Cellular Onc. (2011) 34:33–44 DOI 10.1007/s13402-010-0003-7

ORIGINAL PAPER

Effect of BIBF 1120 on reversal of ABCB1-mediated multidrug resistance Qing-feng Xiang & Fang Wang & Xiao-dong Su & Yong-ju Liang & Li-sheng Zheng & Yan-jun Mi & Wei-qiang Chen & Li-wu Fu

Accepted: 10 October 2010 / Published online: 28 January 2011 # International Society for Cellular Oncology 2011

Abstract Background: The overexpression of ATP-binding cassette (ABC) transporters is one of the main causes of multi-drug resistance (MDR) which represents a major obstacle to the success of cancer chemotherapy. In this study, we examined the effect of BIBF 1120, an inhibitor of vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs) and fibroblast growth factor receptors (FGFRs) tyrosine kinases, on the reversal of multidrug resistance in vitro. Methods: The doxorubicin and rhodamine 123 retention assay was performed by flowcytometry. Western blot were employed to identify ABCB1 expression level and the effect of BIBF 1120 on the blockade of Akt and ERK1/2 phosphorylation. The expression of mdr1 mRNA was determined by RT-PCR analysis. The ATPase activity of ABCB1 was investigated using Pgp-Glo™ assay systems. Results: BIBF 1120 significantly enhanced the cytotoxicity of doxorubicin and paclitaxel and increased the accumulation of ABCB1 substrates in ABCB1-overexpressing

cancer cells, whereas it had no effect on the parental cells. On the other hand, BIBF 1120 did not alter the cytotoxicity of non-ABCB1 substrates and was unable to reverse ABCC1 or ABCG2-mediated MDR. Furthermore, BIBF 1120 inhibited the ATPase activity of ABCB1 in a concentration-dependent manner. However, no detectable alteration on the expression level of mdr1 mRNA or ABCB1 protein was identified in ABCB1-overexpressing cancer cells by different treatments of BIBF 1120. Interestly, total and phosphorylated forms of AKT and ERK1/2 were not inhibited by BIBF 1120 at the reversal concentrations. Conclusion: Our results suggest that BIBF 1120 is capable of overcoming ABCB1-mediated drug resistance by inhibiting ABCB1 function, which may have clinical significance for BIBF 1120 combinational treatment of certain resistant cancers. Keywords BIBF 1120 . Multidrug resistance . ATP-binding cassette transporters . ABCB1/P-glycoprotein 1 Introduction

Q.-f. Xiang : W.-q. Chen (*) Department of General Surgery, Chen Xing Hai Hospital, Guangdong Medical College, Zhongshan 528415, China e-mail:[email protected] Q.-f. Xiang : F. Wang : X.-d. Su : Y.-j. Liang : L.-s. Zheng : Y.-j. Mi : L.-w. Fu (*) State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China e-mail: [email protected]

Intrinsic or acquired resistance of tumor cells to anticancer drugs remains one of the main causes of suboptimal outcomes in cancer therapy. After developing resistance to a single drug or a class of drugs, cancer cells present crossresistance to other functionally and structurally unrelated drugs. This phenomenon known as multi-drug resistance (MDR) has a profound effect on successful chemotherapy of cancer [1]. A number of cellular and molecular alterations may contribute to the development of the MDR phenotype, and one of the best known mecha-

