Next Article in Journal
Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo
Next Article in Special Issue
Mambalgin-2 Induces Cell Cycle Arrest and Apoptosis in Glioma Cells via Interaction with ASIC1a
Previous Article in Journal
Association between Abdominal Obesity and Incident Colorectal Cancer: A Nationwide Cohort Study in Korea
Previous Article in Special Issue
Chemotherapeutic Agents Sensitize Resistant Cancer Cells to the DR5-Specific Variant DR5-B More Efficiently Than to TRAIL by Modulating the Surface Expression of Death and Decoy Receptors
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cancers
Volume 12
Issue 6
10.3390/cancers12061366
cancers-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Erdafitinib Resensitizes ABCB1-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs

by
Chung-Pu Wu
1,2,3,
Tai-Ho Hung
3,4,
Sung-Han Hsiao
1,
Yang-Hui Huang
1,2,
Lang-Cheng Hung
5,
Yi-Jou Yu
1,
Yu-Tzu Chang
1,2,
Shun-Ping Wang
6,7 and
Yu-Shan Wu
5,*
1
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan
2
Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan
3
Department of Obstetrics and Gynecology, Taipei Chang Gung Memorial Hospital, Taipei 10507, Taiwan
4
Department of Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan
5
Department of Chemistry, Tunghai University, Taichung 40704, Taiwan
6
Department of Orthopedics, Taichung Veterans General Hospital, Taichung 40705, Taiwan
7
Sports Recreation and Health Management Continuing Studies-Bachelor’s Degree Completion Program, Tunghai University, Taichung 40704, Taiwan
*
Author to whom correspondence should be addressed.
Cancers 2020, 12(6), 1366; https://doi.org/10.3390/cancers12061366
Submission received: 11 May 2020 / Revised: 22 May 2020 / Accepted: 22 May 2020 / Published: 26 May 2020
(This article belongs to the Collection Drug Resistance and Novel Therapies in Cancers)
Browse Figures
Versions Notes

Abstract

:
The development of multidrug resistance (MDR) in cancer patients, which is often associated with the overexpression of ABCB1 (MDR1, P-glycoprotein) in cancer cells, remains a significant problem in cancer chemotherapy. ABCB1 is one of the major adenosine triphosphate (ATP)-binding cassette (ABC) transporters that can actively efflux a range of anticancer drugs out of cancer cells, causing MDR. Given the lack of Food and Drug Administration (FDA)-approved treatment for multidrug-resistant cancers, we explored the prospect of repurposing erdafitinib, the first fibroblast growth factor receptor (FGFR) kinase inhibitor approved by the FDA, to reverse MDR mediated by ABCB1. We discovered that by reducing the function of ABCB1, erdafitinib significantly resensitized ABCB1-overexpressing multidrug-resistant cancer cells to therapeutic drugs at sub-toxic concentrations. Results of erdafitinib-stimulated ABCB1 ATPase activity and in silico docking analysis of erdafitinib binding to the substrate-binding pocket of ABCB1 further support the interaction between erdafitinib and ABCB1. Moreover, our data suggest that ABCB1 is not a major mechanism of resistance to erdafitinib in cancer cells. In conclusion, we revealed an additional action of erdafitinib as a potential treatment option for multidrug-resistant cancers, which should be evaluated in future drug combination trials.

1. Introduction

Several members of the human adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily are linked to cancer multidrug resistance (MDR), which is one of the most difficult challenges in cancer chemotherapy [1,2]. ABCB1 (MDR1; P-glycoprotein) and ABCG2 (BCRP; MXR) are two of the most studied and well-characterized ABC drug transporters, capable of using chemical energy derived from ATP hydrolysis to transport cytotoxic anticancer drugs such as anthracyclines, paclitaxel, SN-38, topotecan, and Vinca alkaloids out of cancer cells. Consequently, the decreased intracellular drug accumulation in cancer cells leads to reduced drug sensitivity, treatment failure, and relapse in cancer patients [2,3,4]. Notably, many studies have reported that high expression of ABCB1 and/or ABCG2 is associated with MDR [1,2] and poor clinical outcome in patients with metastatic breast cancer (MBC) [5], blood cancers such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) [6,7,8,9], and multiple myeloma (MM) [10,11,12]. Therefore, it is of great clinical significance to develop therapeutic agents against the activity of ABCB1 and/or ABCG2.
Despite a large number of different classes of novel inhibitors of ABCB1 and/or ABCG2 that have been developed over the years, there are currently no U.S. Food and Drug Administration (FDA)-approved therapeutic agents that can be used to deal with MDR in patients, mostly due to the lack of selectivity and adverse drug–drug interactions [2,13,14,15,16]. Therefore, instead of synthesizing novel modulators, an alternative approach is to explore the possibility of repurposing FDA-approved tyrosine kinase inhibitors (TKIs) to resensitize ABCB1- or ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic agents [17,18]. Erdafitinib is a small-molecule pan-fibroblast growth factor receptor (FGFR) kinase inhibitor that binds and inhibits the activity of FGFR1–4 and the proliferation of cancer cells [19]. It is the first FGFR kinase inhibitor approved by the FDA for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma (mUC), based on data from a trial of erdafitinib in participants with urothelial cancer (ClinicalTrials.gov Identifier: NCT02365597). In addition, erdafitinib is currently being evaluated as monotherapy or combination therapy in clinical trials for patients with advanced solid tumors with FGFR alterations (NCT04083976), metastatic or locally advanced urothelial cancer with selected FGFR gene alterations (NCT03473743), relapsed or refractory advanced solid tumors, non-Hodgkin lymphoma, or histiocytic disorders with FGFR mutations (NCT03210714), double negative prostate cancer (NCT03999515), non-small-cell lung cancer (NSCLC), urothelial cancer, esophageal cancer, and cholangiocarcinoma (NCT02699606), ER+/HER2-/FGFR-amplified metastatic breast cancer (NCT03238196), and advanced hepatocellular carcinoma (HCC) (NCT02421185).
In the present work, the potential chemosensitization effect of erdafitinib on multidrug-resistant cancer cells was assessed in human cell lines overexpressing ABCB1 or ABCG2. Our data indicate that erdafitinib is equally effective in treating ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells as drug-sensitive cancer cells. More importantly, we discovered an additional mode of action of erdafitinib on modulating the transport function of ABCB1 and resensitizing ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic drugs.

2. Results

2.1. Cells Overexpressing ABCB1 or ABCG2 Are Not Resistant to Erdafitinib

Previous reports have demonstrated that ABCB1 and ABCG2 are capable of effluxing small molecule inhibitors of receptor tyrosine kinases (RTKs) such as sunitinib [20,21] and imatinib [22,23,24,25] out of cancer cells. Consequently, ABCB1- and ABCG2-overexpressing cancer cells are less susceptible to treatment with these RTKs inhibitors [26,27,28]. To this end, the cytotoxicity of erdafitinib was determined in drug-sensitive human cancer cells and the ABCB1- or ABCG2-overexpressing multidrug-resistant variants as well as in HEK293 cells and HEK293 cells stably transfected with human ABCB1 or ABCG2. As shown in Table 1, the ABCB1-overexpressing multidrug-resistant KB-V-1 human epidermal cancer cells, NCI-ADR-RES human ovarian cancer cells, ABCB1-transfected MDR19-HEK293 cells, and the corresponding drug-sensitive parental KB-3-1, OVCAR-8, and pcDNA-HEK293 cells are equally sensitive to erdafitinib treatment. Similarly, the ABCG2-overexpressing multidrug-resistant H460-MX20 human lung cancer cells, S1-M1-80 human colon cancer cells, ABCG2-transfected R482-HEK293 cells, and the corresponding drug-sensitive parental H460, S1 and pcDNA-HEK293 cells are also equally sensitive to erdafitinib treatment.

2.2. Erdafitinib Reverses Multidrug Resistance Mediated by ABCB1

Several receptor tyrosine kinase inhibitors (RTKIs) have been reported to interact strongly with ABCB1 and/or ABCG2 and reverse multidrug resistance in cancer cells overexpressing ABCB1 and/or ABCG2 [17,18,29]. Therefore, the effect of erdafitinib on multidrug resistance mediated by ABCB1 and ABCG2 was determined. First, without significantly affecting the drug-sensitive parental cells, the chemosensitivity of ABCB1-overexpressing multidrug-resistant KB-V-1 (Figure 1a), NCI-ADR-RES (Figure 1b), and ABCB1-transfected MDR19-HEK293 cells (Figure 1c) to paclitaxel, a known substrate for ABCB1 [30], was restored by erdafitinib in a concentration-dependent manner. Similarly, erdafitinib reversed ABCB1-mediated resistance to vincristine, another well-known substrate of ABCB1 [30], in these multidrug-resistant cell lines (Table 2). In contrast, erdafitinib had no significant effect on ABCG2-mediated resistance to topotecan or SN-38, known substrates for ABCG2 [31,32], in ABCG2-overexpressing multidrug-resistant H460-MX20, S1-M1-80, and ABCG2-transfected R482-HEK293 cells (Table 3). Of note, verapamil and Ko143 were used as reference inhibitors for ABCB1 and ABCG2, respectively. The extent of chemosensitization by erdafitinib or verapamil or Ko143 in a particular cell line is represented by the fold-reversal (FR) value, calculated by dividing the IC50 value of a particular drug substrate by the IC50 value of the drug substrate in the presence of erdafitinib or verapamil or Ko143, as described previously [33]. Our results revealed that at sub-toxic concentrations, erdafitinib selectively reverses multidrug resistance mediated by ABCB1 in multidrug-resistant human cancer cell lines.

