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Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function S

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Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function S Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2019/05/02/dmd.118.086181.DC1 1521-009X/47/7/715–723$35.00 https://doi.org/10.1124/dmd.118.086181 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 47:715–723, July 2019 U.S. Government work not protected by U.S. copyright Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function s Andrea N. Robinson, Bethelihem G. Tebase, Sonia C. Francone, Lyn M. Huff, Hanna Kozlowski, Dominique Cossari, Jung-Min Lee, Dominic Esposito, Robert W. Robey, and Michael M. Gottesman Laboratory of Cell Biology (A.N.R., B.G.T., S.C.F., L.M.H., H.K., D.C., R.W.R., M.M.G.) and Women’s Malignancies Branch (J.-M.L.), National Institutes of Health, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland; and Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland (D.E.) Received December 27, 2018; accepted April 23, 2019 ABSTRACT Downloaded from Although overexpression of multiple ATP-binding cassette trans- well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor porters has been reported in clinical samples, few studies have prexasertib, both of which were found to be substrates of both examined how coexpression of multiple transporters affected re- ABCB1 and ABCG2. When B1/G2 cells were incubated with both sistance to chemotherapeutic drugs. We therefore examined how rhodamine 123 and pheophorbide a, transport of both compounds coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to was observed, suggesting that ABCB1 and ABCG2, when coex- drug resistance in a cell line model. HEK293 cells were transfected pressed, can function independently to transport substrates. ABCB1 dmd.aspetjournals.org with vector-encoding full-length ABCB1, ABCG2, or a bicistronic and ABCG2 also functioned additively to transport the common vector containing both genes, each under the control of a separate fluorescent substrates mitoxantrone and BODIPY-prazosin, as it promoter. Cells transfected with both transporters (B1/G2 cells) was necessary to inhibit both transporters to prevent efflux from demonstrated high levels of both transporters, and uptake of both B1/G2 cells. ABCG2 expression was also found to decrease the the ABCB1-specific substrate rhodamine 123 and the ABCG2- efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, specific substrate pheophorbide a was reduced when examined ABCB1 and ABCG2 can independently and additively confer re- by flow cytometry. B1/G2 cells were also cross-resistant to the sistance to substrates, underscoring the need to inhibit multiple at ASPET Journals on September 26, 2021 ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as transporters when they are coexpressed. Introduction levels after oral administration of the drug compared with control Overexpression of ABCB1 (P-glycoprotein, encoded by the mice (Zaher et al., 2006). ABCB1 gene) or ABCG2 is known to confer resistance to multiple It has been demonstrated that both ABCB1 and ABCG2 can limit chemotherapeutic drugs, including many targeted therapies un- the oral bioavailability of common substrates. Upon oral adminis- der clinical development (Robey et al., 2018). Additionally, these tration of the mutant BRAF inhibitor vemurafenib, plasma area under transporters are known to limit oral bioavailability, as both are the curve values were 1.6-fold higher in Abcb1a/1b-deficient mice expressed in the gastrointestinal tract (Thiebaut et al., 1987; and 2.3-fold higher in Abcg2-deficient mice, but were 6.6-fold higher Maliepaard et al., 2001; Fetsch et al., 2006). In knockout models, in mice deficient in both transporters compared with wild-type mice deficient in both Abcb1a and Abcb1b (the murine homologs of controls (Durmus et al., 2012). Similarly, plasma areas under the human ABCB1, previously known as mdr1a and mdr1b)demon- curve of the epidermal growth factor receptor/human epidermal strate increased oral bioavailability of ABCB1 substrates such as growth factor receptor 2 inhibitor afatinib were 4.2-fold, 2.4-fold, taxol compared with wild-type mice (Sparreboom et al., 1997). and 7-fold higher in Abcg2, Abcb1a/1b, and double-knockout mice, Similar results have been found with the ABCG2 substrate respectively, compared with wild-type mice (van Hoppe et al., 2017), sulfasalazine, as mice deficient in Abcg2 had higher plasma drug thus suggesting that both transporters contribute to decreased oral bioavailability of substrates. Additionally, ABCB1 and ABCG2 are coexpressed in the brain capillaries that form the blood-brain barrier (BBB) (Cooray et al., 2002; This work