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nisms is the over-expression of ATP-binding cassette (ABC) transporters which are able to efflux drugs out of tumor cells [2]. Forty-nine different ABC transporters have been identified in the human genome and are divided into seven subfamilies (A–G) based on sequence similarities [3], among which ABC transporter-subfamily B member 1 (ABCB1/MDR1), subfamily C member 1 (ABCC1/MRP1) and subfamily G member 2 (ABCG2/BCRP) are the most important members [4]. ABCB1, a 170-kD plasma membrane glycoprotein encoded by the human mdr1 gene, stands out among ABC transporters by conferring the strongest resistance to the widest variety of compounds. Using ATP as the energy source, ABCB1 is known to facilitate the efflux of a broad range of cytotoxic drugs including anthracyclines, vinca alkaloids, epipodophyllotoxins and tanxanes [5]. ABCB1 is overexpressed at baseline in chemotherapy-resistant tumors, such as colon and kidney cancers, and is upregulated after disease progression following chemotherapy in malignacies such as breast cancer and leukemia [6]. ABCC1 is expressed in a wide range of tissues, clinical tumours [7] and cancer cell lines [8]. Apart from resistance to several hydrophobic compounds that are also ABCB1 substrates, ABCC1 can export glutathione (GSH), glucuronate or sulphate conjugates of organic anions [9]. In contrast to ABCB1, ABCG2 is a half transporter that consists of only one transmembrane domain with six helices and one ATPbinding site, and acts as a homodimeric efflux pump, confering resistance to mitoxantrone, indolocarbazole, topoisomerase I inhbitors and anthracyclines, as well as fluorescent dyes such as Hoechst 33,342 [10] . ABCG2 expression overlaps largely with ABCB1, and can be found in tissues like the placenta, prostate, small intestine, brain, colon, liver, and ovary [11]. In addition, the side population (SP) cells are present in diverse tumor types and overexpress ABCG2, producing inherent drug resistance [12]. The agents that fully or partly block ABC transporters activity thus may prevent the undesirable loss of intracellular drugs and may have beneficial effects during chemotherapy. Therefore, development or discovery of safe and effective ABC transporters-mediated MDR reversal agents is urgently required. Up to date, a number of MDR inhibitors or modulators have been demonstrated ability to reverse MDR, some of which are ongoing clinical trials to evaluate the potential circumvent of anticancer drug resistance [13, 14]. Inhibition of tumor angiogenesis through blockade of (vascular endothelial growth factor) VEGF signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term survival benefit may be improved with blockade of additional proangiogenic receptor tyrosine kinases PDGFRs and FGFRs [15]. BIBF 1120, currently entering phase III clinical studies in

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non-small cell lung carcinoma [16] and other cancers, is a novel, orally available, potent multi-targeted tyrosine kinase inhibitor (TKI) that predominantly blocks the vascular endothelial growth factor receptor (VEGFR) 1,2,3, platelet-derived growth factor receptor (FGFR) 1,2,3, and fibroblast growth factor receptor (PDGFR) α and β tyrosine kinases at nanomolar concentrations. Like other TKIs, BIBF 1120 inhibits multiple tyrosine kinases through competitive inhibition of ATP binding, a key step in the phosphorylation activity of the kinases [15]. Recently, it has been shown that several TKIs including lapatinib [17], gefitinib [18], erlotinib [19], cediranib [20], vandetanib [21] and sunitinib [22] can inhibit functions of ABC transporters, thereby overcoming chemotherapy resistance in multidrug-resistance cancer cells. Thus, interaction with ABC transporters seems to be a class effect of these compounds. In preclinical studies, combining BIBF 1120 with paclitaxel or pemetrexed has marked antitumor effects both in vitro and in vivo compared to single-agent treatment [23]. Several phase I and II studies are underway to extensively investigate the combination treatment of BIBF 1120 for a range of tumor types [24, 25]. However, the mechanisms responsible for the BIBF 1120-induced chemosensitivity of conventional chemotherapeutic agents in cancer cells remain unclear. It is conceivable that these enhancing effects of BIBF 1120 in combination with conventional chemotherapeutic drugs may be due in part to interaction with ABC transporters, which leads us to probe the effect of BIBF 1120 on the reversal of multidrug resistance induced by ABCB1, ABCC1 or ABCG2.

2 Materials and methods 2.1 Chemicals and reagents BIBF 1120 was purchased from Selleck Chemicals, with a molecular structure as shown in Fig. 1a. Monoclonal antibodies against ABCB1 and ABCC1 were from Santa Cruz Biotechnology. ABCG2 antibody was obtained from Chemicon International, Inc (Billerica, MA). Akt antibody was a product of Cell Signaling Technology Inc (Danvers, MA). Phosphorylated Akt, phosphorylated extracellular signal-regulated kinase, MAPK1/2 (ERK1/2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Kangchen Co. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 were products of Gibco BRL. Rhodamine 123 (Rho 123), 1-(4, 5-dimethylthiazol-2-yl)-3, 5diphenylformazan (MTT), fumitremorgin C (FTC), paclitaxel, doxorubicin (Dox), vincristine (VCR), mitoxantrone, and other chemicals were purchased from Sigma Chemical Co (St. Louis, MO).

