Overexpression of Human ABCB1 in Cancer Cells Leads to Reduced

Article pubs.acs.org/molecularpharmaceutics

Overexpression of Human ABCB1 in Cancer Cells Leads to Reduced Activity of GSK461364, a Specific Inhibitor of Polo-like Kinase 1 Chung-Pu Wu,*,†,‡,§ Sung-Han Hsiao,‡ Shi-Yu Luo,‡ Wei-Cherng Tuo,‡ Ching-Ya Su,‡ Yan-Qing Li,† Yang-Hui Huang,§ and Chia-Hung Hsieh∥,⊥ †

Department of Physiology and Pharmacology, ‡Graduate Institute of Biomedical Sciences, and §Molecular Medicine Research Center, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan ∥ Graduate Institute of Basic Medical Science and ⊥Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ABSTRACT: Polo-like kinase 1 (Plk1) is a serine/threonine kinase involved in the regulation of mitosis and is overexpressed in many tumor types. Inhibition of Plk1 leads to cell cycle arrest, onset of apoptosis, and cell death, thus Plk1 has emerged as an important target for cancer treatment. GSK461364 is a potent inhibitor of Plk1 that inhibits the proliferation of multiple human cancer cell lines by promoting G2/M cell cycle arrest at low concentrations. However, as is the case for many therapeutic drugs, the risk of developing drug resistance to GSK461364 can present a therapeutic challenge to clinicians. Since the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 is one of the most common mechanisms of drug resistance, we aimed to investigate the effect of ABCB1 on the cellular efficacy of GSK461364. In this study, we observed a significantly reduced activity of GSK461364 in cells overexpressing human ABCB1. We showed that GSK461364 stimulates the ABCB1 ATPase activity and competitively inhibits ABCB1-mediated efflux of calcein-AM in a concentration-dependent manner. Moreover, as a way to assess the impact of ABCB1 on the efficacy of GSK461364, we evaluated the G2/M cell cycle arrest and apoptosis induced by GSK461364. We discovered that, by inhibiting the function of ABCB1, the reduced G2/M cell cycle arrest, apoptosis, and sensitivity to GSK461364 treatment in ABCB1overexpressing cells can be significantly restored. In conclusion, in order to achieve a better therapeutic outcome, combination therapy of GSK461364 with a modulator of ABCB1 should be further investigated as a potential treatment approach. KEYWORDS: ABCB1, multidrug resistance, Polo-like kinase 1, GSK461364

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INTRODUCTION

The overexpression of ATP-binding cassette (ABC) drug transporters such as ABCB1, ABCC1, and ABCG2 is a major obstacle and one of the most common mechanisms for the development of multidrug resistance (MDR) in cancer chemotherapy.22,23 Collectively, these drug transporters can recognize a diverse range of therapeutic agents and utilize ATP hydrolysis to actively efflux drug substrates out of cancer cells, which lead to MDR, cancer relapse, and death.23 Moreover, studies have shown that the reduced effectiveness of many small-molecule kinase inhibitors is associated with the overexpression of ABC drug transporters.24−26 Therefore, it is important to study the pharmacological and biochemical interactions between ABC drug transporters and the Plk1 inhibitors. Human ABCB1, also known as P-glycoprotein (P-gp/MDR1), is the first member of the mammalian ABC protein family identified to transport drug substrates across cell membranes.27 In normal tissues, ABCB1 is highly expressed at sites such as the blood−brain barrier (BBB),

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is overexpressed in various types of human tumor cells, and it has recently emerged as an important target for cancer treatment.1−7 Plk1 is involved in the regulation of multiple key steps of mitosis,1,8,9 including mitotic entry,10 mitotic exit,11 centrosome maturation,12 and bipolar spindle formation.13 Inhibition of Plk1 has been shown to cause significant cell cycle arrest, induction of apoptosis, and decreased cell viability in cancer cells.12,14−16 Therefore, small-molecule inhibitors of Plk1 have been developed to induce G2/M cell cycle arrest, which leads to apoptotic cell death of cancer cells. GSK461364 is a thiophene amide developed by GlaxoSmithKline, U.K., that inhibits Plk1, induces G2/M arrest, displays potent antiproliferative activity in multiple cancer cell lines, and inhibits tumor growth in animal models.5,17 It is structurally unrelated to the first selective Plk1 inhibitor BI 2536, which is a dihydropteridinone derivative designed by Boehringer Ingelheim, Germany.5,18 Recently, GSK461364 showed excellent anticancer activity against bladder cancer17 and glioblastoma,19,20 as well as favorable pharmacological and antitumor properties in phase I clinical trials.2,21 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3727

