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1.
Proc Natl Acad Sci U S A ; 117(32): 19435-19445, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719131

RESUMO

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.


Assuntos
Glutationa S-Transferase pi/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/deficiência , Glutationa S-Transferase pi/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
2.
Nat Chem Biol ; 16(2): 150-159, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31768034

RESUMO

Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.


Assuntos
Sondas Moleculares/química , Proteômica/métodos , Enxofre/química , Triazóis/química , Tirosina/análise , Tirosina/química , Células A549 , Sítios de Ligação , Flúor/química , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Células HEK293 , Humanos , Sondas Moleculares/síntese química , Fosforilação , Fosfotirosina/química , Fosfotirosina/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ácidos Sulfínicos/química , Tirosina/metabolismo
3.
Mol Biol Rep ; 47(1): 393-400, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31650384

RESUMO

Glutathione S-transferase genes, known to be highly polymorphic, are implicated in the process of phase II metabolism of many substrates, including xenobiotics, anticancer and anti-infective drugs. The detoxification activity is linked to individual genetic makeup. Therefore, the identification of alleles and genotypes in these genes within a population may help to better design genetic susceptibility and pharmacogenetic studies. We performed the present study to establish the frequencies of the GSTM1, GSTT1, and GSTP1 c. 313A > G (rs1695) polymorphisms in 206 individuals of the Malian healthy population. GSTM1 and GSTT1 were genotyped by using multiplex polymerase chain reaction, whereas genotypes of GSTP1 were identified by polymerase chain reaction followed by restriction fragment length polymorphism. The frequencies of GSTM1-null and GSTT1-null genotypes were respectively 24.3 and 41.3%. The observed genotype frequencies for GSTP1 were 25.73% homozygous wild-type AA, 49.03% heterozygous AG and 25.24% homozygous mutant GG. The frequency of GSTP1-A allele was 50.24% versus 49.76% for the GSTP1-G allele. The distribution of these three genes was homogeneous between men and women (p > 0.05). We found no statistical association between the presence of a particular profile of GSTM1 or GSTT1 with the genotypes of GSTP1 (p > 0.05). Nevertheless, we noticed that the majority of the individuals harboring the GSTM1-present or the GSTT1-present harbor also the GSTP1-AG genotype. In addition, the triple genotype GSTM1-present/GSTT1-present/AG was the most frequent with 25.2%. Our findings will facilitate future studies regarding genetic associations of multifactorial diseases and pharmacogenetic, thus opening the way to personalized medicine in our population.


Assuntos
Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Desintoxicação Metabólica Fase II/genética , Adolescente , Adulto , Idoso , Alelos , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Mali , Desintoxicação Metabólica Fase II/fisiologia , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Fatores de Risco
4.
J Dermatolog Treat ; 31(3): 300-308, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-30897007

RESUMO

The aim of this study was to investigate if aloe polysaccharide (AP) has the repairing effect on ultraviolet b (UVB) injured nerve cells. The study applied BALB/c female mice as animal model, and NFG-activated PC12 cells as cell model of skin nerve. The cell viability was detected by MTT assay, and cell apoptosis was detected by TUNEL (TdT-mediated dUTP nick-end labeling) and Annexin-V/PI assay, and cell-cycle status in different groups were observed via flow cytometry (FCM). Enzyme-linked immunosorbent assay (ELISA) was applied to analyze oxidative stress and anti-oxidative ability in each group. Real-time PCR and western blot were used to detect the expression levels of Bax, Bcl-2, Caspase-3, Cyclin D1, Keap1, Nrf2, GCLC, and GSTP1. The results showed obvious inhibition of cell viability and cell-cycle progression and promotion of cell apoptosis by UVB irradiation through inducing oxidative stress. In AP treated groups, cell viability and proliferation could be markedly improved and cell apoptosis inhibited with higher anti-oxidative capability and up-regulated expression of Keap1, Nrf2, GCLC, and GSTP1. It suggested that AP was able to repair UVB induced injury on NGF activated skin neural cell PC12, probably through Keap1/Nrf2/ARE signal pathway.