2.3. Erdafitinib Restores the Intracellular Drug Accumulation in ABCB1-Overexpressing Cells

Direct inhibition of the drug efflux function and transient downregulation of drug transporters [34,35,36] are two of the most common ways for a modulator to resensitize multidrug-resistant cancer cells to chemotherapeutic drugs. To this end, the effect of erdafitinib on ABCB1-mediated efflux of calcein, a fluorescent product of an ABCB1 substrate calcein-AM [37], and ABCG2-mediated efflux of Pheophorbide A (PhA), a fluorescent substrate of ABCG2 [38], was determined in cells overexpressing ABCB1 or ABCG2. Erdafitinib at 1 μM significantly increased the intracellular accumulation of calcein in MDR19-HEK293 cells (Figure 2a) and NCI-ADR-RES cells (Figure 2b). In contrast, erdafitinib had minimal effect on the intracellular accumulation of PhA in R482-HEK293 cells (Figure 2c) and S1-M1-80 cells (Figure 2d). Of note, tariquidar at 3 μM was used as a reference inhibitor of ABCB1, whereas Ko143 at 1 μM was used as a reference inhibitor of ABCG2. Erdafitinib, tariquidar, and Ko143 had no significant effect on the intracellular drug accumulation in parental cell lines (Figure 2a–d, right panels). Moreover, erdafitinib inhibited ABCB1-mediated calcein efflux in a concentration-dependent manner in MDR19-HEK293 cells, NCI-ADR-RES, and KB-V-1 cells (Figure 2e) with a calculated IC50 value of approximately 1.8 μM, 3.6 μM, and 9.3 μM, respectively. Next, the potential effect of erdafitinib on the protein expression of ABCB1 was examined in ABCB1-overexpressing NCI-ADR-RES and KB-V-1 cancer cells. Cells were treated with increasing concentrations of erdafitinib (0.1–1.0 μM) for 72 h followed by immunoblotting, as described in the Materials and Methods. As shown in Figure 3a,b, erdafitinib had no significant effect on the protein expression of ABCB1 in either NCI-ADR-RES or KB-V-1 cancer cell lines. Our data suggest that erdafitinib reverses multidrug resistance by inhibiting the drug transport function of ABCB1, and not by downregulating the ABCB1 protein in ABCB1-overexpressing cancer cell lines.

2.4. Erdafitinib Restores the Effect of Drug-Induced Apoptosis in ABCB1-Overexpressing Multidrug-Resistant Cancer Cells

To confirm that erdafitinib reverses ABCB1-mediated multidrug resistance by restoring the cytotoxicity of cytotoxic drugs in ABCB1-overexpressing cancer cells and not by initiating a growth retardation effect, the effect of erdafitinib on drug-induced apoptosis in ABCB1-overexpressing cancer cells was examined. Drug-sensitive parental KB-3-1 and ABCB1-overexpressing KB-V-1 cancer cells were treated with 0.5 μM of colchicine, a known transported substrate of ABCB1 [30] and a known inducer of apoptosis [40], in the presence or absence of 5 μM of erdafitinib for 48 h before processed as detailed in the Materials and Methods. As shown in Figure 4, colchicine substantially induced the level of apoptosis in KB-3-1 cells (from an approximately 7% basal level to 53% of total apoptosis), but not in KB-V-1 cells (from approximately 7% basal level to 9% of total apoptosis). While treatment with erdafitinib alone did not induce significant apoptosis in KB-3-1 and KB-V-1 cells, the colchicine-induced apoptosis in KB-V-1 cells was significantly increased by erdafitinib from 9% to 57% of early and late apoptosis.

2.5. Erdafitinib Stimulates the ATPase Activity of ABCB1

Knowing that drug transport mediated by ABCB1 is coupled to ABCB1-mediated ATP hydrolysis [41,42], the effect of erdafitinib on vanadate (Vi)-sensitive ATPase activity of ABCB1 was examined. As shown in Figure 5, erdafitinib stimulated ABCB1 ATPase activity in a concentration-dependent manner with a 9-fold maximum stimulation and with the half-maximal effective concentration (EC50) value of approximately 0.35 μM (basal, 18.11 ± 2.08 nmoles Pi/min/mg protein).

2.6. Docking Analysis of Erdafitinib with The Structure of ABCB1

The most energy favorable docking pose of erdafitinib with the ABCB1 protein structure showed five probable interactions of erdafitinib with amino acids on transmembrane helices. The protonated nitrogen at the isopropyl amine moiety of erdafitinib was predicted to interact with Glu875 by both hydrogen bonding and charge–charge interaction. Another hydrogen bond was predicted between the protonated amide on the side chain of Gln990 with nitrogen on the quinoxaline ring. Phe343, Trp232, and Met986 were predicted to interact with aromatic rings on erdafitinib via hydrophobic interaction (Figure 6).

3. Discussion

Overexpression of ABCB1 in cancer cells is one of the known factors associated with poor therapeutic response in patients receiving cytotoxic anticancer agents [2,43]. One of the reasons why ABCB1-mediated multidrug resistance remains a substantial challenge for oncologists is due to the lack of clinically active inhibitors for the treatment of multidrug-resistant cancers [2]. Despite the substantial efforts that have been invested by drug developers, problems associated with the lack of selectivity and adverse drug–drug interactions have hindered the development of novel synthetic inhibitors of ABCB1 [16]. For instance, previous clinical trials of tariquidar (XR9576) in combination with vinorelbine (NCT00042315) or paclitaxel (NCT00042302) to treat non-small cell lung cancer (NSCLC) were terminated prematurely due to toxicity. In contrast, recent studies have demonstrated that several FDA-approved therapeutic agents such as osimertinib and midostaurin exhibited an inhibitory effect on ABCB1-mediated drug efflux, thus resensitizing ABCB1-overexpressing multidrug-resistant cancer cells to cytotoxic anticancer agents [29,44,45,46]. Moreover, the advantage of using combination therapy against multidrug-resistant cancers was demonstrated in a recent phase I study of concomitant administration of doxorubicin with nilotinib as a co-adjuvant treatment in sarcoma patients [47]. Similarly, studies have reported that a combination-therapy of lapatinib and capecitabine was superior to capecitabine alone in patients with human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer [48,49], whereas erlotinib significantly improved the survival of patients with advanced pancreatic cancer receiving gemcitabine [50,51]. These findings prompted us to investigate the chemosensitization effect of erdafitinib, the first FDA-approved FGFR kinase inhibitor, in ABCB1-overexpressing multidrug-resistant cancer cells.
The in vitro antiproliferative activity of erdafitinib has been shown in a large panel of human cancer cell lines of diverse tissue origins, with IC50 values ranging from below 1 µM to over 5 µM, dependent on the expression of FGFR in these cell lines [19]. However, the effect of erdafitinib against human multidrug-resistant cancer cells was not determined. Therefore, we first examined the cytotoxicity of erdafitinib in multiple human multidrug-resistant cancer cell lines that overexpress ABCB1 or ABCG2 as well as in the corresponding drug-sensitive parental cancer cell lines. We discovered that unlike other ABCB1- and ABCG2-interacting tyrosine kinase inhibitors such as dasatinib [28] and imatinib [26], erdafitinib was equally cytotoxic in multidrug-resistant cancer cells overexpressing either ABCB1 or ABCG2 as in the corresponding drug-sensitive parental cancer cells (Table 1). Moreover, it is worth noting that erdafitinib did not alter the protein expression of ABCB1 in KB-V-1 or NCI-ADR-RES cancer cells, suggesting that ABCB1 may not be a major contributor to the development of erdafitinib resistance in cancer patients. Nevertheless, the effect of regular treatment with erdafitinib on ABCB1 and ABCG2 in cancer patients remains to be further investigated.
Next, we examined the chemosensitizing effect of erdafitinib in multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2. Results showed that erdafitinib reverses multidrug resistance mediated by ABCB1 (Table 2), but not by ABCG2 (Table 3), in human cancer cell lines at subtoxic concentrations (<1 µM) and in a concentration-dependent manner. Of note, the cytotoxicity of vincristine amplified by verapamil in drug-sensitive OVCAR-8 and KB-3-1 cancer cells (Table 2) was consistent with previous reports describing an increased cytotoxic effect of vincristine by verapamil at non-toxic concentrations [52,53]. Moreover, the chemosensitization effect of Ko143 on H460 cancer cells shown in Table 3 was due to the presence of basal expression of ABCG2 in H460 cancer cells [54,55]. More significantly, results of erdafitinib inhibiting the drug efflux function of ABCB1 (Figure 2) and improving the susceptibility of KB-V-1 cells to drug-induced apoptosis (Figure 4) indicate that erdafitinib reverses ABCB1-mediated multidrug resistance by restoring the cytotoxicity of ABCB1 substrate anticancer drugs in these multidrug-resistant cancer cells. Moreover, ABCB1-mediated ATP hydrolysis was stimulated by erdafitinib in a concentration-dependent manner (Figure 5), which is consistent with previous reports [29,33,46,56,57] where the ATPase activity of ABCB1 can be stimulated by the presence of a competitive inhibitor in the substrate-binding pocket of ABCB1. Together with the results of erdafitinib stimulating ABCB1 ATPase activity, the in silico docking analysis of erdafitinib binding to ABCB1 in the inward-open conformation (Figure 6) supports the notion that erdafitinib interacts with several amino acid residues within the transmembrane domain of ABCB1 and attenuates the binding of another drug substrate by direct competition (Figure 7). It is worth mentioning that platinum-based combination chemotherapy consisting of methotrexate, vinblastine, doxorubicin, and cisplatin remains one of the standard treatment options for metastatic urothelial carcinoma. Knowing that that methotrexate, vinblastine, and doxorubicin are substrates for ABCB1, it would be beneficial to include erdafitinib in combination therapy for metastatic urothelial carcinoma. Moreover, in the event that there are tumors harboring FGFR alterations and ABCB1 overexpression, erdafitinib would remain effective against FGFR, as we have shown that ABCB1 does not confer significant resistance to erdafitinib in cancer cells (Table 1).

