Dataset Information

Screening Multidrug Resistance Reversal Agents in Traditional Chinese Medicines by Efflux Kinetics of D-Luciferin in MCF-7/DOX^Fluc Cells.

ABSTRACT: In this study, we established a simple and rapid in vitro method for screening multidrug resistance (MDR) reversal agents in traditional Chinese medicines (TCMs), which could better correspond to the MDR reversing effect in vivo. Here, D-luciferin, a substrate for the enzyme firefly luciferase and also a substrate for ATP-binding cassette transporters (ABC transporters), was used as the probe to detect its efflux kinetics caused by ABC transporters. First, we established a stable doxorubicin (DOX)-resistant cell line (MCF-7/DOX^Fluc) that overexpressed luciferase. Then, some kinds of TCMs were chosen for the MDR reversal agents to measure its effect on inhibiting the D-luciferin outflow from MCF-7/DOX^Fluc, and the ideal reversal agent with the least D-luciferin efflux from MCF-7/DOX^Fluc was selected to further investigate its effect combined with DOX on MCF-7/DOX^Fluc tumor-bearing mice. The results indicated that quercetin (Qu) could remarkably increase the retention of D-luciferin in MCF-7/DOX^Fluc in vitro and in vivo. Also, the combination of Qu and DOX could exceedingly inhibit the tumor growth, which proved the feasibility of this in vitro screening method. The study proposed a feasible method for mass screening of MDR agents from TCMs in vitro.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC9909823 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Screening Multidrug Resistance Reversal Agents in Traditional Chinese Medicines by Efflux Kinetics of D-Luciferin in MCF-7/DOXFluc Cells.

Zhao Yue Y Tang Chaoyuan C Huang Jingyi J Zhang Hongyan H Shi Jingbin J Xu Shujun S Ma Lisha L Peng Chun C Liu Qi Q Xiong Yang Y

ACS omega 20230123 5

In this study, we established a simple and rapid in vitro method for screening multidrug resistance (MDR) reversal agents in traditional Chinese medicines (TCMs), which could better correspond to the MDR reversing effect in vivo. Here, D-luciferin, a substrate for the enzyme firefly luciferase and also a substrate for ATP-binding cassette transporters (ABC transporters), was used as the probe to detect its efflux kinetics caused by ABC transporters. First, we established a stable d ...[more]

PMID: 36777569

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Project description:UnlabelledThe AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.ImportanceThe AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

Project description:PurposeP-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.MethodsThe in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions.Results3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.ConclusionWe demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

Dataset Information

Screening Multidrug Resistance Reversal Agents in Traditional Chinese Medicines by Efflux Kinetics of D-Luciferin in MCF-7/DOX^Fluc Cells.

Publications

Screening Multidrug Resistance Reversal Agents in Traditional Chinese Medicines by Efflux Kinetics of D-Luciferin in MCF-7/DOX<sup>Fluc</sup> Cells.

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