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Fig. 1 Cytotoxicity of BIBF 1120 in the drug-resistant and parental sensitive cancer cells. The structure of BIBF 1120 a; The protein expression of ABCB1 in Hep G2, Hep G2/adr, MCF-7 and MCF-7/ adr cells, ABCC1 in HL60 and HL60/adr cells, and ABCG2 in S1 and S1-M1-80 cells b; MTT cytotoxicity assay was assessed in pairs of parental and transporter-overexpressing cells: c ABCB1-negative Hep G2 and ABCB1-positive Hep G2/adr cells, d ABCB1-negative MCF-

7 and ABCB1-positive MCF-7/adr cells, e ABCC1-negative HL60 and ABCC1-positive HL60/adr cells and f ABCG2-negative S1 and ABCG2-positive S1-M1-80 cells which were exposed to the indicated concentrations of BIBF 1120 for 72 h. Each point represents the mean ±standard deviations (SDs) for three determinations. Each experiment was performed in three replicate wells

2.2 Cell lines and cell culture

cell lines S1 and its mitoxantrone (MX)-selected ABCG2overexpressing derivative S1-M1-80 which were obtained from Dr. S.E. Bates (National Cancer Institute, NIH) [27]; the human hepatoma cell lines Hep G2 and its Dox-selected ABCB1-overexpressing derivative Hep G2/adr [28]; the human leukemia cell lines HL60 and its Dox-selected ABCC1-overexpressing derivative HL60/adr [29]. All cells

The following cell lines were cultured in DMEM or RPMI1640 supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2: the human breast carcinoma cell lines MCF-7, its Dox-selected ABCB1-overexpressing derivative MCF-7/adr [26]; the human colon carcinoma

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were grown in drug-free culture medium for more than 2 weeks before assay. 2.3 Cell cytotoxicity assay The MTT assay was performed as described previously to assess the sensitivity of cells to drugs [30]. Briefly, cells were plated in 96-well microtiter plates and then various concentrations of BIBF 1120 and/or a full range concentration of conventional chemotherapeutic drug were added to the wells. After 68 h of incubation, MTT (5 mg/mL, 20 μL/well) was added to the wells and the cells were incubated for an additional 4 h (37°C). Subsequently, the medium was discarded and 200 μL of dimethylsulfoxide (DMSO) was added to dissolve the formazan product from the metabolism of MTT. The optical density was measured at 540 nm with background subtraction at 670 nm using a Model 550 Microplate Reader (BIO-RAD, Hercules, CA). The concentration required to inhibit cell growth by 50% (IC50) was calculated from survival curves using the Bliss method [31]. The degree of resistance was estimated by dividing the IC50 for the MDR cells by that of the parental sensitive cells; the fold-reversal factor of MDR was calculated by dividing the IC50 of the anticancer drug in the absence of BIBF 1120 by that obtained in the presence of BIBF 1120. 2.4 Doxorubicin and rhodamine 123 accumulation The effect of BIBF 1120 on the accumulation of Dox and rhodamine 123 was measured by flow cytometry as previously described [26]. Briefly, the cells were treated with BIBF 1120 of various concentration or vehicle at 37°C for 3 h. And then 10 μM doxorubicin or 5 μM rhodamine 123 was added and incubation was continued for additional 3 h or 0.5 h, respectively. The cells were then collected, washed three times with icecold PBS, and analysed with flow cytometric analysis (Beckman Coulter, Cytomics FC500, USA). Verapamil, a known ABCB1 inhibitor [32], was used as a positive control. 2.5 Reverse transcription PCR After drug treatment for 48 h, total cellular RNA was isolated by Trizol Reagent RNA extraction kit following the manufacturer’s instruction (Molecular Research Center, USA). The first strand cDNA was synthesized by Oligo dT primers with reverse transcriptase (Promega Corp.). PCR primers were 5′-ccc atc att gca ata gca gg-3′ (forward) and 5′-gtt caa act tct gct cct ga-3′ (reverse) for ABCB1; and 5′-ctt tgg tat cgt gga agg a-3′ (forward) and 5′-cac cct gtt gct gta gcc-3′ (reverse) for GAPDH,