July 20, 2014 August 24, 2014 September 5, 2014 September 5, 2014 dx.doi.org/10.1021/mp500492r | Mol. Pharmaceutics 2014, 11, 3727−3736

Molecular Pharmaceutics

Article

on ABCB1-mediated calcein-AM efflux was measured and analyzed as described previously.33 The mean fluorescence intensity was calculated with the histogram stat program in CellQuest software. For the determination of concentration-dependent inhibition of ABCB1-mediated calcein-AM efflux by GSK461364 in KB-V-1 cells, the median value peak of calcein-AM for KB-V-1 cells was defined as fully active ABCB1, whereas the median value peak for KB-V-1 cells in the presence of tariquidar was defined as inactivated ABCB1. Median value peaks for KB-V-1 cells in the presence of various concentrations of GSK461364 were consequently calculated as the ratio within the range, representing the activity of ABCB1 function as percentage control value. ATPase Assay of ABCB1. The vanadate (Vi)-sensitive ABCB1-specific ATPase activities were recorded by using the Pgp-Glo assay system (Promega, WI, USA) according to the manufacturer’s instructions. The drug-stimulated ATPase activity of ABCB1 was determined based on end point Pi assay as described previously.34 Cell Cycle Analysis and Apoptosis Assay. Cell cycle experiments were carried out using standard propidium iodide (PI) staining method and analyzed using a FACSort flow cytometer equipped with CellQuest software. Briefly, cells were treated with the indicated regimens for 24 h before being harvested in PBS and fixed in ethanol overnight. Cells were washed once with PBS and then treated with 0.5% Triton X-100 and 0.05% RNase in PBS at 37 °C for 1 h. Cells were washed, propidium iodide (50 μg/mL) was added, and then the cells were incubated at 4 °C for at least 20 min before analysis. To determine the percentage of apoptotic cells, cells were treated with the indicated regimens for 48 h before being harvested, centrifuged, and resuspended in FACS buffer containing 1.25 μg/mL annexin V−FITC (PharMingen) and 0.1 mg/mL PI and incubated for 15 min at room temperature. The labeled cells (10,000 per sample) were then analyzed by FACScan (BD Biosciences) using the CellQuest software. Cells in the lower right dot-plot quadrant (PS, positive; PI, negative) were counted as apoptotic and have intact plasma membranes, whereas cells in the upper right dot-plot quadrant (PS, positive; PI, positive) have leaky membranes and can be either necrotic or late apoptotic. Statistical Analysis. Data are presented as mean ± SEM, whereas IC50 values were calculated as mean ± SD from at least three independent experiments. Differences between any mean values were analyzed by two-sided Student’s t test, and results were considered statistically significant at P < 0.05.

liver, and intestinal walls, thus contributing greatly to the oral bioavailability and distribution of therapeutic drugs.28,29 In cancer cells, ABCB1 confers resistance to a large number of conventional anticancer agents and newly developed targeted therapy drugs.23 We have previously identified that the effectiveness of BI 2536, the first selective inhibitor of Plk1, is reduced by the overexpression of ABC drug transporters.30 Here, we investigated the potential impact of ABCB1 overexpression on the effectiveness of GSK461364 in ABCB1-overexpressing cells. Surprisingly, we revealed that even though GSK461364 is structurally unrelated to BI 2536, the overexpression of ABCB1 led to reduced G2/M cell cycle arrest, cell apoptosis, and eventual chemosensitivity to GSK461364 in ABCB1-overexpressing cells. Moreover, we demonstrated that the chemosensitivity of ABCB1overexpressing cells to GSK461364 can be restored if cells were treated in combination with an inhibitor or a drug substrate of ABCB1, providing a rationale for development of new combination therapy based on Plk1 inhibitors and modulators of ABCB1.

