Reversal of ABCB1-related multidrug resistance by ERK5-IN-1

Background Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. Methods The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. Results Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. Conclusions Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.


Background
Despite considerable advances in chemotherapies against cancer, multi-drug resistance (MDR) remains a major obstacle to positive therapeutic outcomes [1,2]. One of the major causes of MDR involves the increased expression of drug efflux transporters of the ATP binding cassette (ABC) family [3][4][5][6]. Structurally, ABC transporters are highly conserved and each is composed of at least one hydrophobic membrane-spanning domain (MSD). By coupling ATP binding and hydrolysis, these pumps reduce the intracellular accumulation of many anticancer drugs to sub-therapeutic levels, thus decreasing or abolishing chemotherapy efficacy [7,8]. To date, at least 15 ABC transporters have been implicated to confer resistance to cancer chemotherapy, notably P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), are overexpressed in various cancers and extrude a wide range of chemotherapeutic agents, making them attractive therapeutic targets [9][10][11][12][13][14][15].
The most direct way to restore drug sensitivity in MDR cancer cells caused by ABC transporters is to block or modulate their activity [16]. So far, 4 generations of ABC transporter modulators including verapamil, valspodar, tariquidar, Fumitremorgin C and Ko143 have been developed [17,18]. Unfortunately, Clinical trials using MDR-inhibitors have had only limited success, mainly due to significant toxicity, lack of specificity and drug-interactions [19][20][21]. These limitations have spurred efforts to search for safe and effective inhibitors of these transporters.
Small molecule tyrosine kinase inhibitors (TKIs) are developed to block the uncontrolled activity of intracellular signaling pathways in tumor cells. Our studies have previously revealed that some TKIs are either inhibitors or substrates to alter efflux mechanisms mediated by ABC transporters. TKIs such as EGFR inhibitors (erlotinib, lapatinib), VEGFR inhibitors (axitinib, apatinib) and others can significantly inhibit the efflux function of ABC transporters, thereby sensitizing cancer cells to chemotherapeutic agents [22][23][24][25][26].

Cell cytotoxicity assay
MTT assay was used to assess the cytotoxicity of ERK5-IN-1 and chemotherapeutic agents, individually or in combination as described previously [36]. Bliss method was used to calculate the half maximal (50%) inhibitory concentration (IC 50 ) values. The fold-reversal of MDR was calculated by dividing the IC 50 of chemotherapeutic agents in the absence of ERK5-IN-1 by that obtained in the presence of ERK5-IN-1.

Animal experiments
The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Briefly, 2.5 × 10 6 KBv200 cells were subcutaneously injected into the right flank of mice (Athymic nude mice, 4 to 6 weeks old, weighing 16 to 18 g). When the tumors reached a mean volume of 50mm 3 , mice were randomized into four groups and received various treatments: (a) saline (q3d); (b) paclitaxel (20 mg/kg, i.p. q3d); (c) ERK5-IN-1 (25 mg/kg, p.o. q3d); (d) ERK5-IN-1 (25 mg/kg, p.o. q3d, given 1 h before paclitaxel administration) plus paclitaxel (20 mg/kg, i.p. q3d). The body weight and two perpendicular tumor diameters (A and B) were measured every 2 days, and the tumor volume (V) was calculated according to the following formula: V = (π/6)[(A + B)/2] 3 . When the average tumor volume reached 2000 mm 3 in the saline group, mice were euthanized and the xenografts were excised and weighed. The ratio of tumor growth inhibition (IR) was estimated according to the following formula [37].
Mean tumor weight of control group Mean tumor weight of experiment group Þ Â 100 All experiments were conducted with the approval of the Sun Yat-sen University.
Institutional Animal Care and Use Committee.

