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

Figures & data

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.

Table 1 CI and DRI values for combined treatment of crizotinib and chemotherapeutic drugs resulting in 50% reduction in growth of multiple breast cancer cell lines

Breast cancer cell line	Classification	Receptor status	CI		DRI
			Crizotinib and paclitaxel	Crizotinib and doxorubicin	Crizotinib	Paclitaxel	Doxorubicin
MDA-MB-231	Triple-negative	ER−, PR−, HER2−	0.427	0.68	2.5	25	3.5
MCF-7	Luminal A	ER+, PR±, HER2−	0.518	0.355	3	5.4	45.3
SK-BR-3	HER2-enriched	ER−, PR−, HER2+	1.423	1.06	ND	ND	ND

Abbreviations: CI, combination index; DRI, dose reduction index; ND, not determined.

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.