Assuntos
Aloe/metabolismo , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclina D1/metabolismo , Feminino , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Crescimento Neural/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos da radiação
5.
Sci Rep ; 9(1): 14867, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619723

RESUMO

We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Prometáfase/efeitos dos fármacos , Administração Oral , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Curcumina/análogos & derivados , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Células HeLa , Humanos , Células K562 , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células MCF-7 , Camundongos Nus , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Prometáfase/genética , Espécies Reativas de Oxigênio/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Sci Rep ; 9(1): 14764, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31611630

RESUMO

Antitumor drug development based on the concept of intervening in the antioxidant system of cancer cells has been gaining increased interest. In this study, we propose a promising strategy for cancer treatment using modulation of oxidative stress by suppression of glutathione S-transferases (GSTs), a typical antioxidant enzyme. siRNA which can be applied to the development of nucleic acid drugs, enabling them to eliminate unwanted side effects, increase specificity, and avoid the problem of drug resistance, was employed for GSTP-silencing at the transcriptional level. The silencing of the pi class of GST (GSTP) that displayed the most characteristic expression profile in 13 kinds of cancer cell lines has shown significant impairment in the growth of cancer cells due to oxidative stress caused by excess ROS accumulation. Comparative proteomics between normal cells and GSTP-silenced pancreatic cancer cell PANC-1 suggested that GSTP-silencing facilitated the mitochondrial dysfunction. These findings show promise for the development of strategies toward cancer therapy based on the mechanism that allows genetic silencing of GSTP to promote oxidative stress through mitochondria dysfunction.


Assuntos
Glutationa S-Transferase pi/genética , Mitocôndrias/genética , Estresse Oxidativo , Neoplasias Pancreáticas/genética , Linhagem Celular Tumoral , Proliferação de Células , Glutationa S-Transferase pi/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Terapêutica com RNAi
7.
Arch Toxicol ; 93(11): 3249-3260, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31552474

RESUMO

Sunitinib malate is a multi-targeted tyrosine kinase inhibitor used extensively for treatment of human tumors. However, cardiovascular adverse effects of sunitinib limit its clinical use. It is pivotal to elucidate molecular targets that mediate sunitinib-induced cardiotoxicity. Sirtuin 3 (Sirt3) is an effective mitochondrial deacetylase that has been reported to regulate sensitivity of different types of cells to chemotherapies, but roles of Sirt3 in sunitinib-induced cardiotoxicity have not been investigated. In the present study, we established wild type, Sirt3-knockout, and Sirt3-overexpressing mouse models of sunitinib (40 mg kg-1 day-1 for 28 days)-induced cardiotoxicity and examined cardiovascular functions and pathological changes. We further cultured wild type, Sirt3-knockout, and Sirt3-overexpressing primary mouse cardiac pericytes and analyzed sunitinib (10 µMol for 48 h)-induced alterations in cellular viability, cell death processes, and molecular pathways. Our results show that sunitinib predominantly induced hypertension, left ventricular systolic dysfunction, and cardiac pericyte death accompanied with upregulation of Sirt3 in cardiac pericytes, and these cardiotoxicities were significantly attenuated in Sirt3-knockout mice, but aggravated in Sirt3-overexpressing mice. Mechanistically, sunitinib induced cardiac pericyte death through inhibition of GSTP1/JNK/autophagy pathway and Sirt3 interacted with and inhibited GSTP1, further inhibiting the pathway and aggravating sunitinib-induced pericyte death. Conclusively, we demonstrate that Sirt3 promotes sensitivity to sunitinib-induced cardiotoxicity via GSTP1/JNK/autophagy pathway. Our results suggest Sirt3 might be a potential target for developing cardioprotective therapies for sunitinib-receiving patients.