Dox accumulation assay
The effect of ERK5-IN-1 on the intracellular accumulation of Dox was measured by flow cytometry as previously described [24]. Briefly, cells were incubated with different concentrations of ERK5-IN-1 or vehicle at 37°C for 3 h. Then, 10 μM Dox was added and cells were cultured for another 3 h. Finally, the cells were collected, washed 3 times with chilled phosphate-buffered saline (PBS) and analyzed by flow cytometry (Cytomics FC500, Beckman Coulter Inc., Brea, CA, USA). VRP and FTC were used as positive control.

Dox efflux assay
Dox efflux assay was performed as described formerly [37]. KB and KBv200 cells were treated with 10 μM

ABCB1 ATPase assay
A colorimetric ATPase assay was performed as previously described with minor modification [38].

Photo-affinity labeling of ABCB1 with [ 125 I]iodoarylazidoprazosin (IAAP)
Crude membrane from High Five insect cells expressing ABCB1 (50 μg) was incubated with 0-5 μM ERK5-IN-1 for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). [ 125 I]-IAAP (2200 Ci/nmole, 3 nM) was then added and incubated for another 5 min under subdued light. The samples were cross-linked by UV illumination (365 nm) on ice and labeled ABCB1 was immunoprecipitated using the C219 antibody. Finally, samples were subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY) at -80°C for 4 h. The radioactivity incorporated into the transporter protein was quantified using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).

Western blot analysis
Western blot was performed as previously described [39]. Reverse transcription-polymerase chain reaction (PCR) and real-time quantitative PCR Cells were pre-treated with indicated concentrations of ERK5-IN-1 for 48 h and total cellular RNA was isolated by Trizol RNA extraction reagent (#15596026, Thermo Fisher Scientific Inc., MA, USA). The first strand cDNA was synthesized by cDNA reverse transcription Kit (Promega Corp., WI, USA) under the guidance of instructions. The PCR primers used were 5 ′ -CAGGCTTGCT GTAATTACCCA-3 ′ (forward) and 5 ′ -TCAAAGAAAC AACGGTTCGG-3 ′ (reverse) for ABCB1; 5 ′ -GAGT-CAAGGATTTGGTCGT-3 ′ (forward) and 5′-GATCTC GCTCCTGGAAGATG-3′ (reverse) for GAPDH, respectively. Amplification reactions were carried out at 94°C for 3 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. The products were resolved and examined by 1.5% agarose gel electrophoresis. Real-time qPCR was performed using SYBR Green qPCR Master Mix according to manufacturer's protocol. The reaction was under the following conditions: 50°C for 2 min, 95°C for 5 min and 40 cycles at 95°C for 15 s, 60°C for 30 s. Data was analyzed using the 2 -ΔΔCt method after normalization with the GAPDH expression level in each sample.

Cell surface expression of ABCB1 examined by flow cytometry
Cells were treated for 48 h with ERK5-IN-1 at 0.4 μM and then collected and washed three times with chilled PBS (supplemented with 0.5% bovine serum albumin). 1 × 10 6 cells (25 μL) were incubated with 10 μL FITCconjugated ABCB1 antibody for 45 min at 4°C, washed twice and resuspended in 400 μL PBS. Isotype control samples were treated with mouse IgG2b/κ antibody in parallel. All stained samples were analyzed by flow cytometry.

Immunofluorescence and confocal microscopy
KBv200 cells seeded at 5 × 10 5 per well on glass bottom cell culture dish and treated for 48 h in the absence and presence of ERK5-IN-1 at 0.4 μM. Cells were then washed three times with cold PBS and fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100. Subsequently, cells were washed three times with PBS, blocked for 1 h with 1% BSA in PBST and immunolabeled with ABCB1 antibody at 1:100 (cell signaling, #13342) overnight. Finally, cells were incubated with second antibody Alexa Fluor Plus 594-conjugated goat anti-rabbit IgG at 1:500 (invitrogen, A32740) for 1 h. Nuclei were counterstained with the blue fluorescent DNA stain 4′, 6-diamidino-2phenylindole (DAPI, 1:1000; Invitrogen, Molecular Probes) for 10 min. Images were acquired by a Zeiss LSM 880 microscope using 40x oil immersion objective lens and analyzed by Zens2 Software.

