Calcein accumulation as a fluorometric functional assay of the multidrug transporter

doi:10.1016/0005-2736(94)90190-2

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 1191, Issue 2, 11 May 1994, Pages 384-388

https://doi.org/10.1016/0005-2736(94)90190-2 Get rights and content

Abstract

Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein - Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493–21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.

References (20)

L. Homolya et al.
J. Biol. Chem.
(1993)
M.M. Gottesman et al.
Annu. Rev. Biochem.
(1993)
R.J. Arceci
Blood
(1993)
L.J. Goldstein et al.
Crit. Rev. Oncol./Haematol.
(1992)
H. Herweijer et al.
Cytometry
(1989)
A.A. Neyfakh
Exp. Cell Res.
(1988)
P.W. Wigler et al.
Biochim. Biophys. Acta
(1994)
S.V. Egudina et al.
FEBS Lett.
(1993)
D.M. Wall et al.
J. Natl. Cancer Inst.
(1991)
D.M. Wall et al.
Eur. J. Cancer
(1993)

There are more references available in the full text version of this article.

Cited by (298)

Overcoming multi drug resistance mediated by ABC transporters by a novel acetogenin- annonacin from Annona muricata L.
2024, Journal of Ethnopharmacology
Multi-Drug Resistance (MDR), mediated by P-glycoprotein (P-gp) is one of the barriers to successful chemotherapy in colon cancer patients. Annona muricata L. (A.muricata), commonly known as soursop/Graviola, is a medicinal plant that has been traditionally used in treating diverse diseases including cancer. Phytochemicals of A.muricata (Annonaceous Acetogenins-AGEs) have been well-reported for their anti-cancer effects on various cancers.
The study aimed to examine the effect of AGEs in reversing MDR in colorectal cancer cells.
Based on molecular docking and molecular dynamic simulation, the stability of annonacin upon P-gp was investigated. Further in vitro studies were carried in oxaliplatin-resistant human colon cancer cells (SW480^R) to study the biological effect of annonacin, in reversing drug resistance in these cells.
Molecular docking and simulation studies have indicated that annonacin stably interacted at the drug binding site of P-gp. In vitro analysis showed that annonacin was able to significantly reduce the expression of P-gp by 2.56 folds. It also induced apoptosis in the drug-resistant colon cancer cells. Moreover, the intracellular accumulation of P-gp substrate (calcein-AM) was observed to increase in resistant cells upon treatment with annonacin.
Our findings suggest that annonacin could inhibit the efflux of chemotherapeutic drugs mediated by P-gp and thereby help in reversing MDR in colon cancer cells. Further in vivo studies are required to decipher the underlying mechanism of annonacin in treating MDR cancers.
Fluorescence-based methods for studying activity and drug-drug interactions of hepatic solute carrier and ATP binding cassette proteins involved in ADME-Tox
2023, Biochemical Pharmacology
In humans, approximately 70% of drugs are eliminated through the liver. This process is governed by the concerted action of membrane transporters and metabolic enzymes. Transporters mediating hepatocellular uptake of drugs belong to the SLC (Solute carrier) superfamily of transporters. Drug efflux either toward the portal vein or into the bile is mainly mediated by active transporters of the ABC (ATP Binding Cassette) family. Alteration in the function and/or expression of liver transporters due to mutations, disease conditions, or co-administration of drugs or food components can result in altered pharmacokinetics. On the other hand, drugs or food components interacting with liver transporters may also interfere with liver function (e.g., bile acid homeostasis) and may even cause liver toxicity. Accordingly, certain transporters of the liver should be investigated already at an early stage of drug development. Most frequently radioactive probes are applied in these drug-transporter interaction tests. However, fluorescent probes are cost-effective and sensitive alternatives to radioligands, and are gaining wider application in drug-transporter interaction tests. In our review, we summarize our current understanding about hepatocyte ABC and SLC transporters affected by drug interactions. We provide an update of the available fluorescent and fluorogenic/activable probes applicable in in vitro or in vivo testing of these ABC and SLC transporters, including near-infrared transporter probes especially suitable for in vivo imaging. Furthermore, our review gives a comprehensive overview of the available fluorescence-based methods, not directly relying on the transport of the probe, suitable for the investigation of hepatic ABC or SLC-type drug transporters.
The WD repeat-containing protein 5 (WDR5) antagonist WDR5-0103 restores the efficacy of cytotoxic drugs in multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2
2022, Biomedicine and Pharmacotherapy
Citation Excerpt :
Knowing that WDR5–0103 reverses MDR mediated by ABCB1 (Fig. 2) and ABCG2 (Fig. 3), we next examined the effect of WDR5–0103 on the drug transport function of ABCB1 and ABCG2 by performing a short-term drug accumulation assay as described in Materials and methods. As expected, the intracellular accumulation of calcein, a known fluorescent substrate of ABCB1 [75], in ABCB1-overexpressing multidrug-resistant cell lines (Fig. 4A - C, left panels) was substantially lower than in the respective drug-sensitive parental cell lines (Fig. 4A-C, right panels). Similarly, the intracellular accumulation of PhA, a known fluorescent substrate of ABCG2 [62], in ABCG2-overexpressing multidrug-resistant cell lines (Fig. 4D - F, left panels) was substantially lower than in the respective drug-sensitive parental cell lines (Fig. 4D - F, right panels).