4. Materials and Methods

4.1. Chemicals

Erdafitinib (JNJ-42756493) was purchased from Selleckchem (Houston, TX, USA). The Tools Cell Counting (CCK-8) Kit was obtained from Biotools Co. Ltd. (Taipei, Taiwan). All other chemicals were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Dulbecco’s Modified Eagle’s Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), Rosewell Park Memorial Institute (RPMI) 1640 medium, fetal calf serum (FCS), phosphate-buffered saline (PBS), trypsin-EDTA, penicillin, and streptomycin were obtained from Gibco, Invitrogen (Carlsbad, CA, USA). The FITC Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, CA, USA).

4.2. Cell Culture Conditions

The human embryonic kidney cell line HEK293, HEK293 cells stably transfected with human ABCB1 (MDR19-HEK293), and HEK293 cells stably transfected with wild-type human ABCG2 (R482-HEK293); the human epidermal cancer cell line KB-3-1; and its ABCB1-overexpressing variant KB-V-1 were maintained in DMEM supplemented with 10% FCS, l-glutamine and penicillin and streptomycin. The human ovarian cancer cell line OVCAR-8 and its ABCB1-overexpressing variant NCI-ADR-RES; the human non-small cell lung cancer (NSCLC) cell line H460 and its ABCG2-overexpressing variant H460-MX20; and the human colon cancer cell line S1 and its ABCG2-overexpressing variant S1-M1-80 were maintained in RPMI-1640 supplemented with 10% FCS, l-glutamine, and penicillin and streptomycin. HEK293 transfectants and KB-V-1 cells were maintained in the presence of 2 mg/mL of G418 [39] or 1 mg/mL of vinblastine [58], respectively. The S1-M1-80 and H460-MX20cells were maintained in the presence of 80 μM of mitoxantrone [59] or 20 nM of mitoxantrone [60], respectively. All cells were cultured at 37 °C in 5% CO2 humidified air and maintained in a drug-free medium for seven days before the assay.

4.3. Cell Viability Assay

The viability of cells treated with erdafitinib or other drug combinations was determined using the Cell Counting Kit-8 (CCK-8) assay or MTT assay based on the method described by Ishiyama et al. [61]. Briefly, cells were plated in 96-well flat-bottom plates and allowed to attach for 24 h at 37 °C in 5% CO2 humidified air, then incubated with erdafitinib or other drug combinations for an additional 72 h. At least three independent experiments were performed to obtain the IC50 values, calculated using the fitted concentration-response curve of each drug regimen. The extent of chemosensitization by a modulator was presented as a fold-reversal (FR) value, determined by adding a non-toxic concentration of erdafitinib or a reference inhibitor to the cytotoxicity assays as described previously [33,39].

4.4. Fluorescent Substrate Accumulation and Flow Cytometry Analysis

The intracellular drug accumulation experiments were carried out and analyzed according to the method described by Gribar et al. [62] using the CellQuest software and the FlowJo software (Tree Star, Inc., Ashland, OR, USA). The accumulation of the fluorescent ABCB1 substrate drug calcein [37] in ABCB1-overexpressing cells or the fluorescent ABCG2 substrate drug pheophorbide A (PhA) [38] in ABCG2-overexpressing cells was determined in the presence of DMSO (control), 1 μM of erdafitinib, 3 μM of tariquidar, or 1 μM of Ko143 using a Becton-Dickinson FACSort flow cytometer as described previously [38,63].

4.5. Immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting for ABCB1 and tubulin were performed as described previously [39]. ABCB1-overexpressing KB-V-1 and NCI-ADR-RES cancer cells were treated with DMSO (control) or erdafitinib at 100 nM, 200 nM, 500 nM, or 1μM for 72 h before being harvested and subjected to SDS-polyacrylamide electrophoresis and western blotting. The anti-P-glycoprotein antibody C219 (1:3000 dilution) and anti-alpha tubulin antibody (1:100,000 dilution) (Abcam, Cambridge, MA, USA) were used as the primary antibodies to detect ABCB1 and the positive loading control tubulin, respectively. The horseradish peroxidase-conjugated goat anti-mouse IgG (1:100,000 dilution) was used as the secondary antibody. The signals were detected and visualized using the enhanced chemiluminescence (ECL) kit (Merck Millipore, Billerica, MA, USA).

4.6. Apoptosis Assay

The effect of erdafitinib, alone or in combination with colchicine, a known apoptotic inducer, in drug-sensitive and ABCB1-overexpressing multidrug-resistant cancer cells was determined based on the concurrent annexin V–FITC and propidium iodide (PI) staining method, according to the manufacturer’s instructions (BD Pharmingen) and as previously described [64]. Briefly, drug-sensitive parental KB-3-1 and ABCB1-overexpressing KB-V-1 cells were treated with DMSO, erdafitinib alone, colchicine alone, or the combination of colchicine and erdafitinib as indicated for 48 h before stained with annexin V–FITC (1.25 µg/mL) and PI (0.1 mg/mL) for 15 min at room temperature. FACScan equipped with CellQuest software (Becton-Dickinson Biosciences, San Jose, CA, USA) was used to analyze the labeled cells as described previously [29].

4.7. ATPase Assay

The effect of erdafitinib on the vanadate (Vi)-sensitive ATPase activity of ABCB1 was determined based on the endpoint Pi assay [65] using the Pgp-Glo assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol and as previously described [66,67].

4.8. In Silico Docking of Erdafitinib in The Drug-Binding Pockets of ABCB1

The energy of the three-dimensional structure of ABCB1(PDB:6QEX) [68] was minimized using Acclerys Discovery Studio 4.0. The preparation of the structure of erdafitinib and docking was performed by the CDOCKER module of the same software.

4.9. Quantification and Statistical Analysis

The experimental data were expressed as mean ± standard deviation (S.D.) from at least three independent experiments unless stated otherwise. GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used for curve plotting, and KaleidaGraph software (Synergy Software, Reading, PA, USA) was used for statistical analysis. The difference between the mean values of the experimental and control or improvement in fit was analyzed by the two-tailed Student’s t-test and labeled with asterisks as “statistically significant” if the probability, p, was less than 0.05.

5. Conclusions

In summary, without excluding the possibility that other mechanisms may also contribute to the reversal of multidrug resistance and that adverse drug reactions have been reported in combination therapy [69,70,71], our study demonstrated that erdafitinib is an FDA-approved therapeutic agent that could be utilized to fight multidrug-resistant cancers associated with the overexpression of ABCB1, and the concomitant administration of cytotoxic anticancer drugs with erdafitinib warrants further investigation.

Supplementary Materials

The following are available online at https://www.mdpi.com/2072-6694/12/6/1366/s1, Figure S1: Expression of ABCB1 in human epidermal cancer KB-3-1 and KB-V-1 cancer cell lines, Figure S2: Expression of ABCB1 in human ovarian cancer OVCAR-8 and NCI-ADR-RES cancer cell lines.

Author Contributions

Conceptualization, C.-P.W., T.-H.H., and Y.-S.W.; Methodology, S.-H.H., Y.-H.H., L.-C.H., Y.-J.Y., and Y.-T.C.; Software, S.-H.H., Y.-H.H., L.-C.H., Y.-J.Y., and Y.-T.C.; Validation, C.-P.W., S.-H.H., and Y.-S.W.; Formal analysis, C.-P.W., S.-H.H., L.-C.H., and Y.-S.W.; Investigation, S.-H.H., Y.-H.H., L.-C.H., Y.-J.Y. and Y.-T.C.; Resources, C.-P.W., T.-H.H., and Y.-S.W.; Data curation, C.-P.W., S.-H.H., L.-C.H., and Y.-S.W.; Writing—original draft preparation, C.-P.W., T.-H.H., and Y.-S.W.; Writing—review and editing, C.-P.W.,T.-H.H., and Y.-S.W.; Visualization, C.-P.W.; Supervision, C.-P.W. and Y.-S.W.; Project administration, C.-P.W. and Y.-S.W.; Funding acquisition, C.-P.W., T.-H.H., S.-P.W., and Y.-S.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by the Ministry of Science and Technology of Taiwan (MOST-107-2314-B-182A-099, MOST-108-2113-M-029-009, MOST-108-2320-B-182-035, MOST-108-2320-B-182-038-MY3), the Chang Gung Medical Research Program (CMRP3G0281 and CMRPG1J0071 to THH and BMRPC17 and CMRPD1J0281 to CPW), and the Taichung Veterans General Hospital (TCVGH-T1097802 to SPW and YSW).

Acknowledgments

We thank Suresh V. Ambudkar (National Cancer Institute, NIH, USA) for the cell lines.

Conflicts of Interest

The authors have no conflicts of interest to declare.

