Ceritinib potentiated the cytotoxicity of conventional chemotherapeutic agents in ABCB1 and ABCG2-overexpressing cells

The IC₅₀ values of the anticancer drugs in sensitive and resistant cells in the absence or presence of ceritinib are shown in Table ​Table11 and Table ​Table2.2. Ceritinib produced a concentration-dependent decrease in the IC₅₀ values of doxorubicin and paclitaxel (both are ABCB1 substrates) in KBv200 cells and MCF-7/adr cells but did not alter the cytotoxicity of cisplatin which is not an ABCB1 substrate. The similar results were observed in the stably transfected HEK293/ABCB1, but not in the parental sensitive cells, even at the maximum concentration used. Ceritinib also significantly enhanced the cytotoxicity of mitoxantrone and topotecan (ABCG2 substrate drugs) in ABCG2 overexpressing S1-M1-80 and transfected HEK293/ABCG2-R2 cells, while there was also no obvious effect on the parental S1 and HEK293/pcDNA3.1 cells. These results suggest that ceritinib enhances the sensitivity of ABCB1 and ABCG2 overexpressing MDR cells to conventional chemotherapeutic agents, but has negligible effect on their parental cell lines.

Ceritinib enhanced the efficacy of conventional chemotherapeutic agents in ABCB1-mediated MDR in nude mouse xenografts

An ABCB1-mediated multidrug resistant KBv200 cell xenograft model was established in nude mice to evaluate whether ceritinib could reverse the resistance to paclitaxel in vivo. We did not find significant difference in tumor size between animals treated separately with saline, ceritinib or paclitaxel. However, there was a significant inhibition of tumor growth in the group with a combination of ceritinib and paclitaxel compared with other groups (P < 0.05; Figure ​Figure2).2). The mean weights of tumors excised from mice were 1.91 ± 0.52, 1.63 ± 0.54, 1.60 ± 0.66, 0.99 ± 0.44g for saline, paclitaxel, Ceritinib and combination group, respectively. Furthermore, we did not observe any death or apparent decrease in body weight in the combination treatment group at the doses tested, suggesting that the combination regimen did not increase toxicity.

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Figure 2

Ceritinib enhanced the anticancer effect of paclitaxel in the KBv200 cell xenograft model in nude mice

A. the changes in tumor volume over time after the KBv200 cell implantation. Data shown are mean ± SD of tumor volumes for each group. n = 8. B. the image of tumors size in four groups excised from the mice on the 21th day after implantation. C. Average percentage change in body weight after treatments. D. mean tumor weight (n = 8) after excising from the mice on the 21th day after implantation. The four treatment groups were: (1) saline (q3d × 4); (2) paclitaxel (20 mg/kg, i.p., q3d × 4); (3) Ceritinib (25 mg/kg, p.o., q3d × 4); and (4) Ceritinib (25 mg/kg, p.o., q3d × 4 given 1 h before injecting paclitaxel) + paclitaxel (20 mg/kg, i.p., q3d × 4).

Ceritinib enhanced the accumulation of DOX and Rho123 in cells overexpressing ABCB1 and ABCG2

The results described above revealed that ceritinib could enhance the sensitivity of ABCB1 and ABCG2-overexpressing cells to the transporter substrate anticancer agents in vitro and in vivo. To investigate the potential mechanisms for the MDR reversal by ceritinib, we examined the intracellular accumulation of DOX and Rho 123 in the presence or absence of ceritinib in ABCB1- and ABCG2- overexpressing MDR cells and their parental cells. Our results showed that the intracellular accumulation of DOX or Rho123 in resistant cells was significantly lower than that in sensitive cells in the absence of ceritinib. Importantly, ceritinib was found to significantly increase the intracellular accumulation of DOX (fluorescent substrate drug of both ABCB1 and ABCG2) and Rho123 (fluorescent probe substrate of ABCB1 and ABCG2) in a concentration-dependent manner in KBv200, MCF-7/adr and S1-MI-80, while no notable change was observed in the parental cells (Figure ​(Figure33 & 4). These results suggested that ceritinib could increase intracellular accumulation of chemotherapeutic agents in ABCB1 and ABCG2-overexpressing cells.