The development of multidrug resistance (MDR) is one of the major challenges in the treatment of cancer which is caused by the overexpression of the ATP-binding cassette (ABC) transporters ABCB1 (P-glycoprotein) and/or ABCG2 (BCRP/MXR/ABCP) in cancer cells. These transporters are capable of reducing the efficacy of cytotoxic drugs by actively effluxing them out of cancer cells. Since there is currently no approved treatment for patients with multidrug-resistant tumors, the drug repurposing approach provides an alternative route to identify agents to reverse MDR mediated by ABCB1 and/or ABCG2 in multidrug-resistant cancer cells. WDR5–0103 is a histone H3 lysine 4 (H3K4) methyltransferase inhibitor that disrupts the interaction between the WD repeat-containing protein 5 (WDR5) and mixed-lineage leukemia (MLL) protein. In this study, the effect of WDR5–0103 on MDR mediated by ABCB1 and ABCG2 was determined. We found that in a concentration-dependent manner, WDR5–0103 could sensitize ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to conventional cytotoxic drugs. Our results showed that WDR5–0103 reverses MDR and improves drug-induced apoptosis in multidrug-resistant cancer cells by inhibiting the drug-efflux function of ABCB1 and ABCG2, without altering the protein expression of ABCB1 or ABCG2. The potential sites of interactions of WDR5–0103 with the drug-binding pockets of ABCB1 and ABCG2 were predicted by molecular docking. In conclusion, the MDR reversal activity of WDR5–0103 demonstrated here indicates that it could be used in combination therapy to provide benefits to a subset of patients with tumor expressing high levels of ABCB1 or ABCG2.
Nanoplaform based on ultra-small Au regulating phototoxicity and fluorescence off–on function of Ag<inf>2</inf>S for multi-modal diagnosis and treatment of tumor
2022, Chemical Engineering Journal
Citation Excerpt :
Calcein and PI were used to label cell. Calcein could label living cell to produce green FL [44], while PI could not pass through the living cell membrane but the damaged cell membrane to stain the nucleus and produced red FL [45]. It showed that all cells were survived in the control, US and Laser groups (Fig. 5B), indicating that simple laser irradiation or US treatment had little effect on the cell; a small amount of cell died in probe group owing to the low toxicity of probe; a large number of cell died in the ASAuDP + US group after SDT treatment, and similar phenomenon was observed in the ASAuDP + Laser group after PTT treatment, and the death ratio of the latter was greater than that of the former, indicating that PTT had a better effect than SDT; however, almost all cells died in the ASAuDP + Laser + US group treated with PTT and SDT, indicating that the combined treatment had the most significant effect.
In this paper, a mixture of lecithin, polyethylene glycol monostearate, and polyethylene glycol phospholipid (DSPE-PEG₂₀₀₀) is used to wrap hydrophobic nano silver sulfide (Ag₂S) and 3 ∼ 5 nm ultra-small gold nanoparticle (mAu) to form ∼ 150 nm hydrophilic nanocomposite probe Ag₂S/Au@DSPE-PEG₂₀₀₀ (ASAuDP) with good stability and dispersion. The probe combines the functions of Ag₂S and mAu, while mAu can significantly inhibit the fluorescence (FL) of Ag₂S, thereby reducing the phototoxicity of probe when circulating in the body. When the ASAuDP probe is phagocytosed by tumor cell, Ag₂S and mAu are separated due to the rupture of probe, thereby restoring the ability of FL imaging and ROS production. MTT test, blood analysis and organ pathology test prove that ASAuDP is a safe probe. In vitro and in vivo imaging experiments prove that the ASAuDP probe presents good FL, CT and photoacoustic imaging capabilities. In vitro and in vivo treatments prove that the ASAuDP probe has both photothermal and sonodynamic therapy abilities, and the synergy between them has the best therapeutic effect. Therefore, ASAuDP probe is a kind of integrated diagnostic and therapeutic probe with good safety, and it has a good application prospect in the treatment of tumor.
Design and Synthesis of Cyclolipopeptide Mimics of Dysoxylactam A and Evaluation of the Reversing Potencies against P-Glycoprotein-Mediated Multidrug Resistance
2024, Journal of Medicinal Chemistry
Epidermal Growth Factor Receptor Inhibitor Mobocertinib Resensitizes Multidrug-Resistant Cancer Cells by Attenuating the Human ATP-Binding Cassette Subfamily B Member 1 and Subfamily G Member 2
2024, ACS Pharmacology and Translational Science

View all citing articles on Scopus

View full text

Rapid reportCalcein accumulation as a fluorometric functional assay of the multidrug transporter

Abstract

J. Biol. Chem.

Annu. Rev. Biochem.

Blood

Crit. Rev. Oncol./Haematol.

Cytometry

Exp. Cell Res.

Biochim. Biophys. Acta

FEBS Lett.

J. Natl. Cancer Inst.

Eur. J. Cancer

Rapid report
Calcein accumulation as a fluorometric functional assay of the multidrug transporter