References

  1. Gottesman, M.M.; Ambudkar, S.V. Overview: ABC transporters and human disease. J. Bioenerg. Biomembr. 2001, 33, 453–458. [Google Scholar] [CrossRef] [PubMed]
  2. Robey, R.; Pluchino, K.M.; Hall, M.D.; Fojo, A.T.; Bates, S.E.; Gottesman, M.M. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat. Rev. Cancer 2018, 18, 452–464. [Google Scholar] [CrossRef] [PubMed]
  3. Szakács, G.; Paterson, J.K.; Ludwig, J.A.; Booth-Genthe, C.; Gottesman, M.M. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006, 5, 219–234. [Google Scholar] [CrossRef] [PubMed]
  4. Wu, C.-P.; Hsieh, C.-H.; Wu, Y.-S. The Emergence of Drug Transporter-Mediated Multidrug Resistance to Cancer Chemotherapy. Mol. Pharm. 2011, 8, 1996–2011. [Google Scholar] [CrossRef]
  5. Kovalev, A.A.; Tsvetaeva, D.A.; Grudinskaja, T.V. Role of ABC-cassette transporters (MDR1, MRP1, BCRP) in the development of primary and acquired multiple drug resistance in patients with early and metastatic breast cancer. Exp. Oncol. 2013, 35, 287–290. [Google Scholar]
  6. Ross, D.D.; Karp, J.E.; Chen, T.T.; Doyle, L.A. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood 2000, 96, 365–368. [Google Scholar] [CrossRef]
  7. Steinbach, D.; Sell, W.; Voigt, A.; Hermann, J.; Zintl, F.; Sauerbrey, A. BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia 2002, 16, 1443–1447. [Google Scholar] [CrossRef]
  8. Uggla, B.; Ståhl, E.; Wågsäter, D.; Paul, C.; Karlsson, M.-G.; Sirsjö, A.; Tidefelt, U. BCRP mRNA expression v. clinical outcome in 40 adult AML patients. Leuk. Res. 2005, 29, 141–146. [Google Scholar] [CrossRef]
  9. Matthews, C.; Catherwood, M.A.; Larkin, A.; Clynes, M.; Morris, T.C.; Alexander, H.D. MDR-1, but not MDR-3 gene expression, is associated with unmutated IgVHgenes and poor prognosis chromosomal aberrations in chronic lymphocytic leukemia. Leuk. Lymphoma 2006, 47, 2308–2313. [Google Scholar] [CrossRef]
  10. Schwarzenbach, H. Expression of MDR1/P-Glycoprotein, the Multidrug Resistance Protein MRP, and the Lung-Resistance Protein LRP in Multiple Myeloma. Med. Oncol. 2002, 19, 87–104. [Google Scholar] [CrossRef]
  11. Tsubaki, M.; Satou, T.; Itoh, T.; Imano, M.; Komai, M.; Nishinobo, M.; Yamashita, M.; Yanae, M.; Yamazoe, Y.; Nishida, S. Overexpression of MDR1 and survivin, and decreased Bim expression mediate multidrug-resistance in multiple myeloma cells. Leuk. Res. 2012, 36, 1315–1322. [Google Scholar] [CrossRef] [PubMed]
  12. Turner, J.G.; Gump, J.L.; Zhang, C.; Cook, J.M.; Marchion, D.; Hazlehurst, L.; Munster, P.; Schell, M.J.; Dalton, W.S.; Sullivan, D.M. ABCG2 expression, function, and promoter methylation in human multiple myeloma. Blood 2006, 108, 3881–3889. [Google Scholar] [CrossRef]
  13. Kannan, P.; Telu, S.; Shukla, S.; Ambudkar, S.V.; Pike, V.W.; Halldin, C.; Gottesman, M.M.; Innis, R.B.; Hall, M.D. The “Specific” P-Glycoprotein Inhibitor Tariquidar Is Also a Substrate and an Inhibitor for Breast Cancer Resistance Protein (BCRP/ABCG2). ACS Chem. Neurosci. 2010, 2, 82–89. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Weidner, L.D.; Zoghbi, S.S.; Lu, S.; Shukla, S.; Ambudkar, S.V.; Pike, V.W.; Mulder, J.; Gottesman, M.M.; Innis, R.B.; Hall, M.D. The Inhibitor Ko143 Is Not Specific for ABCG2. J. Pharmacol. Exp. Ther. 2015, 354, 384–393. [Google Scholar] [CrossRef] [Green Version]
  15. Toyoda, Y.; Takada, T.; Suzuki, H. Inhibitors of Human ABCG2: From Technical Background to Recent Updates with Clinical Implications. Front. Pharmacol. 2019, 10, 208. [Google Scholar] [CrossRef] [Green Version]
  16. Dong, J.; Qin, Z.; Zhang, W.-D.; Cheng, G.; Yehuda, G.A.; Ashby, C.R.; Chen, Z.-S.; Cheng, X.-D.; Qin, J.-J.; Jinyun, D.; et al. Medicinal chemistry strategies to discover P-glycoprotein inhibitors: An update. Drug Resist. Updat. 2020, 49, 100681. [Google Scholar] [CrossRef] [PubMed]
  17. Beretta, G.L.; Cassinelli, G.; Pennati, M.; Zuco, V.; Gatti, L. Overcoming ABC transporter-mediated multidrug resistance: The dual role of tyrosine kinase inhibitors as multitargeting agents. Eur. J. Med. Chem. 2017, 142, 271–289. [Google Scholar] [CrossRef]
  18. Wu, S.; Fu, L. Tyrosine kinase inhibitors enhanced the efficacy of conventional chemotherapeutic agent in multidrug resistant cancer cells. Mol. Cancer 2018, 17, 25. [Google Scholar] [CrossRef]
  19. Perera, T.P.; Jovcheva, E.; Mevellec, L.; Vialard, J.; De Lange, D.; Verhulst, T.; Paulussen, C.; Van De Ven, K.; King, P.; Freyne, E.; et al. Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small molecule FGFR family inhibitor. Mol. Cancer Ther. 2017, 16, 1010–1020. [Google Scholar] [CrossRef] [Green Version]
  20. Mizuno, T.; Fukudo, M.; Terada, T.; Kamba, T.; Nakamura, E.; Ogawa, O.; Inui, K.-I.; Katsura, T. Impact of genetic variation in breast cancer resistance protein (BCRP/ABCG2) on sunitinib pharmacokinetics. Drug Metab. Pharmacokinet. 2012, 27, 631–639. [Google Scholar] [CrossRef]
  21. Sato, H.; Siddig, S.; Uzu, M.; Suzuki, S.; Nomura, Y.; Kashiba, T.; Gushimiyagi, K.; Sekine, Y.; Uehara, T.; Arano, Y.; et al. Elacridar enhances the cytotoxic effects of sunitinib and prevents multidrug resistance in renal carcinoma cells. Eur. J. Pharmacol. 2015, 746, 258–266. [Google Scholar] [CrossRef] [PubMed]
  22. Thomas, J.; Wang, L.; Clark, R.E.; Pirmohamed, M.; Chen, G.; Zeng, W.; Miyazato, A.; Billings, E.; Maciejewski, J.P.; Kajigaya, S.; et al. Active transport of imatinib into and out of cells: Implications for drug resistance. Blood 2004, 104, 3739–3745. [Google Scholar] [CrossRef] [Green Version]
  23. Breedveld, P.; Pluim, D.; Cipriani, G.; Wielinga, P.; Van Tellingen, O.; Schinkel, A.H.; Schellens, J. The Effect of Bcrp1 (Abcg2) on theIn vivoPharmacokinetics and Brain Penetration of Imatinib Mesylate (Gleevec): Implications for the Use of Breast Cancer Resistance Protein and P-Glycoprotein Inhibitors to Enable the Brain Penetration of Imatinib in Patients. Cancer Res. 2005, 65, 2577–2582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Burger, H.; Van Tol, H.; Brok, M.; Wiemer, E.A.C.; De Bruijn, E.A.; Guetens, G.; De Boeck, G.; Sparreboom, A.; Verweij, J.; Nooter, K. Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Boil. Ther. 2005, 4, 747–752. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Nakanishi, T.; Shiozawa, K.; Hassel, B.A.; Ross, D.D. Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL–expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression. Blood 2006, 108, 678–684. [Google Scholar] [CrossRef] [Green Version]
  26. Mahon, F.-X.; Belloc, F.; Lagarde, V.; Chollet, C.; Moreau-Gaudry, F.; Reiffers, J.; Goldman, J.M.; Melo, J.V. MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models. Blood 2003, 101, 2368–2373. [Google Scholar] [CrossRef]
  27. Mahon, F.-X.; Hayette, S.; Lagarde, V.; Belloc, F.; Turcq, B.; Nicolini, F.; Belanger, C.; Manley, P.W.; Leroy, C.; Etienne, G.; et al. Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression. Cancer Res. 2008, 68, 9809–9816. [Google Scholar] [CrossRef] [Green Version]
  28. Hiwase, D.K.; Saunders, V.; Hewett, D.; Frede, A.; Zrim, S.; Dang, P.; Eadie, L.; To, L.B.; Melo, J.; Kumar, S.; et al. Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications. Clin. Cancer Res. 2008, 14, 3881–3888. [Google Scholar] [CrossRef] [Green Version]
  29. Hsiao, S.-H.; Lu, Y.-J.; Li, Y.-Q.; Huang, Y.-H.; Hsieh, C.; Wu, C.-P. Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro. Mol. Pharm. 2016, 13, 2117–2125. [Google Scholar] [CrossRef]
  30. Kartner, N.; Riordan, J.R.; Ling, V. Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science 1983, 221, 1285–1288. [Google Scholar] [CrossRef]
  31. Bates, S.E.; Medina-Pérez, W.Y.; Kohlhagen, G.; Antony, S.; Nadjem, T.; Robey, R.; Pommier, Y. ABCG2 Mediates Differential Resistance to SN-38 (7-Ethyl-10-hydroxycamptothecin) and Homocamptothecins. J. Pharmacol. Exp. Ther. 2004, 310, 836–842. [Google Scholar] [CrossRef] [Green Version]
  32. Maliepaard, M.; Van Gastelen, M.A.; De Jong, L.A.; Pluim, D.; Van Waardenburg, R.C.; Ruevekamp-Helmers, M.C.; Floot, B.G.; Schellens, J.H. Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer Res. 1999, 59, 4559–4563. [Google Scholar]
  33. Dai, C.-L.; Tiwari, A.K.; Wu, C.-P.; Su, X.-D.; Wang, S.-R.; Liu, D.-G.; Ashby, C.R.; Huang, Y.; Robey, R.; Liang, Y.-J.; et al. Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res. 2008, 68, 7905–7914. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Cuestas, M.L.; Castillo, A.I.; Sosnik, A.; Mathet, V.L. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles. Bioorg Med. Chem. Lett. 2012, 22, 6577–6579. [Google Scholar] [CrossRef] [PubMed]
  35. Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z.-S.; Ambudkar, S.V.; Baer, M. The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms. Biochem. Pharmacol. 2012, 85, 514–524. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Wang, S.; Liu, S.-T.; Zhao, B.-X.; Yang, F.-H.; Wang, Y.-T.; Liang, Q.-Y.; Sun, Y.-B.; Liu, Y.; Song, Z.-H.; Cai, Y.; et al. Afatinib reverses multidrug resistance in ovarian cancer via dually inhibiting ATP binding cassette subfamily B member 1. Oncotarget 2015, 6, 26142–26160. [Google Scholar] [CrossRef] [PubMed]