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respectively. Using the GeneAmp PCR system 9700 (PE Applied Biosystems, USA), reactions were carried out at 94°C for 2 min for initial denaturation, and then at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. After 35 cycles of amplification, additional extensions were carried out at 72°C for 10 min. Products were resolved and examined by 1.5% agarose gel electrophoresis. Expected PCR products were 157 bp for ABCB1 and 475 bp for GAPDH, respectively. 2.6 Western blot analysis To identify whether BIBF 1120 affects the expression of ABCB1, the cells were incubated with different concentrations of BIBF 1120 for 48 h. To determine whether BIBF 1120 is able to block Akt or Erk1/2 phosphorylation, we incubated cells with different concentrations of BIBF 1120 for 24 h. Then, whole cells were harvested and washed twice with ice-cold PBS. Cell extracts were collected in cell lysis buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/mL phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/ml leupeptin). Equal amounts of cell lysate from various treatments were resolved by sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE). After blocked in TBST (10 mmol/L Tris-HCL, 150 mmol/L NaCl, and 0.1% Tween20 pH 8.0) with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with appropriately diluted primary antibodies overnight at 4°C. The membranes were then washed thrice with TBST and incubated with HRP-conjugated secondary antibody at 1:5000 dilution for 2 h at room temperature. After washed thrice with TBST, 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 (Carestream Health, USA). GAPDH was used as a loading control. 2.7 ABCB1 ATPase activity assay The changes of ATPase activity were estimated by PgpGlo™ assay systems (Promega, USA). The inhibitory effects of BIBF 1120 were examined against a verapamilstimulated ABCB1 ATPase activity. Sodium orthovanadate (Na3VO4) was used as an ABCB1 ATPase inhibitor. Various concentrations of BIBF 1120 diluted with assay buffer were incubated in 0.1 mM verapamil, 5 mM MgATP and 25 μg recombinant human ABCB1 membranes at 37°C for 40 min. Luminescence was initiated by ATP detection buffer. After incubated at room temperature for 20 min to allow luminescent signal to develop, the untreated white opaque 96-well plate (corning, USA) was

Reversal of MDR by BIBF1120

read on luminometer (spectraMax M5, molecular devices, USA). The changes of relative light units (ΔRLU) were determined by comparing Na3VO4-treated samples with BIBF 1120 and verapamil combination-treated samples, and hence, the ATP consumed was obtained by comparing to a standard curve.

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effects of BIBF 1120 were observed in their parental cells. Meanwhile, BIBF 1120 had no significant reversal effect on ABCC1-mediated drug resistance in HL60/adr cells or ABCG2-mediated drug resistance in S1-M1-80 cells. These results suggest that BIBF 1120 significantly sensitizes ABCB1-overexpressing cells to antineoplastic drugs that are substrates of ABCB1.

2.8 Statistics 3.3 Doxorubicin and rhodamine 123 accumulation All experiments were repeated at least thrice and the differences were determined by using the Student’s t-test. The significance was determined at P<0.05.

3 Results 3.1 Determination of multidrug resistance ABCB1 is overexpressed in Hep G2/adr and MCF-7/ adr cells, while ABCC1 and ABCG2 were overexpressed in HL60/adr and S1-M1-80 cells, respectively (Fig. 1b). The basal expressions of ABCB1, ABCC1 and ABCG2 in the parental cell lines were nearly undetectable. MTT assay showed that four MDR cell lines exerted much higher tolerance to multiple anticancer drugs compared with their parental, drug-sensitive cell lines (Table 1). 3.2 Modulation of multidrug resistance in MDR cell lines by BIBF 1120 The intrinsic in vitro toxicity of BIBF 1120 on different cells was determined using the MTT assay. The IC50 values were 15.28±0.73, 18.94±0.79, 25.16±0.65, 28.54±0.66, 7.64±0.73, 9.12±0.59, 4.35±0.34 and 6.54±0.56 μM, for Hep G2, Hep G2/adr, MCF-7, MCF-7/adr cells, HL60, HL60/adr S1 and S1-M1-80 respectively (Fig. 1). As determined by the dose-effect curve, more than 90% of cells were viable at the concentrations of 3 μM BIBF 1120 in Hep G2, Hep G2/adr, MCF-7 and MCF-7/adr cells and 1.5 μM in HL60, HL60/adr, S1 and S1-M1-80 cells. Therefore, BIBF 1120 at a concentration of 3 μM (in Hep G2, Hep G2/adr, MCF-7 and MCF-7/adr) or 1.5 μM (in HL-60, HL60/adr, S1 and S1-M1-80) was chosen for combination treatment with known ABCB1 (Dox and paclitaxel), ABCC1 (VCR) or ABCG2 (mitoxantrone) substrate anticancer drugs. The IC50 values of the antineoplastic drugs in sensitive and resistant cells at different concentrations of BIBF 1120 are shown in Table 1. BIBF 1120 significantly dose-dependently sensitized Hep G2/adr and MCF-7/adr cells to Dox and paclitaxel but did not alter the cytotoxicity of cisplatin which is not ABCB1 substrate. However, no enhancement