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Figure 3

Effect of ceritinib on the intracellular accumulation of Dox in MDR cells and their parental cells

The accumulation of DOX A, B, C. in KBv200, MCF-7/adr, S1-MI-80 cells and their parental cells were measured by flow cytometric analysis as described in Materials and Methods, The results were presented as fold change in fluorescence intensity relative to control MDR cells. Columns, means of triplicate determinations; bars, SD. “*” P < 0.05, “**” P < 0.01 significantly different from control group.

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Figure 4

Effect of ceritinib on the intracellular accumulation of Rho123 in MDR cells and their parental cells

The accumulation of Rho123 A, B, C. in KBv200, MCF-7/adr, S1-MI-80 cells and their parental cells were measured by flow cytometric analysis as described in Materials and Methods, The results were presented as fold change in fluorescence intensity relative to control MDR cells. Columns, means of triplicate determinations; bars, SD. “*” P < 0.05, “**” P < 0.01 significantly different from control group.

Ceritinib inhibited the efflux of DOX in MDR cells overexpressing ABCB1

Ceritinib increased intracellular accumulation of DOX and Rho123 in ABCB1-overexpression MDR cells; Next, we examined whether the increased accumulation of anticancer agents was due to inhibition of efflux of anticancer agents. The efflux of DOX over 2 h after an initial drug accumulation was monitored and the result is shown in Figure ​Figure5A.5A. As expected, due to ABCB1 overexpression in KBv200 cells, DOX retention dropped remarkably from 100% (0 h efflux) to about 46.4% (2 h efflux). The decrease in DOX retention was much less in the parental KB cells (69.4% retention at 2 h). Importantly, ceritinib (0.5 μM) was found to significantly increase DOX retention (p < 0.05) in KBv200 cells to 63.0% of the level attained at the 2 h time point. The result shows that ceritinib inhibited drug efflux of ABCB1 in KBv200 cells but did not influence drug efflux in sensitive KB cells.

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Figure 5

Effect of ceritinib on the efflux of DOX, the ATPase activity of ABCB1 and ABCG2 and the [125I]-IAAP photoaffinity labeling of ABCB1 and ABCG2

A. Time course of Dox efflux was measured in KB and KBv200 cells, with or without 0.5 μM Ceritinib. B, C. Effect of ceritinib on ATPase activity of ABCB1 and ABCG2. The vanadate-sensitive ABCB1 or ABCG2 ATPase activity in the presence of the indicated concentrations of ceritinib was evaluated. The mean and standard error values from three independent experiments are shown. D, E. Ceritinib competed for photolabeling of ABCB1 or ABCG2 by [¹²⁵I]-IAAP. Crude membranes from High Five insect cells expressing ABCB1 or ABCG2 were incubated with [¹²⁵I]-IAAP and increasing concentration (0 – 5 μM) of ceritinib. The samples were then cross-linked by UV illumination, subjected to electrophoresis, and analyzed as outlined under Materials and Methods. A representative autoradiogram from three independent experiments is shown. The relative amount of [¹²⁵I]-IAAP incorporated is plotted against the concentration of ceritinib present. 100% incorporation refers to the absence of ceritinib.

Ceritinib stimulated the ATPase activity of ABCB1 and ABCG2

The drug-efflux function of ABCB1 and ABCG2 is linked to ATP hydrolysis which is stimulated in the presence of ABCB1 and ABCG2 substrates. To understand whether ceritinib influenced the ATPase activity of ABCB1 and ABCG2, we measured the vanadate-sensitive ATPase activity of ABCB1 and ABCG2 in the presence of a range of different concentrations of ceritinib. While ceritinib activated the ATPase activity of ABCB1 in a concentration-dependent manner, it stimulated the ATPase activity of ABCG2 at low concentrations but the stimulated ABCG2 ATPase activity dropped at higher concentration of ceritinib (Figure 5B, 5C). These results indicated that ceritinib may be a substrate of ABCB1 and ABCG2.

Ceritinib bound to substrate binding site of ABCB1 and ABCG2

The substrate binding site of ABCB1 or ABCG2 could be photo-labeled by [¹²⁵I]-IAAP. It is known that substrate of ABCB1 or ABCG2 could compete for [¹²⁵I]-IAAP labeling of the two transporters [12]. To further ascertain the interaction of ceritinib with the substrate binding sites of ABCB1 and ABCG2, we examined the photo-labeling of ABCB1 and ABCG2 with [¹²⁵I]-IAAP by incubating membrane vesicles in the presence of various concentrations of ceritinib. As showed in (Figure 5D, 5E), ceritinib strongly inhibited the photoaffinity labeling of ABCB1and ABCG2 with [¹²⁵I]-IAAP in a concentration-dependent manner. The results suggest that ceritinib binds with high affinity to both the ABCB1 and ABCG2 substrate-binding sites.