  37. Holló, Z.; Homolya, L.; Davis, C.; Sarkadi, B. Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim. Biophys. Acta (BBA) Biomembr. 1994, 1191, 384–388. [Google Scholar] [CrossRef]
  38. Robey, R. Pheophorbide a Is a Specific Probe for ABCG2 Function and Inhibition. Cancer Res. 2004, 64, 1242–1246. [Google Scholar] [CrossRef] [Green Version]
  39. Wu, C.-P.; Shukla, S.; Calcagno, A.M.; Hall, M.D.; Gottesman, M.M.; Ambudkar, S.V. Evidence for dual mode of action of a thiosemicarbazone, NSC73306: A potent substrate of the multidrug resistance linked ABCG2 transporter. Mol. Cancer Ther. 2007, 6, 3287–3296. [Google Scholar] [CrossRef] [Green Version]
  40. Riordan, J.R.; Ling, V. Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J. Boil. Chem. 1979, 254, 12701–12705. [Google Scholar]
  41. Ambudkar, S.V.; Dey, S.; Hrycyna, C.A.; Ramachandra, M.; Pastan, I.; Gottesman, M.M. Biochemical, cellular and pharmacologocial aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 361–398. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Ambudkar, S.V.; Kimchi-Sarfaty, C.; Sauna, Z.E.; Gottesman, M.M. P-glycoprotein: From genomics to mechanism. Oncogene 2003, 22, 7468–7485. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Gillet, J.-P.; Gottesman, M.M. Mechanisms of Multidrug Resistance in Cancer. Adv. Struct. Saf. Stud. 2009, 596, 47–76. [Google Scholar] [CrossRef]
  44. Hsiao, S.-H.; Lusvarghi, S.; Huang, Y.-H.; Ambudkar, S.V.; Hsu, S.-C.; Wu, C.-P. The FLT3 inhibitor midostaurin selectively resensitizes ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic agents. Cancer Lett. 2019, 445, 34–44. [Google Scholar] [CrossRef]
  45. Wu, Z.-X.; Teng, Q.-X.; Cai, C.-Y.; Wang, J.-Q.; Lei, Z.-N.; Yang, Y.; Fan, Y.-F.; Zhang, J.-Y.; Li, J.; Chen, Z.-S. Tepotinib reverses ABCB1-mediated multidrug resistance in cancer cells. Biochem. Pharmacol. 2019, 166, 120–127. [Google Scholar] [CrossRef]
  46. Wu, C.P.; Lusvarghi, S.; Wang, J.C.; Hsiao, S.H.; Huang, Y.H.; Hung, T.H.; Ambudkar, S.V. Avapritinib: A Selective Inhibitor of KIT and PDGFRalpha that Reverses ABCB1 and ABCG2-Mediated Multidrug Resistance in Cancer Cell Lines. Mol. Pharm. 2019, 1, 3040–3052. [Google Scholar] [CrossRef]
  47. Alemany, R.; Moura, D.; Redondo, A.; Martinez-Trufero, J.; Calabuig, S.; Saus, C.; Obrador-Hevia, A.; Ramos, R.F.; Villar, V.H.; Valverde, C.; et al. Nilotinib as Coadjuvant Treatment with Doxorubicin in Patients with Sarcomas: A Phase I Trial of the Spanish Group for Research on Sarcoma. Clin. Cancer Res. 2018, 24, 5239–5249. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Geyer, C.E.; Forster, J.; Lindquist, D.; Chan, S.; Romieu, C.G.; Pienkowski, T.; Jagiello-Gruszfeld, A.; Crown, J.; Chan, A.; Kaufman, B.; et al. Lapatinib plus Capecitabine for HER2-Positive Advanced Breast Cancer. N. Engl. J. Med. 2006, 355, 2733–2743. [Google Scholar] [CrossRef] [Green Version]
  49. Cetin, B.; Benekli, M.; Türker, I.; Koral, L.; Ulaş, A.; Dane, F.; Öksüzoğlu, B.; Kaplan, M.A.; Koca, D.; Boruban, C.; et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer: A multicentre study of Anatolian Society of Medical Oncology (ASMO). J. Chemother. 2013, 26, 300–305. [Google Scholar] [CrossRef]
  50. Moore, M.J.; Goldstein, D.; Hamm, J.; Figer, A.; Hecht, J.R.; Gallinger, S.; Au, H.J.; Murawa, P.; Walde, D.; Wolff, R.A.; et al. Erlotinib Plus Gemcitabine Compared With Gemcitabine Alone in Patients With Advanced Pancreatic Cancer: A Phase III Trial of the National Cancer Institute of Canada Clinical Trials Group. J. Clin. Oncol. 2007, 25, 1960–1966. [Google Scholar] [CrossRef]
  51. Yang, Z.-Y.; Yuan, J.-Q.; Di, M.-Y.; Zheng, D.-Y.; Chen, J.-Z.; Ding, H.; Wu, X.-Y.; Huang, Y.-F.; Mao, C.; Tang, J. Gemcitabine Plus Erlotinib for Advanced Pancreatic Cancer: A Systematic Review with Meta-Analysis. PLoS ONE 2013, 8, e57528. [Google Scholar] [CrossRef] [Green Version]
  52. Tsuruo, T.; Iida, H.; Naganuma, K.; Tsukagoshi, S.; Sakurai, Y. Promotion by verapamil of vincristine responsiveness in tumor cell lines inherently resistant to the drug. Cancer Res. 1983, 43, 808–813. [Google Scholar] [PubMed]
  53. Tsuruo, T.; Iida, H.; Yamashiro, M.; Tsukagoshi, S.; Sakurai, Y. Enhancement of vincristine- and adriamycin-induced cytotoxicity by verapamil in P388 leukemia and its sublines resistant to vincristine and adriamycin. Biochem. Pharmacol. 1982, 31, 3138–3140. [Google Scholar] [CrossRef]
  54. Zhang, G.-N.; Zhang, Y.-K.; Wang, Y.-J.; Gupta, P.; Ashby, C.R.; Alqahtani, S.; Deng, T.; Bates, S.E.; Kaddoumi, A.; Wurpel, J.N.; et al. Epidermal growth factor receptor (EGFR) inhibitor PD153035 reverses ABCG2-mediated multidrug resistance in non-small cell lung cancer: In vitro and in vivo. Cancer Lett. 2018, 424, 19–29. [Google Scholar] [CrossRef] [PubMed]
  55. Cai, C.-Y.; Zhai, H.; Lei, Z.-N.; Tan, C.-P.; Chen, B.-L.; Du, Z.-Y.; Wang, J.-Q.; Zhang, Y.-K.; Wang, Y.-J.; Gupta, P.; et al. Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance. Eur. J. Med. Chem. 2019, 179, 849–862. [Google Scholar] [CrossRef]
  56. Mi, Y.-J.; Liang, Y.-J.; Huang, H.-B.; Zhao, H.-Y.; Wu, C.-P.; Wang, F.; Tao, L.-Y.; Zhang, C.-Z.; Dai, C.-L.; Tiwari, A.K.; et al. Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res. 2010, 70, 7981–7991. [Google Scholar] [CrossRef] [Green Version]
  57. Shukla, S.; Chen, Z.-S.; Ambudkar, S.V. Tyrosine kinase inhibitors as modulators of ABC transporter-mediated drug resistance. Drug Resist. Updat. 2012, 15, 70–80. [Google Scholar] [CrossRef] [Green Version]
  58. Shen, D.; Fojo, A.; Chin, J.; Roninson, I.; Richert, N.; Pastan, I.; Gottesman, M. Human multidrug-resistant cell lines: Increased mdr1 expression can precede gene amplification. Science 1986, 232, 643–645. [Google Scholar] [CrossRef]
  59. Honjo, Y.; Hrycyna, C.A.; Yan, Q.W.; Medina-Pérez, W.Y.; Robey, R.W.; Van De Laar, A.; Litman, T.; Dean, M.; Bates, S.E. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res. 2001, 61, 6635–6639. [Google Scholar]
  60. Henrich, C.J.; Bokesch, H.R.; Dean, M.; Bates, S.E.; Robey, R.; Goncharova, E.I.; Wilson, J.A.; McMahon, J.B. A High-Throughput Cell-Based Assay for Inhibitors of ABCG2 Activity. J. Biomol. Screen. 2006, 11, 176–183. [Google Scholar] [CrossRef] [Green Version]
  61. Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K. A Combined Assay of Cell Viability and in Vitro Cytotoxicity with a Highly Water-Soluble Tetrazolium Salt, Neutral Red and Crystal Violet. Boil. Pharm. Bull. 1996, 19, 1518–1520. [Google Scholar] [CrossRef] [Green Version]
  62. Gribar, J.; Ramachandra, M.; Hrycyna, C.; Dey, S.; Ambudkar, S. Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system. J. Membr. Boil. 2000, 173, 203–214. [Google Scholar] [CrossRef] [PubMed]
  63. Wu, C.-P.; Hsiao, S.-H.; Sim, H.-M.; Luo, S.-Y.; Tuo, W.-C.; Cheng, H.-W.; Li, Y.-Q.; Huang, Y.-H.; Ambudkar, S.V. Human ABCB1 (P-glycoprotein) and ABCG2 mediate resistance to BI 2536, a potent and selective inhibitor of Polo-like kinase 1. Biochem. Pharmacol. 2013, 86, 904–913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Anderson, H.A.; Maylock, C.A.; Williams, J.A.; Paweletz, C.P.; Shu, H.; Shacter, E. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat. Immunol. 2002, 4, 87–91. [Google Scholar] [CrossRef]
  65. Ambudkar, S.V. Drug-stimulatable ATPase activity in crude membranes of human MDR1-transfected mammalian cells. Methods Enzymol. 1998, 292, 504–514. [Google Scholar] [CrossRef] [PubMed]
  66. Wu, C.-P.; Hsiao, S.-H.; Luo, S.-Y.; Tuo, W.-C.; Su, C.-Y.; Li, Y.-Q.; Huang, Y.-H.; Hsieh, C.-H. Overexpression of Human ABCB1 in Cancer Cells Leads to Reduced Activity of GSK461364, a Specific Inhibitor of Polo-like Kinase 1. Mol. Pharm. 2014, 11, 3727–3736. [Google Scholar] [CrossRef]
  67. Wu, C.-P.; Hsieh, C.-H.; Hsiao, S.-H.; Luo, S.-Y.; Su, C.-Y.; Li, Y.-Q.; Huang, Y.-H.; Huang, C.-W.; Hsu, S.-C. Human ATP-Binding Cassette Transporter ABCB1 Confers Resistance to Volasertib (BI 6727), a Selective Inhibitor of Polo-like Kinase 1. Mol. Pharm. 2015, 12, 3885–3895. [Google Scholar] [CrossRef] [PubMed]
  68. Alam, A.; Kowal, J.; Broude, E.; Roninson, I.; Locher, K.P. Structural insight into substrate and inhibitor discrimination by human P-glycoprotein. Science 2019, 363, 753–756. [Google Scholar] [CrossRef] [Green Version]
  69. Stewart, C.F.; Leggas, M.; Schuetz, J.D.; Panetta, J.C.; Cheshire, P.J.; Peterson, J.; Daw, N.; Jenkins, J.J.; Gilbertson, R.; Germain, G.S.; et al. Gefitinib Enhances the Antitumor Activity and Oral Bioavailability of Irinotecan in Mice. Cancer Res. 2004, 64, 7491–7499. [Google Scholar] [CrossRef] [Green Version]
  70. Leggas, M.; Panetta, J.C.; Zhuang, Y.; Schuetz, J.D.; Johnston, B.; Bai, F.; Sorrentino, B.; Zhou, S.; Houghton, P.J.; Stewart, C.F. Gefitinib Modulates the Function of Multiple ATP-Binding Cassette Transporters In vivo. Cancer Res. 2006, 66, 4802–4807. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Shukla, S.; Wu, C.-P.; Ambudkar, S.V. Development of inhibitors of ATP-binding cassette drug transporters—Present status and challenges. Expert Opin. Drug Metab. Toxicol. 2008, 4, 205–223. [Google Scholar] [CrossRef] [PubMed]
