study, we characterized the inflammatory gene signatures altered in glioma cell lines and tumor specimens of differing histological and molecular subtypes. The results showed that glioblastoma multiforme (GBM) shows upregulation of a subset of inflammatory genes when compared to astrocytomas and oligodendrogliomas. With molecular subtypes of GBM, the expression of inflammatory genes is heterogeneous, being enriched in mesenchymal and downregulated in Proneural/GCIMP. Other inflammation-associated processes such as tumor-associated macrophage (TAM) signatures are upregulated in mesenchymal, and a subset of 33 mesenchymal-enriched inflammatory and TAM markers showed correlation with poor survival. We found that various GBM tumor-upregulated genes such as IL6, IL8 and CCL2 are also actively expressed in glioma cell lines, playing differential and cooperative roles in promoting proliferation, invasion, angiogenesis and macrophage polarization in vitro. These genes can be stimulated by pathways typically altered in GBM, including the EGFR, PDGFR, MEK1/2-ERK1/2, PI3K/Akt and NFκB cascades. Taken together, the results presented herein depict some inflammatory pathways altered in gliomas and highlight potentially relevant targets to therapy improvement.


Assuntos
Neoplasias Encefálicas/fisiopatologia , Glioblastoma/fisiopatologia , NF-kappa B/metabolismo , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Glioblastoma/imunologia , Humanos , Mesoderma/fisiopatologia , NF-kappa B/genética , NF-kappa B/imunologia , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos
12.
Mol Cancer Ther ; 15(12): 3000-3014, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27638861

RESUMO

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa/metabolismo , Homeostase/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Compostos de Sulfidrila/metabolismo , Apoptose/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Homeostase/genética , Humanos , Modelos Biológicos , Mutação , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , eIF-2 Quinase/metabolismo
13.
J Nutr Biochem ; 32: 181-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27142750

RESUMO

Menopause occurs gradually and is characterized by increased susceptibility to developing mood disorders. Several studies have suggested treatments based on the antioxidant properties of vitamins and herbal compounds as an alternative to hormone replacement therapies, with few or none reporting toxicity. The present study was performed to explore the effects of curcumin oral supplementation on anxiety-like behavior and oxidative stress parameters in different central nervous system (CNS) areas of ovariectomized (OVX) rats. Female Wistar rats were randomly divided into either sham-operated or OVX groups. Sham-operated group (n=8) and an OVX group (n=11) were treated with vehicle, and the other two OVX groups received curcumin at 50 or 100mg/kg/day doses (n=8/group). Elevated plus maze (EPM) test was performed on the 28th day of treatment. On the 30th day, animals were killed and the dissected brain regions were removed and stored at-80°C until analysis. Ovariectomy induced deficit in the locomotor activity and increased anxiety-like behavior. Moreover, OVX rats showed increased lipid oxidized in the frontal cortex and striatum, increased hippocampal and striatal carbonylated protein level, and decreased striatal thiol content of non-protein fraction indicative of a glutathione (GSH) pool. Curcumin oral treatment for 30days reduced oxidative stress in the CNS areas as well as the behavior alterations resulting from ovariectomy. Curcumin supplementation attenuated most of these parameters to sham comparable values, suggesting that curcumin could have positive effects against anxiety-like disturbances and brain oxidative damage due to hormone deprivation.


Assuntos
Antioxidantes/uso terapêutico , Disfunção Cognitiva/prevenção & controle , Curcumina/uso terapêutico , Suplementos Nutricionais , Neurônios/metabolismo , Estresse Oxidativo , Pós-Menopausa , Animais , Antioxidantes/administração & dosagem , Ansiedade/metabolismo , Ansiedade/prevenção & controle , Comportamento Animal , Biomarcadores/metabolismo , Disfunção Cognitiva/metabolismo , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Curcumina/administração & dosagem , Feminino , Lobo Frontal/crescimento & desenvolvimento , Lobo Frontal/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Aprendizagem em Labirinto , Transtornos do Humor/metabolismo , Transtornos do Humor/prevenção & controle , Ovariectomia , Carbonilação Proteica , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/prevenção & controle , Distribuição Aleatória , Ratos Wistar
14.
PLoS One ; 11(4): e0153970, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27100653

RESUMO

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.


