ORIGINAL ARTICLE

PD173074, a selective FGFR inhibitor, reverses ABCB1-mediateddrug resistance in cancer cells

Atish Patel • Amit K. Tiwari • Eduardo E. Chufan •

Kamlesh Sodani • Nagaraju Anreddy • Satyakam Singh •

Suresh V. Ambudkar • Ralph Stephani • Zhe-Sheng Chen

Received: 13 February 2013 / Accepted: 1 May 2013! Springer-Verlag Berlin Heidelberg 2013

AbstractPurpose Specific tyrosine kinase inhibitors were recently

reported to modulate the activity of ABC transporters,

leading to an increase in the intracellular concentration oftheir substrate drugs. In this study, we determine whether

PD173074, a specific fibroblast growth factor receptor

(FGFR) inhibitor, could reverse ABC transporter-mediatedmultidrug resistance.

Methods 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapati-

nibrazolium bromide assay was used to determine theeffect of PD173074 on reversal of ABC transporter-medi-

ated multidrug resistance (MDR). In addition, [3H]-paclit-

axel accumulation/efflux assay, western blotting analysis,ATPase, and photoaffinity labeling assays were done to

study the interaction of PD173074 on ABC transporters.

Results PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this

transporter to substrate anticancer drugs colchicine, paclit-

axel, and vincristine. This effect of PD173074 is specific toABCB1, as no significant interaction was detected with other

ABC transporters such as ABCC1 and ABCG2. Theobserved reversal effect seems to be primarily due to the

decreased active efflux of [3H]-paclitaxel in ABCB1 over-

expressing cells observed in efflux assay. In addition, nosignificant change in the ABCB1 expression was observed

when ABCB1 overexpressing cells were exposed to 5 lM

PD173074 for up to 3 days, thereby further suggesting itsrole in modulating the function of the transporter. In addi-

tion, PD173074 stimulated the ATPase activity of ABCB1 in

a concentration-dependent manner, indicating a directinteraction with the transporter. Interestingly, PD173074 did

not inhibit photolabeling of ABCB1 with [125I]-iodoaryla-

zidoprazosin (IAAP), showing that it binds at a site differentfrom that of IAAP in the drug-binding pocket.

Conclusions Here, we report for the first time, PD173074,

an inhibitor of the FGFR, to selectively reverse ABCB1transporter-mediated MDR by directly blocking the efflux

function of the transporter.

Keywords PD173074 ! ABC transporter ! ABCB1 !Fibroblast growth factor receptor ! Multidrug resistance !Tyrosine kinase inhibitor

Introduction

Cancer cells through the years have continuously evolved to

resist its treatments by adaptations that alter the gene

machinery toward abnormal amplification of genes withinresistant cancer cells. Multidrug resistance (MDR) has been

associated with this type of acquired resistance [1]. It is a

phenomenon where cells develop resistance to structurallyand mechanistically unrelated drugs. Efflux of anticancer

drugs out of the cells is one of the many factors responsible for

the development of MDR, predominantly known as typical

A. Patel ! A. K. Tiwari ! K. Sodani ! N. Anreddy ! S. Singh !R. Stephani ! Z.-S. Chen (&)Department of Pharmaceutical Sciences,College of Pharmacy and Health Sciences,St. John’s University, Queens, NY 11439, USAe-mail: chenz@stjohns.edu

Present Address:A. K. TiwariDepartment of Biomedical Sciences, College of VeterinaryMedicine, Nursing and Allied Health, Tuskegee University,Tuskegee, AL 36088, USA

E. E. Chufan ! S. V. AmbudkarLaboratory of Cell Biology, Center for Cancer Research,National Cancer Institute, NIH, Bethesda, MD 20892, USA

123

Cancer Chemother Pharmacol

DOI 10.1007/s00280-013-2184-z

MDR. This often leads to lower therapeutic concentrations of

the substrate drugs within the cytosol [2]. The ATP-bindingcassette (ABC) transporters a family of 48 members is among

the key factors responsible for the development of such

resistance in cancer cells [3] They are divided into seven sub-families A through G. Physiologically they serve as a defense

mechanism, wherein they efflux harmful substances out of

normal cells. Similarly, cancer cells tend to overexpress thesetransporters in order to protect themselves from cytotoxic

anticancer drugs. These transporters function by utilizing theenergy produced via hydrolysis of ATP [3]. The first member

of the ABC transporter family to be discovered was P-gly-

coprotein (P-gp) also known as ABCB1 (ABC transporterbelonging to subfamily B member 1). It has a molecular

weight of 170 kDa and is known to efflux a variety of sub-

stances in an ATP-dependent manner [3]. It is widelyexpressed on the plasma membrane of cells and overexpressed

in tumorous tissues that have originated from the epithelium of

colon, kidney, adrenal, pancreas, and liver, thereby renderingthem resistant to cytotoxic drugs before chemotherapy is

initiated. On the other hand, tumorous tissues with lower basal

levels of ABCB1 tend to overexpress ABCB1 via repeatedexposure to chemotherapeutic agents [4]. Substrate anticancer