Cancers 12 01366 g001 550
Figure 1. Erdafitinib sensitizes ABCB1-overexpressing multidrug-resistant cells to the ABCB1 substrate drug paclitaxel. The effect of erdafitinib on ABCB1-mediated paclitaxel resistance was examined in (a) ABCB1-overexpressing multidrug-resistant KB-V-1 human epidermal cancer cells (left panel) and the corresponding drug-sensitive parental KB-3-1 cancer cells (right panel), (b) ABCB1-overexpressing NCI-ADR-RES human ovarian cancer cells (left panel) and the corresponding drug-sensitive parental OVCAR-8 cancer cells (right panel) as well as (c) HEK293 cells transfected with human ABCB1 (MDR1, left panel) and parental HEK293 cells (right panel). Cells were treated with increasing concentrations of paclitaxel, a well-known substrate of ABCB1, in the presence of DMSO (open squares) or erdafitinib at 100 nM (open circles), 200 nM (filled circles), 500 nM (open triangles), or at 1.0 μM (filled triangles). Points, mean values from at least three independent experiments; bars; S.E.M.
Figure 1. Erdafitinib sensitizes ABCB1-overexpressing multidrug-resistant cells to the ABCB1 substrate drug paclitaxel. The effect of erdafitinib on ABCB1-mediated paclitaxel resistance was examined in (a) ABCB1-overexpressing multidrug-resistant KB-V-1 human epidermal cancer cells (left panel) and the corresponding drug-sensitive parental KB-3-1 cancer cells (right panel), (b) ABCB1-overexpressing NCI-ADR-RES human ovarian cancer cells (left panel) and the corresponding drug-sensitive parental OVCAR-8 cancer cells (right panel) as well as (c) HEK293 cells transfected with human ABCB1 (MDR1, left panel) and parental HEK293 cells (right panel). Cells were treated with increasing concentrations of paclitaxel, a well-known substrate of ABCB1, in the presence of DMSO (open squares) or erdafitinib at 100 nM (open circles), 200 nM (filled circles), 500 nM (open triangles), or at 1.0 μM (filled triangles). Points, mean values from at least three independent experiments; bars; S.E.M.
Cancers 12 01366 g001
Cancers 12 01366 g002 550
Figure 2. Erdafitinib increases the intracellular accumulation of ABCB1 fluorescent substrate drug in ABCB1-overexpressing cells. The intracellular accumulation of calcein, a fluorescent product of a known ABCB1 substrate calcein-AM, was determined in the (a) ABCB1-transfected MDR19-HEK293 cells (left panel) and the corresponding drug-sensitive parental HEK293 cells (right panel), and the (b) ABCB1-overexpressing NCI-ADR-RES cancer cells (left panel) and the corresponding drug-sensitive parental OVCAR-8 cancer cells (right panel), whereas the intracellular accumulation of pheophorbide A (PhA), a known ABCG2 fluorescent substrate drug, was determined in (c) ABCG2-transfected R482-HEK293 cells (left panel) and the corresponding drug-sensitive parental HEK293 cells (right panel), and the (d) ABCG2-overexpressing S1-M1-80 cancer cells (left panel) and the corresponding drug-sensitive parental S1 cancer cells (right panel). Cells were treated with DMSO (solid line), 1 μM of erdafitinib (filled solid line) or a reference inhibitor (dotted line) for ABCB1 (3 μM tariquidar, a,b) or for ABCG2 (1 μM Ko143, c,d) as a positive control. The respective fluorescent signals were analyzed by flow cytometry as described previously [39]. Representative histograms of at least three independent experiments are shown. (e) Concentration-dependent inhibition of ABCB1-mediated calcein efflux in MDR19-HEK293 cells (open squares), NCI-ADR-RES (filled circles) and KB-V-1 cancer cells (open circles). Points, mean values from at least three independent experiments; bars; S.D.
Figure 2. Erdafitinib increases the intracellular accumulation of ABCB1 fluorescent substrate drug in ABCB1-overexpressing cells. The intracellular accumulation of calcein, a fluorescent product of a known ABCB1 substrate calcein-AM, was determined in the (a) ABCB1-transfected MDR19-HEK293 cells (left panel) and the corresponding drug-sensitive parental HEK293 cells (right panel), and the (b) ABCB1-overexpressing NCI-ADR-RES cancer cells (left panel) and the corresponding drug-sensitive parental OVCAR-8 cancer cells (right panel), whereas the intracellular accumulation of pheophorbide A (PhA), a known ABCG2 fluorescent substrate drug, was determined in (c) ABCG2-transfected R482-HEK293 cells (left panel) and the corresponding drug-sensitive parental HEK293 cells (right panel), and the (d) ABCG2-overexpressing S1-M1-80 cancer cells (left panel) and the corresponding drug-sensitive parental S1 cancer cells (right panel). Cells were treated with DMSO (solid line), 1 μM of erdafitinib (filled solid line) or a reference inhibitor (dotted line) for ABCB1 (3 μM tariquidar, a,b) or for ABCG2 (1 μM Ko143, c,d) as a positive control. The respective fluorescent signals were analyzed by flow cytometry as described previously [39]. Representative histograms of at least three independent experiments are shown. (e) Concentration-dependent inhibition of ABCB1-mediated calcein efflux in MDR19-HEK293 cells (open squares), NCI-ADR-RES (filled circles) and KB-V-1 cancer cells (open circles). Points, mean values from at least three independent experiments; bars; S.D.
Cancers 12 01366 g002
Cancers 12 01366 g003 550
Figure 3. Erdafitinib does not alter the protein expression of ABCB1 in ABCB1-overexpressing cancer cells. (a) KB-V-1 cancer cells and (b) NCI-ADR-RES cancer cells were treated with DMSO (vehicle control) or erdafitinib at 100 nM, 200 nM, 500 nM, or 1 μM for 72 h and processed for western blotting as described in the Materials and Methods. α-tubulin was used as an internal loading control. The representative immunoblots (upper panel) and the corresponding quantification (lower panel) human ABCB1 protein are shown. Values are presented as mean ± S.D. calculated from at least three independent experiments. Detailed information about western blot can be found in Figures S1 and S2.
Figure 3. Erdafitinib does not alter the protein expression of ABCB1 in ABCB1-overexpressing cancer cells. (a) KB-V-1 cancer cells and (b) NCI-ADR-RES cancer cells were treated with DMSO (vehicle control) or erdafitinib at 100 nM, 200 nM, 500 nM, or 1 μM for 72 h and processed for western blotting as described in the Materials and Methods. α-tubulin was used as an internal loading control. The representative immunoblots (upper panel) and the corresponding quantification (lower panel) human ABCB1 protein are shown. Values are presented as mean ± S.D. calculated from at least three independent experiments. Detailed information about western blot can be found in Figures S1 and S2.
Cancers 12 01366 g003
Cancers 12 01366 g004 550
Figure 4. Erdafitinib sensitizes ABCB1-overexpressing multidrug-resistant cancer cells to apoptosis induction by an ABCB1 substrate drug. The representative flow cytometric dot plots (upper panel) and quantification (lower panel) of drug-sensitive KB-3-1 cells and the ABCB1-overexpressing multidrug resistant KB-V-1 cells treated with either DMSO (control), 5 μM of erdafitinib (+erdafitinib), 500 nM of colchicine (+colchicine), or a combination of colchicine and erdafitinib (+colchicine +erdafitinib). Cells were treated with the respective drug regimens and analyzed by flow cytometry as described in the Materials and Methods. The corresponding quantification data are presented as the mean ± SD calculated from at least three independent experiments. ** p < 0.01; *** p < 0.001, versus the same treatment in the absence of erdafitinib.
Figure 4. Erdafitinib sensitizes ABCB1-overexpressing multidrug-resistant cancer cells to apoptosis induction by an ABCB1 substrate drug. The representative flow cytometric dot plots (upper panel) and quantification (lower panel) of drug-sensitive KB-3-1 cells and the ABCB1-overexpressing multidrug resistant KB-V-1 cells treated with either DMSO (control), 5 μM of erdafitinib (+erdafitinib), 500 nM of colchicine (+colchicine), or a combination of colchicine and erdafitinib (+colchicine +erdafitinib). Cells were treated with the respective drug regimens and analyzed by flow cytometry as described in the Materials and Methods. The corresponding quantification data are presented as the mean ± SD calculated from at least three independent experiments. ** p < 0.01; *** p < 0.001, versus the same treatment in the absence of erdafitinib.
Cancers 12 01366 g004
Cancers 12 01366 g005 550
Figure 5. Erdafitinib stimulates ABCB1 ATPase activity. The effect of erdafitinib on vanadate-sensitive ATPase activity of ABCB1 was determined by the endpoint Pi assay as described in the Materials and Methods. Data are presented as a mean ± S.D. from three independent experiments.
Figure 5. Erdafitinib stimulates ABCB1 ATPase activity. The effect of erdafitinib on vanadate-sensitive ATPase activity of ABCB1 was determined by the endpoint Pi assay as described in the Materials and Methods. Data are presented as a mean ± S.D. from three independent experiments.