The decrease of intracellular drug concentrations, a result of the efflux of anticancer drugs from tumor cells into the surrounding tissue, is believed to be a common cause of MDR. Several modulators have been reported to reverse MDR by inhibiting cellular drug efflux [33–35]. To investigate whether BIBF 1120 inhibits the function of ABCB1 as an efflux transporter, the intracellular accumulation of Dox and rhodamine 123 in the presence or absence of BIBF 1120 was examined using ABCB1overexpressing MDR cells and their parental cells. The intracellular accumulation of Dox or rhodamine-123 in drug-resistant Hep G2/adr and MCF-7/adr cells was decreased compared with that for the parental cells, suggestting that ABCB1-overexpression results in decreased intracellular substract accumulation. BIBF 1120 enhanced the intracellular accumulation of Dox and rhodamine 123 in MDR cells in a dose-dependent manner, but not in the parental sensitive cells (Fig. 2). The fluorescent index of Dox was increased by 1.21-, 1.63-, 1.98-fold in Hep G2/adr cells and 1.98-, 2.25-, 2.88-fold in MCF-7/adr cells in the presence of 0.75, 1.5 and 3 μM of BIBF 1120, respectively (Fig. 2b). As shown in Fig. 2d, BIBF 1120 at 0.75, 1.5 and 3 μM increased the intracellular accumulation of rhodamine 123 by 3.12-, 4.23-, 5.78-fold in Hep G2/adr cells and 2.53-, 3.78-, 6.15-fold in MCF-7/ adr cells, respectively. These results suggest that BIBF 1120 increases the accumulation of the anticancer drugs which may relate to modulating ABCB1-mediated transport in MDR cells. 3.4 BIBF 1120 does not alter the expression of mdr1 gene and ABCB1 The reversal of ABCB1-mediated MDR can usually be achieved either by down-regulating ABCB1 expression or inhibiting its function. To assess the effect of BIBF 1120 on mdr1 mRNA and ABCB1 protein expression levels, reverse transcription-PCR and Western blot analysis were performed. Our results showed that the expression level of mdr1 mRNA or ABCB1/P-gp protein (Fig. 3) was not significantly altered. These results indicate that inhibiting the expression of ABCB1 is not involved in the reversal of ABCB1-mediated MDR by BIBF 1120.

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Table 1 Effect of BIBF 1120 on reversing ABCB1-, ABCC1- and ABCG2-mediated MDR in drug selection cell lines Compounds

IC50±SD (μM) (fold-reversal) Hep G2

Hep G2/adr (ABCB1)

Doxorubicin + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 3.0 μM BIBF 1120 + 10 μM Verapamil

0.167±0.004 0.168±0.014 0.159±0.023 0.149±0.022 0.154±0.002

(1.00) (0.99) (1.05) (1.12) (1.08)

26.35±1.198 12.08±0.335** 6.808±0.124** 3.599±0.041** 1.577±0.012**

(1.00) (2.18) (3.87) (7.32) (16.7)

Paclitaxel + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 3.0 μM BIBF 1120 + 10 μM Verapamil Cisplatin + 3.0 μM BIBF 1120

0.051±0.032 0.052±0.043 0.046±0.002 0.050±0.009 0.045±0.067 6.895±0.012 6.384±0.002 MCF-7 0.339±0.037 0.342±0.031 0.325±0.029 0.322±0.006 0.335±0.035 0.028±0.025 0.027±0.014 0.029±0.028 0.026±0.098 0.026±0.019

(1.00) (0.97) (1.09) (1.01) (1.11) (1.00) (1.08)

6.298±0.115 2.460±0.057** 1.412±0.066** 0.790±0.045** 0.292±0.038** 8.093±1.002 7.781±0.002 MCF-7/adr (ABCB1) 12.50±1.106 3.561±0.176** 2.367±0.217** 1.479±0.056** 0.553±0.076** 1.120±0.136 0.375±0.036** 0.221±0.039** 0.148±0.024** 0.043±0.065**

(1.00) (2.56) (4.46) (7.89) (21.5) (1.00) (1.04)

4.875±0.036 4.826±0.148 HL60 0.039±0.022 0.040±0.032 0.038±0.057 0.035±0.064 0.037±0.033 S1 0.254±0.037 0.259±0.032 0.235±0.054 0.203±0.026 0.249±0.018

(1.00 ) (1.01 )

6.531±1.048 6.664±0.848 HL60/adr (ABCC1) 6.104±0.418 6.165±0.278 5.651±0.256 5.450±0.319 0.992±0.433** S1-M1-80 (ABCG2) 15.671±0.952 15.515±0.782 16.839±0.363 13.746±0.464 1.051±0.062**

