Expression of recombinant human interleukin-29-Fc fusion protein in HEK293F cells and analysis of its anti-tumor activity in vitro / 中国生物制品学杂志

@#Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.

Curcumin-rich extract from Central Brazil Curcuma longa protects human umbilical vein endothelial cells under oxidative stress / Extrato rico em Curcumina do Brasil Central Curcuma longa protege células endoteliais de veias umbilicais humanas sob estresse oxidativo

INTRODUCTION: In vascular diseases, the interruption of the local blood flow and the subsequent reperfusion of oxygen can cause deleterious oxidative effects on the cells. Turmeric (Curcuma longa L.) presents the capacity to neutralize free radicals along with preventive and therapeutic effects for several diseases. OBJECTIVE: To analyze the bioactive compounds and the antioxidant capacity of the ethanolic extract of Curcuma (EEC), to evaluate its effect on human umbilical vein endothelial cells, and to analyze its effect on cellular signaling pathways. METHODS: Cells were exposed to different concentrations of EEC for 24, 48, and 72 h. Folin-Ciocalteau test, HPLC-Fluorescence analysis, and DPPH method were used to determine the phenolic compounds, curcumin content, and antioxidant action, respectively; the tetrazolium salt reduction to obtain cell viability, cytotoxicity, and the concentration that inhibits 50% of cell viability; and the immunocytochemistry technique to analyze the expression of caspase3, SIRT1, and mTOR. RESULTS: We found the presence of polyphenols in the classes of phenolic acids and curcuminoids in EEC, with 16.7% curcumin content. The number of antioxidants needed to reduce the initial DPPH concentration by 50% was 18.1 µmol/g. The extract mitigated cell damage at a dosage of 100 µg/ml, decreased the immunoexpression of caspase3, and promoted the signaling of the SIRT1 and mTOR survival pathways. CONCLUSION: EEC had a protective effect on human umbilical vein endothelial cells, subjected to oxidative stress, with decreased apoptosis (caspase3) at lower concentrations, cytoprotection by maintaining essential cell functions (mTOR), and signaling of the survival pathway (SIRT1).

INTRODUÇÃO: Em doenças vasculares, a interrupção do fluxo sanguíneo locale subsequente reperfusão de oxigênio pode causar efeitos deletérios e danos irreparáveis às células. Curcuma (Curcuma longa L.) neutraliza radicais livres além de apresentar efeitos preventivos e terapêuticos. OBJETIVO: Caracterizar os compostos bioativos e a capacidade antioxidante do extrato etanólico de cúrcuma (EEC); avaliar seu efeito nas células endoteliais da veia umbilical humana, e analisar a expressão de vias de sinalização celular. MÉTODOS: As células foram expostas a diferentes concentrações de EEC por 24, 48 e 72 horas. Utilizamos o teste de Folin-Ciocalteau, análise por HPLC-Fluorescência e método DPPH para determinar os compostos fenólicos, conteúdo de curcumina e ação antioxidante, respectivamente; o método de redução de tetrazólio para viabilidade celular, a citotoxicidade e a concentração que inibe 50% da viabilidade celular; e a técnica de imunocitoquímica para analisar a expressão de caspase3, SIRT1 e mTOR. RESULTADOS: Observou-se presença de polifenóis nas classes de ácidos fenólicos e curcuminóides no EEC, com teor de curcumina de 16,7%. A quantidade de antioxidante necessária para reduzir a concentração inicial de DPPH em 50% foi de 18,1 µmol/g. O extrato mitigou o dano celular na dosagem de 100 µg/ml, diminuiu a imunoexpressão da caspase3 e promoveu a sinalização das vias de sobrevivência SIRT1 e mTOR. CONCLUSÃO: O EEC teve efeito protetor nas células endoteliais de veia umbilical humana, submetidas ao estresse oxidativo, com diminuição da apoptose (caspase3) em concentrações mais baixas, citoproteção pela manutenção das funções celulares essenciais (mTOR) e sinalização da via de sobrevivência (SIRT1).

