Sensitization of ABCG2-overexpressing cells to ...

Cancer Letters 279 (2009) 74–83

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Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2 Chun-ling Dai a, Yong-ju Liang a, Yan-sheng Wang a,b, Amit K. Tiwari c, Yan-yan Yan a, Fang Wang a, Zhe-sheng Chen c, Xiu-zhen Tong b, Li-wu Fu a,* a

State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China c Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, New York, NY 11439, USA b

a r t i c l e

i n f o

Article history: Received 16 November 2008 Received in revised form 15 January 2009 Accepted 18 January 2009

Keywords: Multidrug resistance ABCG2/BCRP Tyrosine kinase inhibitor Sunitinib

a b s t r a c t Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5 lM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [3H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ATP-binding cassette (ABC) transporter proteins pump a wide range of structurally and functionally unrelated drugs currently used in cancer chemotherapy with the energy of ATP hydrolysis, which play a key role in the development of multidrug resistance (MDR). Overexpression of ABC transporters is a significant impediment to successful cancer treatment. In the human genome, 48 different ABC transporters have been identified and divided into seven * Corresponding author. Tel.: +86 20 873 431 63; fax: +86 20 873 431 70. E-mail address: [email protected] (L.-w. Fu). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.01.027

subfamilies (A–G) based on sequence similarities [1]. The P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are the major members of the ABC transporters leading to MDR in cancer cells [1]. ABCG2/BCRP, also referred to mitoxantrone resistanceassociated protein (MXR) and placenta-specific ATP-binding cassette transporter (ABCP), was identified independently from drug selected human breast cancer cells (MCF-7) [2], human colon carcinoma cells (S1-M180) [3] and human placenta [4], respectively. Molecular characterization revealed that ABCG2 mRNA encodes a 72.6 kDa membrane protein composed of 655 amino acids. In contrast to ABCB1, which has 12 transmembrane

C.-l. Dai et al. / Cancer Letters 279 (2009) 74–83

domains and two ATP-binding sites [5], ABCG2 is a half-ABC transporter and contains only six transmembrane domains and one ATP-binding site [4]. ABCG2 may form a homodimer to become functionally active [6]. Overexpression of ABCG2 is associated with resistance to a wide range of different anticancer agents including doxorubicin, topotecan, SN-38, mitoxantrone and methotrexate [3,5,7–9]. More recently, ABCG2 has also been shown to confer resistance to some purine analogues such as 9-(2-phosphonylmethoxyethyl) adenine and cladribine [10] and transport some endogenous compound such as sulfate conjugates [11]. It is also reported that some mutations in the open reading frame of ABCG2 are associated with resistance to some anticancer drugs [12]. Wild-type ABCG2, with an arginine at position 482 (R482), facilitated efficient transport of mitoxantrone, but not rhodamine 123 or doxorubicin. In contrast, cells carrying a glycine (R482G) or a threonine (R482T) at position 482 were able to transport rhodamine 123 and doxorubicin, while also maintained their ability to transport mitoxantrone. The ABCG2 variants were found in drug-resistant S1-M1-80 (R482G) and MCF-7 AdVp3000 (R482T) but not in the parental S1 and MCF-7 cell lines, suggesting that these were acquired mutations resulting from drug selection. Therefore, it is possible that certain kinds of single nucleotide polymorphisms (SNPs) of ABCG2 may alter its function and, consequently, affects the disposition of substrate drugs. Tyrosine kinase inhibitors (TKIs) exert their action through competition with ATP for binding at the catalytic domain of tyrosine kinases. In vitro studies using biochemical and cell assays showed that TKIs also interact with and modulate the function of the ABC transporters such as ABCG2, ABCB1 and ABCC1 [13,14]. Canertinib (CI-1033) is a HER family TKI that has been shown to enhance the cytotoxicity of topotecan and SN-38 through inhibition of ABCG2-mediated drug efflux in cancer cells [15]. Imatinib mesylate is a TKI of BCR-ABL, platelet-derived growth factor receptor and stem cell factor/c-Kit and has been observed to reverse ABCG2-mediated resistance of topotecan and SN-38 [16]. Gefitinib, an epidermal growth factor receptor (EGFR) TKI, has been observed to directly inhibit the function of ABCB1 in multidrug resistant cancer cells [17] and to reverse ABCG2-mediated MDR in vitro [18]. In vivo studies indicate that gefitinib modulates the function of ABCB1 and ABCG2 [19]. In our previous study, we also found that erlotinib and lapatinib were also able to antagonize ABCB1- and ABCG2-mediated MDR [20,21]. Sunitinib malate (SUTENTÒ; Pfizer Inc., New York) is an oral, multi-targeted receptor tyrosine kinase inhibitor that selectively and potently inhibits vascular endothelial growth factor receptors (VEGFR-1, -2 and -3), platelet-derived growth factor receptors (PDGFR-a and -b), stem cell factor (KIT), colony-stimulating factor receptor type 1 (CSF-1R), FMS-like tyrosine kinase-3 receptor (FLT3), and glial cell-line derived neurotrophic factor receptor (rearranged during transfection; RET) [22–24]. It is conceivable that sunitinib may inhibit the functions of ABC transporters by binding to their ATP-binding sites. These have spurred on efforts to investigate whether sunitinib can enhance the efficacy of conventional chemotherapeutic drugs via interaction with ABC transporters in MDR cancer cells.

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2. Materials and methods 2.1. Chemicals and reagents 3-(4,5-dimethylthiazol-yl)-2,5-diphenylrazolium bromide (MTT), vincristine, doxorubicin, 6-mercaptopurine (6-MP), topotecan and rhodamine 123 were products of Sigma Chemical Co. [3H]-methotrexate (23 Ci/mmol) was purchased from Moravek Biochemicals, Inc. Sunitinib malate was product of Pfizer Inc. (New York, USA). Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 were products of Gibco BRL. Monoclonal antibody BXP-21 (against ABCG2) was product of Chemicon International Inc. Anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (sc-20357) was products of Santa Cruz Biotechnology Inc. Anti-MAPK1/2 (Erk1/2), phosphorylated extracellular signal-regulated kinase, and phosphorylated Akt antibodies were purchased from Kangchen Co. Akt antibody was a product of Cell Signaling Technology Inc. Other routine laboratory reagents were obtained from commercial sources of analytical grade. 2.2. Cell lines and cell culture The following cell lines were cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum at 37 °C in the presence of 5% CO2: the colon carcinoma cell lines S1 and its mitoxantrone-selected derivative ABCG2-overexpressing S1-M1-80; the human breast carcinoma cell lines MCF-7 and its doxorubicin-selected derivative ABCB1 overexpressing MCF-7/adr; the human epidermoid carcinoma cell lines KB-3-1 and its VCR-selected derivative ABCC1 overexpressing KB-CV60 or doxorubicin-selected derivative ABCC1 overexpressing C-A120; the human lung squamous carcinoma cell lines SW1573 and its doxorubicin-selected derivative LRP overexpressing SW1573/ 2R120; HEK293/pcDNA3.1 and ABCG2-482-R5 cells were established by selection with G418 after transfecting HEK293 with either empty pcDNA3.1 vector or pcDNA3.1 vector containing full length of ABCG2 coding arginine (R) at amino acid 482 position, respectively, and were cultured in medium with 2 mg/ml of G418 [25]. 2.3. Cell proliferation assays Cytotoxicity tests were performed using the MTT assays as described [26]. Briefly, cells were distributed evenly into 96-well microtiter plates. For determining the toxicity of sunitinib, various concentrations of sunitinib diluted with medium were added into the wells; for reversal experiments, sunitinib was added to the medium with full range concentrations of topotecan and doxorubicin in S1 or S1M1-80, doxorubicin in MCF-7 or MCF-7/adr, vincristine in KB-3-1 or KB-CV60, doxorubicin in KB-3-1 or C-A120, and 6-MP in SW1573 or SW1573/2R120 cells, respectively. Cell viability was measured by Model 550 Microplate reader (BIO-RAD, USA) at 540 nm with 655 nm as reference filter. The concentrations required to inhibit growth by 50% (IC50) were calculated from survival curves using the Bliss method [26]. Fold of resistance was calculated by dividing the IC50 for the MDR cells by that of the parental sensitive

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cells. The degree of the reversal of MDR (fold-reversal) was calculated by dividing the IC50 for cells with the anticancer drug in the absence of sunitinib by that obtained in the presence of sunitinib. 2.4. Rhodamine 123 and doxorubicin accumulation The effect of sunitinib on the cellular accumulation of rhodamine 123 or doxorubicin in S1 and S1-M1-80 cells was analyzed by flow cytometry as previously described [21]. In detail, S1 and S1-M1-80 cells (5 105 cells each) were incubated in six-well plates and allowed to attach overnight. The cells were treated with 0.625, 1.25 and 2.50 lM sunitinib or vehicle at 37 °C for 3 h in the medium, and then 5 lg/ml rhodamine 123 was added and further incubated for 30 min or 10 lM doxorubicin was added and incubated for another 3 h. Subsequently, the cells were collected, centrifuged and washed twice with ice-cold PBS, and subjected to fluorescence analysis by flow cytometry (Beckman Coulter, Cytomics FC500, USA). To measure ABCG2 function as a drug efflux transporter, rhodamine 123 was used as a fluorescent probe for predicting the activity of an agent reversing ABCG2-mediated resistance. After incubation with rhodamine 123 containing medium (5 lg/mL) for 30 min at 37 °C, the cells were washed twice with ice-cold DMEM, and then cultured in rhodamine 123 free medium in the presence or absence of 2.5 lM sunitinib at 37 °C. Then the cells were harvested at the indicated times and cellular rhodamine 123 accumulation was analyzed in 10,000 cells by flow cytometry (Beckman Coulter, Cytomics FC500, USA). 2.5. In vitro transport assays Transport assays were performed essentially using the rapid filtration method as previously described [20,27]. Membrane vesicles were prepared by the nitrogen cavitation method as previously described [20]. Membrane vesicles were incubated with 5 lM sunitinib for 1 h on ice, and then transport reactions were carried out at 37 °C for 10 min in a total volume of 50 ll medium (membrane vesicles 10 lg, 0.25 M sucrose, 10 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 4 mM ATP or 4 mM AMP, 10 mM phosphocreatine, 100 lg/ml creatine phosphokinase, and 0.5 lM [3H]-methotrexate). Reactions were stopped by the addition of 3 ml of ice-cold stop solution (0.25 M sucrose, 100 mM NaCl and 10 mM Tris–HCl, pH 7.4). During the rapid filtration step, samples were passed through 0.22 lm GVWP filters (Millipore Corporation, Billerica, MA) presoaked in the stop solution. The filters were washed three times with 3 ml of ice-cold stop solution. Radioactivity was measured by the use of a liquid scintillation counter. 2.6. Reverse transcription-PCR After treatment with sunitinib, total RNA was isolated from cell cultures using Trizol Reagent (Invitrogen) according to the manufacturer instruction. cDNA synethesis reaction was performed with reverse transcriptase (Promega Corp.). Oligonucleotide primers for ABCG2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthe-

sized commercially (Invitrogen Co., China). The PCR assays were performed using primers specific for ABCG2: 50 -TGGCTGTCATGGCTTCAGTA-30 (sense) and 50 -GCCACGT GATTCTTCCACAA-30 (antisense); for GAPDH: 50 -CACCCTGT TGCTGTAGCC-30 (sense) and 50 -CTTTGGTA TCGTGGAAG GA-30 (antisense). The expected length of PCR product for BCRP is 235 bp and for GAPDH is 475 bp, respectively. Using the GeneAmp PCR system 9700 (PE Applied Biosystem, Foster City, CA), reactions were carried out for ABCG2 and GAPDH at 94 °C for 3 min for initial denaturation, and then at 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min. After 35 cycles of amplification, additional extensions were done at 72 °C for 10 min. Products were resolved and examined by 1.5% agarose gel electrophoresis as described [26]. 2.7. Western blot analysis To determine whether sunitinib affects the expression of ABCG2, the cells were incubated with 0.625, 1.25 and 2.5 lM sunitinib for 48 h or with 1.25 lM sunitinib for 24, 48 and 72 h, respectively. To test whether sunitinib blocks Akt or Erk1/2 phosphorylation, the cells were incubated with different concentrations of sunitinib (0.625–2.5 lM) for different periods of time (24–72 h). After treatment, the cells were harvested and rinsed twice with ice-cold PBS and total cell lysates were collected with cell lysates buffer (1 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 lg/ml phenylmethylsulfonyl fluoride, 10 lg/ml aprotinin, 10 lg/ml leupeptin) for 30 min with gentle rocking and clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. Equal amounts (100 lg of protein) of cell lysates were boiled for 20 min and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. After being incubated in blocking solution containing 5% non-fat milk in TBST buffer (10 mM Tris–HCL (pH 8.0), 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature, membranes were immunoblotted overnight with the appropriately diluted primary monoclonal antibodies against ABCG2, Akt, p-Akt, Erk1/2, p-Erk1/2, or GAPDH at 4 °C. Then the membranes were washed thrice with TBST buffer and incubated at room temperature for 2 h with HRP-conjugated secondary antibody at 1:5000 dilutions. After thrice wash with TBST buffer, the protein–antibody complex were visualized by the enhanced Phototope TM-HRP Detection Kit (Cell Signaling, USA) and exposed to Kodak medical X-ray processor (Kodak, USA) [21]. 2.8. Statistical analysis All experiments were done at least thrice. Statistical analysis was done by Student’s t test analyses. The significance was determined at p < 0.05. 3. Results 3.1. Sunitinib reverses ABCG2-mediated MDR in vitro To examine the effect of sunitinib on the reversal of ABCB1-, ABCG2-, ABCC1- and LRP-mediated MDR in cancer cells, we first determined the cytotoxicity of sunitinib alone in different cell lines with the MTT assay.

C.-l. Dai et al. / Cancer Letters 279 (2009) 74–83 More than 90% of cells were viable up to 2.5 lM of sunitinib in all the cell lines used in this study (Fig. 1). So we used 0.625, 1.25 and 2.5 lM sunitinib to reverse MDR in vitro. The IC50 concentration for S1 cells to topotecan or doxorubicin were 0.135 ± 0.056 lM and 0.165 ± 0.009 lM, respectively; however, the concentration of topotecan or doxorubicin required to inhibit S1-M1-80 cells by 50% was about 5.132 ± 2.024 and 6.845 ± 1.035 lM (Table 1). Thus, overexpression of ABCG2 resulted in a significant resistance to topotecan (38-fold) and doxorubicin (41-fold), respectively. The sensitivity to topotecan or doxorubicin was unchanged when S1 cells were coincubated with sunitinib at concentration up to 2.5 lM (Table 1). In contrast, S1-M1-80 cells were sensitized by low concentration of sunitinib. At the 0.625, 1.25 and 2.50 lM sunitinib, the concentrations required to inhibit the growth of S1-M1-80 cells by 50% for topotecan were 1.411 ± 0.042, 0.472 ± 0.136 and 0.091 ± 0.012 lM, and for doxorubicin were 0.919 ± 0.185, 0.320 ± 0.032 and 0.181 ± 0.037 lM, respectively. These results suggested that sunitinib potently reverses ABCG2-mediated resistance to topotecan and doxorubicin in vitro.

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To evaluate transporter specificity, we examined the effects of sunitinib on ABCB1-, ABCC1- and LRP-mediated MDR in cancer cells. As shown in Table 1, sunitinib showed no significant effect on ABCB1-, ABCC1- and LRP-mediated drug resistance in MCF-7/adr, KB-CV60, C-A120 and SW1573/2R120 cell lines, respectively. In addition, a small synergetic effect was observed for the combination of sunitinib with doxorubicin in MCF-7 and MCF-7/adr cells (Table 1). Similar results were observed when sunitinib was coincubated with doxorubicin in KB-3-1 and C-A120 cells. Our data suggest that sunitinib probably specifically reverses ABCG2mediated MDR in drug resistance cancer cells.

3.2. ABCG2 does not confer resistance to sunitinib As shown in Fig. 1A, S1 and S1-M1-80 cells had similar sensitivity to sunitinib. S1-M1-80 cells was slightly more sensitive than S1 cells, and the IC50 concentration for sunitinib inhibiting the growth of S1-M1-80

Fig. 1. Cytotoxicity of sunitinib alone in the drug-sensitive and drug resistance cell lines. Cytotoxicity of sunitinib alone to drug-sensitive cell lines S1, KB-31, MCF-7 and SW1573, and ABCG2-overexpressing S1-M1-80, ABCC1-overexpressing C-A120 and KB-CV60, ABCB1-overexpressing MCF-7/adr, and LRPoverexpressing SW1573/2R120 cell lines was determined by MTT assay as described in Section 2. Data are the means ± SD of at least three independent experiments performed in triplicate.

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Table 1 Effect of sunitinib on reversing ABCG2-mediated drug resistance. S1

S1-M1-80 (ABCG2)

IC50 ± SD (lM) (fold-reversal) Topotecan +0.625 lM Sunitinib +1.25 lM Sunitinib +2.5 lM Sunitinib

0.135 ± 0.056 0.151 ± 0.059 0.134 ± 0.039 0.096 ± 0.064

(1.0) (0.9) (1.0) (1.4)

5.132 ± 2.024 1.411 ± 0.042* 0.472 ± 0.136** 0.091 ± 0.012**

(1.0) (3.6) (10.9) (56.4)

Doxorubicin +0.625 lM Sunitinib +1.25 lM Sunitinib +2.5 lM Sunitinib

0.165 ± 0.009 0.186 ± 0.014 0.195 ± 0.043 0.146 ± 0.023

(1.0) (0.9) (0.8) (1.1)

6.845 ± 1.035 0.919 ± 0.185** 0.320 ± 0.032** 0.181 ± 0.037**

(1.0) (7.4) (21.4) (37.8)

KB-3-1 Vincristine + 0.625 lM Sunitinib + 1.25 lM Sunitinib + 2.5 lM Sunitinib

0.0098 ± 0.0014 0.0079 ± 0.0015 0.0078 ± 0.0021 0.0048 ± 0.0015

KB-CV60 (ABCC1) (1.0) (1.2) (1.2) (2.0)

KB-3-1 Doxorubicin + 0.625 lM Sunitinib + 1.25 lM Sunitinib + 2.5 lM Sunitinib

0.444 ± 0.108 0.367 ± 0.097 0.189 ± 0.106* 0.120 ± 0.067*

0.196 ± 0.020 0.172 ± 0.022 0.166 ± 0.022 0.120 ± 0.018

0.396 ± 0.094 0.304 ± 0.003 0.207 ± 0.043* 0.105 ± 0.037**

22.3 ± 3.81 17.0 ± 1.75 15.3 ± 1.74 8.23 ± 1.72*

(1.0) (1.3) (1.4) (2.7)

SW1573/2R120 (LRP) (1.0) (1.1) (1.2) (1.6)

MCF-7 Doxorubicin + 0.625 lM Sunitinib + 1.25 lM Sunitinib + 2.5 lM Sunitinib

(1.0) (1.0) (1.3) (1.6)

C-A120 (ABCC1) (1.0) (1.2) (2.3) (3.7)

SW1573 6-MP + 0.625 lM Sunitinib + 1.25 lM Sunitinib + 2.5 lM Sunitinib

0.3591 ± 0.0552 0.3425 ± 0.0503 0.2699 ± 0.0504 0.2179 ± 0.0443

2.68 ± 0.58 2.46 ± 0.74 2.51 ± 0.61 2.25 ± 0.61

(1.0) (1.1) (1.1) (1.2)

MCF-7/adr (ABCB1) (1.0) (1.3) (1.9) (3.8)

31.8 ± 2.80 23.8 ± 1.58 12.2 ± 1.52* 7.81 ± 1.26**

(1.0) (1.3) (2.6) (4.1)

Cell survival was determined by MTT assay as described in Section 2. Data are the means ± SDs of at least three independent experiments performed in triplicate. The fold-reversal of MDR was calculated by dividing the IC50 for cells with the anticancer drug in the absence of inhibitor by that obtained in the presence of inhibitor. * p < 0.05, for values vs. that obtained in the absence of inhibitor. ** p < 0.01, for values vs. that obtained in the absence of inhibitor.

and S1 cells by 50% was 5.75 ± 0.23 lM and 8.75 ± 1.03 lM, respectively. Thus, overexpression of ABCG2 does not confer significant resistance to sunitinib.

3.3. Sunitinib modulates ABCG2-mediated transport The results above indicated that sunitinib could enhance the sensitivity of ABCG2-overexpressing cells to certain chemotherapeutic agents. The mechanism by which this occurs is unknown. Therefore, we examined its effects on rhodamine 123 and doxorubicin accumulation in ABCG2-expressing S1-M1-80 cells and parental S1 cells. Rhodamine 123 accumulation was significantly higher (11.5-fold) in the sensitive S1 cells than that of the ABCG2-expressing S1-M1-80 cells (Fig. 2A, B and C). In the absence of sunitinib, the level of rhodamine 123 accumulation was low in S1-M1-80 cells and sunitinib restored the level of rhodamine 123 accumulation in a dose-dependent manner (Fig. 2B). The intracellular accumulation of rhodamine 123 was 3.8-, 5.4- and 9.7-fold higher in S1M1-80 cells in the presence of 0.625, 1.25 or 2.5 lM of sunitinib, respectively (Fig. 2C). In contrast, the level of rhodamine 123 accumulation in the drug-sensitive S1 cells was not significantly affected by sunitinib at the concentration of 0.625, 1.25 or 2.5 lM (Fig. 2A). Similarly, doxorubicin accumulation was enhanced in S1-M1-80 cells by 1.3-, 1.5- and 1.8fold, respectively (Fig. 2E and F). However, in the sensitive S1 cells, sunitinib did not significantly alter the intracellular accumulation of doxorubicin (Fig. 2D). To investigate whether sunitinib inhibits the function of ABCG2 as a drug efflux pump, the extrusion of rhodamine 123 was examined by flow cytometry in the S1 and S1-M1-80 cells. The time course of release of

rhodamine 123 after 30 min incubation at 37 °C was shown (Fig. 3A). S1-M1-80 cells released a higher percentage of accumulated rhodamine 123 than S1 cells. At 30 min, 67% of the accumulated rhodamine 123 was released from S1-M1-80 cells, whereas only 17% was lost from S1 cells. Sunitinib at 2.5 lM significantly inhibited the efflux of rhodamine 123 from the S1-M1-80 cells, but showed no apparent effect on the S1 cells. Taken together, these results suggest that sunitinib inhibits ABCG2-mediated release of established ABCG2 substrates. 3.4. Sunitinib inhibits the transport of [3H]-methotrexate by wild-type ABCG2 To further confirm the effect of sunitinib on the transport activity of ABCG2, we used membrane vesicles prepared from HEK293/pcDNA3 and ABCG2-482-R5 cells to perform inhibition experiments. The effect of sunitinib on the transport of methotrexate by ABCG2 was shown in Fig. 3B. Though the inhibitory effect of sunitinib on methotrexate transport by ABCG2 membrane vesicles was slightly weaker than that of erlotinib, the rates of [3H]-methotrexate uptake was significantly inhibited by sunitinib at 5 lM compared with control. These transport results suggest that sunitinib inhibits the transport of [3H]-methotrexate in wild-type ABCG2-482-R5 expressing cells. 3.5. Sunitinib does not alter the expression of ABCG2 at mRNA and protein levels To study the effect of sunitinib on the expression of ABCG2 in mRNA and protein levels, we incubated S1-M1-80 cells with sunitinib at 1.25 lM for 24, 48 and 72 h, or at 0.625, 1.25 and 2.5 lM for 48 h, respec-


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Fig. 2. Effect of sunitinib on the accumulation of rhodamine 123 and doxorubicin in S1 and S1-M1-80 cells. Cellular rhodamine 123 or doxorubicin was measured by flow cytometry as described in Section 2. (A and D) Cellular rhodamine 123 and doxorubicin was not significant affected by sunitinib in S1 cells; (B and E) The accumulation of rhodamine 123 and doxorubicin was increased in a dose-dependent manner in S1-M1-80 cells. Red filled, control; black, 0.625 lM; green, 1.25 lM; blue, 2.5 lM. (C and F) Rhodamine 123 and doxorubicin levels were expressed as the units of mean fluorescent intensity. Data were shown as means ± SD of triplicate determinations. **p < 0.05 vs. the control.

tively. Our results indicated that no marked difference of ABCG2 expression in the mRNA (Fig. 4A) or protein (Fig. 4B) level was observed in S1M1-80 cells treated with sunitinib compared with untreated cells. These results suggest that sunitinib does not affect the expression of ABCG2 at the levels of mRNA and protein. 3.6. Sunitinib does not block Akt and Erk1/2 phosphorylation Accumulating evidence suggest that blockade of Akt or Erk1/2 may decrease resistance to doxorubicin and paclitaxel in cancer cells [28,29]. To determine whether the concentrations of sunitinib used in our experiments attenuates cell survival signaling pathways, we studied Akt and Erk1/2 signal transduction in S1 and S1-M1-80. As shown in Fig. 5, sunitinib did not significantly block the phosphorylation of Akt and Erk1/2 in S1 (Fig. 5A) and S1-M1-80 cells (Fig. 5B), respectively. This result suggests that sunitinib-induced enhancement of the cytotoxicity of topotecan and doxorubicin in S1-M1-80 cells is independent of the blockade of Akt or Erk1/2 phosphorylation.

4. Discussion Tyrosine kinase inhibitors (TKIs) exert their action through competition with ATP for binding at the catalytic domain of tyrosine kinases, thus preventing activation of kinase activity. In vitro studies using biochemical and cell assays showed that TKIs also interact with and modulate the function of the ABC transporters [13,14]. CI-1033, an irreversible inhibitor of ERBB1, reversed ABCG2-mediated resistance to camptothecin and SN-38 [15]. We also re-

ported that erlotinib (TKI of EGFR) and lapatinib (TKI of EGFR and Her-2) both reversed ABCB1- and ABCG2-mediated MDR in cancer cells through directly inhibiting the drug efflux function of ABCB1 and ABCG2 [20,21]. ZD6474, a small molecule inhibitor of VEGFR, EGFR and RET tyrosine kinases, reversed ABCB1-mediated MDR [30]. Sunitinib malate (SUTENTÒ; Pfizer Inc., New York, NY) is an oral, multi-targeted receptor tyrosine kinase inhibitor of VEGFR-1, -2 and -3, PDGFR-a and -b, KIT, CSF-1R, FLT3 and RET [22–24]. Clinical trials with sunitinib are showing promising antitumor activity against metastatic renal cell carcinoma [31], gastrointestinal stromal tumor [32], metastatic colorectal cancer [33], breast cancer [34,35] and advanced non-small-cell lung cancer [36]. Recently, the FDA approved sunitinib for the treatment of metastatic renal cell carcinoma (mRCC) and gastrointestinal stromal tumor (GIST) in patients who have failed to respond to imatinib or were unable to tolerate it [37]. In combination studies in xenograft models, additive or synergistic effects of sunitinib were observed when combined with docetaxel, fluorouracil, or doxorubicin in breast cancer [34], and with cisplatin in a small-cell lung cancer model [22]. Similarly, when MV4-11 acute myeloid leukemia cells hemizygous for the FLT3-ITD mutation were treated with cytarabine or daunorubicin combined with sunitinib, additive to synergistic levels of inhibition were observed, along with

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Fig. 3. Effect of sunitinib on the efflux of rhodamine 123 and the transport of [3H]-methotrexate: (A) efflux of rhodamine 123 in ABCG2-overexpressing S1M1-80 and parental S1 cells and (B) the transport of [3H]-methotrexate in membrane vesicles from HEK293/pcDNA3.1 and ABCG2-482-R2 cells. All were measured as described in Section 2. Points or columns, means of at least three independent experiments; bars, SEs. p < 0.05 vs. control group.

Fig. 4. Sunitinib does not affect ABCG2 expression at mRNA or protein levels: (A) S1-M1-80 cells treated with sunitinib at 1.25 lM for 24, 48 and 72 h, or at 0.625, 1.25 and 2.5 lM for 48 h, respectively. The mRNA levels of ABCG2 were determined by RT-PCR as described in Section 2, S1 cells as negative control and (B) S1-M1-80 cells treated with sunitinib at 1.25 lM for 24, 48 and 72 h, or at 0.625, 1.25 and 2.5 lM for 48 h, respectively. Equal amounts of total cell lysates were used for loading and detected by Western blotting as described in Section 2. Results from a representative experiment; similar results were obtained in two other trials.

induction of apoptosis [38]. Studies on the mechanism behind the supra-additive effects of sunitinib indicate that

the agent may potentiate cell death induced by chemotherapy through inhibition of compensatory prosurvival


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Fig. 5. The effect of sunitinib on the blockade of Akt and Erk1/2 phosphorylation. S1 and S1-M1-80 cells were treated with sunitinb at 1.25 lM for 24, 48 and 72 h, or at 0.625, 1.25 and 2.5 lM for 48 h, respectively. After treatment, cells were harvested and the proteins were extracted and subjected to Western blotting analysis as described in Section 2. The experiments were repeated at least three times independently, and a representative experiment is shown in each panel.

pathways operative in tumor, stromal and endothelial cells [38,39]. In the present study, we examined the ability of sunitinib to reverse MDR in drug-resistant cell lines. Our data showed that S1 and S1-M1-80 cells had similar sensitivity to sunitinib, and the IC50 values were 8.75 ± 1.03 and 5.75 ± 0.23 lM, respectively; thus, overexpression of functional ABCG2 did not confer significant resistance to sunitinib. In contrast, overexpression of functional ABCG2 conferred 38-fold and 41-fold resistance to topotecan and doxorubicin in S1-M1-80 cells, respectively (Table 1). Coincubation with non-toxic concentrations of sunitinib reversed resistance to topotecan or doxorubicin in S1M1-80 cells but not in parental S1 cells (Table 1). Furthermore, sunitinib had no significant reversal in ABCB1-, ABCC1- and LRP-mediated MDR cancer cells, though there was a slightly synergetic effect when coincubated with doxorubicin in drug-sensitive MCF-7 and KB-3-1 cells, or in drug resistance MCF-7/adr and C-A120 cells, respectively, which may be generated by a non-specific cytotoxic mechanism or other unknown actions of the drug. Recently, Shukla and coworkers [14] reported that sunitinib slightly reversed P-gp mediated resistance to depsipeptide and had little effect on resistance to doxorubicin in ABCB1transfected HEK-293 cells, but completely inhibited resistance to topotecan and SN-38 in ABCG2-transfected HEK-293 cells. Their results are not only consistent with our findings that sunitinib is more effective in inhibiting ABCG2 than P-gp function, but also in agreement with our findings that sunitinib at 2.5 lM has no effect on MRP1-mediated resistance. Taken as a whole, these data suggest that sunitinib may specifically reverse ABCG2mediated drug resistance.

Sunitinib increased rhodamine 123 and doxorubicin accumulation in S1-M1-80 cells in a dose-dependent manner, which was not observed in S1 cells (Fig. 2). In addition, sunitinib at 2.5 lM significantly inhibited rhodamine123 efflux in S1-M1-80 cells. Furthermore, at the concentration of 5 lM, sunitinib significantly inhibited the transport of [3H]-methotrexate by wild-type ABCG2 in vitro. Guerin et al. [40] reported a supra-additive antitumor effect of sunitinib combined with docetaxel in hormone refractory prostate cancer. Though they found that sunitinib did not modify the expression of ABCB1, which can modulate the activity of docetaxel, there was somewhat counterintuitive since that the inhibition of tumor angiogenesis may decrease delivery of drugs and, therefore, antagonize chemotherapy. The enhancement mechanisms, interactions between sunitinib and the function of ABC transporters were not analyzed in the report. As shown in our study, the enhancement effect of sunitinib on established ABCG2 substrates is probably due to the inhibition of ABCG2-mediated drug efflux. Taken together, these results suggest that sunitinib interacts with ABCG2 and inhibits the function of ABCG2-mediated drug efflux. Receptor tyrosine kinases such as c-KIT, FLT3, PDGFR and VEGFR play important roles in regulating cell proliferation, differentiation and survival by activating downstream effectors such as signal transducers and activators of transcription (STAT), protein kinase B/AKT and extracellular signal-regulated kinase 1/2 (Erk1/2) [41,42]. Overexpression and/or structural alteration of different RTKs are generally associated with cancer and, when RTKs-mediated signal transduction pathways are abnormally activated, cancer growth, angiogenesis and metastatization generate. Several recent investigations have shown that

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PI3 K/Akt or Erk pathway activation is concerned with resistance to conventional chemotherapeutic agents in various cancer cells [43,44]. Sunitinib inhibits the proliferation of GIST-T1 cells in conjunction with blockade of c-KIT and its downstream effectors Akt and Erk [45]. In our study, sunitinib up to 2.5 lM did not block the phosphorylation of Akt and Erk1/2 both in S1 and S1-M1-80 cells. Our results suggest that sunitinib-induced enhancement of the cytotoxicity of chemotherapeutic agents in S1-M180 cells is not due to its antagonism of the phosphorylation of Akt or Erk1/2. Our study suggests that sunitinib significantly inhibits the function of ABCG2 in vitro. Hence, the reversal effects shown in the present study would be sufficient for reversing ABCG2-mediated drug resistance in vivo. However, ABCG2 is highly expressed in the placenta, liver, intestine and the blood–brain barrier [46], which indicates a possible role of ABCG2 in the regulation of drug uptake and excretion. Therefore, when combined with ABCG2 substrate agents in a clinical setting, sunitinib may affect the plasma concentrations of these drugs, leading to adverse effects because sunitinib may inhibit ABCG2-mediated drug transport in normal cells. In fact, coadministration of the ABCG2 inhibitor GF120918 has significantly increased the plasma levels of topotecan in mice and humans [9,47], and gefitinib reduced the clearance of topotecan [19]. In conclusion, sunitinib significantly reverses ABCG2mediated drug resistance by inhibiting the drug efflux function of ABCG2 and increasing the intracellular accumulation of cytotoxic agents in ABCG2-overexpressing cells, but not depending on the blockade of Akt or Erk1/2 phosphorylation. However, whether sunitinib could be used with established ABCG2 substrate anticancer agents to improve clinical outcome is worthy of further study. 5. Conflicts of interest statement None declared. Acknowledgements This work was supported by Grants from China National Natural Sciences Foundation No. 30672407 and 863 Project Foundation No. 2006AA09Z419. References [1] M. Dean, A. Rzhetsky, R. Allikmets, The human ATP-binding cassette (ABC) transporter superfamily, Genome Res. 11 (2001) 1156–1166. [2] L.A. Doyle, W. Yang, L.V. Abruzzo, T. Krogmann, Y. Gao, A.K. Rishi, et al, A multidrug resistance transporter from human MCF-7 breast cancer cells, Proc. Natl. Acad. Sci. USA 95 (1998) 15665–15670. [3] K. Miyake, L. Mickley, T. Litman, Z. Zhan, R. Robey, B. Cristensen, et al, Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes, Cancer Res. 59 (1999) 8–13. [4] R. Allikmets, L.M. Schriml, A. Hutchinson, V. Romano-Spica, M. Dean, A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance, Cancer Res. 58 (1998) 5337–5339. [5] T. Litman, T.E. Druley, W.D. Stein, S.E. Bates, From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance, Cell. Mol. Life Sci. 58 (2001) 931–959.

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