drugs of ABCB1 include vinca alkaloids, anthracyclines,

epipodophyllotoxins, and taxanes [5]. ABCG2, another keytransporter in the ABC family, also contributes to the devel-

opment of MDR in cancer cells. The spectrum of chemo-

therapeutic agents transported by ABCG2 includesanthracyclines, mitoxantrone, methotrexate, flavopiridol,

camptothecin-derived, and indolocarbazole topoisomerase I

inhibitors [6, 7]. ABCC1 a member of the C subfamily of ABCtransporters can also transport various hydrophobic drugs;

some anionic drugs and its drug conjugates, including antif-

olates, certain nucleotides, and also vinca alkaloids [8–10].Hence, due to the vast overexpression pattern and sub-

strate profile of these ABC transporters in various tumor

types, it poses a serious threat toward successful chemo-therapy. Thus, resensitizing MDR tumorous tissues to che-

motherapeutic drugs using agents that inhibit this efflux

system could lead to a chemotherapeutic success. Using thisconcept, various drugs have been screened for their potential

to reverse MDR due to these transporters. Verapamil,

cyclosporin A, tamoxifen, and several calmodulin antago-nists were among the first, identified to have ABCB1

inhibitory action, and are hence called the first-generation

modulators [11]. But with their low binding affinities, thesedrugs produced rather disappointing results when tested

in vivo. Moreover, some agents were substrates of ABCB1

themselves and required higher concentrations in order tohave inhibitory potential thereby rendering them toxic [12].

This lead to a search for drugs with lower toxicity and greater

potency and resulted in the development of second-genera-tion inhibitory agents, where drugs such as dexverapamil,

valspodar (PSC 833), and biricodar (VX-710) were more

potent and less toxic than their predecessors [13–15]. How-ever, many of them were substrates of cytochrome P450 3A4

resulting in increased plasma concentration of the co-

administered chemotherapeutic agent leading to increasedtoxicity. Also, some of these drugs were non-specific and

inhibited other ABC transporters leading to increased

adverse effects of the chemotherapeutic agents [16, 17].Later using quantitative structure–activity relationship drugs

such as elacridar (GF120918), laniquidar (R101933), zos-uquidar (LY335979), and tariquidar (XR9576) were devel-

oped so as to effectively block the ABCB1 function at

nanomolar concentration [18–20]. They too failed duringclinical trials due to their excessive toxicity and non-speci-

ficity [21, 22]. Thus, increased toxicity and non-specificity of

these reported compounds constantly urge the researchers tosearch for newer inhibitors of ABC transporters.

Previous reports have demonstrated TKIs of different

classes, such as lapatinib an EGFR inhibitor, nilotinib aBcr-Abl tyrosine kinase inhibitor (TKI), imatinib another

Bcr-Abl TKI, and apatinib a VEGFR inhibitor, to inhibit

various ABC transporters among others [23–25]. However,they lack the specificity toward inhibiting a single ABC

transporter. Also, there have been no reports so far of any

fibroblast growth factor receptor (FGFR) TKI interactingwith ABC transporters. Therefore, we explored the poten-

tial of PD173074 an FGFR inhibitor for its ability to

reverse ABC transporter-mediated MDR. FGFRs signalthrough fibroblast growth factors (FGFs) and regulate

essential bodily functions such as proliferation, angiogen-

esis, and differentiation [26]. However, FGFs and FGFRscan also act as oncogenes when their signaling is deregu-

lated leading to mitogenic, anti-apoptotic, and angiogenic

responses in cells each of which are considered hallmarksof cancer [27]. PD173074 a pyrido-[2,3-d]pyrimidine is a

small-molecule TKI that selectively inhibits FGFR family-

related signaling [28, 29].

Materials and methods

Chemicals and equipment

[3H]-Paclitaxel (23 Ci/mmol) was purchased from Moravek

Biochemicals, Inc (Brea, CA). Dulbecco’s modified Eagle’s

medium (DMEM), RPMI 1640 medium, fetal bovine serum(FBS), penicillin/streptomycin, and trypsin 0.25 % were

products of Hyclone, Thermo Scientific (Logan, UT).

Monoclonal antibody against b-actin was purchased fromSanta Cruz Biotechnology, Inc (Santa Cruz, CA). Antibody

against ABCB1 was purchased from Signet Laboratories Inc

(Dedham, MA). MK571 was purchased from BiomolResearch Laboratories (Plymouth Meeting, PA).

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Fumitremorgin C (FTC) was synthesized by Thomas

McCloud Developmental Therapeutics Program, NaturalProducts Extraction Laboratory, NCI, NIH (Bethesda, MD),

and it was a gift from Dr. Susan E Bates (NCI, NIH,

Bethesda, MD). Mitoxantrone, paclitaxel, cisplatin, andPD173074 were purchased from Tocris Bioscience (Ellis-

ville, MO). 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapati-

nibrazolium bromide (MTT), dimethyl sulfoxide (DMSO),verapamil, and other chemicals were obtained from Sigma

Chemical (St. Louis, MO). [125I]-Iodoarylazidoprazosin(IAAP) (2200 Ci/mmol) was purchased from PerkinElmer

Life Sciences (Waltham, MA). OPSYS microplate reader

was purchased from Dynex Technologies (Chantilly, VA).