Assuntos
Antineoplásicos/toxicidade , Glutationa S-Transferase pi/metabolismo , Hipertensão/induzido quimicamente , MAP Quinase Quinase 4/metabolismo , Pericitos/efeitos dos fármacos , Sirtuína 3/genética , Sunitinibe/toxicidade , Animais , Autofagia/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cardiotoxicidade , Sobrevivência Celular/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/patologia , Camundongos , Camundongos Knockout , Pericitos/metabolismo , Pericitos/patologia , Transdução de Sinais
8.
Chem Biol Drug Des ; 94(6): 2094-2102, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31452310

RESUMO

Quantum dots (QD) are being evaluated as inorganic nanoparticles for both in vitro and in vivo optical imaging. They are also used as sensors or vehicles for targeted drug delivery combined with optical imaging. In this study, we demonstrated that glutathione-coated Ag2 S QDs (GSH-Ag2 S QDs) act as a substrate analogue of glutathione S-transferase (GST) enzymes for the first time in the literature. The GSTs belong to a major group of detoxification enzymes involved in the detoxification metabolism responsible for the protection of cells against reactive oxygen species (ROS) or electrophiles. GST isozymes are impaired in the various diseases such as neurological diseases and cancer. We evaluated the interaction of GST-pi enzyme with GSH-Ag2 S QDs, which have never been studied in the literature before, using both fluorometric and spectrophotometric methods. Our data showed that GSH-Ag2 S QDs gave reaction with GST enzyme as a substrate analogue. In conclusion, our data may help to guide researchers for further development of sensing systems for GST activity which is impaired in various diseases including cancer.


Assuntos
Glutationa S-Transferase pi/metabolismo , Glutationa/química , Pontos Quânticos/química , Compostos de Prata/química , Animais , Linhagem Celular Tumoral , Glutationa S-Transferase pi/química , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Fígado/enzimologia , Fígado/metabolismo , Pontos Quânticos/metabolismo , Ratos , Ratos Wistar , Especificidade por Substrato
9.
Artigo em Inglês | MEDLINE | ID: mdl-31301398

RESUMO

In fish of freshwaters environments, the accumulation and toxic effects of arsenite (AsIII) can be attenuated by detoxification proteins such as GST and ABCC transporters. We studied the effects of AsIII on the middle intestine of O. mykiss in ex-vivo and in vivo/ex vivo assays. For the ex vivo assays, we measured the transport rate of the ABCC substrate DNP-SG and GST activity in intestinal strips and everted sacs. AsIII inhibited DNP-SG transport in a concentration-dependent manner, specifically when we applied it on the basolateral side. GST activity increased when we applied a maximum concentration of AsIII. For the in vivo/ex vivo assays, we kept fish in water with or without 7.7 µmol L-1 of AsIII for 48 h. Then, we measured DNP-SG transport rate, GST activity, and PP1 activity in intestine strips during one hour. For PP1 activity, we incubated the strips with or without microcystin-LR (MCLR), a toxin excreted through ABCC2 proteins. We also analyzed Abcc2 and Gst-π mRNA expression in intestine and liver tissue. In the group exposed in vivo to AsIII, DNP-SG transport rate and GST activity were higher and the effect of MCLR over PP1 activity was attenuated. AsIII significantly induced only Abcc2 mRNA expression in both middle intestine and liver. Our results suggest that, in the middle intestine of O. mykiss, AsIII is absorbed mainly at the basolateral side of the enterocytes, excreted to the lumen by ABCC2 transporters, and is capable of modulating Abcc2 mRNA expression by a transcriptional mechanism.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Arsenitos , Glutationa S-Transferase pi/metabolismo , Intestinos/enzimologia , Fígado/metabolismo , Oncorhynchus mykiss/metabolismo , Animais , Arsenitos/metabolismo , Arsenitos/farmacocinética , Arsenitos/toxicidade , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro , Xenobióticos/metabolismo , Xenobióticos/farmacocinética , Xenobióticos/toxicidade
10.
Redox Biol ; 26: 101256, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31229842