Estrategias para la evaluación de extractos de polifenoles en modelos in vitro de cáncer de vías digestivas / Strategies for Evaluation of Polyphenol Extracts on in vitro Models of Digestive Tract Cancer / Estratégias para a avaliação de extratos de polifenóis em modelos in vitro de câncer do sistema digestivo

Introducción: Los polifenoles son compuestos que se encuentran naturalmente en alimentos como frutas, verduras, té, vino y chocolates, a los que se les atribuyen beneficios a la salud humana por su capacidad antioxidante. El cáncer de las vías digestivas se encuentra entre la tercera y quinta causas de muerte para la población, por lo que se ha incrementado el interés por realizar los estudios dirigidos a encontrar compuestos polifenólicos que ayuden en su prevención o tratamiento. Objetivo: Identificar las estrategias disponibles para la evaluación de polifenoles en células de cáncer de vías digestivas. Metodología: Búsqueda de literatura en bases de datos como Ovid, Pubmed, Science Direct y Elsevier Journal. Se seleccionaron artículos en los cuales se reporta el efecto biológico de los polifenoles sobre líneas celulares de cáncer de vías digestivas publicados entre 2012 y 2022. Resultados: Varios estudios han reportado el uso de un buen número de líneas celulares como modelos in vitropara estudios de polifenoles en cáncer y han resaltado las líneas AGS y HT-29, además de técnicas para la caracterización de los polifenoles, como el ensayo 2,2-Difenil-I-Picril Hidrazilo (DPPH). Sin embargo, para evaluar el efecto biológico se identifican diversas pruebas que deben analizarse antes de su implementación. Conclusiones: En la literatura se identifica que existen varias alternativas y estrategias para la evaluación de extrac-tos vegetales en cultivos in vitro de cáncer de vías digestivas; no obstante, antes de pasar al diseño experimental, deben tenerse en cuenta una serie de consideraciones para garantizar la utilidad de los resultados.

Introduction: Polyphenols are compounds naturally found in foods such as fruits, vegetables, tea, wine and chocolates, and it was attributed with benefits to human health due to their antioxidant capacity. Cancer of the digestive tract is between the third and fifth cause of death for the population, increasing the interest in carrying out studies aimed at finding polyphenolic compounds that help in their prevention or treatment. Objective: Identify the available strategies for the evaluation of polyphenols in digestive tract cancer cells. Method: A literature search was performed in databases such Ovid, Pubmed, Science Direct and Elsevier Journal and selected articles reporting the biological effect of polyphenols on digestive tract cancer cell lines, published between 2012 and 2022. Results: Currently studies report the use of a good number of cell lines as in vitro models for poly-phenol studies in cancer highlighting the AGS and HT-29 lines, in addition to techniques for the characterization of polyphenols such as the α, α-diphenyl-ß-picrylhydrazyl DPPH assay, however, to evaluate the biological effect various tests are identified that should be analyzed before implemen-tation. Conclusions: The literature identifies that there are many alternatives and strategies for the evaluation of plant extracts in in vitro cultures of digestive tract cancer, however, before moving on to the experimental design, a number of considerations should be taken into account to ensure the usability of the results

Introdução: Os polifenóis são compostos encontrados naturalmente em alimentos como frutas, legumes, chá, vinho e chocolates, aos quais são atribuídos benefícios para a saúde humana devido à sua capacidade antioxidante. O câncer do sistema digestivo está entre a terceira e a quinta principais causas de morte na população, o que levou a um interesse crescente em estudos destinados a encon-trar compostos polifenólicos que ajudem a prevenir ou tratar esse tipo de câncer. Objetivo: Identificar as estratégias disponíveis para a avaliação dos polifenóis nas células cancerosas do sistema digestivo. Metodologia: Pesquisa bibliográfica em bases de dados como Ovid, Pubmed, Science Direct e Elsevier Journal. Foram selecionados artigos que relatam o efeito biológico dos polifenóis em linhas celulares de câncer do sistema digestivo, publicados entre 2012 e 2022. Resultados: Vários estudos relataram a utilização de várias linhas celulares como modelos in vitro para estudos de polifenóis no câncer destacando as linhas AGS e HT-29, bem como técnicas para a ca-racterização de polifenóis, como o ensaio 2,2-Difenil-I-Picril Hidrazil (DPPH). No entanto, para avaliar o efeito biológico, são identificados vários testes que devem ser analisados antes da sua aplicação. Conclusões: A literatura identifica que existem várias alternativas e estratégias para a avaliação de extratos de plantas em culturas in vitro de câncer do sistema digestivo; no entanto, antes de passar à concepção experimental, é necessário ter em conta uma série de considerações para garantir a uti-lidade dos resultados

Investigation on the in vitro antioxidant capacity of methanol extract, fractions and flavones from Oroxylum indicum Linn bark