Cell lines and cell culture

The KB-C2 cell line was established by a stepwise selection of

KB-3-1, a parental human epidermoid carcinoma cell line, by

exposure to increasing concentrations of colchicine up to2 lg/mL [30]. Another ABCB1 overexpression cell line KB-

V1 was isolated from KB-3-1 cells and maintained in a

medium with 1 lg/mL of vinblastine. Human primaryembryonic kidney cell line HEK293 and its pcDNA3.1,

ABCB1 stable gene-transfected cell lines HEK293/

pcDNA3.1, and HEK293/ABCB1 [31] were obtained fromDr. Susan.E. Bates (NCI, NIH, Bethesda, MD). HEK293/

ABCC1 was generated in Dr. Ambudkar’s laboratory at NCI

[32]. Parental human non-small cell lung cancer cell H460was grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA)

supplemented with 10 % heat-inactivated fetal bovine serum.

Resistant H460/MX20 cells were cultured in the aforemen-tioned medium with the addition of 20 nM mitoxantrone. Both

H460 and H460/MX20 cells were provided by Dr. Susan E

Bates (NCI, NIH, Bethesda, MD). All other cell lines werecultured under 37 "C, 5 % CO2 with DMEM containing 10 %

of FBS and 1 % penicillin/streptomycin.

Cell sensitivity by tetrazolium dye assay

Cells in 96-well plates were pre-incubated with or withoutthe inhibitory agents for 1 h (h), and then different con-

centrations of chemotherapeutic drugs were added into

designated wells. After 72 h of incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)

solution (4 mg/mL) was added to each well, and the plate

was further incubated for 4 h, allowing viable cells tochange the yellow-colored MTT into dark-blue formazan

crystals. Subsequently, the medium was discarded, and

100 lL of dimethylsulfoxide (DMSO) was added into eachwell to dissolve the formazan crystals. The absorbance was

determined at 570 nm by an OPSYS MR Microplate

Reader from Dynex Technologies, Inc.

Preparation of cell lysates

Approximately 1 9 106 cells were harvested and sus-pended in PBS followed by centrifugation at 2,000 rpm for

2 min, then by washing twice with PBS. Later, lysis buffer

and 1 % aprotinin were added to the suspension followedby vortexing. The resuspended cells were kept on ice for

30 min followed by centrifugation at 10,000 rpm for

20 min. The supernatant was separated and was stored in-80 "C until required for the experiment. Protein con-

centration was determined by Bradford method [33].

Immunoblot analysis

Cell lysates were resolved by 4–12 % SDS-polyacrylamidegel electrophoresis and transferred to PVDF (polyvinylidene

difluoride) filters. Forty micrograms of KB-C2 cell lysates

was loaded in each lane. ABCB1 was determined fromC-219 mouse monoclonal antibody, and b-actin was used as

internal loading control as previously mentioned in [34].

[3H]-Paclitaxel accumulation assay

The KB-3-1 and KB-C2 cell lines were harvested at thecondition of around 80 % confluence for this experiment.

Approximately, 1 9 106 cells were incubated at 37 "C in

DMEM (10 % FBS added) with and without PD173074 atconcentrations of 2.5 and 5 lM for 1 h in 24-well plates.

After 1 h, cells were treated with 0.1 lM [3H]-paclitaxel and

incubated further for another 2 h. Later, the medium wasremoved, and the cells were rinsed three times with cold

PBS. The cells were then lysed by adding 200 lL lysis buffer

and transferred to scintillation vials. Each sample was placedin scintillation fluid, and radioactivity was measured [34].

[3H]-Paclitaxel efflux assay

To measure the drug efflux, the cells were treated in the same

manner as in drug accumulation experiment and then incu-bated in fresh medium at 37 "C at various time points in the

presence or absence of reversal agents in 24-well plates.

After washing three times with ice-cold PBS, the cells werethen trypsinized and placed in the scintillation fluid to

measure the radioactivity as previously described [34].

ABCB1 ATPase assay

The vanadate-sensitive ATPase activity of ABCB1 incrude membranes of High-five insect cells, in the presence

of concentrations of PD173074 ranging from 0 to 20 lM,was measured as previously described [35].

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Photoaffinity labeling of ABCB1

with [125I]-iodoarylazidoprazosin

We incubated ABCB1-transfected High-five insect cell

membranes expressing ABCB1 (70 lg protein/100 ll)

with varying concentrations of PD173074 (0–20 lM) orwith 10 lM cyclosporine A for 10 min at 37 "C in 50 mM

MES–Tris pH 6.8. [125I]-IAAP (2,200 Ci/mmol; 4–6 nM)

was added and membranes incubated for an additional5 min under minimal exposure to light. The samples were

then illuminated with 365 nm UV lamp, and photoaffinity

labeling of ABCB1 with [125I]-IAAP was determined aspreviously described [36].