(1.00) (0.98)

Doxorubicin + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 3.0 μM BIBF 1120 + 10 μM Verapamil Paclitaxel + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 3.0 μM BIBF 1120 + 10 μM Verapamil Cisplatin + 3.0 μM BIBF 1120 Doxorubicin + 0.345 μM BIBF 1120 + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 50 μM MK571 Mitoxantrone + 0.345 μM BIBF 1120 + 0.75 μM BIBF 1120 + 1.5 μM BIBF 1120 + 2.5 μM FTC

(1.00 ) (0.99) (1.04) (1.05 ) (1.01) (1.00 ) (1.03) (0.96) (1.04) (1.07)

(1.00) (0.97) (1.02) (1.09) (1.04) (1.00) (0.98) (1.08) (1.25) (1.02)

(1.00) (3.51) (5.28) (8.45) (22.6) (1.00 ) (2.98) (5.05) (7.55) (25.7)

(1.00) (0.99) (1.08) (1.12) (6.15) (1.00) (1.01) (0.99) (1.14) (14.9)

Cell survival was determined by MTT assay as described in “Materials and Methods”. Data are the mean±standard deviation (SD) of at least three independent experiments performed in triplicate. The fold-reversal of MDR (values given in parenthesis in last column) was calculated by dividing the IC50 for cells with the anticancer drugs in the absence of BIBF 1120 by that obtained in the presence of BIBF 1120. ** represent P<0.01 for values versus that obtained in the absence of BIBF 1120

3.5 BIBF 1120 did not block the phosphorylation of AKT and ERK1/2 The phosphorylation of AKT and ERK1/2, the downstream markers of BIBF 1120 targets, are usually

utilized to test the targeted activity of BIBF 1120. Several studies proved that inhibiting AKT and ERK1/ 2 pathways may enhance the efficacy of chemotherapeutic agents in cancer cells [36, 37]. Here, total and phosphorylated forms of AKT and ERK1/2 were mea-

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Fig. 2 Effect of BIBF 1120 on the accumulation of doxorubicin (Dox) and rhodamine 123. The accumulations of doxorubicin a, b and rhodamine 123 c, d were measured by flow cytometric analysis as described in

“Materials and Methods”. The results are presented as fold change in fluorescence intensity relative to control MDR cells. Columns, means of triplicate determinations; bars, SDs. **P<0.01 versus control group

sured to determine whether the ABCB1 reversal activity of BIBF 1120 was related to the blockade of the phosphorylation of AKT and ERK1/2. As illustrated in Fig. 4, after treated with 0.75–3 μM of BIBF 1120 for

24 h, no detectable effect of BIBF 1120 on total and phosphorylated AKT and ERK1/2 in all cells were found (Fig. 4). The results suggest that the ABCB1 reversal effect of BIBF 1120 in drug-resistant Hep G2/adr and

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activity of ABCB1, we measured ABCB1-mediated ATP hydrolysis with various concentrations of BIBF 1120. We found that BIBF 1120 was an inhibitor of ABCB1 ATPase. As shown in Fig 5, BIBF 1120 reduced verapamilstimulated ATPase activity in a dose-dependent manner.

4 Discussion

Fig. 3 Effect of BIBF 1120 on the expression of ABCB1 in MDR cells. Hep G2/adr and MCF-7/adr cells were treated with BIBF 1120 at various concentrations for 48 h. a The mRNA level of ABCB1 was determined by RT-PCR as described in “Materials and Methods”; b Equal amounts of total cell lysates were loaded and detected by Western blot. A representative result is shown from at least three independent experiments

MCF-7/adr cells is independent of inhibition of AKT and ERK1/2 phosphorylation. 3.6 BIBF 1120 inhibits the ATPase activity of ABCB1 The drug-efflux function of ABCB1 is linked to ATP hydrolysis, and thus ATP consumption reflects ATPase activity. To assess the effect of BIBF 1120 on the ATPase Fig. 4 Effect of BIBF 1120 on blockade of AKT and ERK1/2 phosphorylation. Hep G2/adr and MCF-7/adr cells were treated with drugs for 24 h. Equal amount of protein was loaded for Western blot as described in “Materials and Methods”. All these experiments were repeated at leas thrice, and a representative experiment is shown in each pane