Ceritinib did not alter the expression level of ABCB1 and ABCG2

ABC transporter-mediated MDR may be circumvented by downregulating the expression of the transporters or by inhibiting their transport function. Therefore, we determined the effect of ceritinib on the mRNA and protein expression of ABCB1 and ABCG2 using RT-PCR and Western blot analysis, respectively. Our results showed that no significant difference in ABCB1 or ABCG2 expression at the mRNA or protein level was observed in KBv200, MCF-7/adr cells or S1-M1-80 cells treated with 0.125, 0.25, 0.5 μM ceritinib for 48 h compared to untreated control cells (Figure 6A–6C). Therefore, it is likely that ceritinib reversed MDR by inhibiting the transport function of ABCB1 and ABCG2, but not by downregulating the expression of the transporters.

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Figure 6

Effect of ceritinib on the expression of ABCB1 or ABCG2 in cells

A. The protein level of ABCB1 and ABCG2 were measured by Western blot analysis, and B. mRNA level were measured by RT-PCR, Ceritinib did not alter the mRNA and protein levels in MDR cells. C. q-PCR was further applied to confirm the unaltered mRNA levels in MDR cells. D, E. the cell surface expression of ABCB1 and ABCG2 were measured by Flow Cytometer. All these experiments were repeated at least three times, and data from a representative experiment is shown in each panel.

Ceritinib did not change the cell surface expression of ABCB1 and ABCG2

To further understand whether the localization of ABCB1 and ABCG2 were influenced by ceritinib, we analyzed the cell surface expression of ABCB1 and ABCG2 in the presence or absence of 0.5 μM ceritinib in ABCB1- or ABCG2- overexpressing MDR cells and their parental cells. We found that the expression of ABCB1 or ABCG2 was no obvious change in KBv200, MCF-7/adr and S1-MI-80 in the presence or absence of 0.5 μM ceritinib, and their parental cells no expression of ABCB1 or ABCG2, respectively (Figure 6D, 6E). These suggest ceritinib did not alter the localization of ABCB1 or ABCG2.

Cell culture

The human oral epidermoid carcinoma cell line KB and its vincristine-selected ABCB1 overexpressing derivative KBv200 cells [33]; the human breast carcinoma cell line MCF-7, its doxorubicin selected ABCB1 overexpressing derivative MCF-7/adr cells [34]; the human colon carcinoma cell line S1 and its mitoxantrone-selected ABCG2-overexpressing derivative S1-M1-80 cells [35] and the human embryonic kidney cell line HEK293 and its stable pcDNA3.1, ABCB1 and ABCG2 stable gene transfect cell lines HEK293/pcDNA3.1, HEK293/ABCB1, HEK293/ABCG2-R2 were kind gift provided by Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD, U.S.A) [36]. The transfected cells were cultured in medium containing 2 mg/mL G418. All cell lines were cultured in DMEM or RPMI 1640 supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. All cells were grown in drug-free culture medium for more than 2 weeks before assay.

Cell cytotoxicity assay

The MTT assay was used to assess cytotoxicity as described previously [37]. Briefly, cells growing in logarithmic phase were seeded at a density of 2000 ~ 6000 cells per well in 96-well plates. When the cells became adherent 24 h later, a range of different concentrations of conventional chemotherapeutic drugs with or without a fixed combination of ceritinib were added to the wells. After 68 h, MTT (5 mg/ml, 20 μL) was added into each well, and 4 h later, the medium was discarded and 150 μL DMSO was added into the wells to dissolve the formazan product from the metabolism of MTT. Finally, optical density was measured at 540 nm, with background subtraction at 670 nm by a Model 550 Microplate Reader (Bio-Rad, Hercules, CA, USA). The half maximal (50%) inhibitory concentration (IC₅₀) value of a substance was calculated by the Bliss method [38]. The fold reversal of MDR was calculated as previously described [29]. All experiments were repeated at least three times.