Statistical analysis

Differences of the parameters between two cell groups

were analyzed by two-tailed Student’s t test. P \ 0.05 wasconsidered as statistically significant.

Results

Effect of PD173074 in combination with ABCB1substrates in cell lines overexpressing ABCB1

To determine the minimal toxic concentrations ofPD173074, a cell survival MTT assay at different con-

centrations of PD173074 was performed and observed that

more than 80 % of the KB-3-1, KB-C2, KB-V1, HEK293/pcDNA3.1, and HEK293/ABCB1 cells survived at 2.5 and

5 lM concentrations (data not shown). With these results,

2.5 and 5 lM of PD173074 were used to study its ability toinhibit ABC transporters. The IC50 values obtained using

PD173074 with various substrate anticancer drugs of

ABCB1 are summarized in Table 1. The KB-C2 cellsshowed resistance folds of 197.9 toward colchicine, 182.6

toward paclitaxel, and 121.4 toward vincristine as com-

pared to KB-3-1 its parental cell line (Table 1). These arethree different substrates of ABCB1, thereby displaying

MDR mediated via ABCB1. However, the resistance was

overcome in the presence of PD173074 at 2.5 and 5 lMwhen combined with the aforementioned ABCB1 sub-

strates. However, the parental KB-3-1 cells remained

unaffected in the presence of the inhibitor. This effect wassimilar to the one obtained when using verapamil at 5 lM,

a widely accepted ABCB1 inhibitor. KB-V1 another

ABCB1 overexpressing drug-selected cell line displayed aresistance fold of 165.8 as compared to KB-3-1 parental

cells toward vincristine. However, this resistance fold was

overcome in the presence of PD173074 at 2.5 and 5 lM.Again the parental KB-3-1 cells remained unaffected in the

presence or absence of PD173074. However, there may be

various factors responsible for the development of MDR in

cancer cells [1]. In order to limit those factors to only onemediated via ABCB1, HEK293/ABCB1 stably transfected

cell line was used along with its empty vector-transfected

parental cell line HEK293/pcDNA3.1. The HEK293/ABCB1 displayed a resistance fold of 157.4 to colchicine,

149.2 to paclitaxel, and 146.3 to vincristine as compared to

its parental HEK293/pcDNA3.1. PD173074 again at 2.5and 5 lM was able to resensitize the HEK293/ABCB1

cells to the aforementioned substrates (Table 1). It wasshown that the parental HEK293/pcDNA3.1 cells remained

unaffected in the presence of PD73074. When cisplatin a

non-substrate of ABCB1 was used to investigate theinhibitory activity of PD173074, the KB-3-1, KB-V1, and

KB-C2 cell lines showed similar sensitivity to PD173074.

Similarly, HEK293/pcDNA3.1 and HEK293/ABCB1 cellsshowed similar IC50 values for cisplatin in the presence or

absence of PD173074,. thereby confirming its specific

modulatory effect on ABCB1 transporter function.

The effect of PD173074 on cell lines overexpressing

ABCG2 and ABCC1

In order to study whether PD173074 could modulate the

activity of ABCG2, we treated the parental H460 andH460/MX20 cells with 5 lM of PD173074 using

mitoxantrone as a substrate. PD173074 at 5 lM when

combined with mitoxantrone displayed a resistance fold of43.1 in the resistant H460/MX20 cells as compared to the

parental H460 cells, thereby indicating that there was no

change in the resistance fold as compared to the controlwhere the resistance fold observed was 50. Thus,

PD173074 does not inhibit the ABCG2 function (Table 2).

In another instance, parental HEK293/pcDNA3.1 andHEK293/ABCC1 cells transfected with ABCC1 were used

to study the effect of PD173074 on ABCC1. It was found

that in the presence of PD173074 combined with vincris-tine a substrate of ABCC1, the resistance fold in the

resistant HEK293/ABCC1 was 32.8 as compared to the

control where the resistance fold was 33.9. This showedthat PD173074 can not reverse ABCC1-mediated MDR

(Table 2).

The effect of PD173074 on the expression levels

of ABCB1

In order to confirm whether the reversal of ABCB1-med-

iated MDR through PD173074 was not due to the decrease

in the protein expression, we performed an immunoblotanalysis. PD173074 at 5 lM was incubated with KB-C2

cells overexpressing ABCB1 for 0, 24, 48, and 72 h and the

cells were collected, and analyzed for the expression levelsof ABCB1 on the respective cell lysates. It was observed

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Table 1 PD173074 reverses the ABCB1-mediated drug resistance to colchicine, paclitaxel, and vincristine

Treatments KB-3-1 KB-C2

IC50a (nM) RFb IC50 (nM) RFb

Colchicine 23.4 ± 0.251a [1.0] 2291.0 ± 382.027 [197.9]

?PD173074 (2.5 lM) 25.7 ± 0.572 [0.9] 21.7 ± 0.062* [0.9]

?PD173074 (5 lM) 21.6 ± 0.357 [1.1] 19.6 ± 0.029* [0.8]