Assuntos
Reparo do DNA/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas/metabolismo , Interferência de RNA , Transdução de Sinais , Alquilação , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Estresse do Retículo Endoplasmático/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Genoma , Ensaios de Triagem em Larga Escala , Humanos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , Proteínas/genética , Resposta a Proteínas não Dobradas/genética
15.
Ecotoxicol Environ Saf ; 129: 16-24, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26970882

RESUMO

Tropospheric ozone (O3) background concentrations have increased since pre-industrial times, reaching phytotoxic concentrations in many regions globally. However, the effect of high O3 concentrations on quality of fruit and vegetables remains unknown. Here, we evaluated whether O3 pollution alters the quality of Capsicum baccatum peppers by changing the secondary compound profiles and biological activity of the fruit. C. baccatum pepper plants were exposed to ozone for 62 days in an open-top chamber at a mean O3 concentration of 171.6µg/m(3). Capsaicin levels decreased by 50% in the pericarp, but remained unchanged in the seeds. In contrast, the total carotenoid content increased by 52.8% in the pericarp. The content of total phenolic compounds increased by 17% in the pericarp. The total antioxidant potential decreased by 87% in seeds of O3-treated plants. The seeds contributed more than the pericarp to the total radical-trapping antioxidant potential and total antioxidant reactivity. O3 treatment impaired the ferric-reducing antioxidant power of the seeds and reduced NO(•)-scavenging activity in the pericarp. However, O3 treatment increased ferrous ion-chelating activity and hydroxyl radical-scavenging activity in the pericarp. Our results confirm that O3 alters the secondary metabolite profile of C. baccatum pepper fruits and, consequently, their biological activity profile.


Assuntos
Poluentes Atmosféricos/toxicidade , Capsicum/efeitos dos fármacos , Oxidantes/toxicidade , Ozônio/toxicidade , Anti-Inflamatórios/metabolismo , Antioxidantes/metabolismo , Capsaicina/metabolismo , Capsicum/metabolismo , Carotenoides/metabolismo , Frutas/efeitos dos fármacos , Frutas/metabolismo , Fenóis/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo
16.
Mol Neurobiol ; 53(9): 6124-6135, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-26541884

RESUMO

Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox® as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tretinoína/farmacologia , Acetilcisteína/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo
17.
Oxid Med Cell Longev ; 2016: 5719291, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26640615

RESUMO

The aim of this study was to investigate the potential of curcumin oral supplementation (50 and 100 mg/Kg/day, for 30 days) in circumventing menopause-associated oxidative stress and lipid profile dysfunctions in a rat ovariectomy (OVX) model. Female Wistar rats were operated and randomly divided into either sham-operated or OVX groups. Sham-operated group (n = 8) and one OVX group (n = 11) were treated with vehicle (refined olive oil), and the other two OVX groups received curcumin at 50 or 100 mg/Kg/day doses (n = 8/group). OVX vehicle-treated animals presented a higher deposition of intestinal adipose tissue as well as increased serum levels of IL-6, LDL, and total cholesterol when compared to sham-operated rats. In addition, several oxidative stress markers in serum, blood, and liver (such as TBARS, carbonyl, reduced-sulphydryl, and nonenzymatic antioxidant defenses) were altered toward a prooxidant status by OVX. Interestingly, curcumin supplementation attenuated most of these parameters to sham comparable values. Thus, the herein presented results show that curcumin may be useful to ameliorate lipid metabolism alterations and oxidative damage associated with hormone deprivation in menopause.


Assuntos
Adiposidade/efeitos dos fármacos , LDL-Colesterol/sangue , Curcumina/farmacologia , Menopausa/sangue , Ovariectomia , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Interleucina-6/sangue , Ratos , Ratos Wistar
18.
Appl Physiol Nutr Metab ; 40(12): 1253-61, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26566243

RESUMO

Exercise training induces reactive oxygen species production and low levels of oxidative damage, which are required for induction of antioxidant defenses and tissue adaptation. This process is physiological and essential to improve physical conditioning and performance. During exercise, endogenous antioxidants are recruited to prevent excessive oxidative stress, demanding appropriate intake of antioxidants from diet or supplements; in this context, the search for vitamin supplements that enhance the antioxidant defenses and improve exercise performance has been continuously increasing. On the other hand, excess of antioxidants may hinder the pro-oxidant signals necessary for this process of adaptation. The aim of this study was to investigate the effects of vitamin A supplementation (2000 IU/kg, oral) upon oxidative stress and parameters of pro-inflammatory signaling in lungs of rats submitted to aerobic exercise (swimming protocol). When combined with exercise, vitamin A inhibited biochemical parameters of adaptation/conditioning by attenuating exercise-induced antioxidant enzymes (superoxide dismutase and glutathione peroxidase) and decreasing the content of the receptor for advanced glycation end-products. Increased oxidative damage to proteins (carbonylation) and lipids (lipoperoxidation) was also observed in these animals. In sedentary animals, vitamin A decreased superoxide dismutase and increased lipoperoxidation. Vitamin A also enhanced the levels of tumor necrosis factor alpha and decreased interleukin-10, effects partially reversed by aerobic training. Taken together, the results presented herein point to negative effects associated with vitamin A supplementation at the specific dose here used upon oxidative stress and pro-inflammatory cytokines in lung tissues of rats submitted to aerobic exercise.