Angiogenesis is crucial for the growth of malignant tumors and metastases. Targeted drugs interfering with the formation and maintenance of tumor blood vessels provide clinical benefit to cancer patients, including tumor regressions and prolonged survival [38, 39]. Monoclonal antibodies to vascular endothelial growth factor (VEGF), notably bevacizumab, as well as small molecule inhibitors targeting the VEGF receptor (VEGFR) kinases, such as sunitinib and sorafenib, have been introduced into clinical practice and continue to be profiled in additional indications, alone or in combination with other treatment modalities. Unfortunetly, clinical trials involving a variety of antiangiogenesis agents alone or in combination with chemotherapy have largely been disappointing [40, 41]. In addtion, preclinical animal models reveal that targeting VEGF-VEGFR signaling and focusing on endothelial cells is only beneficial at the start of treatment, but with continued drug treatment and the pressure of VEGF signaling blockade resulting in increased hypoxia and malnutrition in the tumor cells, other signaling molecules and their cognate receptors provide alternate mechanisms to drive disease progression [42]. Among the potential compensatory mechanisms, the PDGF and FGF pathways have been identified as promising targets for optimized drug candidates.

Reversal of MDR by BIBF1120

Fig. 5 BIBF 1120 inhibition of verapamil-stimulated ABCB1 ATPase activity. ABCB1 ATPase assays were performed according to the instruction of Pgp-Glo™ Assay Systems. Each point represents the mean±SDs for triplated independent determinations

BIBF 1120 is an orally available triple angiokinase inhibitor that simultaneously and potently inhibits VEGFRs, PDGFRs and FGFRs. In vitro, BIBF 1120 inhibits growth factor-induced intracellular signaling in endothelial and smooth muscle cells, as well as pericytes, resulting in inhibition of cell proliferation and induction of apoptosis. BIBF 1120 is effective in mice with established human head and neck squamous cell carcinoma FaDu tumor xenografts, as demonstrated by rapid effect on tumor perfusion and permeability and significant inhibition of tumor growth [15]. Similar inhibitory effects of BIBF 1120 were demonstrated in other in vivo human tumor xenograft models, including hepatoma (Hep G2), renal cell carcinoma (Caki1), colorectal (HT29), ovarian (SKOV3), NSCLC (Calu6) and prostate carcinoma (PAC120) [43]. In a phase I clinical study in patients with advanced solid tumors, BIBF 1120 had a favorable safety profile as twice-daily (b.i.d.) dosing up to the maximum tolerated dose (MTD) of 250 mg b.i.d [44]. Phase II evaluation in patients with advanced refractory NSCLC demonstrated improvements in progression-free survival [45]. Based on encouraging results from phase I/II trials, BIBF 1120 has entered phase III clinical development. Modulators of multidrug resistance ABC transporters are regarded as potential clinically applicable agents to inhibit cancer multidrug resistance, as well as to alter the absorption, tissue distribution, metabolism, and toxicity (ADME-Tox) parameters for various pharmacons [46]. Several TKIs have been found to inhibit the functions of major MDR transporters such as ABCB1, ABCC1 and ABCG2. This modulatory property may make TKIs promising compounds for use in combination with other anticancer drugs, allowing an effective enhancement of various cytotoxic agents. The objectives of this study were to determine the reversal effect of BIBF 1120 on ABC transporters-mediated drug resistance and to gain insight into the mechanisms involved. As demonstrated by MTT assay, both of the two ABCB1overexpressing cell lines had similar sensitivity to BIBF 1120 compared with sensitive parental cells (Fig. 1). Our data also demonstrated the ability of BIBF 1120 to enhance cytotox-