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EXPERIMENTAL SECTION Reagents. DMEM, RPMI medium, fetal calf serum (FCS), trypsin-EDTA, penicillin, streptomycin, and PBS were purchased from Gibco, Invitrogen. MTT dye, Cell Counting Kit-8 (CCK-8), and all other chemicals were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Tariquidar was a generous gift from Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD, USA). GSK461364 (99% purity by HPLC, Chiral HPLC) was purchased from ChemieTek (Indianapolis, IN, USA). Cell Lines and Culture Conditions. KB-3-1 and ABCB1overexpressing sublines KB-8-5-11, KB-C-1, and KB-V-1 as well as OVCAR-8 and NCI-ADR-RES cells were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine, and 100 units of penicillin/streptomycin/mL. KB-V-1 cells were maintained in media containing 1 μg/μL vinblastine.31 pcDNA3.1-HEK293 and MDR19-HEK293 cells were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine, 100 units of penicillin/ streptomycin/mL, and 2 mg/mL G418.25 All cell lines were generous gifts from Dr. Suresh V. Ambudkar (National Cancer Institute, NIH, Bethesda, MD, USA), and were all maintained at 37 °C in 5% CO2 humidified air. Cytotoxicity Assay. CCK-8 and MTT assays were used to determine the general sensitivities of cells to the tested chemicals as described previously.25 For the determination of IC50 values, cells were plated into 96-well plates at 37 °C for 24 h before addition of drug to make a final concentration range between 0 and 100 μM, incubated for an additional 72 h before processing for analysis. For the reversal of cytotoxicity assays, a nontoxic concentration of ABCB1 modulator was added into the cytotoxicity assay, and the extent of reversal was then calculated on the basis of the relative resistance values. Immunoblotting. Antibodies C219 (1:1000) and anti-αtubulin (1:2000) were used to detect ABCB1 and tubulin as positive control for Western blotting, respectively. The secondary antibody used was the horseradish peroxidase-conjugated goat anti-mouse IgG (1:10000). Signals were detected as described previously.32 Fluorescent Drug Accumulation Assay. ABCB1-mediated efflux assay was carried out using a FACSort flow cytometer equipped with CellQuest software (Becton-Dickinson) as described previously.33 The effect of GSK461364 and tariquidar

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RESULTS Cells with Overexpression of Human ABCB1 Are Resistant to GSK461364. In order to evaluate the effect of ABCB1 on the efficacy of GSK461364, the cytotoxicity of GSK461364 was tested against drug sensitive and ABCB1-overexpressing drug resistant cell lines. We found that GSK461364 was effective against drug sensitive human epidermal KB-3-1 cells and human ovary OVCAR-8 cells, with calculated IC50 values of 0.09 μM and 1.56 μM, respectively (Table 1). By dividing the IC50 value of the ABCB1-overexpressing subline by the IC50 value of the respective parental line, the resistance factor (RF) value here represents the degree of resistance to GSK461364 caused by the presence of ABCB1. We discovered that the KB-V-1 cancer cells, a drug resistant ABCB1-overexpressing subline of the drug sensitive human epidermal KB-3-1 cells, were significantly more resistant to doxorubicin, an established substrate 3728