Data analysis
All experiments were repeated at least three times. The SPSS statistical software (SPSS 16.0) was used in statistical analysis. Results were shown as mean values ± standard deviation (SD) and the statistical differences were determined by using the Student's t-test. The statistical significance was determined at p < 0.05.

ERK5-IN-1 reversed ABCB1-mediated MDR in vivo
An established KBv200 cell xenograft model was used to evaluate the MDR reversal activity of ERK5-IN-1 in vivo. Paclitaxel and ERK5-IN-1 alone showed minimal inhibitory activity toward KBv200 tumors. However, the antitumor activity of paclitaxel was significantly enhanced when co-administered with ERK5-IN-1 and the inhibition rate was 46% (Fig. 2a, b and d). No significant body weight loss or mortality was observed, indicating that the combination regimen was tolerated by the mice (Fig. 2c).

ERK5-IN-1 inhibited the transport function of ABCB1
The results above indicated that ERK5-IN-1 effectively sensitized ABCB1 overexpressing MDR cells to chemotherapeutic agents. We further investigated the impact of ERK5-IN-1 on the transport function of ABCB1. The intracellular levels of Dox in MDR cells were much lower than the parental cells, whereas ERK5-IN-1 significantly increased the intracellular accumulation of Dox in a dosedependent manner ( Fig. 3a and b). However, no significant change in the intracellular accumulation of Dox was observed in S1-MI-80 cells suggesting that the reversal effects were dependent on the inhibition of ABCB1. By contrast, in the presence of FTC, cellular accumulation of Dox was significantly increased in S1-MI-80 cells (Fig. 3c).

ERK5-IN-1 inhibited the efflux of Dox and stimulated the ATPase activity of ABCB1
Dox efflux experiment was carried out to testify if the increased accumulation of Dox was due to inhibition of Given that the drug efflux function of ABCB1 is associated with ATP hydrolysis, we then measured the ATP hydrolysis activity mediated by ABCB1 when treated with different concentrations of ERK5-IN-1. As shown in Fig. 4b, ERK5-IN-1 (Fig. 4c). Thus, the [ 125 I]-IAAP photolabeling of ABCB1 was slightly affected by ERK5-IN-1 at 0.4 μM, suggesting that either ERK5-IN-1 binds to a separate site, or that it has low-affinity for the IAAP binding site.

ERK5-IN-1 did not affect ABCB1 expression
Considering that the reversal of MDR in cancers sometimes can be due to lowered expression of the protein, we further investigated the effect of ERK5-IN-1 on the expression of ABCB1 at protein and mRNA levels. Western blot and PCR analysis of KBv200 and MCF7/ Fig. 3 Effect of ERK5-IN-1 on the intracellular accumulation of Dox. The intracellular accumulations of Dox in: a, KB and KBv200 cells; b, MCF7 and MCF7/adr cells; c, S1 and S1-MI-80 cells were measured by flow cytometric analysis, respectively. Right column presents the quantification of Dox fluorescence, which was calculated by dividing the fluorescence intensity of each sample with that of MDR cells. Data shown are means ± SD of triplicate determinations. **P < 0.01 significantly different from control group  (Fig. 5a-c).
Cell surface expression and subcellular localization of ABCB1 in KBv200 and MCF7/adr cells were verified by flow cytometry and immunofluorescence. Our results showed that ERK5-IN-1 at 0.4 μM did not affect the cell surface expression of ABCB1 (Fig. 5d). The glycoprotein was predominantly localized in the transmembrane region in KBv200 cells and ERK5-IN-1 treatment did not change the distribution of ABCB1 (Additional file 1: Figure S1). These data indicate that ERK5-IN-1 exerts its MDR reversal activity via direct inhibition of ABCB1mediated efflux, rather than modulation of its expression.

