ABSTRACT
Introduction. Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania ( Viannia ) species . Materials and methods. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. Results. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed K m and V max values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H 2 F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity ( Ki = 22.0 µM) than trimethoprim ( Ki = 33 µM) and pyrimethamine ( Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. Conclusion. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.
Introducción. La dihidrofolato reductasa (DHFR) se ha utilizado como blanco molecular en tratamientos antibacterianos, anticancerígenos y antipalúdicos. También, actúa como blanco molecular en Leishmania ; sin embargo, no existen reportes de la enzima bifuncional en especies de Leishmania ( Viannia ). Materiales y métodos. Se ha aislado y expresado en Escherichia coli el gen que codifica para la enzima bifuncional DHFR y la timidilato-sintasa (TS) de Leishmania braziliensis . La enzima recombinante se purificó y caracterizó, y se evaluó el efecto inhibitorio de algunos antifolatos, así como de cuatro alcaloides aporfínicos. Resultados. El gen se compone de aproximadamente 1.560 pb y codifica un péptido de 58 kDa que contiene los dominios DHFR y TS ligados en una sola cadena polipeptídica. La enzima recombinante DHFR-TS, utilizando el dihidrofolato (H2F) como sustrato, presentó valores de K m y V max de 55,35 ± 4,02 (media ± el error estándar de la media) y de 0,02 ± 5,34 x 10 -4 , respectivamente. La enzima rDHFR-TS de L. braziliensis presentó valores de Ki para los antifolatos en el rango de micras. El metotrexato fue el inhibidor más potente de la actividad enzimática ( Ki =22,0 mM) en comparación del trimetoprim ( Ki =33 mM) y la pirimetamina ( Ki =68 mM). Estos valores de Ki son significativamente más bajos en comparación con los obtenidos para los alcaloides aporfínicos. Conclusión. Los resultados muestran el efecto inhibitorio de los antifolatos sobre la actividad enzimática, lo cual indica que la rDHFR-TS de L. braziliensis podría ser un modelo para estudiar moléculas antiprotozoarias potenciales.
Subject(s)
Folic Acid Antagonists/pharmacology , Leishmania/enzymology , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , Tetrahydrofolate Dehydrogenase/chemistry , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/chemistryABSTRACT
Objective: To identify the differentially expressed proteins after activation of CCR5 by its ligand using two-dimensional electrophoresis. Methods: The HEK-293 cells stably expressing CCR5 were constructed. The global protein analysis in 293-CCR5 stables and mock cells was performed after treatment with RANTES. After analysis with the PDQuest image software, the differential spots were identified by MOLDI-TOF MS/MS. RT-PCR was performed to further analyze the changes at mRNA level. Results: Flow cytometry revealed that HEK-293 cells stably expressed CCR5. In this study, we analyzed the proteomic results to reveal the proteins which were significantly modulated by the activation of CCR5 with RANTES. Expression of dihydrofolate reductase was found in HEK-293 cells after RANTES treatment, which was confirmed by RT-PCR at the mRNA level. Conclusion: This proteomic study suggests that CCR5 activated by its ligand RANTES inhibits the expression of dihydrofolate reductase.
ABSTRACT
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.
Subject(s)
Humans , Blotting, Western , Cell Line , Folic Acid , Genetic Variation , Genotype , HCT116 Cells , Inhibitory Concentration 50 , Methotrexate , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain Reaction , Reduced Folate Carrier Protein , Rhodamines , RNA, Messenger , Tetrahydrofolate DehydrogenaseABSTRACT
Objective To develop a method for the determination of the dynamic parameters of interaction between methotrexate(MTX)enantiomer and dihydrofolate reductase(DHFR).Methods An affinity capillary electrophoresis(ACE)method was adopted.Using bare fused-silica capillary,the electrophoresis buffer was 50 mmoL/L sodium tetraborate with 0.2%Brij-35,pH 9.50.The temperature of separation was controlled at 25℃ and a voltage of 25 kV was applied.The separation of the reaction mixture was performed at a wavelength of 254 nm.The difference of peak areas about the product was used to calculate the inhibitory rate and IC50 values.Results We establish the detection method for the dynamic parameters of interaction between MTX enantioner and DHFR.The separation of the reaction mixture could be achieved within 30 min.The IC50 value of D-(-)-MTX and L-(+)-MTX were 3.17×10-7 and 2.48×10-8mol/L,respective.The IC50 value of the D-(-)-MTX was 31.67×10-8mol/L,the L-(+)-MTX was2.48×10-8mol/L.The IC50 value of the D-(-)-MTX was about 13 times higher than that of the L-(+)-MTX.Conclusions ACE is a rapid.simple and accurate method that can be used to monitor DHFRdynamic reaction.The IC50 values of MTX enantiomer were quite different.The result first indicated that reaction between MTX enantiomer and DHFR had three-dimensional selection.
ABSTRACT
Objectives To study the relationships between methylenetetrahydrofolate reductase(MTHFR) gene polymorphism and diabetic retinopathy (DR) in Chinese Han race. Methods With polymerase chain reaction and restriction fragment length polymorphism (PCR RFLP), MTHFR gene 677 T mutation (cytosine is replced by thymine in No.677 site) was detected in 85 health controls, 62 with DR and 117 without DR of type 2 diabetics comfrimed by ophthalmoscope. Results The frequency of MTHFR variant genotypes and alleles of DR in Chinese Han race.patients were signigicantly higher than those without retinopathy and healthy controls (P