was supported in part by the Intramural Research Program of the Fetsch et al., 2006) and serve to keep toxins and some chemotherapeutic National Institutes of Health, the National Cancer Institute. agents out of the brain (Robey et al., 2010). Mouse knockout models The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade point to a compensatory and cooperative role for ABCB1 and ABCG2 at names, commercial products, or organizations imply endorsement by the U.S. the BBB. Brain concentrations of the PARP inhibitor rucaparib were Government. increased by 2-fold, 5.2-fold, and 32.6-fold in mice deficient in Abcg2, https://doi.org/10.1124/dmd.118.086181. Abcb1a/1b, or both transporters, respectively, compared with wild-type s This article has supplemental material available at dmd.aspetjournals.org. controls, suggesting a cooperative role for the two transporters at the BBB ABBREVIATIONS: BBB, blood-brain barrier; CMV, cytomegalovirus; FTC, fumitremorgin; MEF, mouse embryonic fibroblast; PhA, pheophorbide a; VAL, valspodar. 715 716 Robinson et al. (Durmus et al., 2015b). ABCB1 and ABCG2 were found to cooperatively transporters may therefore confer greater resistance to chemotherapy exclude the Janus kinase 1/2 inhibitor momelotinib from the brain, as than expression of a single transporter. 8 hours after oral administration of the drug, mice deficient in Abcg2, Despite evidence suggesting a cooperative and potentially com- Abcb1a/b, or Abcg2;Abcb1a/b were found to have 6.5-fold, 3-fold, or pensatory role for ABCB1 and ABCG2, few studies have addressed 48-fold higher brain levels compared with controls (Durmus et al., how these transporters might be working together to render chemo- 2013). This apparent synergism from deleting both of the murine therapy less effective. We thus generated HEK293 cell lines that homologs for ABCB1 and ABCG2 results from the fact that transport express both transporters and find that the transporters function both due to ABCB1 and ABCG2 is much higher than passive diffusion of independently and additively to transport substrates. the drugs across the BBB (Kusuhara and Sugiyama, 2009; Kodaira et al., 2010). Coadministration of the dual ABCB1/ABCG2 inhibitor Materials and Methods elacridar resulted in significantly increased brain levels of the kinase inhibitors tandutinib (Yang et al., 2010), pazopanib (Minocha et al., Chemicals. Doxorubicin, mitoxantrone, paclitaxel, etoposide, and rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO). SN-38 and 2012), and sunitinib (Tang et al., 2010). ABCB1 and ABCG2 are thus topotecan were from LKT Laboratories (St. Paul, MN). Valspodar (VAL) major obstacles to overcome when treating brain cancers or metas- was obtained from Apex Biotechnology (Houston, TX). Pheophorbide a tases to the brain. (PhA) was purchased from Frontier Scientific (Logan, UT). Zeocin was ABCB1 and ABCG2 are found to be coexpressed in some cancers, from InvivoGen (San Diego, CA), and prexasertib from Selleck Chemicals particularly leukemia. Wilson et al. (2006) obtained gene expression (Houston, TX). BODIPY-prazosin was obtained from Life Technologies Downloaded from profiles of 170 pretreated samples of acute myelogenous leukemia (Eugene, OR). Tariquidar was a gift of Xenova Group (Slough, UK). by microarray analysis. Using unsupervised clustering, the patients Fumitremorgin (FTC) was synthesized by the National Institutes of Health Chemical Biology Laboratory (Bethesda, MD). clustered into six groups; the cluster characterized by the highest Cell Lines. Human embryonic kidney cells (HEK293) were obtained from levels of resistant disease showed increased expression of ABCB1 American Type Culture Collection (Manassas, VA) and cultured in Eagle’s and ABCG2 (Wilson et al., 2006). Profiling 380 drug-resistance– minimum essential medium (Gibco/Life Technologies, Carlsbad, CA) with related genes in a set of 11 paired samples obtained at diagnosis and 10% FBS, 1% glutamine, and 1% penicillin. Cells were transfected with empty dmd.aspetjournals.org again at relapse identified two patients with increases in both ABCB1 vector with zeocin resistance alone, vectors encoding full-length human and ABCG2 at relapse (Patel et al., 2013). Liu et al. (2018) examined ABCB1 or ABCG2, or a bicistronic vector in which each gene is under the control of a separate cytomegalovirus (CMV) promoter. ABCB1 and ABCG2 expression of ABCB1, ABCB4, ABCC1, ABCC4, and ABCG2 in expression constructs were generated by Gateway cloning using human cDNA bone marrow mononuclear cells from 96 de novo acute myelogenous templates. Both genes were cloned with Kozak translational initiation sites leukemia patients and found that coexpression of multiple trans- prior
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