ERK5-IN-1 did not block the phosphorylation of Akt and Erk1/2
Studies have shown that inhibition of Akt and Erk1/2, the downstream target kinases of ERK5-IN-1, may decrease resistance to antineoplastic drugs. To determine whether the MDR reversal activity of ERK5-IN-1 was related to inhibition of Akt or Erk1/2 phosphorylation, total and phosphorylated forms of Akt and Erk1/2 were measured after pretreatment with ERK5-IN-1 at concentrations used in the MTT assays. As shown in Fig. 6, ERK5-IN-1 treatment did not alter the total or phosphorylated forms of Akt and Erk1/2 in KB, KBv200, MCF7 and MCF7/adr cells. The results indicate that the MDR reversal activity of ERK5-IN-1 was independent of the blockage of Akt and Erk signaling pathways.

Discussion
Constitutive and/or acquired MDR is a major obstacle to successful chemotherapy and overexpression of some of the ABC transporters was correlated with poor prognosis of patients [40]. Inhibition of ABC transporters appears a logical approach to circumvent MDR and improve patient's outcome [41,42]. To date, three generations of ABCB1 inhibitors have been tested in clinical trials. Both, the first-generation modulators (e.g., verapamil, quinidine, and cyclosporin A), and second Representative results from three independent experiments were shown in each panel generation agents (e.g., dexverapamil, valspodar and cinchonine) in combination with chemotherapy failed to improve outcomes in clinical trials due to unacceptable toxicities and unexpected pharmacokinetic interactions, respectively. Consequently, the third-generation agents (e.g., laniquidar, zosuquidar and biricodar) were developed using structure-activity, which showed high affinity and low pharmacokinetic interaction due to a limited CYP3A inhibition. Nevertheless disappointingly, no overall satisfactory results have been obtained in clinical trials. Studies in the development of fourth-generation agents including natural compounds, peptidomimetics and surfactants are still ongoing. The development of safe and effective MDR-reversing agents still remains an unmet need in clinic.
Tyrosine kinase inhibitors (TKIs) such as imatinib, gefitinib and erlotinib are small molecules developed to inhibit the uncontrolled activity of various tyrosine kinases involved in cancer [43,44]. Many of them are either substrates, inhibitors or both for ABC transporters [22,45,46]. Some combination therapies of TKIs with cytotoxic drugs have been performed to overcome MDR in the clinic, including the combination of gefitinib with 5-fluorouracil, leucovorin, and irinotecan in patients with colorectal cancer, the combination of imatinib and cytarabine in newly diagnosed patients with chronic myeloid leukemia, and the combination of gemcitabine/ cisplatin with gefitinib in NSCLC [47][48][49].
ERK5, also known as BMK1, is the least studied member of mitogen-activated protein kinases (MAPKs) family, but has been reported to play an important role in tumor neovascularization, tumor cell invasion and migration. Scientists have also identified MEK5/ERK5 pathway as a novel target implicated in chemoresistance by gene expression profiling analysis [50]. ERK5-IN-1 was discovered as a novel ERK5 inhibitor scaffold, which shows excellent kinase selectivity and extraordinary anticancer activities. In this study, we explored the effects of ERK5-IN-1 on reversal of MDR and its interaction with ABC transporters.
The in vitro modulating potency was first studied by the potentiation of cytotoxic drug activity using the MTT assay. The cytotoxic activity of ERK5-IN-1 were evaluated in vitro using a panel of cancer cell lines stabling expressing ABCB1 or ABCG2 and ERK5-IN-1 at non-toxic concentrations up to 0.4 μM were used for reversal assay in vitro (Fig. 1). Our results showed that significant reversal of resistance in ABCB1-overexpressing cells to chemotherapy drugs was achieved in the presence of ERK5-IN-1, but no such effect in the drugsensitive parental cell lines was observed. Additionally, ERK5-IN-1 did not affect the cytotoxicity of non-Pgp substrates such as cisplatin (Table 1). However, parallel results were not observed in S1-MI-80, HL60/adr, HEK293/MRP7-2 and SW1573/2R120 cells indicating that ERK5-IN-1 could not reverse ABCG2-, ABCC1-, MRP7-and LRP-mediated MDR (Table 1 and S1).