ABSTRACT Antioxidants from natural sources hold high values regarding their indispensible roles in the development of nutraceuticals, pharmaceuticals and cosmetic products. Oroxylum indicum L. is a common medicinal plant with a wide range of therapeutic properties, including a notable antioxidant potency that was reported, yet has not been subjected to more detailed studies. The present study evaluated the potency of Oroxylum indicum methanol stem bark extract, along with its hexane, ethyl acetate, methanol fractions, three flavones including baicalein, oroxylin A and chrysin using DPPH assay. In terms of IC50 values, the crude extract (65,48 µg/mL) exhibited moderate inhibitory activity which was as half potent as that of its ethyl acetate fraction (32,94 µg/mL). This fraction was also superior to the methanol and hexane fractions, as their IC50 were 57,19 and 137,95 µg/mL respectively. Remarkably, a yellow powdery sub-fraction consisted of isolated compounds showed powerful activity (32,89 µg/mL) compared to those of its components, revealing the intriguing effect of synergism while giving evidence for the theory of structure-activity relationship between some flavones and their antioxidant capability. Perpetual search for new radical scavenging agents in Oroxylum indicum is emboldened considering its partially exploited potential in this study

Establishment of a new screening method for anti-HIV-1 drugs / 中华临床感染病杂志

Objective To establish and assess a new screening method for anti-HIV-1 drugs.Methods JLTRG cells were co-cultured with different proportions of H9/HTLV-Ⅲ B cells for 24,48,72 and 96 h.Intensity and density of green fluorescent protein were observed under fluorescence microscope,and were tested using flow cytometry.The optimal proportion of cells in co-culture system and the culture time were determined.The effectiveness of Enfuvirtide (T20) and Efavirenz (EFV),and their half maximal inhibitory concentrations (IC50) were determined by using cell co-culture system and half life of drugs.HIV load was detected using RT-PCR for HIV-1 p24 antigen,and its correlations with drug concentration and mean fluorescent intensity were analyzed by Spearman rank correlation analysis.Results Experiments demonstrated that JLTRG cells co-cultured with H9/HTLV-ⅢB cells at the proportion of 10 ∶ 1 for 72 hours was the best.Along with the concentrations of T20 and EFV changed,JLTRG cells were infected with HIV-1 in different degrees,and the IC50s of T20 and EFV were 10 nmol/L and 5 nmol/L,respectively.The concentrations of T20 and EFV were negatively correlated with mean fluorescent intensity and viral load (r =-1,-0.986 and-1,-1,P ＜ 0.01); and mean fluorescent intensity was positively correlated with viral load (r =0.986 and 1,P ＜ 0.01).Conclusion The drug screening method established in this study is efficient and easy to operate,which provides a new option for anti-HIV-1 drug screening.

Actividad antioxidante de clones de cacao (Theobroma cacao l. ) finos y aromáticos cultivados en el estado de Chiapas, México / Antioxidant activity of clones of cocoa (Theobroma cacao L.) fine and aromatic grown in the state of Chiapas-México

Objetivo: determinar la capacidad antioxidante y contenido de grasa de clones de cacao, provenientes de especies nativas del Estado de Chiapas, México. Materiales y métodos: en extractos de 34 muestras semillas de cacao diluidas en metanol al 95% y clasificadas según pH en tres grupos: I (5,52-5,90), II (5,91- 6,28) y III (6,29-6,67), se realizaron ensayos analíticos de variables físicas y químicas. Se evaluó la inhibición de radicales 2,2-difenil-2-picrilhidrazil (DPPH) a 517 nm, el contenido de polifenoles mediante el reactivo de folin-ciocalteu (FCR) a 765 nm y cantidad de grasas totales según método AOAC. Se aplicó un ANOVA al nivel del 95%, con post prueba Tukey para comparar de medias muestrales y determinar diferencias significativas (p<0.05), además de sus correlaciones lineales. Resultados: El Grupo I con menor pH, mostró menor contenido calórico (26,3±3,6% de grasas), posee la mejor actividad antioxidante con menor valor EC50 de 4182 ppm y mayor contenido de polifenoles 6,6±0,32 equivalentes de ácido gálico por 100 g de muestra seca. Conclusión: El cacao chiapaneco posee importantes ventajas competitivas en el mercado, por su calidad como alimento funcional-nutracéutico y como materia prima para la industria alimentaria, fuente de antioxidantes y aditivos naturales, con potencial consumo en la industria farmacéutica y cosmética internacional.