RESUMO

Environmental proteases have been widely associated to the pathogenesis of allergic disorders. Der p 1, a cysteine-protease from house dust mite (HDM) Dermatophagoides pteronyssinus, constitutes one of the most clinically relevant indoor aeroallergens worldwide. Der p 1 protease activity depends on the redox status of its catalytic cysteine residue, which has to be in the reduced state to be active. So far, it is unknown whether Der p 1-protease activity could be regulated by host redox microenvironment once it reaches the lung epithelial lining fluid in addition to endogenous mite components. In this sense, Glutathione-S-transferase pi (GSTpi), an enzyme traditionally linked to phase II detoxification, is highly expressed in human lung epithelial cells, which represent the first line of defence against aeroallergens. Moreover, GSTpi is a generalist catalyst of protein S-glutathionylation reactions, and some polymorphic variants of this enzyme has been associated to the development of allergic asthma. Here, we showed that human GSTpi increased the cysteine-protease activity of Der p 1, while GSTmu (the isoenzyme produced by the mite) did not alter it. GSTpi induces the reduction of Cys residues in Der p 1, probably by rearranging its disulphide bridges. Furthermore, GSTpi was detected in the apical medium collected from human bronchial epithelial cell cultures, and more interesting, it increased cysteine-protease activity of Der p 1. Our findings support the role of human GSTpi from airways in modulating of Der p 1 cysteine-protease activity, which may have important clinical implications for immune response to this aeroallergen in genetically susceptible individuals.


Assuntos
Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína/metabolismo , Dermatophagoides pteronyssinus/química , Células Epiteliais/enzimologia , Glutationa S-Transferase pi/metabolismo , Animais , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Brônquios/citologia , Brônquios/enzimologia , Brônquios/imunologia , Linhagem Celular , Cisteína/imunologia , Cisteína Endopeptidases/imunologia , Dermatophagoides pteronyssinus/enzimologia , Dermatophagoides pteronyssinus/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Glutationa S-Transferase pi/imunologia , Humanos , Isoenzimas/imunologia , Isoenzimas/metabolismo , Cinética , Oxirredução , Proteólise , Especificidade da Espécie
11.
Chem Commun (Camb) ; 55(56): 8122-8125, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31237279

RESUMO

Pi-class glutathione S-transferase (GSTP1) is a molecular marker enzyme whose expression level is altered in various malignant tumour tissues. Herein, we report the first highly selective fluorogenic GSTP1 substrate, Ps-TG, and its membrane-permeable derivative Ps-TAc, for specific visualization of intracellular GSTP1 activity in cancer cells or epigenetically regulated GSTP1 expression.


Assuntos
Epigênese Genética , Corantes Fluorescentes/metabolismo , Glutationa S-Transferase pi/metabolismo , Glutationa/metabolismo , Humanos , Células MCF-7 , Espectrometria de Fluorescência , Especificidade por Substrato
12.
J Enzyme Inhib Med Chem ; 34(1): 1131-1139, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31169043

RESUMO

The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Glutationa S-Transferase pi/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
13.
Exp Mol Pathol ; 108: 156-163, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30951700

RESUMO

As the fifth most common cancer and the second leading cause of cancer related deaths worldwide, hepatocellular carcinoma (HCC) causes up to one million deaths annually. Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) are becoming the two major risk factors because both may develop liver fibrosis and hepatocellular carcinoma (HCC) if left untreated. However, compared with 3-10% of patients with ASH may progress to HCC annually, about only 0.5% NASH patients may progress to HCC annually. The present study is to clarify the protein expression differences of tumor suppressor genes (TSGs) between ASH and NASH. In liver biopsied specimens from NASH and ASH patients, using an immunofluorescence method and morphometrically quantitating the fluorescence intensity, we studied the protein expression within hepatocytes cytoplasm of candidate TSGs including RUNX3, GSTP1, and RASSF1A. Compared with the control group of patients, the expression levels of all three proteins were upregulated in the ASH group of patients (p < .001 in all molecules). While RUNX3 was upregulated, GSTP1 and RASSF1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three TSG proteins, RUNX3, GSTP1, and RASSF1, were significantly lower in the NASH group of patients (p < .001 in all three molecules). These results confirmed our previous finding that there are significant differences of many molecules including TSGs that changed in NASH compared to ASH. Thus, we conclude that there are significantly different TSGs and pathways involved during the pathogenesis of HCC development in NASH compared to ASH that may help to develop different strategies for prevention and treatment of NASH and ASH patients.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Fígado Gorduroso Alcoólico/metabolismo , Glutationa S-Transferase pi/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Progressão da Doença , Fígado Gorduroso Alcoólico/diagnóstico , Imunofluorescência/métodos , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/diagnóstico
14.
Anticancer Res ; 39(4): 1795-1805, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30952719