Assuntos
Suplementos Nutricionais/toxicidade , Pulmão/efeitos dos fármacos , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Esforço Físico , Vitamina A/toxicidade , Animais , Glutationa Peroxidase/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Carbonilação Proteica/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Superóxido Dismutase/metabolismo , Natação , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
19.
Stem Cells Dev ; 24(22): 2700-8, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26159230

RESUMO

Misexpression of stem cell-related genes may occur in some cancer cells, influencing patient's prognosis. This is the case of medulloblastoma, a common and clinically challenging malignant tumor of the central nervous system, where expression of the pluripotency factor, OCT4, is correlated with poor survival. A downstream target of OCT4, L1TD1 (LINE-1 type transposase domain-containing protein 1 family member), encodes a novel embryonic stem cell (ESC)-related protein involved in pluripotency and self-renewal of ESCs. L1TD1 is still poorly characterized and its expression pattern and function in cancer cells are virtually unknown. Although normally restricted to non-neoplastic undifferentiated cells and germ cells, we found that high L1TD1 expression also occurs in medulloblastoma cells, reaching levels similar to those found in ESCs, and is correlated with poor prognosis. Conversely to what is reported during normal cell differentiation, when differentiated cells remain healthy, despite L1TD1 downregulation, depletion of L1TD1 protein levels by targeted gene silencing significantly reduced medulloblastoma cell viability, inhibiting cell proliferation and inducing apoptosis. More strikingly, L1TD1 depletion downregulated expression of the neural stem cell markers, CD133 and Nestin, inhibited neurosphere generation capability, and sensitized medulloblastoma cells to temozolomide and cisplatin, two chemotherapeutic agents of clinical relevance in medulloblastoma treatment. Our findings provide insights about the contribution of pluripotency-related genes to a more aggressive tumor phenotype through their involvement in the acquisition of stem-like properties by cancer cells and point out L1TD1 as a potential therapeutic target in malignant brain tumors.


Assuntos
Apoptose , Meduloblastoma/metabolismo , Células-Tronco Neurais/citologia , Proteínas/metabolismo , Antígeno AC133 , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Meduloblastoma/patologia , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/genética , Temozolomida
20.
Cancer Lett ; 358(2): 220-231, 2015 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-25542083

RESUMO

Glioblastoma is a devastating primary brain tumor resistant to conventional therapies. In this study, we tested the efficacy of combining temozolomide with curcumin, a phytochemical known to inhibit glioblastoma growth, and investigated the mechanisms involved. The data showed that synergy between curcumin and temozolomide was not achieved due to redundant mechanisms that lead to activating protective autophagy both in vitro and in vivo. Autophagy preceded apoptosis, and blocking this response with autophagy inhibitors (3-methyl-adenine, ATG7 siRNA and chloroquine) rendered cells susceptible to temozolomide and curcumin alone or combinations by increasing apoptosis. While curcumin inhibited STAT3, NFκB and PI3K/Akt to affect survival, temozolomide-induced autophagy relied on the DNA damage response and repair components ATM and MSH6, as well as p38 and JNK1/2. However, the most interesting observation was that both temozolomide and curcumin required ERK1/2 to induce autophagy. Blocking this ERK1/2-mediated temozolomide and curcumin induced autophagy with resveratrol, a blood-brain barrier permeable drug, improved temozolomide/curcumin efficacy in brain-implanted tumors. Overall, the data presented demonstrate that autophagy impairs the efficacy of temozolomide/curcumin, and inhibiting this phenomenon could provide novel opportunities to improve brain tumor treatment.


Assuntos
Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Curcumina/farmacologia , Dacarbazina/análogos & derivados , Glioblastoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos , Temozolomida
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