?Verapamil (5 lM) 17.5 ± 0.106 [1.3] 30.7 ± 0.433* [1.3]

Paclitaxel 15.8 ± 0.123 [1.0] 2884.5 ± 124.021 [182.6]

?PD173074 (2.5 lM) 13.6 ± 0.736 [0.9] 43.5 ± 0.439* [2.8]

?PD173074 (5 lM) 15.7 ± 0.327 [1.0] 20.9 ± 0.245* [1.3]

?Verapamil (5 lM) 15.1 ± 0.226 [1.0] 33.0 ± 0.742* [2.1]

Vincristine 1.66 ± 0.162 [1.0] 202.56 ± 42.481 [121.4]

?PD173074 (2.5 lM) 1.61 ± 0.049 [0.9] 2.5849 ± 0.315* [1.5]

?PD173074 (5 lM) 1.42 ± 0.144 [0.8] 1.8876 ± 0.019* [1.3]

?Verapamil (5 lM) 1.49 ± 0.136 [0.9] 1.7360 ± 0.095* [1.4]

Cisplatin 735 ± 21.04 [1.0] 643.0 ± 32.463 [0.9]

?PD173074 (5 lM) 742 ± 34.061 [0.9] 612 ± 45.831 [0.8]

Treatments KB-3-1 KB-V1

IC50a (nM) RFb IC50 (nM) RFb

Vincristine 1.26 ± 0.027a [1.0] 208.85 ± 20.4 [165.8]

?PD173074 (2.5 lM) 1.31 ± 0.009 [1.0] 5.96 ± 0.061* [4.7]

?PD173074 (5 lM) 1.86 ± 0.020 [1.3] 3.56 ± 0.013* [2.8]

?Verapamil (5 lM) 1.01 ± 0.564 [1.2] 24.51 ± 2.536* [19.5]

Cisplatin 738 ± 41.01 [1.0] 678.7 ± 51.76 [0.9]

?PD173074 (5 lM) 731.2 ± 50.04 [1.0] 653.4 ± 40.53 [0.9]

Treatments HEK293/pcDNA3.1 HEK293/ABCB1

IC50a (nM) RFb IC50 (nM) RFb

Colchicine 23.2 ± 0.241a [1.0] 3662.1 ± 390.569 [157.4]

?PD173074 (2.5 lM) 23.5 ± 0.723 [1.0] 19.6 ± 0.129* [0.8]

?PD173074 (5 lM) 25.4 ± 0.533 [1.1] 9.6 ± 0.026* [0.4]

?Verapamil (5 lM) 25.1 ± 0.124 [1.1] 83.1 ± 1.953* [3.6]

Paclitaxel 26.4 ± 0.821 [1.0] 3941.3 ± 310.452 [149.2]

?PD173074 (2.5 lM) 27.9 ± 0.261 [0.9] 171.7 ± 23.035* [6.1]

?PD173074 (5 lM) 27.8 ± 0.832 [0.9] 90.7 ± 29.095* [3.4]

?Verapamil (5 lM) 27.6 ± 0.716 [1.0] 97.7 ± 31.042* [3.7]

Vincristine 24.1 ± 0.204 [1.0] 3512.8 ± 378.391 [146.3]

?PD173074 (2.5 lM) 26.4 ± 0.424 [1.0] 36.8 ± 1.029* [1.5]

?PD173074 (5 lM) 25.9 ± 0.319 [0.9] 24.9 ± 2.047* [1.0]

?Verapamil (5 lM) 23.7 ± 0.014 [1.1] 87.8 ± 9.387* [3.7]

Cisplatin 735.0 ± 21.56 [1.0] 643.0 ± 32.41 [0.9]

?PD173074 (5 lM) 742.0 ± 34.41 [1.0] 612.0 ± 45.67 [0.8]

a IC50: concentration that inhibited cell survival by 50 % (mean ± SD)b RF: resistance fold was determined by dividing the IC50 values of substrate in KB-C2 cells in the absence or presence of reversal agents, orKB-3-1 cells with reversal agents, by the IC50 of substrate in KB-3-1 cells without reversal agents. The RF for KB-3-1 and KB-V1 and theHEK293/pcDNA3.1 and HEK293/ABCB1 cells was obtained in the similar manner. *P \ 0.01 versus that obtained in the absence of PD173074

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that there was no significant change in the expression levels

of the ABCB1 transporter in the KB-C2 cell line as shownin Fig. 1. The result suggested that the reversal effect was

not caused by the decrease in ABCB1 protein expression.

Effect of PD173074 on cellular accumulation

of [3H]-paclitaxel

To further analyze the effect of PD173074 on the drug

efflux function of ABCB1, an intracellular accumulation of[3H]-paclitaxel study was performed. The intracellular

accumulation of [3H]-paclitaxel was significantly lower in

the drug-resistant KB-C2 cells than that in the parental KB-3-1 cells. However, upon 2-h incubation with PD173074 at

2.5 and 5 lM concentrations, [3H]-paclitaxel intracellular

accumulation was significantly increased in drug-resistant

KB-C2 cells (Fig. 2). The results obtained were compara-

ble to 5 lM verapamil treatment group, a known inhibitorof ABCB1. However, the parental KB-3-1 showed no

significant change in the accumulation of [3H]-paclitaxel.