Objective: To determine antioxidant capacity and fat content of cacao clones from the State of Chiapas, Mexico. Materials and Methods: 34 cocoa beans samples were diluted in 95% methanol and classified by pH in: group I (5.52-5.90), group II (5.91- 6.28), and group III (6.29-6.67). Chemical and physical analysis were developed. 2,2- diphenyl-2-picrylhydrazyl (DPPH) inhibition was evaluated at 517 nm. Polyphenol and fat contents were evaluated by Folin-Ciocalteu Reagent at 765 nm and AOAC method, respectively. Differences in variables were evaluated by ANOVA and Tukey test, in addition to its linear correlations. Results: Group I showed the lowest caloric content (26.3±3.6% fat), the best antioxidant activity with lower EC50 value of 4182 ppm and the highest polyphenol content (6.6±0.3 gallic acid/100 g dry sample). Conclusion: Chiapas cocoa has significant competitive advantages due to its quality as a functional and nutraceutical food. It is a good raw material for food industry because it is a good source of antioxidants and natural additives. Furthermore, it is a potential ingredient in the international pharmaceutical and cosmetic industry.

Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The aim of this study is to investigate the effect of genetic variations and the expression of the reduced folate carrier (RFC) and dihydrofolate reductase (DHFR) on the drug sensitivity to methotrexate (MTX) in different cancer cell lines. MATERIALS AND METHODS: We examined the six human cancer cell lines (MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2). The cytotoxicity of MTX was measured by sulforhodamine B (SRB) assay. The expressions of the DHFR and RFC were evaluated by real-time PCR and western blotting. Four single nucleotide polymorphisms (SNPs) of the DHFR and two SNPs of the RFC were genotyped. RESULTS: The IC50s of MTX was in an extensively broad range from 6.05+/-0.81 nM to>1,000 nM in the cell lines. The Saos-2 (>1,000 nM) and MCF-7 (114.31+/-5.34 nM) cells were most resistant to MTX; in contrast, the AGS and HCT-116 cells were highly sensitive to MTX with an IC50 of 6.05+/-0.81 nM and 13.56+/-3.76 nM, respectively. A reciprocal change of the RFC and DHFR mRNA expression was found between the MTX-sensitive AGS and MTX-resistant Saos-2 cells. There was no significant difference in the expression levels of RFC protein in both the AGS and Saos-2 cells, whereas DHFR protein was more increased in the MTX-resistant Saos-2 cells treated with MTX. The genotype of the MTX-sensitive AGS cells were mutant variants of the DHFR; in contrast, the Saos-2 cells had the wild-type of the DHFR. CONCLUSION: In conclusion, this study showed that inverse change of the RFC and DHFR mRNA and protein expression was associated with RFC and DHFR polymorphisms and it is postulated that this phenomenon might play an important role in sensitivity of certain cancers to MTX.

Effects of Mitomycin C on Lens: in Vitro Study

PURPOSE: Cataract could occur as a complication of mitomycin C (MMC) used to increase the success rate of glaucoma surgery. Thus, the effects of MMC on cellular growth inhibition and apoptosis induction were examined by the culturing lens epithelial cells and the morphologic change in cells when MMC induced apoptosis. METHODS: Cellular morphology was observed after treating the mouse lens epithelial cell line, alpha-TN4, with MMC at different concentrations. Cellular toxicity was measured using LDH. Furthermore, IC50 inhibiting cellular proliferation and IC50 inducing apoptosis were measured. Staining and TUNEL assay were done to define apoptosis. The lens shape was observed under a transmission electron microscope, and electrophoresis was performed investigate the change in protein level when cataract was induced by MMC. RESULTS: The lens epithelial cells were atrophic even at low concentrations of MMC and more severe cellular changes along with reduced survival rate were seen at higher concentrations. According to the results of measuring cellular toxicity using LDH, the amount of LDH increased with increasing concentrations of MMC. The results of staining showed the findings of apoptosis of cells with orange-colored, compressed nuclei at low concentrations of MMC. Also on microscopic observation, the compression of chromosomes and fragmentation of nuclei were seen. Furthermore, the number of the high molecularr 53 KDa protein was increased among lens proteins of fertilized egg. CONCLUSIONS: These results demonstrated that MMC induced apoptosis of lens epithelial cells; the formation of cataract occurred in subcapsular area and the equator region; and denatuation of lens proteins and high molecular weight 53 KDa protein were increased.