A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.

Keywords: Met; SRC; glioblastoma multiforme; invasion; tyrosine kinase inhibitors.

Figure 1. Cytotoxicity of a combination of dasatinib and crizotinib on GBM cell lines and GBM tumor spheroids

A. Cell number was measured following 72 h of treatment at concentrations of 0.2 μM dasatinib and 4 μM crizotinib using an SRB assay in I: U87, II: LN-18, III: U373, IV: A172, V: NZG1003 and VI: NZG0906 cells. Data are expressed as the mean ± SEM. Experiments were conducted in triplicate and repeated independently three times. B. U87 spheroids I: before treatment, II: following 4 days treatment and III: Ki67 staining of spheroids treated for 4 days. Experiments were conducted in sextuplicate and repeated independently three times, representative images are shown. C. NZG1003 spheroids I: before treatment and II: following 4 days treatment. Experiments were conducted in sextuplicate and repeated independently three times, representative images are shown. D. Acid phosphatase assay of I: U87 and II: NZG1003 spheroids following 4 days of treatment. Data are expressed as the mean ± SEM. Experiments were conducted in sextuplicate and repeated independently three times. Scale bars denote 100 μm, * denotes p ≤ 0.05 as determined by an ANOVA with a Bonferroni post-hoc test.

Figure 2. Combination of dasatinib and crizotinib decreases the activity and/or expression of Met, SRC and related proteins

GBM established and primary cell lines were treated with dasatinib 0.2 μM, crizotinib 4 μM or their combination for 48 h. Total lysates were analyzed by western blotting with antibodies as indicated.

Figure 3. Immunocytochemistry of SRC and Met localization

Cellular localization of SRC and Met following treatment for 48 h with dasatinib, crizotinib or combination in A. LN-18 and B. U373 cells. Scale bar denotes 10 μm. Nuclei were visualized by DAPI while SRC and Met were labeled with a fluorescein and Texas-red conjugated secondary antibodies, respectively.

Figure 4. Combination treatment promotes apoptosis in GBM cell lines

Cells were treated with dasatinib 0.2 μM, crizotinib 4 μM or their combination for up to 72 h followed by a washout period of up to 72 h, cell death was assessed at 48 and 72 h and every 24 h during the washout period. Bars denote necrotic; ■ (PI stained) and apoptotic; □ (Annexin V stained). A. U87, B. LN-18, C. U373, and D. A172 cells. Experiments were conducted in triplicate and independently repeated three times, p ≤ 0.05 relative to control for annexin V (*) and PI (ƚ).

Figure 5. Combination treatment triggered cell cycle arrest in G2/M phase and increased occurrence of polyploid cells

Cells were treated with dasatinib 0.2 μM, crizotinib 4 μM or their combination for up to 72 h followed by a washout period of up to 72 h, cell cycle was assessed at 48 and 72 h and every 24 h during the washout period. Bars denote cells with 1X DNA , 2X DNA , 4X DNA , 8X DNA and the transition phases between the respective DNA content ■. A. U87, B. LN-18, C. U373, and D. A172 cells. Data are expressed as the mean ± SEM, experiments were conducted in triplicate and repeated independently three times. Statistical analysis is available in Supplementary Table S1–S4.

Figure 6. Combination of dasatinib and crizotinib impedes the formation of the α-tubulin-labeled mitotic spindle

LN-18 cells were treated with dasatinib 0.2 μM, crizotinib 4 μM or their combination for 48 h. Mitotic spindle were visualized using α-tubulin antibody conjugated to a fluorescein-labeled secondary antibody. Nuclei were stained by DAPI. The scale bar denotes 10 μm.

Figure 7. Migration and invasion of GBM cell lines

A. Migration of LN-18 cells following 20 h incubation and treatment with dasatinib (0.2 μM) and/or crizotinib (0.75 μM). The experiments were conducted in triplicate and repeated independently three times, representative pictures are shown. The scale bar denotes 200 μm. B. Invasion of LN-18 cells through a Geltrex basement membrane following 24 h of incubation and treatment with dasatinib (0.2 μM) and/or crizotinib (0.75 μM). The experiments were conducted in triplicate and repeated independently three times, * denotes p ≤ 0.05 relative to control.

Figure 8. Tube formation of HUVEC cells and tube-like formation of U87, U373 and A172 cells

Cells were seeded onto Geltrex matrix and treated for 20 h before representative were pictures taken. The scale bar denotes 100 μm.