These results suggested that PD173074 might affect theefflux of paclitaxel by ABCB1.

The effect of PD173074 on the efflux of [3H]-paclitaxel

To ascertain whether the increase in the intracellular

accumulation of [3H]-paclitaxel in the presence ofPD173074 was due to the inhibition of [3H]-paclitaxel

efflux by ABCB1, we performed a time course study to

measure intracellular [3H]-paclitaxel levels in the presenceof PD17074. The resistant KB-C2 cells effluxed intracel-

lular [3H]-paclitaxel, with increasing time compared withparental KB-3-1 cells. When PD173074 at 5 lM was

added to both the cells, they significantly blocked the [3H]-

paclitaxel efflux at different time periods (0, 60, and120 min) from resistant KB-C2 cells, but not in the

parental KB-3-1 cells (Fig. 3). Furthermore, PD173074

showed an effect similar to that obtained when incubatingthe cells with verapamil at 5 lM. These results herein

further validate the ability of PD173074 to inhibit the

extruding drug function of ABCB1.

Effect of PD173074 on ABCB1 ATP hydrolysis

To determine whether PD173074 modulates the activity of

ABCB1 by directly interacting with this transporter, we

Table 2 PD173074 does not show any effect on ABCG2 andABCC1 transporters

Treatment H460 H460/MX20

IC50 (lM) RFb IC50 (lM) RFb

Mitoxantrone 0.1031 ± 0.067a [1.0] 5.4538 ± 0.563 [50]

?PD173074 (5 lM) 0.1064 ± 0.020 [1.0] 5.1513 ± 0.608 [43.1]

?FTC (5 lM) 0.1141 ± 0.088 [1.1] 0.1081 ± 0.086 [1.0]

Treatment HEK293/pcDNA3.1 HEK/ABCC1

IC50 (lM) RFb IC50 (lM) RFb

Vincristine 0.0224 ± 0.001a [1.0] 0.7599 ± 0.039 [33.9]

?PD173074 (5 lM) 0.0298 ± 0.003 [0.8] 0.7368 ± 0.045 [32.8]

?MK571 (5 lM) 0.0256 ± 0.009 [0.9] 0.0270 ± 0.019 [1.2]

a IC50: concentration that inhibited cell survival by 50 % (mean ± SD)b RF: resistance fold was determined by dividing the IC50 values ofmitoxantrone in H460/MX20 cells in the absence or presence of reversalagents, or H460 cells with reversal agents, by the IC50 of mitoxantrone in H460cells without reversal agents. The RF for HEK293/pcDNA3.1 and ABCC1were obtained in a similar manner

Fig. 1 Immunoblot analysis showing the expression of ABCB1 atdifferent time points after treatment with PD173074. Cell lysates ofABCB1 overexpression KB-C2 cells exposed to PD173074 (5 lM)were prepared at different time points (0, 24, 48, and 72 h), and equalamount samples (40 lg protein) were loaded into each well andsubjected to immunoblot analysis as described in ‘‘Materials andmethods,’’ representative result is shown, and similar results wereobtained in two additional independent trials

Fig. 2 Effect of PD173074 on the accumulation of [3H]-paclitaxel.The accumulation of [3H]-paclitaxel was measured after the cells(parental KB-3-1 and KB-C2) were pre-incubated with or withoutPD173074 at 5 lM or verapamil for 1 h at 37 "C and then incubatedwith 0.1 lM [3H]-paclitaxel for another 2 h at 37 "C. Columns are themean of triplicate determinations; bars represent SD. AsteriskP \ 0.05 versus the control group. The figure is a representative ofthree independent experiments each done in triplicates

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determined the effect of this compound on ATPase activity

of ABCB1. As shown in Fig. 4a, PD173074 stimulatesATPase activity of ABCB1 in a concentration-dependent

manner. The concentration required for maximal stimulation

(2.5–5 lM) is in the same range as that required for sensi-tization of cells to anticancer drugs (Table 1). These data

demonstrate that similar to a majority of tyrosine kinase

inhibitors, PD173074 also modulates ABCB1 function bydirectly interacting with the transporter [25, 37].

Effect of PD17073 on photo-crosslinking

of [125I]-IAAP to ABCB1

A number of modulators including TKIs interact at the

drug-binding pocket of ABCB1 and inhibit photolabeling

of this transporter with [125I]-IAAP. To further understandthe interaction of PD173074 with the drug-binding pocket

of ABCB1, the incorporation of [125I]-IAAP into ABCB1

in the presence of increasing concentration of PD173074was measured. Interestingly, as shown in Fig. 4b,

PD173074 had no effect on the photolabeling of ABCB1

with [125I]-IAAP. On the other hand, cyclosporine A at10 lM (a known inhibitor of ABCB1) interacting with the

[125I]-IAAP-binding site of the transporter inhibited the

photolabeling. These data indicate that PD173074 interactsat a site on ABCB1 that is different from the [125I]-IAAP-

binding site.

