Crizotinib (PF-02341066) reverses multidrug resistance in cancer cells by inhibiting the function of P-glycoprotein

Abstract

Background and purpose: Besides targeting the well-known oncogenic c-Met, crizotinib is the first oral tyrosine kinase inhibitor inhibiting anaplastic lymphoma kinase (ALK) in clinical trials for the treatment of non-small cell lung cancer. Here, we assessed the possible reversal of multidrug resistance (MDR) by crizotinib in vitro and in vivo.

Experimental approach: 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan was used in vitro and xenografts in nude mice were used in vivo to investigate reversal of MDR by crizotinib. To understand the mechanisms for MDR reversal, the alterations of intracellular doxorubicin or rhodamine 123 accumulation, doxorubicin efflux, ABCB1 expression level, ATPase activity of ABCB1 and crizotinib-induced c-Met, Akt and ERK1/2 phosphorylation were examined.

Key results: Crizotinib significantly enhanced the cytotoxicity of chemotherapeutic agents which are also ABCB1 substrates, in MDR cells with no effect found on sensitive cells in vitro and in vivo. Additionally, crizotinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and inhibited the drug efflux in ABCB1-overexpressing MDR cells. Further studies showed that crizotinib enhanced the ATPase activity of ABCB1 in a concentration-dependent manner. However, expression of ABCB1 was not affected, and reversal of MDR by crizotinib was not related to the phosphorylation of c-Met, Akt or ERK1/2. Importantly, crizotinib significantly enhanced the effect of paclitaxel against KBv200 cell xenografts in nude mice.

Conclusions and implications: Crizotinib reversed ABCB1-mediated MDR by inhibiting ABCB1 transport function without affecting ABCB1 expression or blocking the Akt or ERK1/2 pathways. These findings are useful for planning combination chemotherapy of crizotinib with conventional chemotherapeutic drugs.

Figure 1

Cytotoxicity of crizotinib in the drug-resistant and parental, sensitive, cancer cells. A, the structure of crizotinib; MTT cytotoxicity assay was assessed in pairs of parental and transporter-overexpressing cells: B, ABCB1-negative KB and ABCB1-positive KBv200 cells. C, ABCB1-negative MCF-7 and ABCB1-positive MCF-7/adr cells. D, ABCG2-negative S1 and ABCG2-positive S1-M1-80 cells. E, ABCC1-negative HL60 and ABCC1-positive HL60/adr cells. F, ABCB1-negative HEK293 and HEK293/ABCB1 cells. Data shown are means ± SD for three determinations. Each experiment was performed in three replicate wells.

Figure 2

Potentiation of the antitumour effects of paclitaxel by crizotinib in a KBv200 xenograft model in athymic nude mice. A, changes of body weight after tumour cell inoculation. Data shown are means ± SD for each group of seven mice after implantation. B, changes in tumour volume with time after tumour cell inoculation. Data shown are means ± SD for each group of seven mice after implantation. C, tumour size. The photograph was taken on the 24th day after implantation. The various treatments were as follows: control (vehicle alone); paclitaxel (18 mg·kg⁻¹, i.p., q3d × 4); crizotinib (25 mg·kg⁻¹, p.o., q3d × 4) and paclitaxel (18 mg·kg⁻¹, i.p., q3d × 4) plus crizotinib (25 mg·kg⁻¹, p.o., q3d × 4, given 1 h before paclitaxel administration).

Figure 3

Effect of crizotinib on the accumulation of doxorubicin and rhodamine 123. The accumulation of doxorubicin (Dox; A, B) and rhodamine 123 (C, D) were measured by flow cytometric analysis. The results are presented as fold change in fluorescence intensity relative to control MDR cells. Data shown are means ± SD of triplicate determinations. **P < 0.01 significantly different from control group.

Figure 4

Effect of crizotinib on the efflux of doxorubicin and on ABCB1 ATPase activity. A, Time course of doxorubicin (Dox) efflux was measured in KB and KBv200 cells, with or without 1.5 µM crizotinib. B, ABCB1 ATPase assays were performed according to the instruction of Pgp-Glo™ Assay Systems. All these experiments were repeated at least three times. Data shown are means ± SD for independent determinations in triplicate.

Figure 5

Effect of crizotinib on the expression of ABCB1 in MDR cells. KBv200 cells were treated with crizotinib at various concentrations for 48 h: A, equal amounts of total cell lysates were loaded and detected by Western blot; B, the mRNA level of ABCB1 was determined by RT-PCR. C, summary data from the mRNA level of ABCB1 determined by RT real-time-PCR. A representative result is shown from at least three independent experiments. All these experiments were repeated at least three times, and a representative experiment is shown in each panel.

Figure 6

Effect of crizotinib on phosphorylation of c-Met, Akt and ERK1/2. KB and KBv200 cells were treated with crizotinib at various concentrations for 24 h and for various times at 1.5 µM. 10 µM crizotinib was used as a positive control for blockade of c-Met phosphorylation. 10 µM lapatinib was used as a positive control for blockade of Akt and ERK1/2 phosphorylation. Equal amounts of protein were loaded for Western blot analysis. Independent experiments were performed at least three times and results from a representative experiment are shown.