Cancers 12 01366 g005
Cancers 12 01366 g006 550
Figure 6. Docking of erdafitinib in the drug-binding pocket of ABCB1. Binding modes of erdafitinib with ABCB1 protein structure (PDB: 6QEX) was predicted by BIOVIA (Accelrys) Discovery Studio 4.0 software (San Diego, CA, USA) as described in the Materials and Methods. Erdafitinib is shown as a molecular model with the highlighted yellow color and the atoms for interacting amino acid residues are colored as carbon, gray; hydrogen, light gray; nitrogen, blue; and oxygen, red. Dotted lines indicate proposed interactions.
Figure 6. Docking of erdafitinib in the drug-binding pocket of ABCB1. Binding modes of erdafitinib with ABCB1 protein structure (PDB: 6QEX) was predicted by BIOVIA (Accelrys) Discovery Studio 4.0 software (San Diego, CA, USA) as described in the Materials and Methods. Erdafitinib is shown as a molecular model with the highlighted yellow color and the atoms for interacting amino acid residues are colored as carbon, gray; hydrogen, light gray; nitrogen, blue; and oxygen, red. Dotted lines indicate proposed interactions.
Cancers 12 01366 g006
Cancers 12 01366 g007 550
Figure 7. Simplified schematic diagram of erdafitinib modulating the drug efflux function of ABCB1 in multidrug-resistant cancer cells. In the absence of erdafitinib (white triangle), ABCB1 substrate chemotherapy drugs (red circles) are effluxed out of multidrug-resistant cancer cells by ABCB1, reducing the efficacy of these drugs. In contrast, binding of erdafitinib to the drug-binding pocket of ABCB1 outcompetes the binding of ABCB1 substrate chemotherapy drugs to the same site, consequently increasing the intracellular accumulation and efficacy of these drugs in ABCB1-overexpressing multidrug-resistant cancer cells.
Figure 7. Simplified schematic diagram of erdafitinib modulating the drug efflux function of ABCB1 in multidrug-resistant cancer cells. In the absence of erdafitinib (white triangle), ABCB1 substrate chemotherapy drugs (red circles) are effluxed out of multidrug-resistant cancer cells by ABCB1, reducing the efficacy of these drugs. In contrast, binding of erdafitinib to the drug-binding pocket of ABCB1 outcompetes the binding of ABCB1 substrate chemotherapy drugs to the same site, consequently increasing the intracellular accumulation and efficacy of these drugs in ABCB1-overexpressing multidrug-resistant cancer cells.
Cancers 12 01366 g007
Table
Table 1. Cytotoxicity of erdafitinib in human cell lines overexpressing ABCB1 or ABCG2.
Table 1. Cytotoxicity of erdafitinib in human cell lines overexpressing ABCB1 or ABCG2.
Cell LineTypeTransporter ExpressedIC50 (μM) †
KB-3-1epidermal-3.18 ± 0.48
KB-V-1epidermalABCB14.92 ± 1.19
OVCAR-8ovarian-10.80 ± 2.83
NCI-ADR-RESovarianABCB16.21 ± 1.15
H460lung-3.46 ± 0.37
H460-MX20lungABCG24.66 ± 0.71
S1colon-4.31 ± 1.19
S1-M1-80colonABCG25.60 ± 1.21
pcDNA-HEK293--6.02 ± 1.25
MDR19-HEK293-ABCB16.51 ± 1.27
R482-HEK293-ABCG25.16 ± 0.80
† IC50 values are the mean ± SD calculated from dose-response curves obtained from at least three independent experiments using the cytotoxicity assay as described in the Materials and Methods.
Table
Table 2. The effect of erdafitinib on ABCB1-mediated multidrug resistance in ABCB1-overexpressing human cell lines.
Table 2. The effect of erdafitinib on ABCB1-mediated multidrug resistance in ABCB1-overexpressing human cell lines.
TreatmentConcentration
(μM)		Mean IC50 † ± SD and (FR ‡)
OVCAR-8 (Parental)
[nM]		NCI-ADR-RES (Resistant)
[μM]
Paclitaxel-3.74 ± 0.67 (1.0)7.96 ± 1.46 (1.0)
+ erdafitinib0.13.57 ± 0.62 (1.0)6.08 ± 0.88 (1.3)
+ erdafitinib0.23.66 ± 0.63 (1.0)4.76 ± 0.64 * (1.7)
+ erdafitinib0.53.46 ± 0.63 (1.1)2.80 ± 0.34 ** (2.8)
+ erdafitinib1.03.00 ± 0.48 (1.2)0.68 ± 0.10 *** (11.7)
+ verapamil5.02.92 ± 0.55 (1.3)0.31 ± 0.04 *** (25.7)
[nM][μM]
Vincristine-69.30 ± 10.38 (1.0)4.36 ± 0.96 (1.0)
+ erdafitinib0.169.17 ± 12.60 (1.0)3.41 ± 0.63 (1.3)
+ erdafitinib0.264.38 ± 10.45 (1.1)3.23 ± 0.61 (1.3)
+ erdafitinib0.561.65 ± 11.86 (1.1)1.73 ± 0.24 * (2.5)
+ erdafitinib1.054.37 ± 9.90 (1.3)0.57 ± 0.06 ** (7.6)
+ verapamil5.019.20 ± 2.97 ** (3.6)0.18 ± 0.04 ** (24.2)
TreatmentConcentration
(μM)		KB-3-1 (Parental)
[nM]		KB-V-1 (Resistant)
[μM]
Paclitaxel-1.72 ± 0.66 (1.0)2.84 ± 0.54 (1.0)
+ erdafitinib0.11.71 ± 0.61 (1.0)2.31 ± 0.20 (1.2)
+ erdafitinib0.21.74 ± 0.63 (1.0)1.11 ± 0.12 ** (2.6)
+ erdafitinib0.52.16 ± 0.84 (0.8)246.89 ± 39.24 ** [nM] (11.5)
+ erdafitinib1.02.10 ± 0.81 (0.8)83.93 ± 10.28 *** [nM] (33.9)
+ verapamil5.01.71 ± 0.68 (1.0)43.36 ± 5.94 *** [nM] (65.5)
[nM][nM]
Vincristine-0.79 ± 0.30 (1.0)954.77 ± 150.75 (1.0)
+ erdafitinib0.10.76 ± 0.23 (1.0)714.49 ± 172.29 (1.3)
+ erdafitinib0.20.75 ± 0.23 (1.1)341.99 ± 75.71 ** (2.8)
+ erdafitinib0.50.80 ± 0.20 (1.0)77.57 ± 19.24 *** (12.3)
+ erdafitinib1.00.78 ± 0.18 (1.0)20.47 ± 4.51 *** (46.6)
+ verapamil5.00.15 ± 0.05 * (5.3)8.74 ± 1.41 *** (109.2)
TreatmentConcentration
(μM)		pcDNA-HEK293
(Parental)
[nM]		MDR19-HEK293
(Resistant)
[nM]
Paclitaxel-3.25 ± 0.69 (1.0)532.02 ± 75.55 (1.0)
+ erdafitinib0.12.72 ± 0.56 (1.2)254.49 ± 41.07 ** (2.1)
+ erdafitinib0.22.61 ± 0.53 (1.2)110.74 ± 19.20 *** (4.8)
+ erdafitinib0.52.35 ± 0.50 (1.4)29.55 ± 5.21 *** (18.0)
+ erdafitinib1.02.30 ± 0.50 (1.4)10.10 ± 1.58 *** (52.7)
+ verapamil5.02.13 ± 0.49 (1.5)7.87 ± 1.71 *** (67.6)
[nM][nM]
Vincristine-1.89 ± 0.23 (1.0)363.39 ± 41.69 (1.0)
+ erdafitinib0.11.99 ± 0.38 (0.9)156.57 ± 21.81 ** (2.3)
+ erdafitinib0.21.92 ± 0.38 (1.0)95.18 ± 19.98 *** (3.8)
+ erdafitinib0.51.74 ± 0.31 (1.1)18.15 ± 2.53 *** (20.0)
+ erdafitinib1.01.50 ± 0.30 (1.3)5.59 ± 0.95 *** (65.0)
+ verapamil5.00.65 ± 0.18 ** (2.9)3.70 ± 0.57 *** (98.2)
FR, fold-reversal. † IC50 values are mean ± SD calculated from dose-response curves obtained from at least three independent experiments using cytotoxicity assay as described in the Materials and Methods. ‡ FR values were calculated by dividing the IC50 values of cells treated with a particular therapeutic drug in the absence of erdafitinib or verapamil by IC50 values of cells treated with the same therapeutic drug in the presence of erdafitinib or verapamil. * p < 0.05; ** p < 0.01; *** p < 0.001.
Table
Table 3. The effect of erdafitinib on ABCG2-mediated multidrug resistance in ABCG2-overexpressing human cell lines.
Table 3. The effect of erdafitinib on ABCG2-mediated multidrug resistance in ABCG2-overexpressing human cell lines.
TreatmentConcentration
(μM)		Mean IC50 † ± SD and (FR ‡)
H460 (Parental)
[nM]		H460-MX20 (Resistant)
[μM]
Topotecan-64.14 ± 13.91 (1.0)1.43 ± 0.26 (1.0)
+ erdafitinib0.161.83 ± 13.83 (1.0)1.29 ± 0.23 (1.1)
+ erdafitinib0.261.07 ± 15.77 (1.0)1.23 ± 0.22 (1.2)
+ erdafitinib0.556.76 ± 12.36 (1.1)1.10 ± 0.19 (1.3)
+ erdafitinib1.046.20 ± 10.58 (1.4)1.13 ± 0.21 (1.3)
+ Ko1431.022.94 ± 5.53 ** (2.8)99.10 ± 18.86 *** [nM] (14.4)
[nM][nM]
SN-38-7.48 ± 1.36 (1.0)126.16 ± 11.15 (1.0)
+ erdafitinib0.17.54 ± 1.41 (1.0)122.92 ± 11.13 (1.0)
+ erdafitinib0.27.24 ± 1.37 (1.0)120.46 ± 10.80 (1.0)
+ erdafitinib0.56.40 ± 1.21 (1.2)110.08 ± 10.57 (1.1)
+ erdafitinib1.05.56 ± 1.13 (1.3)100.69 ± 12.37 (1.3)
+ Ko1431.03.19 ± 0.91 * (2.3)6.79 ± 1.60 *** (18.6)
TreatmentConcentration
(μM)		S1 (Parental)
[nM]		S1-M1-80 (Resistant)
[μM]
Topotecan-19.51 ± 2.34 (1.0)6.35 ± 1.14 (1.0)
+ erdafitinib0.120.40 ± 2.54 (1.0)8.00 ± 1.60 (0.8)
+ erdafitinib0.221.80 ± 2.96 (0.9)6.25 ± 1.11 (1.0)
+ erdafitinib0.522.31 ± 2.96 (0.9)6.25 ± 1.50 (1.0)
+ erdafitinib1.023.90 ± 3.55 (0.8)6.65 ± 1.53 (1.0)
+ Ko1431.020.88 ± 2.52 (0.9)0.19 ± 0.05 *** (33.4)
[nM][nM]
SN-38-3.03 ± 0.23 (1.0)1036.58 ± 119.08 (1.0)
+ erdafitinib0.13.07 ± 0.34 (1.0)1083.89 ± 94.65 (1.0)
+ erdafitinib0.23.23 ± 0.31 (0.9)1397.68 ± 195.28 (0.7)
+ erdafitinib0.53.37 ± 0.34 (0.9)1251.31 ± 108.50 (0.8)
+ erdafitinib1.02.76 ± 0.21 (1.1)1128.26 ± 228.23 (0.9)
+ Ko1431.02.64 ± 0.19 (1.1)42.43 ± 9.66 *** (24.4)
TreatmentConcentration
(μM)		pcDNA-HEK293 (Parental)
[nM]		R482-HEK293 (Resistant)
[nM]
Topotecan-25.56 ± 6.29 (1.0)295.26 ± 25.68 (1.0)
+ erdafitinib0.126.19 ± 6.47 (1.0)274.81 ± 25.32 (1.1)
+ erdafitinib0.223.67 ± 5.79 (1.1)256.80 ± 27.11 (1.1)
+ erdafitinib0.522.65 ± 5.25 (1.1)240.19 ± 25.26 (1.2)
+ erdafitinib1.020.77 ± 5.18 (1.2)229.64 ± 35.28 (1.3)
+ Ko1431.023.80 ± 5.79 (1.1)20.59 ± 3.66 *** (14.3)
[nM][nM]
SN-38-2.93 ± 0.85 (1.0)83.53 ± 9.80 (1.0)
+ erdafitinib0.12.54 ± 0.49 (1.2)81.56 ± 12.19 (1.0)
+ erdafitinib0.22.77 ± 0.72 (1.1)91.97 ± 18.20 (0.9)
+ erdafitinib0.52.45 ± 0.55 (1.2)74.69 ± 13.69 (1.1)
+ erdafitinib1.02.39 ± 0.58 (1.2)74.46 ± 14.87 (1.1)
+ Ko1431.03.09 ± 0.98 (0.9)4.50 ± 0.70 *** (18.6)
FR, fold-reversal. † IC50 values are mean ± SD calculated from dose-response curves obtained from at least three independent experiments using the cytotoxicity assay as described in the Materials and Methods. ‡ FR values were calculated by dividing the IC50 values of cells treated with a particular therapeutic drug in the absence of erdafitinib or Ko143 by IC50 values of cells treated with the same therapeutic drug in the presence of erdafitinib or Ko143. * p < 0.05; ** p < 0.01; *** p < 0.001.