Discussion

The overexpression of ABC transporters has become more

and more prevalent with the extensive use of chemother-

apeutic agents. Cancer cells exploit these transporters toprotect themselves from the chemotherapeutic drugs. Thus,

inhibition of these transporters still remains a priority.

More than three decades into the discovery of the first ABCtransporter (ABCB1) and after three generations of inhib-

itory drugs, there has been no reports of a single drug being

approved clinically that can reverse MDR caused by ABCtransporters due to toxicity issues [5]. Recently various

TKIs have shown promising results both in vitro and

in vivo wherein they block the function of ABC trans-porters [23, 38, 39]. TKIs mainly act on the catalytic site of

tyrosine kinase domain by competing with ATP binding,

thereby blocking the kinase activity [40, 41]. Nilotinib aBcr-Abl inhibitor approved for the use of chronic mye-

logenous leukemia (CML) has been shown to reverse

ABCB1-, ABCG2-, and ABCC10-mediated drug resistance[23, 24]. Lapatinib an EGFR inhibitor approved for the use

of breast cancer was shown to inhibit ABCB1- and

ABCG2-mediated MDR in cancer cells [25]. Apatinib,another TKI, directed toward VEGFR has been reported to

reverse ABCB1- and ABCG2-mediated MDR in cancer

cells [42]. These being a few of the many TKIs studied fortheir drug reversal capacity. However, they lack selectivity

Fig. 3 Effect of PD173074 on the efflux of [3H]-paclitaxel. Theeffects of PD173074 on the efflux of [3H]-paclitaxel from KB-3-1 andKB-C2 cells were measured. Briefly, cells were pre-treated with orwithout PD173074 at 5 lM for 1 h at 37 "C and further incubatedwith 0.1 lM [3H]-paclitaxel at 37 "C for 2 h. Cells were thenincubated in the fresh medium with or without the reversal agents for

different time periods at 37 "C. Cells were then collected, and theintracellular levels of [3H]-paclitaxel were determined by scintillationcounting. A time course versus percentage of intracellular [3H]-paclitaxel was plotted (0, 30, 60, 120 min). Verapamil (5 lM) wasused as a positive control. The figure is a representative of threeindependent experiments each done in triplicates

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toward ABC transporters. Therefore, we selected

PD173074 a selective FGFR-TKI to investigate whether itwould modulate a specific ABC transporter. So far, there

has been no report of any compound targeting FGFRs to

modulate ABC transporters. PD173074 has shown prom-ising results both in vitro as well as in vivo to block the

growth of small cell lung cancer (SCLC) [43]. FGFR sig-

naling has been related to neoangiogenesis [44, 45], andFGF-2 has been reported to induce SCLC cell proliferation

and resistance to cytotoxic drugs [46, 47]. When usedin vivo, PD173074 was shown to inhibit FGF-driven neo-

angiogenesis while being exempt of general toxicity [29,

48]. Here, for the first time, we show the potential of thissmall-molecule TKI to selectively modulate ABCB1. It

was seen that PD173074 at relatively non-toxic concen-

trations of 2.5 and 5 lM was able to selectively reverseABCB1-mediated MDR in both drug-selected cancer cells

and ABCB1-gene-transfected cell lines (Table 1). It also

increased the intracellular accumulation of [3H]-paclitaxelby directly blocking the efflux function of the transporter.

Consistent with these findings, PD173074 stimulates

ATPase activity of ABCB1 (Fig. 4a), indicating that it

directly interacts with this transporter. Interestingly it was

seen that PD173074 did not inhibit the binding of [125I]-IAAP, which is known to bind to the ABCB1 drug-binding

pocket, thereby suggesting it binds to a different site within

the large hydrophobic drug-binding site of ABCB1.PD173074 was initially subjected to cytotoxicity assays

to determine its toxicity. With the data obtained, a rela-

tively non-toxic concentration of 2.5 and 5 lM was used tostudy its potential to inhibit the ABC transporters. It was

seen that the compound sensitized the KB-C2 and KB-V1,ABCB1 overexpressing drug-selected cancer cell lines

when used in combination with ABCB1 substrates col-

chicine, paclitaxel, and vincristine (Table 1). Moreover,there was also no change observed in the cytotoxicity

results when using cisplatin a non-substrate of ABCB1 in

both the parental and drug resistant cells, thus suggestingthat PD173074 inhibits ABCB1-mediated MDR in cancer

cells. However, there are multiple factors contributing

toward resistance in cancer cells, in order to limit thosefactors to only the one mediated via ABCB1 transporter,

we used ABCB1 stably transfected HEK293 cells. The

cytotoxicity data obtained indicated that the compound

Fig. 4 PD173074 stimulates ATPase activity of ABCB1 but does notinhibit the photolabeling of ABCB1 with [125I]-IAAP. a Effect ofPD173074 on ATP hydrolysis by ABCB1. Crude membranes (100 lgprotein/mL) from High-five cells expressing ABCB1 were incubatedwith increasing concentrations of PD173074 (0–20 lM), in thepresence and absence of sodium orthovanadate (Vi) (0.3 mM), inATPase assay buffer as described in ‘‘Materials and methods.’’ Themean values are plotted and error bars depict SD (n = 4). b IAAPlabeling of ABCB1 in the presence of increasing concentrations ofPD173074. Crude membranes of High-five insect cells expressing