dx.doi.org/10.1021/mp500492r | Mol. Pharmaceutics 2014, 11, 3727−3736


Article

with the parental KB-3-1 cells (Table 1). Similarly, we found that the ABCB1-overexpressing human ovary NCI-ADR-RES cells were also more resistant to GSK461364 as compared with the parental OVCAR-8 cells (RF ≈ 4, P < 0.01). In order to confirm our findings further, we examined the cytotoxicity of GSK461364 in human embryonic kidney HEK293 cells and HEK293 cells transfected with human ABCB1, MDR19HEK293 cells. As shown in Figure 1D,E, the ABCB1transfected MDR19-HEK293 cells were significantly more resistant to doxorubicin and GSK461364, with resistant factors of approximately 55 and 17 (P < 0.01), respectively. GSK461364 Inhibits ABCB1-Mediated Efflux of CalceinAM. Knowing that ABCB1 confers resistance to GSK461364, we next evaluated how GSK461364 affects ABCB1-mediated efflux of calcein-AM, an established fluorescent substrate of ABCB1,25 in short-term drug accumulation assays as described in the Experimental Section. The efflux assays were carried out in the absence (solid lines) or presence of GSK461364 (shaded, solid lines) or a reference ABCB1 inhibitor tariquidar36 (dotted lines) in KB cancer cell lines, as well as in pcDNA-HEK293 and MDR19-HEK293 cells. At 10 μM, GSK461364 inhibited ABCB1-mediated transport of calcein-AM from ABCB1expressing KB-V-1 (Figure 2A) and MDR19-HEK293 cells (Figure 2B) to the same extent as 3 μM tariquidar. Moreover, we discovered that GSK461364 inhibited the function of ABCB1 in KB-V-1 cancer cells in a concentration dependent manner, with calculated IC50 value of approximately 9 μM (Figure 2C). Next, we examined the effect of GSK461364 on ABCB1-mediated resistance to doxorubicin in ABCB1-overexpressing MDR19HEK293 cells. Previous studies have shown that some drug substrates of ABCB1 can reverse drug resistance in ABCB1overexpressing cells by competing with the transport of another drug substrate.25,37,38 In the absence of GSK461364 or tariquidar, we observed a significant doxorubicin resistance mediated by ABCB1 (relative resistance value of approximately 58) in MDR19HEK293 cells. In the presence of tariquidar at 1 μM, the relative resistance value was reversed from 58 to approximately 3, whereas GSK461364 at tested concentrations had no significant effect on ABCB1-mediated doxorubicin resistance in MDR19-HEK293 cells (Table 2). GSK461364 Stimulates ABCB1 ATPase Activity. Next, we examined the effect of GSK461364 on ABCB1-mediated ATP hydrolysis as described in the Experimental Section. GSK461364 stimulated the Vi-sensitive ABCB1 ATPase activity in a concentration dependent manner to approximately 5-fold maximum stimulation, and the concentration required for 50% maximal stimulation was approximately 100 nM (Figure 3). Our results here showed that GSK461364 stimulated ABCB1 ATPase activity in the same manner as other well-established substrates of ABCB1. Tariquidar and Nilotinib Can Restore GSK461364Induced G2/M Cell Cycle Arrest and Apoptosis in ABCB1-Overexpressing Cancer Cells. Given that G2/M cell cycle arrest is one of the key signatures of Plk1 inhibitors,9,18,39 and that KB-V-1 cells were the least sensitive to GSK461364 among all cell lines tested (Table 1), we decided to investigate the role of ABCB1 overexpression on GSK461364-induced cell cycle arrest in KB-V-1 cells. Within 24 h, we observed substantial cell cycle arrest in the G2/M phase induced by GSK461364 (500 nM) in drug sensitive KB-3-1 cells (upper panels), from 12% basal level to approximately 86% (Figure 4A,B). In contrast, GSK461364 had no significant effect on G2/M arrest in ABCB1-overexpressing KB-V-1 cells (Figure 5B, lower left panel) at the same concentration.

Table 1. Sensitivity of Various Cell Lines to Polo-like Kinase 1 Inhibitor GSK461364 cell line KB-3-1 KB-8-5-11 KB-C-1 KB-V-1 OVCAR-8 NCI-ADR-RES pcDNA-HEK293 MDR19-HEK293

cancer origin epidermal epidermal epidermal epidermal ovary ovary

transporter expressed ABCB1 ABCB1 ABCB1 ABCB1 ABCB1

GSK461364 IC50a (μM)

RFb

0.09 ± 0.01 0.41 ± 0.07** 1.01 ± 0.22** 1.83 ± 0.37** 1.56 ± 0.35 6.15 ± 1.58** 0.07 ± 0.03 1.23 ± 0.29**

1.00 4.56 11.22 20.33 1.00 3.94 1.00 17.57

IC50 values are mean ± SD calculated from dose−response curves obtained from three independent experiments using cytotoxicity assay as described in Experimental Section. **P < 0.01. bRF: resistance factor. RF values were calculated by dividing IC50 values of ABC transporter overexpressing cells by IC50 values of respective parental cells. a