The promising activity of ERK5-IN-1 demonstrated in vitro was further confirmed in vivo, we examined the effect of ERK5-IN-1 on enhancing the antitumor activity of paclitaxel in KBv200 xenograft model. Consistent with the in vitro results, our results indicated that ERK5-IN-1 in combination with paclitaxel resulted in markedly enhanced antitumor activity of paclitaxel in mice (Fig. 2).
To elucidate the mechanism of reversal of ABCB1mediated MDR by ERK5-IN-1, ABCB1 transport activity and expression were examined. Consistent with the high expression level of ABCB1, KBv200 and MCF7/adr cells showed lower intracellular accumulation of Dox than KB and MCF7 cells. ERK5-IN-1 treatment effectively inhibited the efflux function of ABCB1 and significantly increased the intracellular accumulation of Dox, while no significant change was found in the parental KB, MCF7, S1 and ABCG2-overexpressing S1-MI-80 cells (Fig. 3).
ABC transporters exert their drug efflux function by utilizing energy derived from the hydrolysis of ATP, and thus the change of ATPase activity of the transporters directly reflects their transport activity [51]. We found that ERK5-IN-1 significantly stimulated ATPase activity at low concentrations (0-1 μM). This is further confirmed by the results that ERK5-IN-1 competed with [ 125 I]-IAAP for the photoaffinity labeling of ABCB1 (Fig.  4). These data suggested that ERK5-IN-1 did circumvent MDR by inhibiting the efflux function of ABCB1. However, it's worth noting that that ERK5-IN-1 was less potent in inhibiting photolabeling of ABCB1 with [ 125 I]-IAAP than other TKIs (e.g., lapatinib and neratinib) which could also reverse ABCB1-mediated MDR effectively [23,52]. This indicates that either ERK5-IN-1 binds to a separate site, or that it has low-affinity for the IAAP binding site. In drug efflux assay and uptake assay ERK5-IN-1 at 0.4 μM showed comparable results to verapamil, while in cytotoxicity assay verapamil showed much higher enhancement than ERK5-IN-1 at 0.4 μM. The divergence between these two results might be partly explained by the complex competition behavior exhibited by ERK5-IN-1.
The reversal of MDR may be achieved by downregulation of the ABC transporter expression, thus the possible regulation of expression of ABCB1 by ERK5-IN-1 was also examined [53,54]. Our results showed that neither the expression of ABCB1 at protein or mRNA level nor the cell surface localization of ABCB1 was affected in the presence of ERK5-IN-1 ( Fig. 5 and Additional file 1: Figure S1).
As a kinase inhibitor, ERK5-IN-1 inhibited cell proliferation via blocking downstream signaling pathways such as phosphorylation of Akt and Erk1/2, which could also enhance the efficacy of chemotherapeutic agents. To elucidate whether the reversal effect in ABCB1mediated MDR cells was related to the altered activation of downstream targets, cells were pretreated with ERK5-IN-1 and then subjected to Western blot analysis. Our data revealed no significant alteration of the phosphorylation of Akt and Erk1/2 in the tested cell lines, suggesting that the reversal activity of ERK5-IN-1 was not associated with the inhibition Akt and Erk pathways (Fig. 6). A schematic model illustrating the reversal of MDR by ERK5-IN-1 is shown in Fig. 7.

Conclusions
Reversal of MDR by ERK5-IN-1 was through selective and potent inhibition of ABCB1 function. These findings provide strong support for further development of ERK5-IN-1 or its derivates as candidate inhibitors of MDR in cancer treatment.