ABCB1 (700 lg protein/mL) were incubated in 50 mM MES–TrispH 6.8 with increasing concentrations of PD173074 (0–20 lM) or10 lM cyclosporine A, at 37 "C for 10 min. Samples were thentransferred to 4 "C bath, and [125I]-IAAP (4-6 nM) was added undersubdued light. The samples were photo-crosslinked with 365 nm UVlight for 10 min at room temperature followed by electrophoresis andquantification of incorporation of [125I]-IAAP into ABCB1 bands asdescribed previously. The autoradiogram from a typical experiment isshown. Similar results were obtained in one more experiment

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resensitized the resistant HEK293-ABCB1 cells to their

substrates colchicine, paclitaxel, and vincristine when usedin combination with the compound (Table 1), thereby

indicating that the resistance to the substrates was mainly

mediated via ABCB1 and this resistance was overcomewhen the substrates were combined with PD173074.

We further extended our investigation of PD173074 to

include ABCG2 and ABCC1 transporters. The resultsindicated that PD173074 could not reverse ABCG2- and

ABCC1-mediated MDR (Table 2), thereby suggesting thespecificity of PD173074 toward modulating ABCB1-

mediated MDR in cancer cells.

Later to understand how PD173074 interacts withABCB1, we performed a [3H]-paclitaxel accumulation

assay in the presence of PD173074 wherein PD173074

could significantly enhance the intracellular concentrationof [3H]-paclitaxel in the drug-resistant KB-C2 cells

(Fig. 2). To further confirm that the increase in paclitaxel

accumulation was due to ABCB1 inhibition, we performeda [3H]-paclitaxel efflux assay where PD173074 reduced the

rapid efflux of the [3H]-paclitaxel in drug-resistant KB-C2

cells on a time scale as compared to the control where therewas no addition of inhibitor to the resistant KB-C2 cells

(Fig. 3). Pointing toward the fact that the intracellular

increase in the [3H]-paclitaxel levels corresponds to theinhibition of the efflux function of ABCB1 and not due to

the increase in the uptake of the substrate. Using immu-

noblotting techniques, we indicated that PD173074 showedno effect on the expression of ABCB1 transporter, ruling

out the possibility of downregulation of ABCB1 by

PD173074 (Fig. 1), thereby also confirming that thereversal action of PD173074 was mainly due to the inhi-

bition of the efflux function of ABCB1. Generally, sub-

strates of ABCB1 have been reported to stimulate theATPase activity of ABC transporters. Several TKIs were

reported to stimulate the ATPase activity of ABCB1,

suggesting that they might also be substrates of ABCB1[41, 49]. Hence, we studied the effect of PD173074 on the

ATPase activity of ABCB1 wherein PD173074 stimulated

the ATPase activity in a concentration-dependent manneras shown in Fig. 4a, suggesting that PD173074 interacts

directly with the transporter possibly at the drug-binding

site in the transmembrane domains. Further to understandas to where PD173074 binds in the large hydrophobic drug-

binding pocket of ABCB1, we performed a photoaffinity

labeling study where PD173074 was incubated along with[125I]-IAAP which is known to bind to ABCB1. Interest-

ingly, it was seen that PD173074 did not interact with the

[125I]-IAAP-binding site within the transporter (Fig. 4b),implying that it may be binding to a different site than the

one for [125I]-IAAP.

In conclusion, PD173074 at a lower concentration (2.5and 5 lM) significantly and specifically inhibits ABCB1-

mediated MDR in cancer cells by inhibiting the efflux

function of the transporter and increases the intracellularconcentration of the substrate anticancer drugs. However, it

does not have any effect on the total expression level of the

transporter. Collectively, the data presented here suggestthat PD173074 has potential to selectively inhibit ABCB1-

mediated MDR and could be used clinically to inhibit

MDR in cancer cells.

Acknowledgments We thank Drs. Michael M. Gottesman (NCI,NIH, Bethesda, MD, USA) for KB-3-1 cells, Shinichi Akiyama(Kagoshima University, Japan) for the KB-C2 cell lines, Susan E.Bates and Robert W. Robey (NIH, USA) for the FTC, HEK293/pcDNA3.1, HEK293/ABCB1, H460, and H460/MX20 cell lines. Dr.Tanaji Talele and Rishil J Kathawala (St. John’s University) forcarefully reading and editing the manuscript. This work was sup-ported by funds from NIH (No. 1R15CA143701) and RayBiotech, Incto Z.S. Chen. E.E. Chufan and S.V. Ambudkar were supported by theIntramural Research Program of the NIH, National Cancer Institute,Center for Cancer Research.

Conflict of interest There are no conflicts of interest.

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