Crizotinib, a MET inhibitor, inhibits growth, migration, and invasion of breast cancer cells in vitro and synergizes with chemotherapeutic agents

Abstract

MET is a receptor tyrosine kinase known for its pleiotropic effects in tumorigenesis. Dysregulations of MET expression and/or signaling have been reported and determined to be associated with inferior outcomes in breast cancer patients rendering MET a versatile candidate for targeted therapeutic intervention. Crizotinib is a multi-targeted small-molecule kinase inhibitor for MET, ALK, and ROS1 kinases. This study evaluated the anti-proliferative, cytotoxic, anti-migratory, and anti-invasive effects of crizotinib in breast cancer cells in vitro. Cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. In vitro wound-healing assay was used to examine the effect of crizotinib on breast cancer cell migration. The expressions of Ki-67, MET, and phospho-MET receptors were characterized using immunofluorescence staining. Results showed that crizotinib has significant anti-proliferative activity on all mammary tumor cells with IC₅₀ values of 5.16, 1.5, and 3.85 µM in MDA-MB-231, MCF-7, and SK-BR-3 cells, respectively. Crizotinib induced cytotoxic effects in all breast cancer cells examined. Combined treatment of small dose of crizotinib with paclitaxel or doxorubicin exhibited a highly synergistic inhibition of growth of MDA-MB-231 and MCF-7 cells with combination index values <1 while no significant effect was observed in SK-BR-3 cells compared with individual compounds. Treatment with crizotinib demonstrated a remarkable reduction in the expression of Ki-67 protein in all 3 tested cell lines. Crizotinib inhibited migration and invasion of MDA-MB-231 cells in a dose-dependent fashion. Crizotinib reduced MET receptor activation in MDA-MB-231 cells when treated at effective concentrations. In conclusion, crizotinib suppressed proliferation, migration, and invasion of breast cancer cells in vitro. The results of this study demonstrated that combined treatment of crizotinib with chemotherapeutic agents resulted in a synergistic growth inhibition of specific breast cancer cell lines.

Keywords: MET receptor; breast cancer; chemotherapy; crizotinib; invasion; migration.

Figure 1

Growth and cytotoxic effects of crizotinib on breast cancer cells. Notes: (A) Antiproliferative effects of crizotinib on breast cancer cells in vitro. (B) Cytotoxic effects of crizotinib on breast cancer cells in vitro. Vertical bars indicate the mean cell count ± SEM in each treatment group. ^*P<0.05 as compared with vehicle-treated control group.

Figure 2

Effects of chemotherapeutic agents on growth of breast cancer cells. Notes: (A) Antiproliferative effects of paclitaxel on breast cancer cells in vitro. (B) Antiproliferative effects of doxorubicin on breast cancer cells in vitro. Vertical bars indicate the mean cell count ± SEM in each treatment group. ^*P<0.05 as compared with vehicle-treated control group.

Figure 3

Effects of combined treatment of crizotinib and chemotherapeutic agents on growth of breast cancer cells. Notes: (A) Effects of combined crizotinib and paclitaxel treatment on growth of breast cancer cells after 48 h of treatment duration. (B) Effects of combined crizotinib and doxorubicin treatment on growth of breast cancer cells after 48 h of treatment duration. Vertical bars represent the mean cell count ± SEM in each treatment group. ^*P<0.05 compared with respective vehicle-treated control group, and ^**P<0.05 compared to respective group with individual chemotherapy treatment. Abbreviation: ND, not detectable.

Figure 4

Isobolograms for the anti-proliferative effects of combined treatment of crizotinib and chemotherapeutic drugs in breast cancer cells. Notes: (A) Isobolograms of crizotinib and paclitaxel anti-proliferative effects on breast cancer cells. (B) Isobolograms of crizotinib and doxorubicin anti-proliferative effects on breast cancer cells. The data point on each isobologram represents the actual concentrations of crizotinib and chemotherapeutic drug which induced 50% inhibition of cell growth when used in combination.

Figure 5

Effect of crizotinib treatment on Ki-67 labeling in breast cancer cells. Notes: Effect of crizotinib treatment on Ki-67 expression in (A) MDA-MB-231 cells, (B) MCF-7 cells, and (C) SK-BR-3 cells. Upper panel: immunofluorescent staining for Ki-67 in breast cancer cells. Red color indicates positive fluorescence staining for Ki-67, while blue color represents counterstaining of cell nuclei with DAPI. Magnification of each image is 20×. Bottom panel: percentage of positive Ki-67 cancer cells in proportion to the total number of cells. Vertical bars represent percentage of cells with positive Ki-67 staining ± SEM in each treatment group. ^*P<0.05 compared to vehicle-treated control group.

Figure 6

Effect of crizotinib treatment on migration and invasion of breast cancer cells. Notes: (A) Photomicrographs of crizotinib treatment on migration of MDA-MB-231 cancer cells using the in vitro wound-healing assay. Photomicrographs (4× magnification) were taken at the beginning and end of the treatment period after cell fixation. (B) Quantitative analysis of wound closure in each experimental group. Vertical bars represent percent migration ± SEM. ^*P<0.05 compared to vehicle-treated control group. (C) Anti-invasive effect of crizotinib on MDA-MB-231 cells using Trevigen Cultrex BME cell invasion assay. Bars represent average percentage ± SEM of invading cells out of 4 replicates for each treatment group. ^*P<0.05 compared to vehicle-treated control group. Abbreviation: BME, basement membrane extract.

Figure 7

Effect of crizotinib treatment on total levels of MET and phospho-MET (P-MET) in breast cancer cells. Notes: Effect of crizotinib treatment on levels of (A) MET and (B) P-MET in MDA-MB-231 cells. Upper panel: immunofluorescent staining for (A) MET and (B) P-MET in MDA-MB-231 cells. Red color in the photomicrographs indicates positive fluorescence staining for (A) MET and (B) P-MET, while blue color represents counterstaining of cell nuclei with DAPI. Magnification of each image is 20×. Bottom panel: percentage of cancer cells with positive staining for (A) MET and (B) P-MET in proportion to the total number of cells. Vertical bars represent percentage of cells with positive staining ± SEM in each treatment group. ^*P<0.05 compared to vehicle-treated control group.