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icity of known ABCB1 substrates in ABCB1-overexpressing cells (Table 1). For example, BIBF 1120 at 3 μM significantly increased the sensitivity of Hep G2/adr and MCF-7/ adr cells to Dox by 7.32 and 8.45-fold, respectively. The action of BIBF 1120 is probably specific against ABCB1mediated MDR. Firstly, the treatment concentrations chosen to study the reversal effect on MDR cells was weakly cytotoxic (inhintition rate<10%). Secondly, there was no synergistic effect between BIBF 1120 and non-ABCB1 substrate such as cisplatin. Additionally, BIBF 1120 did not affect the cytotoxicity of chemotherapeutic drugs in parental cell lines. Finally, BIBF 1120 had no reversal effect on ABCC1 or ABCG2-mediated MDR cancer cells. Reduction of the intracellular concentration of chemotherapeutic agent is a major cause of MDR. It is a widely held hypothesis that intracellular levels of anticancer drugs are reduced below lethal thresholds by active extrusion, through the operation of ATP-dependent pumps such as ABCB1 [2]. Thus, the activity of ABCB1 can be studied by measuring the transportation of ABCB1 substrates. In our study, we found that BIBF 1120 dose-dependently increased the accumulation of Dox and rhodamine 123 in ABCB1-overexpressing MDR cells. However, no significant change was found in the parental Hep G2 and MCF-7 cells. These dates were consistent with our cytotoxic results, collectively suggesting that BIBF 1120 can inhibit the transport function of ABCB1, thereby increasing the intracellular concentration of its substrate anticancer drugs. Modulators or agents to reverse MDR phenotype can be achieved by reduction of ABCB1 either in the transcriptional or protein level [47]. Since incubation of MDR cells with BIBF 1120 up to 48 h did not significantly change the expressions levels of mdr1 mRNA and ABCB1 protein, it is unlikely that BIBF 1120 reverses ABCB1-mediated MDR via the decrease of mdr1/ABCB1 expression either the transcriptional or the protein level. There are data implicated that PI3K/AKT and/or ERK pathway activation is related with resistance to conventional chemotherapeutic agents [48, 49]. In addition, PI3K/ AKT and ERK1/2 are constitutively activated in cancer cells and are the potential targets for enhancing the cytotoxicity of chemotherapeutic agents in cancer treatment [48–50]. Preclinical study demonstrated that BIBF 1120 inhibits proliferation and induce apoptosis in endothelial cells, pericytes and smooth muscle cells via blocking phosphorylation of AKT and/or ERK1/2 [15]. So, it is important to clarify whether these pathways are related to the reversal effect of BIBF 1120 on ABCB1-mediated MDR. Our results showed that treatment with BIBF 1120 of 3 μM for 24 h did not affect the phosphorylation of AKT and ERK1/2 (Fig. 4). Furthermore, we used pure ABCB1-expressing membrane to exclude the intracellular signaling pathways and found BIBF 1120

42

inhibited the ATPase activity in the ABCB1-expressing membrane (Fig. 5). Taken together, these suggest that BIBF 1120 directly interacts with ABCB1 and inhibits its function. Several non-cytotoxic agents can sensitize MDR cells to chemotherapeutic drug in vitro and in vivo. Combined therapy with MDR-related cytotoxins and modulators could inhibit tumor growth and prolong the life span in animal models. Unfortunately, the data regarding the clinical effecacy is not yet available in early clinical trials. Alternatively, the MDR reversal agent may expose the patient to unacceptable side effects or toxicity at doses required for effectiveness and/or affect on the pharmacokinetics of anticancer drug [51]. The profile of the drugstimulated ATPase activity in the ABCB1-expressing membrane is thought to reflect its direct interaction of transporter pumps with drug substrates [52]. We have previously reported that some TKIs such as lapatinib, sunitinib and erlotinib, at low concentrations can stimulate the ATPase activities of the transporters such as ABCB1 and ABCG2, whereas inhibited their ATPase activities at higher concentrations [17, 19, 22]. These TKIs maybe the substrates of ABC transporters and may alter the pharmacokinetic of conventional chemotherapeutic drugs. Importantly, BIBF 1120 inhibited the ABCB1 ATPase activity assay in dose-dependent manner. This suggests that BIBF 1120 might not be a substrate of ABCB1 transporters. On the other hand, BIBF 1120 has no clinically significant effect on the pharmacokinetic profile of paclitaxel [24]. Therefore, there was no evidence of an increase in conventional chemotherapeutic agent associated toxicity induced by BIBF 1120. We speculate based on these findings that BIBF 1120 has a direct interaction with ABCB1. In conclusion, this study provides the first in vitro evidence that BIBF 1120 significantly enhances the efficacy of chemotherapeutic drugs in ABCB1overexpressing MDR cells, which is achieved by inhibiting ABCB1 ATPase activity and function. In addition, the reversal of MDR by BIBF 1120 is independent of the blockade of AKT and ERK1/2 signal transduction pathways. This interaction of BIBF 1120 with the drug transporter may affect treatment outcome of combinational chemotherapy of BIBF 1120 and conventional chemotherapeutic drugs. Acknowledgements We thank Drs S.E. Bates and R.W. Robey (National Cancer Institute, NIH) for the ABCG2 expressing cell line S1-M1-80 and their parental sensitive cell line S1. The work was supported by grants from China National Natural Sciences Foundation No. 81072669 and No. 81061160. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

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