PMC

Cells were treated with indicated concentrations of BMX for 24^cip/Waf1, cyclinD1, CDK4 and survivin were then determined by immunoblotting. Figures shown in (A) are representative of four independent experiments with similar results. The compiled results of p21^cip/Waf1 (B), cyclinD1 (C), CDK4 (D), and survivin (E) levels are shown. Each column represents the mean ± S.E.M. of four independent experiments. *p<0.05, compared with the control group

Cells were pretreated with indicated concentrations of BMX for 30(20 ng/ml) for another 5 (VEGFR2 and Src) or 30 (FAK, Akt and ERK1/2) min. Phosphorylation status of VEGFR2, Src, FAK, Akt, and ERK1/2 were then determined by immunoblotting. Figures shown in (A) are representative of four independent experiments with similar results. The compiled results of VEGFR2 Y1175 and Y1214 (B), Src Y416 (C), FAK Y397 (D), Akt S473 (E), and ERK1/2 T202/Y204 (F) phosphorylations are shown. Each column represents the mean ± S.E.M. of four independent experiments. *p<0.05, compared with the control group; #p<0.05, compared with the group treated with VEGF alone.

(A) HCT116 cells were treated with vehicle or BMX at indicated concentrations for 24 or 28 h. Cell viability was then determined by MTT assay. Each column represents the mean ± S.E.M. of seven independent experiments performed in duplicate. (^*p<0.05 as compared with the vehicle-treated control group) (B) After starvation in serum free medium for 24 h, HCT116 cells were treated with indicated concentrations of BMX in the presence of serum (10% FBS) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± S.E.M. of five independent experiments performed in duplicate. (^*p<0.05 as compared with the vehicle-treated control group in the presence of serum) Cells were treated for 24 h with vehicle or BMX at indicated concentrations. Protein levels of p21 (C) and survivin (D) were then determined by immunoblotting. Compiled results are shown at the bottom of the chart. Each column represents the mean ± S.E.M. of five independent experiments. (^*p<0.05 as compared with the vehicle-treated control group).

(A) HUVECs were starved in 2% FBS containing medium without ECGS for 16 h. Cell monolayer were then scratched and treated with vehicle or indicated concentrations of BMX in the presence of VEGF for another 16 h. The number of migrated cells was then determined. Each column represents the mean ± S.E.M. of four independent experiments. *p<0.05, compared with the group treated with VEGF alone. (B) After starvation as described in (A), Cells were then seeded in the top chamber in the absence or presence of BMX (5 µM) using VEGF as chemo-attractant. After 16 h, invaded cells through the gelatin-coated membrane were stained and quantified. Each column represents the mean ± S.E.M. of four independent experiments. *p<0.05, compared with the group treated with VEGF alone. (C) HUVECs were seeded on Matrigel in the presence of VEGF (20 ng/ml) with or without BMX at indicated concentrations. Cells were then photographed under phase-contrast microscopy after 16 h. Bar graphs show compiled data of average sprout arch numbers (n = 4). *p<0.05, compared with the group treated with VEGF alone. (D) Rat aortic rings were placed in Matrigel and treated with VEGF (20 ng/ml) in the presence or absence of BMX (5 µM). The effect of BMX on formation of vessel sprout from various aorta samples was examined on day 8. Bar graphs show compiled data of average microvessels area (n = 4). *p<0.05, compared with the group treated with VEGF alone.

(A) HUVECs were starved in 2% FBS containing medium without ECGS for 16 h. After starvation, cells were pretreated with indicated concentrations of BMX followed by the stimulation with VEGF (20 ng/ml) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± S.E.M. of five independent experiments performed in duplicate *p<0.05, compared with the group treated with VEGF alone. (B) HUVECs were treated as in (A), and cell proliferation was determined as described in the “Materials and Methods” section. Each column represents the mean ± SEM of five independent experiments performed in duplicate. *p<0.05, compared with the group treated with VEGF alone. (C) HUVECs were treated as in (A), and cytotoxicity of BMX was determined by LDH assay. Cells were also treated with cell lysis buffer (total lysis) to serve as positive control. (D) HUVECs were treated as in (A), the percentage of apoptotic cells was then analyzed by flow-cytometric analysis as described in the “Materials and Methods” section. The lower left quadrant of each panel (annexin V⁻PI⁻) shows the viable cells, which exclude PI and are negative for annexin V binding. The lower right quadrant (annexin V⁺PI⁻) represents the early apoptotic cells, annexin V positive and PI negative, demonstrating cytoplasmic membrane integrity. The upper right quadrant (annexin V⁺PI⁺) contains advanced apoptotic cells and necrotic cells, which are positive for annexin V binding and for PI uptake. Results shown are representative of four independent experiments.

(A) HCT 116 colorectal cancer cells were mixed with Matrigel and then injected subcutaneously into the right flank of nude mice. After implantation, animals were treated intraperitoneally with vehicle or BMX 20 mg/kg/day for 10 days. Matrigel plugs removed from the mice administered intraperitoneally with vehicle or BMX were shown. (B) The blood vessels in the Matrigel plug were stained with anti-CD31 antibody as described in the “Materials and Methods” section. Images of immunohistochemical staining representative of three independent Matrigel plug with similar results are shown. (C) Hemoglobin levels in the Matrigel plug shown in (A) were quantified. Each column represents the mean ± S.E.M. of six plugs in each group (^*p<0.05 as compared with the vehicle-treated control group, n = 5). (C) Nude mice bearing xenografts of HCT116 colorectal cancer cells were treated intraperitoneally with BMX 20 mg/kg/day for 22 days. The control group received vehicle only. Tumor volumes were calculated as described in the Materials and Methods section. Values represents the mean ± S.E.M. (^*p<0.05 as compared with the vehicle-treated control group, n = 5). (D) After 22 days of treatment as in (C), mice were sacrificed and tumors were dissected and weighted. Each column represents the mean ± S.E.M. (^*p<0.05 as compared with the vehicle-treated control group, n = 5). (E) The body weights of the nude mice were examined within 22 days treatment of vehicle or BMX. Values represent the mean ± S.E.M.