Figure 1. Overexpression of human ABCB1 reduces the cytotoxicity of GSK461364. (A) The representative immunoblots of ABCB1 in total cell lysate protein (10 μg) in parental human epidermal KB-3-1, ABCB1-overexpressing resistant KB-V-1 cells, drug sensitive pcDNAHEK293, and ABCB1-transfected MDR19-HEK293 cells. α-Tubulin was used as an internal control for equal loading. Sensitivity of KB-3-1 (○) and KB-V-1 (●) cells to (B) doxorubicin or (C) GSK461364, as well as pcDNA-HEK293 (○) and MDR19-HEK293 (●) cells to (D) doxorubicin or (E) GSK461364, was determined as described previously.25 Points, mean from at least three independent experiments; bars, SEM.

of ABCB1,35 and to Plk1 inhibitor GSK461364 (RF value of 20, P < 0.01) (Figure 1A−C). In addition, ABCB1-overexpressing KB-8-5-11 and KB-C-1 cells were also more resistant to GSK461364 (RF values of 5 and 11, P < 0.01) as compared 3729



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Figure 2. GSK461364 inhibits ABCB1-mediated efflux of calcein-AM. The accumulation of fluorescent calcein in (A) KB-3-1 (left panel) and KB-V-1 (right panel) and (B) pcDNA-HEK293 (left panel) and MDR19-HEK293 (right panel) cells was measured in the presence (shaded, solid lines) or absence (solid lines) of 10 μM GSK461364 or 3 μM of a reference ABCB1 inhibitor tariquidar (dotted lines), and analyzed immediately by flow cytometry as described in the Experimental Section. Representative histograms of three independent experiments are shown. (C) Concentrationdependent inhibition of ABCB1-mediated calcein-AM efflux by GSK461364 in KB-V-1 cells was also determined. Data points represent the mean ± SEM from at least three independent experiments. The IC50 values were calculated as the concentration that inhibited the efflux to 50% of the control values.

(Figure 4B, lower middle panel) and nilotinib (Figure 4B, lower right panel). The overall effect of GSK461364, tariquidar, and nilotinib on the phases of cell cycle in KB cells is summarized in Table 3. Since GSK461364-induced G2/M arrest in KB-V-1 cells can be reversed by inhibiting the function of ABCB1, we next examined the effect of tariquidar and nilotinib on GSK461364induced apoptosis in KB cancer cells. The basal level of apoptosis

Next, we investigated the effect of competitive inhibitor or substrate of ABCB1 on GSK461364-induced G2/M arrest in KB cancer cells. We discovered that in KB-V-1 cells, while the reference ABCB1 inhibitor tariquidar (1 μM) and ABCB1 substrate nilotinib (5 μM) alone had no effect on cell cycle phase distribution (Figure 4A), the reduced GSK461364-induced G2/M cell cycle arrest was significantly restored by tariquidar 3730



