BMS-536924, an ATP-competitive IGF-1R/IR inhibitor, decreases viability and migration of temozolomide-resistant glioma cells in vitro and suppresses tumor growth in vivo

Figures & data

Figure 1 BMS-536924 reduces viability of both TMZ-sensitive and -resistant glioma cells.

Notes: (A and B) M059K and U87MG cells were induced to become TMZ-resistant cells (M059K-R and U87MG-R) by incubation with gradually increased doses (2~20 μM) of TMZ for 4 months. The TMZ sensitivity was then examined using a cell viability assay. (C and D) Both TMZ-sensitive and -resistant cells were treated with BMS-536924 (12.5~800 nM) for 72 hours, followed by measurement of cell viability. (E and F) Both TMZ-sensitive and -resistant cells were treated with BMS-536924 (400 nM) for 24, 48, or 72 hours, followed by measurement of cell viability.
Abbreviation: TMZ, temozolomide.

Figure 2 BMS-536924 induces apoptosis of both TMZ-sensitive and -resistant glioma cells.

Notes: (A) TMZ-resistant and -sensitive cells were treated with BMS-536924 (100~800 nM) for 48 hours, followed by PI staining and flow cytometry analysis. (B) TMZ-resistant and -sensitive cells were incubated with BMS-536924 (100~800 nM) for 48 hours. The nuclei were stained with DAPI and analyzed using a fluorescent microscope. The representative images are shown (the fragmented nuclei were labeled by arrow heads). (C) The number of cells with condensed/fragmented nuclei was quantitated by counting in five random fields, and the inhibition was calculated.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; TMZ, temozolomide.

Figure 3 BMS-536924 suppresses phosphorylation of IGF-1R and IR in both TMZ-sensitive and -resistant glioma cells.

Notes: M059K-R and U87MG-R cells were seeded in six-well plates and incubated for 24 hours. Then the medium was replaced with serum-free medium, and BMS-536924 (200 and 800 nM) was added and incubated for 6 hours. Finally, the cells were stimulated with 30 ng/mL IGF-1 or 3 μg/mL insulin for 30 minutes, then were lysed and analyzed by western blot, using the indicated antibodies.

Figure 4 BMS-536924 inhibits migration of both TMZ-sensitive and -resistant glioma cells.

Notes: (A) TMZ-resistant and -sensitive cells were treated with BMS-536924 (10, 40, or 160 nM) for 8 hours. The nonmigrated cells on the upper surface of the filter were removed, and the migrated cells on the lower side were stained and photographed. The representative images are shown. Then, cells were lysed, and colorimetric determination was made at 595 nm. (B) Quantitation of the inhibition from Transwell^® assay. (C) A scratch was introduced into a monolayer of TMZ-resistant and -sensitive cells, followed by treatment with BMS-536924 (10, 40, or 160 nM) for 8 hours. The width of the wounded cell monolayer was measured in five random fields, and representative images are shown. (D) Quantitation of the inhibition from the wound healing assay.
Abbreviation: TMZ, temozolomide.

Figure 5 BMS-536924 suppresses M059K-R and M059K tumor growth in vivo.

Notes: (A and B) After inoculation of M059K-R and M059K cells, BMS-536924 (20 and 40 mg/kg) was injected in mice every day. The tumors were measured every other day, and the tumor volumes are shown. (C) BMS-536924-induced apoptosis of M059K-R tumor cells in vivo was examined with TUNEL assay (green), DAPI staining (blue), or H&E staining (purple); the apoptotic cells were labeled with arrowheads. (D) BMS-536924 had no significant cytotoxic effects on the body weight of mice during the treatments.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.