Construction and identification of a HEK293 cell line with stable TrxR1 overexpression / 南方医科大学学报

OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

Estudo do potencial de reposicionamento do composto auranofina no controle de infecções por Candida albicans / Study on the potential for the repositioning of auranofin in the control of Candida albicans infections

A ocorrência crescente de resistência antifúngica, a toxicidade, além do pequeno espectro de ação dos antifúngicos convencionais, limita o número de alternativas terapêuticas para doenças causadas por leveduras do gênero Candida. Uma das frentes de pesquisa é a proposta de novos usos para drogas existentes chamada de reposicionamento, que diminui o tempo e esforço na busca de novos compostos eficazes. Neste contexto, o presente estudo tem como objetivo avaliar o potencial antifúngico e os mecanismos de ação do composto auranofina contra a espécie Candida albicans, além da toxicidade in vivo. Foram utilizadas cepas padrões de C. albicans (SC5314, ATCC 18804) e as concentrações inibitória mínima (CIM) e fungicida mínima (CFM) foram determinadas. Os mecanismos de ação dos compostos foram avaliados sobre a estrutura celular de C. albicans, com verificação de alterações na morfologia, na parede celular, sobre os fatores de virulência de C. albicans, como transição levedura-hifa e produção de exoenzimas, além do efeito do composto sobre o metabolismo de C. albicans e efeito antifúngico sob condições de estresse osmótico. Para avaliação da toxicidade das concentrações efetivas de auranofina in vivo, foi utilizado o modelo invertebrado, Drosophila melanogaster. Os dados obtidos no ensaio de transição levedura-hifa foram avaliados pelos testes ANOVA e de Tukey e os testes Kruskal-Wallis e de Dunn. foram utilizados na análise do efeito de auranofina sobre o metabolismo fúngico. O nível de significância para todos os testes foi de 5%. Foram verificadas concentrações inibitória e fungicida de auranofina sobre C. albicans. Apesar da ausência de efeitos sobre fatores de virulência, auranofina causou redução no metabolismo fúngico e no crescimento fúngico sob estresse osmótico, sugerindo, nesse caso, um possível efeito direto ou indireto na membrana celular fúngica. Além disso, nas concentrações efetivas não foi observada toxicidade relevante in vivo. Os resultados demonstraram, portanto, que auranofina tem potencial para seu reposicionamento como antifúngico, no entanto, mais estudos são necessários para mais esclarecimentos e utilização adequada do medicamento (AU).

The increasing occurrence of antifungal resistance, toxicity, in addition to the small spectrum of action of conventional antifungals, limits the number of therapeutic alternatives for diseases caused by yeasts of the genus Candida. One of the research fronts is the proposal of new uses for existing drugs called repositioning, which reduces the time and effort in the search for new effective compounds. In this context, the present study aims to evaluate the antifungal potential and mechanisms of action of the auranofin compound against Candida albicans, in addition to in vivo toxicity. Standard strains of C. albicans (SC5314, ATCC 18804) were used and minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined. The mechanisms of action of the compounds were evaluated on the cellular structure of C. albicans, with verification of changes in morphology, in the cell wall, on the virulence factors of C. albicans, such as yeast-hypha transition and production of exoenzymes, in addition to the effect of the compound on the metabolism of C. albicans and antifungal effect under osmotic stress conditions. To evaluate the toxicity of effective concentrations of auranofin in vivo, the invertebrate model, Drosophila melanogaster, was used. The data obtained in the yeast-hypha transition assay were evaluated by the ANOVA and Tukey tests and the KruskalWallis and Dunn tests. were used to analyze the effect of auranofin on fungal metabolism. The significance level for all tests was 5%. Inhibitory and fungicidal concentrations of auranofin were verified on C. albicans. Despite the absence of effects on virulence factors, auranofin caused a reduction in fungal metabolism and fungal growth under osmotic stress, suggesting, in this case, a possible direct or indirect effect on the fungal cell membrane. Furthermore, at effective concentrations, no relevant in vivo toxicity was observed. The results showed, therefore, that auranofin has the potential for its repositioning as an antifungal, however, more studies are needed for further clarification and proper use of the drug (AU).

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Compatibilidad química por calorimetría diferencial de barrido y termogravimetría del auranofin tabletas 3 mg / Chemical compatibility of 3 mg Auranofin tablets demonstrated by differential scanning calorimetry and thermogravimetry

Como parte de la pre-estabilidad de la preformulación de auranofin tabletas, se realizó un estudio de compatibilidad química, para lo cual se emplearon técnicas de análisis térmico como la calorimetría diferencial de barrido y la termogravimetría. Previo a dichos estudios se caracterizó térmicamente por calorimetría diferencial de barrido el principio activo y cada uno de los excipientes. Posteriormente se procedió a la realización del estudio de compatibilidad química, mediante la preparación de mezclas físicas binarias entre el principio activo y cada uno de los excipientes. Se detectó por ambos métodos que el principio activo tuvo una transición física de fusión, no reportada en la literatura, lo que permitió poder calcular su pureza por calorimetría diferencial de barrido. Mediante la técnica calorimétrica fue posible inferir la ausencia de incompatibilidad química entre el principio activo y los excipientes estudiados. Además, mediante el cálculo de la energía de activación se estableció el siguiente orden de estabilidad térmica: auranofin:PVP> auranofin:lactosa> auranofin:explotab> auranofin:estearato> auranofin:aerosil> auranofin:celulosa, por lo que se recomienda el uso de estos excipientes en la elaboración de la formulación farmacéutica

As part of the pre-stability study of the Auranofin tablet pre-formulation, a chemical compatibility study was conducted using thermal analysis techniques such as the differential scanning calorimetry and the thermogravimetry. Prior to these studies, the active principle and each of the excipients were thermally characterized with the aid of the differential scanning calorimetry. Then, there proceeded to carry out the chemical compatibility study by preparing binary physical mixtures between the active principle and each of the excipients. Both methods showed that the active principle had a melting physical transition, not reported in the literature, which allowed calculating its purity aided by the differential scanning calorimetry. It was possible to infer from the calorimetry technique that there was not chemical incompatibility between the active principle and the studied excipients. By means of the activation energy estimation, the following order of thermal stability was set: Auranofin:PVP> Auranofin: lactose> Auranofin:explotab> Auranofin:estearate> Auranofin:aerosil> Auranofin:celullose. The use of these excipients was recommended for the preparation of the pharmaceutical formulation

Opções terapêuticas na Asma grave / Therapeutic options for severe asthma

Estima-se que 10 dos asmáticos sofram de asma persistente grave. Estes doentes apresentam controle insatisfatório da sua asma, com sintomas persistentes graves e maior risco de exacerbações, hospitalizações e morte e geralmente com marcada deterioração da qualidade de vida. A terapêutica baseia-se na avaliação do grau de gravidade e otimização da terapêutica com minimização de efeitos secundários. Os doentes mantêm controle inadequado dos sintomas e apesar de administradas altas doses de corticosteróides inalados e agonistas p2 de longa duração, freqüentemente necessitam de medicação adicional com teofilina de libertação lenta, anti-Ieucotrienos e/ou corticosteróides oral. Nos doentes com asma córtico-dependente ou córtico-resistente outras opções terapêuticas devem ser equacionadas como os fármacos poupadores de corticosteróides: ciclosporina, metotrexato, imunoglobulina endovenosa e sais de ouro. O omalizumabe, anticorpo monoclonal anti-IgE, foi recentemente incluído dentro das alternativas terapêuticas disponíveis. Para se conseguir um controle mais eficaz desta doença complexa é fundamental a caracterização futura dos diferentes fenótipos desta síndrome

The Effect of Auranofin on Thrombomodulin Expression in Acute Promyelocytic Leukemia Cell

BACKGROUND: Acute promyelocytic leukemia (APL) is distinguished from other forms of leukemia by its association with bleeding diatheses. All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), which have been effectively used in the treatment of the APL, promptly improve coagulation/bleeding syndromes by regulating the expressions of tissue factor (TF) and thrombomodulin (TM). We have previously shown a novel activity of auranofin (AF), in that it induced apoptosis and differentiation of NB4 cells. To study whether AF also possesses similar anticoagulant effects to those of ATRA and As2O3, its effects on the expressions of TM and TF were investigated. METHODS: NB4 cells derived from APL were incubated with 1 micrometer of AF. After incubation for 12, 24 and 48 hours, the AF-regulated expressions of TM and TF were analyzed by RT-PCR, Northern blot and Western blot. The assay for the TM antigen on the cell surface was performed using a flow cytometry. RESULTS: The expression of the TM gene was increased for upto 12 hours after the AF treatment, but no change was observed in the expression of the TF gene. Western blot analysis also demonstrated that AF increased the level of TM proteinin a time-dependent manner. FACS data showed the TM antigen on the cell surface to gradually increase for upto 48 hours in AF-treated cells. CONCLUSION: The results of this study indicate that AF can have an antithrombotic function via the up-regulation of the expression of TM, which suggests it may partially contribute to the improvement of coagulopathies in APL.

Gold compound auranofin inhibits I kappaB kinase (IKK) by modifying Cys-179 of IKK beta subunit

Antirheumatic gold compounds have been shown to inhibit NF-kB activation by blocking IkB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKk alpha and beta subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKK beta with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKK alpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKK alpha and beta and variants; IKK alpha (S176E, S180E) or IKK beta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKK alpha (C178A) and IKK beta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF- kB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKK beta.

Control evolutivo del penfigo con aureoterapia oral / Evolutive control of the pemphigus with oral aureotherapy

Entre 1985 y 1989, 32 pacientes con pénfigo (19 vulgares, 9 eritematodes, 3 foliáceos y un vegetante) recibieron auranofin como tratamiento coadyuvante. En dos dos casos se usó como única medicación y en el resto asocicado a corticoides. El seguimiento fue de 42 a 2 meses, y la dosis administrada osciló de 360 a 2700 mg, con una media de 1538 mg equivalentes a poco más de 8 meses de tratamiento. Los efectos colaterales fueron mínimos: 3 presentaron diarrea, 2 prurito, uno exantema maculoso y otro intolerancia digestiva; en ningún caso fue necesario suspender el oro. El tiempo transcurrido hasta la completa remisión fue de 2 a 8 meses, y los corticoides se eliminaron 1 a 2 meses después. En 10 pacientes se presentaron recaídas: una vez finalizado el tratamiento en 6, y en 4 cuanhdo recibían oro. Todos se controlaron con dosis bajas de corticoides. Mantener tratamientos más prolongados no nos asegura la ausencia de rebrotes y sí mayor probabilidad de efectos adversos. En la actualidad 5 pacientes presentan lesiones, de los que 3 están todavía entre su segundo y cuarto mes de tratamiento, tiempo todavía insuficiente para considerarlos como fracaso terapéutico. El resto (27) se encuentran clínicamente curados

Aureoterapia oral en el penfigo / Oral aurotherapy in pemphigus

El pénfigo constituye una enfermedad ampollar grave, de etiopatogenia autoinmune. Su control terapéutico es difícil y las drogas utilizadas presentan múltiples efectos colaterales. Evaluamos en este trabajo diecisiete pacientes con pénfigo (once vulgares, cinco eritematodes y un foliáceo) tratados con un compuesto áurico por vía oral: auranofín. Los resultados muestran, luego de administrar la droga, en dos tomas diarias de tres miligramos cada una, durante quince días a doce meses pocos efectos colaterales (dos pacientes con diarrea y uno con exantema pruriginoso). Una paciente fue tratada exclusivamente con oro con buen resultado, otra sólo con oro luego de suspender el tratamiento coricoideo y reaparecer el signo de Nikolsky. El resto recibió ora junto con metilprednisona. La dosis media requerida de ésta última fue de 45 mg diarios, menor a la necesaria para controlar el pénfigo con corticoides exclusivamente. Observamos tres fracasos terapéuticos luego de seis y nueve meses de la administración del auranofín. Consideramos en los catorce pacientes restantes una mejoría clínica y desaparición del signo de Nikolsly. Se sugiere la utilidad del auranofín para tratar ciertos pacietnes estables o como droga coadyuvante para disminuir los requerimientos y descender más rápidamente la corticoterapia

Acción de la auroterapia oral sobre la respuesta inmune de pacientes con artritis reumatoidea / Action of oral aurotherapy in immune response of rheumatoid arthritis patients

Se han estudiado las respuestas inmunes celular (RIC) y humoral (RIH) en 13 pacientes afectados de artritis reumatoides (AR) "definida" o "clásica", antes y después de 6 a 12 meses de tratamiento con 6mg/día de auranofin. Se ha observado un aumento en la frecuencia de positividad de algunas pruebas cutáneas (PPD, PHA, candidina) y de estimulación cutánea con DNCB, una reducción o negativización de los títulos de factor reumatoideo y del número de linfocitos B (rosetas EAC) y una normalización de los niveles séricos de IgG y complemento. Se concluye que el auranofin es capaz de influir positivamente en la RIC y normalizar algunos parámetros de la RIH, actuando como un inmunomodulador en el tratamiento de la AR

Evaluation of safety and efficacy of auranofin in 20 Iraqi patients with rheumatoid arthritis

To investigate the safety and efficacy of auranofin in the treatment of rheumatoid arthritis in twenty Iraqi patients [eighteen women and two men] with a mean age of 44.9 +/- 9.2 years and with a confirmed diagnosis of rheumatoid arthritis were treated with oral gold [auranofin] 3 mg bid for a period of six months. All patients were thoroughly assessed and physically examined on entry and one monthly intervals during the study. The study showed a pronounced improvement in morning stiffness, walking time and reduction in the number of tender and pain intensity. Adverse reactions were noted in all patients and ranged from mild symptoms to persistent side effects. One patient developed thrombocytopenia on the six month of therapy which necessitated discontinuation of therapy. The commonest adverse effects were nausea [7/20], visual symptoms [7/20], abdominal pain [6/20] and pruritis [4/20] The study confirms the effectiveness of auranofin in ameliorating the severity of the disease and the lack of serious side effects during the course of this study