RESUMO

BACKGROUND/AIM: The glutathione S-transferase pi gene (GSTP1) is a polymorphic gene encoding functionally different Gstp1 isoenzyme proteins. These seem to contribute to xenobiotic metabolism and might also play a role in susceptibility to cancer. The aim of this study was to elucidate the potential role of GSTP1 as a biomarker for disease progression and predictor of chemotherapeutic effect in glioma. MATERIALS AND METHODS: Using quantitative real time PCR and western blotting analysis, a total of 61 astrocytic tumor samples from WHO grade II-IV were investigated. RESULTS: There were no differences in GSTP1 mRNA expression between diffuse astrocytomas, anaplastic astrocytomas, or GBM. No difference was seen between secondary GBM before and after radio-/chemotherapy. CONCLUSION: The expression of GSTP was highly heterogeneous within the surgical specimens. No significant differences in gene and protein expression were detected between the different types of gliomas, suggesting that glioma chemoresistance is probably multifactorial and GSTP1-independent.


Assuntos
Antineoplásicos/uso terapêutico , Astrocitoma/tratamento farmacológico , Astrocitoma/enzimologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Glutationa S-Transferase pi/genética , Transcriptoma , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Glutationa S-Transferase pi/metabolismo , Humanos , Isoenzimas , Gradação de Tumores , Resultado do Tratamento
15.
Cell Death Dis ; 10(5): 351, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31024008

RESUMO

F-box only protein 8 (FBX8), as a critical component of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases, has been associated with several malignancies through interacting with a member of proteins. However, the substrates of FBX8 for destruction in the progression of colorectal carcinoma (CRC) need to be explored. Here, we show that loss of FBX8 accelerates chemical-induced colon tumorigenesis. FBX8 directly targets GSTP1 for ubiquitin-mediated proteasome degradation in CRC. GSTP1 promotes the proliferation, invasion, and metastasis of CRC cells. Furthermore, GSTP1 is upregulated in CRC tissue samples and predicts poor prognosis of CRC patients. The inactivation of FBX8 negatively correlated with increased levels and stability of GSTP1 in clinical CRC tissues and FBX8 knockout transgenic mice. These findings identify a novel ubiquitination pathway as FBX8-GSTP1 axis that regulates the progression of CRC, which might be a potential prognostic biomarker for CRC patients.


Assuntos
Neoplasias Colorretais/patologia , Proteínas F-Box/metabolismo , Glutationa S-Transferase pi/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/mortalidade , Regulação para Baixo , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitinação
16.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823661

RESUMO

The developing cardiovascular system of zebrafish is a sensitive target for many environmental pollutants, including dioxin-like compounds and pesticides. Some polychlorinated biphenyls (PCBs) can compromise the cardiovascular endothelial function by activating oxidative stress-sensitive signaling pathways. Therefore, we exposed zebrafish embryos to PCB126 or to several redox-modulating chemicals to study their ability to modulate the dysmorphogenesis produced by PCB126. PCB126 produced a concentration-dependent induction of pericardial edema and circulatory failure, and a concentration-dependent reduction of cardiac output and body length at 80 hours post fertilization (hpf). Among several modulators tested, the effects of PCB126 could be both positively and negatively modulated by different compounds; co-treatment with α-tocopherol (vitamin E liposoluble) prevented the adverse effects of PCB126 in pericardial edema, whereas co-treatment with sodium nitroprusside (a vasodilator compound) significantly worsened PCB126 effects. Gene expression analysis showed an up-regulation of cyp1a, hsp70, and gstp1, indicative of PCB126 interaction with the aryl hydrocarbon receptor (AhR), while the transcription of antioxidant genes (sod1, sod2; cat and gpx1a) was not affected. Further studies are necessary to understand the role of oxidative stress in the developmental toxicity of low concentrations of PCB126 (25 nM). Our results give insights into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of environmental pollutants.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Antagonistas de Estrogênios/toxicidade , Coração/efeitos dos fármacos , Estresse Oxidativo , Bifenilos Policlorados/toxicidade , Animais , Antioxidantes/farmacologia , Cardiotoxicidade , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Endotélio Vascular/metabolismo , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Coração/embriologia , Nitroprussiato/farmacologia , Tocoferóis/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Toxicol Sci ; 169(1): 122-136, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690589

RESUMO

This study examined hypermethylated and downregulated genes specific to carbon tetrachloride (CCl4) by Methyl-Seq analysis combined with expression microarray analysis in the liver of rats treated with CCl4 or N-nitrosodiethylamine (DEN) for 28 days, by excluding those with DEN. Among 52 genes, Ldlrad4, Proc, Cdh17, and Nfia were confirmed to show promoter-region hypermethylation by methylation-specific quantitative PCR analysis on day 28. The transcript levels of these 4 genes decreased by real-time reverse transcription-PCR analysis in the livers of rats treated with nongenotoxic hepatocarcinogens for up to 90 days compared with untreated controls and genotoxic hepatocarcinogens. Immunohistochemically, LDLRAD4 and PROC showed decreased immunoreactivity, forming negative foci, in glutathione S-transferase placental form (GST-P)+ foci, and incidences of LDLRAD4- and PROC- foci in GST-P+ foci induced by treatment with nongenotoxic hepatocarcinogens for 84 or 90 days were increased compared with those with genotoxic hepatocarcinogens. In contrast, CDH17 and NFIA responded to hepatocarcinogens without any relation to the genotoxic potential of carcinogens. All 4 genes did not respond to renal carcinogens after treatment for 28 days. Considering that Ldlrad4 is a negative regulator of transforming growth factor-ß signaling, Proc participating in p21WAF1/CIP1 upregulation by activation, Cdh17 inducing cell cycle arrest by gene knockdown, and Nfia playing a role in a tumor-suppressor, all these genes may be potential in vivo epigenetic markers of nongenotoxic hepatocarcinogens from the early stages of treatment in terms of gene expression changes. LDLRAD4 and PROC may have a role in the development of preneoplastic lesions produced by nongenotoxic hepatocarcinogens.


Assuntos
Tetracloreto de Carbono/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Metilação de DNA/efeitos dos fármacos , Dietilnitrosamina/toxicidade , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/induzido quimicamente , Fígado/efeitos dos fármacos , Lesões Pré-Cancerosas/induzido quimicamente , Animais , Caderinas/genética , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteína C/genética , Proteína C/metabolismo , Ratos Endogâmicos F344 , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Tempo
18.
Cell Death Differ ; 26(10): 2086-2099, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30683915

RESUMO

Glutathione S-transferases P1 (GSTP1) is a phase II detoxifying enzyme and increased expression of GSTP1 has been linked with acquired resistance to anti-cancer drugs. However, most anticancer drugs are not good substrates for GSTP1, suggesting that the contribution of GSTP1 to drug resistances might not be dependent on its capacity to detoxify chemicals or drugs. In the current study, we found a novel mechanism by which GSTP1 protects human breast cancer cells from adriamycin (ADR)-induced cell death and contributes to the drug resistance. GSTP1 protein level is very low in human breast cancer cell line MCF-7 but is high in ADR-resistant MCF-7/ADR cells. Under ADR treatment, MCF-7/ADR cells showed a higher autophagy level than MCF-7 cells. Overexpression of GSTP1 in MCF-7 cells by using the DNA transfection vector enhanced autophagy and down-regulation of GSTP1 through RNA interference in MCF-7/ADR cells decreased autophagy. When autophagy was prevented, GSTP1-induced ADR resistance reduced. We found that GSTP1 enhanced autophagy level in MCF-7 cells through interacting with p110α subunit of phosphatidylinositol-3-kinase (PI3K) and then inhibited PI3K/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) activity. Proline123, leucine160, and glutamine163, which located in C terminal of GSTP1, are essential for GSTP1 to interact with p110α, and the following autophagy and drug resistance regulation. Taken together, our findings demonstrate that high level of GSTP1 maintains resistance of breast cancer cells to ADR through promoting autophagy. These new molecular insights provide an important contribution to our better understanding the effect of GSTP1 on the resistance of tumors to chemotherapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Doxorrubicina/farmacologia , Glutationa S-Transferase pi/metabolismo , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Humanos , Células MCF-7 , Espectrometria de Massas/métodos , Microscopia Eletrônica de Transmissão/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transfecção
19.
Chest ; 155(1): 94-102, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616740

RESUMO

BACKGROUND: Glutathione S-transferase (GST) genes are involved in the management of oxidative stress in the lungs. We aimed to determine whether they modify the associations between early life smoke exposure and adverse lung health outcomes. METHODS: The Melbourne Atopy Cohort study (a high-risk birth cohort) enrolled 620 children and followed them prospectively from birth. We recorded perinatal tobacco smoke exposure, asthma, and lung function at 12 (59%) and 18 years (66%) and genotyped for GSTM1, GSTT1, and GSTP1 (69%). RESULTS: GST genotypes were found to interact with tobacco smoke exposure on lung function outcomes (P interaction ≤ .05). Only among children with GSTT1 null genotypes was exposure to mother's, father's, or parental tobacco smoke in early life associated with an increased risk of reductions in prebronchodilator (BD) FEV1 and FVC at both 12 and 18 years. These associations were not seen in children with GSTT1 present. Similarly, only among children with GSTM1 null genotypes was exposure to father's or parental smoking associated with reductions in pre- and post-BD FEV1 and FVC at 18 years. Only among children with Ile/Ile genotypes of GSTP1 was exposure to mother's smoking associated with increased risk of reduced FEV1 at 18 years, but this was not the case among children with Val/Val or Ile/Val genotypes. CONCLUSIONS: Our study provides evidence of interaction between early tobacco smoke exposure and GST genotypes on lung function. Carriers of GST null mutations and GSTP1 Ile/Ile alleles may be more susceptible when exposed to tobacco smoke in early life. These findings support stronger recommendations to protect all infants from tobacco smoke exposure. TRIAL REGISTRY: Australian and New Zealand Clinical Trials Registry; No.: ACTRN12609000734268; URL: http://www.anzctr.org.au/.


Assuntos
Asma/fisiopatologia , Previsões , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Polimorfismo Genético , Poluição por Fumaça de Tabaco/efeitos adversos , Adolescente , Asma/genética , Asma/metabolismo , Criança , DNA/genética , Feminino , Seguimentos , Fluxo Expiratório Forçado/fisiologia , Predisposição Genética para Doença , Genótipo , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Humanos , Pulmão/fisiopatologia , Masculino , Estudos Retrospectivos , Fatores de Risco
20.
Biochim Biophys Acta Gen Subj ; 1863(1): 130-143, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30290218

RESUMO

The glutathione (GSH) S-transferase family of detoxification and signalling proteins represents a major hub for the metabolism of Selenium-derived compounds. At the same time, these compounds can be used to modulate the expression and multiple activities of GSTs and other glutathione-dependent genes, that are important aspects in both the chemoprevention and therapy of drug-resistant cancers. In this context, the isoform GSTP-1 (GSTP) appears to play a fundamental role. Besides promoting GSH-dependent detoxification of cellular electrophiles, GSTP physically interacts with a number of small molecules and cellular proteins producing regulatory effects across the main signal transduction and transcription pathways (identified as the "regulatory interactome of GSTP"). An emerging molecular mechanism behind such regulatory function is the activity of GSTP as a redox chaperonine responsible for the selective glutathionylation of protein Cys residues in the different subcellular compartments. The redox-sensitive transcription factor Nrf2 was recently identified as one of the regulatory nodes of this interactome at the interface between inflammation, adaptive stress response, and cell death pathways. The influence of Nrf2 in the stress response to cellular electrophiles and its regulatory interaction with GSTP are discussed in this review suggesting the hypothesis that this interaction may represent the actual pharmacological target of Se compounds with thiol peroxidase activity. These points are critically evaluated with a view to further development of these compounds in cancer prevention and the chemotherapy of drug-resistant tumours.


Assuntos
Glutationa S-Transferase pi/metabolismo , Neoplasias/metabolismo , Selênio/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Glutationa/química , Humanos , Peróxido de Hidrogênio/química , Inflamação , Lipídeos/química , Camundongos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Oxidantes/química , Oxirredução , Estresse Oxidativo , Oxigênio/química , Ligação Proteica , Compostos de Selênio/farmacologia , Transdução de Sinais , Compostos de Sulfidrila/química
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