Effect of ceritinib (LDK378) on enhancement of chemotherapeutic agents in ABCB1 and ABCG2 overexpressing cells in vitro and in vivo

Abstract

Multidrug resistance (MDR) is the leading cause of treatment failure in cancer chemotherapy. The overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1, ABCC1 and ABCG2, play a key role in mediating MDR by pumping anticancer drugs out from cancer cells. Ceritinib (LDK378) is a second-generation tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) currently in phase III clinical trial for the treatment of non-small cell lung cancer. Here, we found that ceritinib remarkably enhanced the efficacy of chemotherapeutic drugs in ABCB1 or ABCG2 over-expressing cancer cells in vitro and in vivo. Ceritinib significantly increased the intracellular accumulation of chemotherapeutic agents such as doxorubicin (DOX) by inhibiting ABCB1 or ABCG2-mediated drug efflux in the transporters-overexpressing cells. Mechanistically, ceritinib is likely a competitive inhibitor of ABCB1 and ABCG2 because it competed with [(125)I]-iodoarylazidoprazosin for photo affinity labeling of the transporters. On the other hand, at the transporters-inhibiting concentrations, ceritinib did not alter the expression level of ABCB1 and ABCG2, and phosphorylation status of AKT and ERK1/2. Thus the findings advocate further clinical investigation of combination chemotherapy of ceritinib and other conventional chemotherapeutic drugs in chemo-refractory cancer patients.

Keywords: ABCB1; ABCG2; ATP-binding cassette transporters; ceritinib; multidrug resistance.

Figure 1. The structure of Ceritinib and cytotoxicity of Ceritinib

A. the structure of Ceritinib; B. MTT cytotoxicity assay was assessed ABCB1 and ABCG2 over-expressing cells and their parental sensitive cells: ABCB1-negative KB and ABCB1-overexpressing KBv200 cells; C. ABCB1-negative MCF-7 and ABCB1-overexpressing MCF-7/adr cells; D. ABCG2-negative S1 and ABCG2-overexpressing S1-M1-80 cells; E. ABCB1-negative HEK293/pcDNA3.1 and ABCB1-overexpressing HEK293/ABCB1 cells; F. ABCG2- negative HEK293/pcDNA3.1 and ABCG2-overexpressing HEK293/ABCG2-R2 cells. All cells were treated with a range of different concentrations of ceritinib for 72 hours. Results from three independent experiments are presented as the mean ± SD.

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).

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.

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.

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.

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.

Figure 7. Effect of ceritinib on the phosphorylation of AKT and ERK1/2

KBv200, MCF-7/adr and S1-MI-80 and their parental cells were treated with different concentrations of ceritinib for 48 h. Equal amount of protein was loaded for Western blot analysis as described in “Materials and Methods”. Independent experiments were performed at least three times.