Evaluation of Drug Transport in MDCKII-Wild Type, MDCKII-MDR1, MDCKII-BCRP and Caco-2 Cell Lines

Mukkavilli, Rao; Jadhav, Gajanan Raosaheb; Vangala, Subrahmanyam

doi:10.2174/1389201019666180308091855

Cited by 10 publications

(13 citation statements)

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“…The human breast cancer cell lines MCF-7 and MDA-MB-231, and Madin-Darby Canine Kidney (MDCKII) cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). MDCKII cells are an epithelial cell line of canine kidney origin that are frequently used to investigate drug transporters in vitro , as they exhibit low expression levels of transporter proteins and low metabolic activity (32). MCF-7 and MDCKII cells were cultured in complete DMEM (Thermo Fisher Scientific Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells

Wen

Huang

et al. 2019

Mol Med Report

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Doxorubicin is one of the most widely used chemotherapy agents for the treatment of breast cancer. However, the development of doxorubicin resistance limits the long-term treatment benefits in patients with breast cancer. Curcumin, a well-known dietary polyphenol derived from the rhizomes of turmeric ( Curcuma longa ), enhances the sensitivity of breast cancer cells to chemotherapeutic agents; however, the mechanisms underlying this phenomenon remain unclear. The aim of the present study was to evaluate the effect of curcumin on chemoresistance in doxorubicin-resistant breast cancerMCF-7/DOX and MDA-MB-231/DOX cell lines. Cell Counting Kit-8, monolayer transport, western blot and ATPase activity assays were performed during the study. The results revealed that curcumin significantly enhanced the effect of doxorubicin in doxorubicin-resistant breast cancer cells. The intracellular accumulation of doxorubicin was substantially increased following curcumin treatment in doxorubicin-resistant breast cancer cells, in a manner that was inversely dependent on the activity of ATP binding cassette subfamily B member 4 (ABCB4). Treatment with a combination of curcumin and doxorubicin decreases the efflux of doxorubicin in ABCB4-overexpressing cells. Furthermore, curcumin inhibited the ATPase activity of ABCB4 without altering its protein expression. In conclusion, curcumin reversed doxorubicin resistance in human breast cancer MCF-7/DOX and MDA-MB-231/DOX cells by inhibiting the ATPase activity of ABCB4. The study highlights the promising use of curcumin as a chemosensitizer in the treatment of breast cancer.

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Section: Methodsmentioning

confidence: 99%

Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells

Wen

Huang

et al. 2019

Mol Med Report

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“…The solution recovery of 23 in the A – B direction of all assays was lower than 75%, which may be due to cellular retention, metabolism, adsorption, or other issues . After transport assays, Lucifer yellow leakage assays were used to examine the integrity of the cell monolayer; in all wells, the recovery of Lucifer yellow in the basolateral side was less than 1%, indicating that cell monolayers were not damaged …”

Section: Resultsmentioning

confidence: 99%

“…61 After transport assays, Lucifer yellow leakage assays were used to examine the integrity of the cell monolayer; in all wells, the recovery of Lucifer yellow in the basolateral side was less than 1%, indicating that cell monolayers were not damaged. 62 We then measured unbound fractions of 23 in mouse blood plasma and brain homogenate in vitro by equilibrium dialysis (for details, please see the Experimental Section). The fractions unbound of 23 in the brain (f u,brain ) and plasma (f u,plasma ) were 0.25 and 0.73% (Table 3), respectively, indicating that 23 has high plasma protein and brain tissue binding.…”

Section: Journal Of Medicinal Chemistrymentioning

confidence: 99%

X-ray Structure-Guided Discovery of a Potent, Orally Bioavailable, Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/hTDO) Inhibitor That Shows Activity in a Mouse Model of Parkinson’s Disease

Ning

Huo

et al. 2021

J. Med. Chem.

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Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan 2,3-dioxygenase (hTDO) have been closely linked to the pathogenesis of Parkinson's disease (PD); nevertheless, development of dual hIDO1 and hTDO inhibitors to evaluate their potential efficacy against PD is still lacking. Here, we report biochemical, biophysical, and computational analyses revealing that 1H-indazole-4-amines inhibit both hIDO1 and hTDO by a mechanism involving direct coordination with the heme ferrous and ferric states. Crystal structure-guided optimization led to 23, which manifested IC 50 values of 0.64 and 0.04 μM to hIDO1 and hTDO, respectively, and had good pharmacokinetic properties and brain penetration in mice. 23 showed efficacy against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse motor coordination deficits, comparable to Madopar, an anti-PD medicine. Further studies revealed that different from Madopar, 23 likely has specific anti-PD mechanisms involving lowering IDO1 expression, alleviating dopaminergic neurodegeneration, reducing inflammatory cytokines and quinolinic acid in mouse brain, and increasing kynurenic acid in mouse blood.

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“…To reliably characterize the transport parameters of a transporter protein in whole cells, the right cellular model is crucial. Therefore, for our study we selected MDCKII, a polarized epithelial cell line widely used as a model in transporter research (Mukkavilli et al, 2017;Szilagyi et al, 2019). While sublines of these cells overexpressing human ABCG2 have been available for some time, we took advantage of the recent commercial availability of a unique variant (huMDCKII) in which the endogenous canine multidrug transporters (cfABCB1 and cfABCG2) were knocked out by genetic engineering, thus removing an important confounding factor in quantitative analysis.…”

Section: Discussionmentioning

confidence: 99%

“…When values of enzyme kinetic constants are required for ABC proteins, three approaches are predominant in the literature. The first one involves measuring equilibrium concentrations of a substrate on the apical and basolateral sides of a cellular monolayer (Mukkavilli et al, 2017). This assay depends on membrane domain-specific expression of the protein of interest in polarized cell lines.…”

Section: Introductionmentioning

confidence: 99%

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate

Różański

Studzian

Pułaski

2019

J Pharmacol Exp Ther

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Flavonoids are an important part of the human diet since plantderived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembranepermeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney IIderived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 mM for quercetin, 36 mM for kaempferol, and 348 mM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/ distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

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Evaluation of Drug Transport in MDCKII-Wild Type, MDCKII-MDR1, MDCKII-BCRP and Caco-2 Cell Lines

Abstract: The very high efflux ratios of MDR1 and BCRP substrates in transfected MDCKII cells clearly demonstrate the potential usefulness of these models to provide more definitive data to evaluate the transporter involvement compared to Caco-2 or MDCKII-WT cells.

Cited by 10 publications

References 0 publications

Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells

Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells

X-ray Structure-Guided Discovery of a Potent, Orally Bioavailable, Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/hTDO) Inhibitor That Shows Activity in a Mouse Model of Parkinson’s Disease

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate

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