Establishment of KBv200 cell xenograft model and reversal of MDR by ceritinib in vivo

The KBv200 cell xenograft model was established as described previously with minor modification [39], Athymic nude mice (5–6 weeks old) were purchased from the center of experimental animals (Southern Medical University). Briefly, KBv200 cells grown in vitro were harvested and implanted subcutaneously under the shoulder in the nude mice. When the tumors reached a mean diameter of 0.5 cm, the mice were randomized into four groups and treated as follows: (1) saline (q3d × 4); (2) paclitaxel (20 mg/kg, i.p., q3d × 4); (3) Ceritinib (25 mg/kg, p.o., q3d × 4); and (4) Ceritinib (25 mg/kg, p.o., q3d × 4 given 1 h before injecting paclitaxel)+ paclitaxel (20 mg/kg, i.p., q3d × 4). The body weights of the animals and the two perpendicular diameters (A and B) were recorded every 2 days, and tumor volume (V) was estimated according to the following formula:

The curve of tumor growth was drawn according to tumor volume and time of implantation. The mice were anesthetized and sacrificed when the mean of tumor weights was over 1 g in the control group. Tumor tissues were excised from the mice and their weight was measured. The ratio of growth inhibition (IR) was calculated according to the following formula:

Doxorubicin (DOX) and rhodamine 123 (rho 123) accumulation

The effect of ceritinib on the accumulation of DOX and Rho 123 was measured by flow cytometry as previously described [40]. Briefly, the cells were incubated in six-well plates to allow attach to the wall overnight. Then the cells were exposed to different concentrations of ceritinib (0.125, 0.25, 0.5 μM). After 3 h, DOX (10 μM) or Rho 123 (5 μM) was added to the medium for further incubation for another 3 h or 0.5 h, then the cells were collected, centrifuged and washed three times with ice-cold phosphate-buffered saline (PBS) buffer, cells were resuspended in 500 μl PBS buffer for flow-cytometric analysis (Cytomics FC500; Beckman Coulter Inc., Brea, CA, USA). Verapamil, a known ABCB1 inhibitor, was used as a positive control for KBv200, KB, MCF-7/adr, MCF-7 cells [41]. Fumitremorgin C (FTC), a specific ABCG2 inhibitor, was used as a positive control of ABCG2 in S1-MI-80 and S1 cells [42].

DOX efflux test

DOX efflux was performed following a modification of methods described earlier [28]. KB and KBv200 cells were treated with 10 μM DOX for 3 h at 37°C, then the cells were washed three times and subsequently maintained at 37°C with culture media without DOX in the presence or absence of 0.5 μM ceritinib at 0, 15, 30, 60 and 120 min, cells were collected and washed three times with ice-cold PBS. Finally, cells were resuspended in ice-cold PBS buffer for flow cytometric analysis immediately (Beckman Coulter).

ABCB1 and ABCG2 ATPase activity assay

A colorimetric ATPase assay was performed as previously described with minor modification [43]. Briefly, crude membranes isolated from High Five insect cells expressing either ABCB1 or ABCG2 (100 μg protein/mL) were incubated at 37°C with a range of different concentrations of ceritinib in the presence or absence of sodium orthovanadate (0.3 mM for ABCB1 and 1.2 mM for ABCG2) in ATPase assay buffer (50 mM KCl, 5 mM sodium azide, 2 mM EDTA, 10 mM MgCl₂, 1 mM DTT, pH 6.8) for 5 min. The crude membranes were kind gift provided by Dr Suresh Ambudkar (National Cancer Institute, NIH, USA). ATP hydrolysis reaction was then started by the addition of 5 mM Mg-ATP (concentration in a final volume of 60 μL) and incubated for 20 min (for ABCB1) or 10 min (for ABCG2). SDS solution (30 μL of 10% SDS) was added to terminate the reaction. Absorbance was subsequently measured at 750 nm after the addition of a detection reagent (35 mM ammonium molybdate, 15 mM zinc acetate, 10% ascorbic acid) and incubation at 37°C for 20 min. The amount of inorganic phosphate released was quantified by reading from a standard curve. Specific ceritinib-stimulated ABCB1 and ABCG2 ATPase activity (i.e. vanadate-sensitive) was determined as the difference between the amounts of inorganic phosphate released from ATP in the absence and presence of sodium orthovanadate.