Share and Cite

MDPI and ACS Style

Wu, C.-P.; Hung, T.-H.; Hsiao, S.-H.; Huang, Y.-H.; Hung, L.-C.; Yu, Y.-J.; Chang, Y.-T.; Wang, S.-P.; Wu, Y.-S. Erdafitinib Resensitizes ABCB1-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs. Cancers 2020, 12, 1366. https://doi.org/10.3390/cancers12061366

AMA Style

Wu C-P, Hung T-H, Hsiao S-H, Huang Y-H, Hung L-C, Yu Y-J, Chang Y-T, Wang S-P, Wu Y-S. Erdafitinib Resensitizes ABCB1-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs. Cancers. 2020; 12(6):1366. https://doi.org/10.3390/cancers12061366

Chicago/Turabian Style

Wu, Chung-Pu, Tai-Ho Hung, Sung-Han Hsiao, Yang-Hui Huang, Lang-Cheng Hung, Yi-Jou Yu, Yu-Tzu Chang, Shun-Ping Wang, and Yu-Shan Wu. 2020. "Erdafitinib Resensitizes ABCB1-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs" Cancers 12, no. 6: 1366. https://doi.org/10.3390/cancers12061366

APA Style

Wu, C. -P., Hung, T. -H., Hsiao, S. -H., Huang, Y. -H., Hung, L. -C., Yu, Y. -J., Chang, Y. -T., Wang, S. -P., & Wu, Y. -S. (2020). Erdafitinib Resensitizes ABCB1-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs. Cancers, 12(6), 1366. https://doi.org/10.3390/cancers12061366

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop