Cancer Letters

Cancer Letters

Volume 279, Issue 1, 28 June 2009, Pages 74-83
Cancer Letters

Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2

https://doi.org/10.1016/j.canlet.2009.01.027Get rights and content

Abstract

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 μM 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.

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 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 resistance-associated 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-M1-80) [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 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], [8], [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 (SUTENT®; 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-α and -β), 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], [23], [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.

Section snippets

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.

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. More than 90% of cells were viable up to 2.5 μM of sunitinib in all the cell lines used in this study (Fig. 1). So we used 0.625, 1.25 and 2.5 μM sunitinib to reverse MDR in vitro. The IC50 concentration for S1 cells to topotecan or doxorubicin were 0.135 ± 0.056 μM and 0.165 ± 0.009 μM,

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 reported that erlotinib (TKI of EGFR) and

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.

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