Article

multiple human cancer cell lines, and shows favorable pharmacological profiles in phase I clinical trials,2,19,21 warrants further development. Unfortunately, as for most chemotherapy drugs, the risk of developing drug resistance to Plk1 inhibitors can present a significant therapeutic challenge to patients and should be investigated. Given that the overexpression of ABCB1 can often lead to reduced oral bioavailability and CNS penetration of drugs41 as well as the development of MDR in cancers,29 and that ABCB1 was reported to mediate resistance to the first selective Plk1 inhibitor BI 2536,30 we decided to examine the potential impact of ABCB1 overexpression on the cellular effectiveness of GSK461364. Considering that GSK461364 is structurally unrelated to BI 2536, we did not expect ABCB1 to affect GSK461364 in the same manner as BI 2536. However, we later discovered that the function of ABCB1 reduces the cellular efficacy of GSK461364 significantly. First, we found that even though the IC50 values of GSK461364 in drug sensitive cancer cells are consistent with the reported IC50 values,5,17 the ABCB1-overexpressing human cancer cells and ABCB1-transfected cells were significantly more resistant to their respective parental cells (Table 1). The KB-8-5-11, KB-C-1, and KB-V-1 are ABCB1-positive drug resistant sublines of human epidermal KB-3-1 cell line with increasing ABCB1 protein expression levels, and are resistant to various known drug substrates of ABCB1, including doxorubicin, when compared with KB-3-1 cells.25,30 Moreover, the RF values obtained for the KB cell lines suggested that the levels of resistance could be related to the amount of ABCB1 protein expression. Collectively, our result indicates that ABCB1 mediates resistance to GSK461364 in the same manner as to doxorubicin, but with lower RF values to GSK461364 than to doxorubicin (Figure 1). In order to gain more insights into the interaction between ABCB1 and GSK461364, we studied the effect of GSK461364 on ABCB1-mediated transport, drug resistance, and ATP hydrolysis in ABCB1-overexpressing cells. We found that even though GSK461364 can inhibit ABCB1-mediated transport of calceinAM in a concentration dependent manner (Figure 2), GSK461364 was unable to reverse ABCB1-mediated doxorubicin resistance at nontoxic concentrations (Table 2). Possible explanations may be that the nontoxic concentrations of GSK461364 tested were lower than what is required for GSK461364 to reverse ABCB1-mediated doxorubicin resistance, or that GSK461364 and doxorubicin may simply bind to different sites of the ABCB1 substrate binding site. Therefore, it is our opinion that, unlike some drug substrates of ABCB1,25,37,38 GSK461364 is not a therapeutic agent suitable for reversing ABCB1-mediated drug resistance. Knowing that the stimulation of ABCB1 ATP hydrolysis is coupled with ABCB1-mediated substrate transport,42,43 we measure the effect of GSK461364 on the vanadate-sensitive ABCB1 ATPase activity. The fact that GSK461364 stimulated ABCB1 ATPase activity in a concentration-dependent manner supports the idea that GSK461364 is a substrate of ABCB1 (Figure 3). Next, we investigated the effect of ABCB1 overexpression on GSK461364-induced G2/M cell cycle arrest, since the induction of G2/M arrest is one of the key signatures of Plk1 inhibitors.9,18,39 Our data showed clearly that the G2/M cell cycle arrest and subsequent cell apoptosis induced by GSK461364 were significantly reduced in ABCB1-overexpressing KB-V-1 cells compared to the ABCB1-negative KB-3-1 cells (Figure 4). Therefore, our data indicated that the function of ABCB1 leads to reduced effect of GSK461364 in ABCB1-overexpressing cells. Since ABCB1mediated drug resistance in cancer cells can often be reversed by

Table 2. Effect of GSK461364 on ABCB1-Mediated Doxorubicin Resistance in ABCB1-Transfected MDR19-HEK293 Cells IC50a (nM) drug

concn (nM)

pcDNAHEK293

MDR19HEK293

RRb

doxorubicin + GSK461364 + GSK461364 + GSK461364 + tariquidar

5 10 20 1000

6.03 ± 1.14 4.69 ± 0.75 4.14 ± 0.57 4.40 ± 0.65 5.53 ± 0.81

348.06 ± 65.41 247.85 ± 48.67 265.84 ± 47.69 271.36 ± 55.30 17.01 ± 2.46***

57.72 52.85 64.21 61.67 3.08

IC50 values are mean ± SD calculated from dose−response curves obtained from three independent experiments using cytotoxicity assay as described in the Experimental Section. ***P < 0.001. bRR: relative resistance. RR values were obtained by dividing IC50 values of ABC transporter-overexpressing cancer cells by IC50 values of respective sensitive cells. a

Figure 3. GSK461364 stimulates Vi-sensitive ABCB1 ATPase activity. Effect of 0−20 μM GSK461364 and 0−2 μM (inset) on Vi-sensitive ABCB1 ATPase activity was measured as described previously.30 Points, mean from at least three independent experiments; bars, SD.

in KB was approximately 13% (Figure 5A), but after exposure of cells to GSK461364 (500 nM) for 48 h, the percentage of apoptotic cells increased greatly in the KB-3-1 cells (Figure 5B, upper left panel), while a considerably lesser effect was observed for KB-V-1 cells (Figure 5B, lower left panel). Treatment with GSK461364 resulted in approximately 72% of early and late apoptosis in KB-3-1 cells in contrast to 23% in KB-V-1 cells. However, we found that, in the presence of tariquidar (1 μM) or nilotinib (5 μM), the induction of apoptosis by GSK461364 increased significantly in resistant KB-V-1 cells to approximately 85% and 78%, respectively (Figure 5B, lower panels). Moreover, the restored apoptosis in KB-V-1 cells by tariquidar (1 μM) and nilotinib (5 μM) can also be translated into increased sensitivity to GSK461364 in KB-V-1 cells, reducing the RF value from 20 to approximately 1 and 4, respectively (Table 4). We confirmed our finding by examining the effect of tariquidar and nilotinib on the sensitivity of ABCB1-transfected MDR19-HEK293 cells to GSK461364. In the presence of tariquidar or nilotinib, the sensitivity of MDR19-HEK293 to GSK461364 was also improved from RF value of 18 to 2 and 4, respectively (Table 4).

