Metformin sensitises hepatocarcinoma cells to methotrexate by targeting dihydrofolate reductase.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC8487431 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1??M. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.
| S-EPMC4028078 | biostudies-literature
Project description:Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line. Uptake experiments with [3H]MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line. Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line. To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells. Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells. Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme. Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX. These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects.
| S-EPMC6148270 | biostudies-literature
Project description:Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.
| S-EPMC2820606 | biostudies-literature
Project description:The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.
| S-EPMC129699 | biostudies-literature
Project description:Hydrogen atoms play a central role in many biochemical processes yet are difficult to visualize by x-ray crystallography. Spallation neutron sources provide a new arena for protein crystallography with TOF measurements enhancing data collection efficiency and allowing hydrogen atoms to be located in smaller crystals of larger biological macromolecules. Here we report a 2.2-A resolution neutron structure of Escherichia coli dihydrofolate reductase (DHFR) in complex with methotrexate (MTX). Neutron data were collected on a 0.3-mm(3) D(2)O-soaked crystal at the Los Alamos Neutron Scattering Center. This study provides an example of using spallation neutrons to study protein dynamics, to identify protonation states directly from nuclear density maps, and to analyze solvent structure. Our structure reveals that the occluded loop conformation [monomer (mon.) A] of the DHFR.MTX complex undergoes greater H/D exchange compared with the closed-loop conformer (mon. B), partly because the Met-20 and beta(F-G) loops readily exchange in mon. A. The eight-stranded beta sheet of both DHFR molecules resists H/D exchange more than the helices and loops. However, the C-terminal strand, betaH, in mon. A is almost fully exchanged. Several D(2)Os form hydrogen bonds with exchanged amides. At the active site, the N1 atom of MTX is protonated and thus charged when bound to DHFR. Several D(2)Os are observed at hydrophobic surfaces, including two pockets near the MTX-binding site. A previously unidentified D(2)O hydrogen bonds with the catalytic D27 in mon. B, stabilizing its negative charge.
| S-EPMC1664550 | biostudies-literature
Project description:Interaction of curcumin (CUR) with the enzyme dihydrofolate reductase (DHFR) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with DHFR were compared to those of methotrexate (MTX), a known inhibitor of the enzyme. The calculated free energy of binding (ΔG binding) shows that curcumin (ΔG = -9.02 kcal/mol; Ki = 243 nM) binds with affinity comparable to or better than MTX (ΔG = -8.78 kcal/mol; Ki = 363 nM). Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in a bent conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interaction with key active site residues are also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of DHFR, which is a potential target of anti-cancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies.
| S-EPMC5450246 | biostudies-literature
Project description: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 IC??s 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 IC(50) 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.
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Project description:Dihydrofolate reductase (DHFR) cDNA sequences were isolated from a methotrexate-resistant mouse L5178Y cell line previously shown to contain methotrexate-resistant dihydrofolate reductase enzyme activity. Specifically-primed reverse transcription products were amplified using the polymerase chain reaction and then cloned into a mammalian expression plasmid. Candidate clones were identified by restriction analysis and then functionally tested by transfection into mouse 3T3 fibroblasts, selecting for methotrexate-resistant colonies. Sequence analysis of the cDNA clones demonstrated the substitution of tryptophan (TGG) in place of the wild-type phenylalanine (TTC) at codon 31. Sequencing of PCR-amplified genomic DNA extracted from the drug-resistant L5178Y cells confirmed the tryptophan codon at position 31. Transfection of mammalian tissue culture cells with expression plasmids containing the trp31 DHFR sequence resulted in substantial methotrexate-resistant colony formation. Recombinant trp31 DHFR enzyme activity expressed in stably-transfected Chinese hamster ovary cells was approximately 20-fold less sensitive to methotrexate inhibition than wild-type mouse DHFR enzyme activity. We conclude that the cloned Trp31 DHFR sequence encodes an enzyme substantially resistant to methotrexate which confers a drug-resistance phenotype to cells in which it is expressed.
| S-EPMC332765 | biostudies-other
Project description:We report the first peptide based hDHFR inhibitors designed on the basis of structural analysis of dihydrofolate reductase (DHFR). A set of peptides were rationally designed and synthesized using solid phase peptide synthesis and characterized using nuclear magnetic resonance and enzyme immunoassays. The best candidate among them, a tetrapeptide, was chosen based on molecular mechanics calculations and evaluated in human lung adenocarcinoma cell line A549. It showed a significant reduction of cell proliferation and an IC50 of 82?µM was obtained. The interaction of the peptide with DHFR was supported by isothermal calorimetric experiments revealing a dissociation constant Kd of 0.7?µM and ?G of -34?±?1?kJ?mol-1. Conjugation with carboxylated polystyrene nanoparticles improved further its growth inhibitory effects. Taken together, this opens up new avenues to design, develop and deliver biocompatible peptide based anti-cancer agents.
| S-EPMC5816602 | biostudies-literature
Project description:Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ?35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ?30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-?,?-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ?9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.
| S-EPMC5147970 | biostudies-literature