Photo-affinity labeling of ABCB1 and ABCG2 with [¹²⁵I]-IAAP

Crude membrane from High Five insect cells expressing ABCB1 or ABCG2 (50 μg protein) was incubated with 0 – 5 μM ceritinib for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). [¹²⁵I]-IAAP (2200 Ci/nmole, 3 nM) was added and incubation was continued for a further 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. The labeled ABCB1 and ABCG2 was immunoprecipitated using the C219 and BXP21 antibody, respectively. The samples were then 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).

Reverse transcription-polymerase chain reaction (PCR) and Q-PCR

ABCB1 and ABCG2 mRNA expression level was assayed as previously described [27]. Cells were treated with different concentrations of ceritinib for 48 h, after which total cellular RNA was isolated by Trizol Reagent RNA extraction kit following the manufacturer's instruction (Molecular Research Center, Cincinnati, OH, USA). The first strand cDNA was synthesized by Oligo dT primers with reverse transcriptase (Promega Corp.). PCR primers were 5′-CCCATCATTGCAATAGCAGG-3′ (forward) and 5′-GTTCAAACTTCTGCTCCTGA-3′ (reverse) for ABCB1, 5′-TGGCTGTCATGGCTTCAGTA-3′ (forward) and 5′-GCCACGTGATTCTTCCACAA-3′ (reverse) for ABCG2, 5′-CTTTGGTATCGTGGAAGGA-3′ (forward) and 5′-CACCCTGTTGCTGTAGCC-3′ (reverse) for GAPDH, 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. At last products were resolved and examined by 1% agarose gel electrophoresis.

Real-time PCR was performed with Real-time PCR Master Mix containing SYBR GREEN I and hotstart Taq DNA polymerase. SYBR Green Assay kit (Molecular Research Center, Cincinnati, OH, USA) was used for real time PCR reaction, following 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 were analyzed using the 2−ΔΔCt method and normalized by GAPDH expression in each sample.

Western blot analysis

The protein expression of ABCB1, ABCG2 or the phosphorylation of AKT and ERK1/2 were evaluated in the ABCB1 and ABCG2 over-expressing cells and their parental cells after treatment with ceritinib (0.125, 0.25 and 0.5 μM) for 48 h. Western blot analysis was conducted as previously described [27]. After blocking with 5% nonfat milk for 2 hours at room temperature, the membranes were immunoblotted by using antibodies including ABCB1, ABCG2, ERK1/2, p-ERK, AKT and p-AKT overnight at 4°C. The membranes were then washed three times with TBST and incubated with HRP-conjugated secondary antibody at 1:5000 dilution for 2 h at room temperature. After three times washed with TBST, the protein-antibody complexes were visualized by the enhanced Phototope TM-HRP Detection Kit (Cell Signaling) and exposed to Kodak medical X-ray processor (Carestream Health, Atlanta, GA, USA). GAPDH was used as a loading control.

Detection of cell surface expression of ABCB1 and ABCG2 by flow cytometer

KBv200, KB, MCF-7/adr, MCF-7, S1-MI-80 and S1 cells were collected and washed three times with an isotonic PBS buffer (supplemented with 0.5% bovine serum albumin(BSA)). For ABCB1 expression analysis, approximately 1×10⁶ KBv200, KB, MCF-7/adr and MCF-7 cells (100ul) were incubated at 4°C for 45 min with 10 μL of FITC-conjugated anti-human pgp (Beckman Coulter, Fullerton, CA, USA), then the cells were washed twice with PBS buffer (supplemented with 0.5% BSA) and resuspended in 400 μL PBS buffer for flow cytometric analysis, Isotype control samples were treated with mouse IgG2a antibody in parallel For ABCG2 expression analysis, FITC-conjugated anti-human Bcrp1/ABCG2 (Santa Cruz, CA, USA) reagent were mixed with 25 μL of Fc-blocked cells (1 × 10⁶ cells). After incubating for 45 min at 4°C, the cells were washed twice with PBS buffer (supplemented with 0.5% BSA) and resuspended in 400 μL PBS buffer for flow cytometric analysis, Isotype control samples were treated in an identical manner with FITC-labeled mouse immunoglobin G2b (IgG2b) antibody. All experiments were repeated at least three times.

We thank Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD, U.S.A) for the ABCB1 and ABCG2 transfected cell lines and everyone who has gave a help to the research. This work was supported by grants from National Nature Scientific Foundation (No. 81473233) and International Collaboration Science Research Foundation of Guangdong Province (No. 2013B051000046) and Medical Science and Technology Research Fund of Guangdong Province (No. B2013145).