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DISCUSSION Plk1 is now considered as a good target for cancer chemotherapy because Plk1 overexpression is found in many tumor types, and its inhibition leads to induction of G2/M cell cycle arrest, subsequent onset of apoptosis, and eventual cell death.6,18,39,40 GSK461364, a selective inhibitor of Plk1 that blocks the activity of Plk1 at low concentrations, inhibits the proliferation of 3731



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Figure 4. Reduced level of GSK461364-induced G2/M cell cycle arrest in ABCB1-overexpressing KB-V-1 human cancer cells can be restored by inhibiting the function of ABCB1. Human KB-3-1 (top panels) and ABCB1-overexpressing KB-V-1 (lower panels) cells were plated and maintained in the absence or presence of GSK461364 alone, or in combination with a reference inhibitor or a competitive drug substrate of ABCB1 for 24 h before harvest for cell cycle analysis. (A) KB cells were treated with either DMSO (left panels), 1 μM tariquidar (middle panels), or 5 μM nilotinib (right panels) or (B) 500 nM GSK461364 (left panels), combination of 500 nM GSK461364 and 1 μM tariquidar (middle panels), or combination of 500 nM GSK461364 and 5 μM nilotinib (right panels). Representative histograms of three independent experiments are shown.

inhibiting the function of ABCB1,38,44 we explored the possibility of restoring the drug sensitivity of GSK461364 in ABCB1-

overexpressing cells using a reference ABCB1 inhibitor tariquidar or nilotinib, a drug substrate of ABCB1. 3732



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Figure 5. Effect of ABCB1 inhibitor or ABCB1 substrate on cancer cell apoptosis induced by GSK461364. KB-3-1 (top panels) and KB-V-1 (lower panels) cells were isolated and analyzed 48 h after treatment with GSK461364 alone, or in combination with tariquidar or nilotinib. (A) KB cells were treated with either DMSO (left panels), 1 μM tariquidar (middle panels), or 5 μM nilotinib (right panels) or (B) 500 nM of GSK461364 (left panels), combination of 500 nM GSK461364 and 1 μM tariquidar (middle panels), or combination of 500 nM GSK461364 and 5 μM nilotinib (right panels). Representative histograms of three independent experiments are shown. Apoptotic cells were quantified by flow cytometry after staining with annexin V-FITC and PI. Live cells (annexin V− PI−) appear in the lower left quadrant; early apoptotic cells (annexin V+ PI−) appear in the lower right quadrant; necrotic or late apoptotic cells (annexin V+ PI+) appear in the upper right quadrant. Cells in the upper right quadrant were confirmed by fluorescence microscopy to be late apoptotic and not necrotic cells. Data shown are representative of three independent experiments.

In our study, GSK461364 at 500 nM induced substantial G2/M cell cycle arrest and apoptosis in drug sensitive KB-3-1 cells, but not in drug resistant ABCB1-overexpressing KB-V-1 cells.

However, both tariquidar and nilotinib were able to inhibit the function of ABCB1 sufficiently to restore GSK461364-induced G2/M cell cycle arrest and apoptosis in KB-V-1 cells to a comparable 3733



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Table 3. Percentage Distribution of Cells in Cycle Phasesa

In summary, our study showed that the overexpression of human ABCB1 significantly reduces the effect of GSK461364induced G2/M cell cycle arrest, apoptosis and cytotoxicity, indicating that future GSK461364 treatment will likely be less effective against cancers with higher ABCB1 expression level. Furthermore, considering the wide tissue distribution of ABCB1 (such as the BBB and intestinal walls), it will not be surprising if ABCB1 affects the disposition of GSK461364 significantly as well. More importantly, we showed that the action of GSK461364 and reduced sensitivity of ABCB1-overexpressing cells to GSK461364 can be significantly restored by inhibiting the function of ABCB1. Therefore, in order to achieve a better therapeutic outcome, combination therapy of GSK461364 with a competitive inhibitor or drug substrate of ABCB1 should be further investigated as a potential treatment approach for patients.

cell cycle phase G1 (%) control + tariquidar + nilotinib + GSK461364 + GSK461364 + tariquidar + GSK461364 + nilotinib control + tariquidar + nilotinib + GSK461364 + GSK461364 + tariquidar + GSK461364 + nilotinib

KB-3-1 59.61 ± 5.71 64.31 ± 2.34 71.83 ± 3.86 2.66 ± 1.44 3.14 ± 1.54 5.54 ± 3.61 KB-V-1 61.15 ± 2.17 64.77 ± 3.82 65.71 ± 3.50 60.68 ± 6.49 11.57 ± 5.61 14.94 ± 6.66

S (%)

G2/M (%)

28.53 ± 4.20 24.66 ± 1.52 18.14 ± 3.16 10.89 ± 4.85 10.98 ± 5.09 16.28 ± 0.34

11.86 ± 1.79 11.03 ± 1.21 10.03 ± 1.03 86.45 ± 5.96 85.87 ± 6.11 78.19 ± 3.40

25.42 ± 1.17 24.46 ± 3.58 23.05 ± 3.03 24.33 ± 6.14 13.97 ± 1.38 25.22 ± 7.38

13.42 ± 1.97 10.77 ± 0.25 11.24 ± 0.59 14.99 ± 4.11 74.46 ± 6.50 59.84 ± 8.07

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AUTHOR INFORMATION

Corresponding Author

a

*259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: 886-3-2118800, ext 3754. Fax: 886-3-2118700. E-mail: [email protected].

The percentage values were calculated from three independent experiments.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Susan Bates (National Cancer Institute, NIH), for generously providing tariquidar and Dr. Suresh V. Ambudkar (National Cancer Institute, NIH) for KB, NCI-ADRRES, and MDR19-HEK293 cells. This work was supported by funds from the Chang Gung Medical Research Program (CMRPD1D0151) and Ministry of Science and Technology of Taiwan (MOST-103-2320-B-182-011).

Figure 6. Schematic showing ABCB1 reduces the effectiveness of GSK461364 in resistant cancer cells. The function of ABCB1 decreases the efficacy of GSK461364 (GSK) to selectively inhibit Polo-like kinase 1 (Plk1), which leads to reduced level of G2/M cell cycle arrest and apoptosis in ABCB1-overexpressing cancer cells.

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ABBREVIATIONS USED MDR, multidrug resistance; ABC, ATP-binding cassette; Plk1, Polo-like kinase 1; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide

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level as in KB-3-1 cells (Figures 4 and 5). It is worth noting that although tariquidar and nilotinib had no significant effect on apoptosis of KB cells, we observed a more pronounced increase in late apoptosis in KB-V-1 cells when GSK461364 was used in combination with either tariquidar or nilotinib (Figure 5). This hypersensitivity appeared to be KB-V-1 specific, as we do not observe the same phenomenon when ABCB1-overexpressing NCI-ADRRES cells were treated with the same regimen (data not shown). Moreover, our reversal data was in accordance with the cell cycle and apoptosis data, as tariquidar and nilotinib were able to reverse ABCB1-mediated GSK461364 resistance in both ABCB1-overexpressing KB-V-1 cells and ABCB1-transfected MDR19-HEK293 cells (Table 4).

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Table 4. Effect of ABCB1 Modulators on the Chemosensitivity of GSK461364 in ABCB1-Overexpressing Cell Lines IC50a (μM) cell line KB-3-1 KB-V-1 HEK293 MDR19

transporter overexpressed ABCB1 ABCB1

GSK461364

GSK461364 + tariquidar (1 μM)

GSK461364 + nilotinib (0.5 μM)

0.09 ± 0.01 1.83 ± 0.37 0.07 ± 0.03 1.23 ± 0.29

0.27 ± 0.06** 0.14 ± 0.03** 0.05 ± 0.01 0.11 ± 0.03**

0.11 ± 0.03 0.41 ± 0.05** 0.07 ± 0.02 0.29 ± 0.06**

IC50 values are mean ± SD calculated from dose−response curves obtained from three independent experiments using cytotoxicity assay as described in the Experimental Section. **P < 0.01.

a

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Overexpression of Human ABCB1 in Cancer Cells Leads to Reduced

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