Article Figures & Data
Figures
- Fig. 1.
Isobolograms representing the synergy of SU6668 in combination with paclitaxel on endothelial and smooth muscle cells. A, representative of the isobologram method of the combination of drugs A and B. Dashed line, additivity line that separates the antagonistic from the synergistic region. When the combined concentrations of drug A and drug B fall in the lower area, they can be interpreted as synergism; if the data fall in the area above the line, the two drugs are antagonistic. As an example, a datum (0.20, 0.33) is shown, meaning that we obtained 50% growth inhibition by combining one fifth of the IC50 of drug A with one third of the IC50 of drug B. The CI of that datum is CI = 0.2 + 0.33 = 0.53. The isobolograms (B-D) show the synergistic effects of the combination of SU6668 with paclitaxel (PTX) at the IC50 level on HUVEC, HMVEC, and AoSMC, respectively. The cells were exposed for 72 hours to a range of combined concentrations of SU6668 and paclitaxel. Combined concentrations producing IC50 were calculated by fitting dose-response curves of paclitaxel at each tested SU6668 concentration (•) and dose-response curves of SU6668 at each tested paclitaxel concentration (○).
- Fig. 2.
SU6668 in combination with paclitaxel induces apoptosis in HUVEC. HUVEC exposed to SU6668, paclitaxel, or the combination were evaluated for apoptosis by TUNEL and Annexin V staining. A, percentage apoptotic HUVEC in response to SU6668 (10−6 mol/L), paclitaxel (10−8 mol/L), or the combination evaluated by fluorescent TUNEL after 16-hour exposure. Apoptosis was induced by lack of serum and growth factors (control), whereas VEGF alone was used as survival factor. Columns, mean percentage of apoptotic cells of three independent experiments; bars, SD. *, P < 0.05, compared with all conditions (ANOVA with Tukey-Kramer correction). B, representative images obtained with confocal scanning laser microscopy (×400) of HUVEC nuclei labeled with 4′,6-diamidino-2-phenylindole. Serum starvation (control) induced apoptotic or necrotic cells (arrow), in contrast to normal nucleated cells (*) exposed to VEGF. Similar apoptotic morphology was observed with the addition of SU6668. HUVEC exposed to paclitaxel showed multinucleated cells (closed arrowhead) and also a higher number of cells undergoing mitosis (open arrowhead). The combination of paclitaxel and SU6668 resulted in a significant increase in apoptosis and a high number of cells undergoing mitotic stress. C, Annexin V evaluation of HUVEC apoptosis after 48-hour exposure to SU6668 (10−6 mol/L), paclitaxel (10−8 mol/L), or the combination. Fluorescence-activated cell sorting evaluation of apoptosis is represented by the percentage of cells stained with Annexin V (bottom right quadrant). Cellular necrosis was evaluated by double staining with Annexin V antibody and propidium iodide (top right quadrant). Representative of one of three experiment. PTX, paclitaxel.
- Fig. 3.
Effect of SU6668 in combination with paclitaxel on angiogenesis in Matrigel. Matrigel containing FGF-2 (300 ng/plug) was injected s.c. in C57BL/6N mice and paclitaxel (6 mg/kg, daily i.v.), SU6668 (100 mg/kg, daily orally), or the combination was administered from days 1 to 6. A, angiogenic responses evaluated by measuring the hemoglobin content of the plugs (n = 8) at day 7 following treatment. Points, hemoglobin content (g/dL) for each plug. Bars, median. *, P < 0.001, compared with negative control (plugs without FGF-2, mice receiving vehicle); **, P < 0.05 compared with positive control (FGF-2 containing plugs, mice receiving vehicle), SU6668, and paclitaxel-treated plugs (Mann-Whitney U test). B, representative images of H&E (×100 magnification) and anti-CD31 (×200) immunostaining of Matrigel plugs. Paclitaxel induced reduction of cellular infiltration, cords, and vessel formation relative to the positive controls, and reduced CD31-positive vessels. SU6668 did not affect the cellularity of the plugs; however, a strong reduction of CD31-positive vessels was detected. The combination of the two agents induced a maximal reduction of cellular infiltration, cords, and blood vessel formation and CD31-immunostained vessels were absent. C, MVD of CD31-positive vessels counted (n = 5) in four different fields at ×400 magnification. Columns, mean number of vessels; bars, SD. *, P < 0.05 compared with positive vehicle (FGF-2-containing plugs; ANOVA with Tukey-Kramer correction).
- Fig. 4.
Histology of the response of 1A9-PTX22 tumor xenografts to the combination of SU6668 with paclitaxel. Representative images of H&E and anti-CD31 immunostaining of 1A9-PTX22 tumor sections obtained 24 hours after the last treatment (day 22) with SU6668, paclitaxel, or the combination as described in Table 2 (P1). The vehicle-treated group showed intense angiogenesis. In paclitaxel-treated tumors, reduction of the number of vessels was not observed. The stain for CD31-positive vessels was decreased in the SU6668-treated group and more so in tumors from mice treated with the combination of the two drugs. Multifocal to coalescing areas of coagulative necrosis are evident in the tumors of mice treated with the combination (arrows). PTX, paclitaxel. Original magnification, ×100.
Tables
- Table 1.
CI values of SU6668 with paclitaxel on endothelial and smooth muscle cells
Cell line Drugs CI IC30 IC50 IC70 HUVEC SU6668 + PTX 0.35 ± 0.09 0.45 ± 0.09 0.57 ± 0.06 HMVEC SU6668 + PTX 0.25 ± 0.14 0.39 ± 0.23 0.34 ± 0.10 AoSMC SU6668 + PTX 0.40 ± 0.02 0.47 ± 0.11 0.58 ± 0.11 NOTE: The CI was evaluated for the combination of SU6668 with paclitaxel at IC30, IC50, and IC70 of each drug for HUVEC/HMVEC/AoSMC after 72-hour exposure (experiment as in Fig. 1). The CI was calculated as described in Materials and Methods and evaluated such that CI < 1 is synergism, CI > 1 is antagonism, and CI = 1 is additivity. Data are expressed as mean ± SE of all available pairs of concentrations producing the indicated growth inhibition and are representative of one experiment, repeated at least thrice.
Abbreviation: PTX, paclitaxel.
- Table 2.
Evaluation of the response of 1A9-PTX22 tumor xenografts to paclitaxel and SU6668 treatments
Treatment Dose (mg/kg) Schedule (× no. treatments) n* %T/C (day) T − C P1 SU6668 100 Q1 × 10 9 41 (22) 9 PTX 6 Q1 × 10 9 74 (22) 2 PTX + SU6668 6 Q1 × 10 9 34 (22)† 13‡ 100 Q1 × 10 P2 SU6668 100 Q1 × 10 8 40 (20)† 4 PTX 20 Q4 × 3 7 81 (17) 1 PTX + SU6668 20 Q4 × 3 7 30 (20)† 12‡ 100 Q1 × 10 NOTE: In two separate protocols (P1 and P2), 1A9-PTX22 was transplanted s.c. in nude mice and treatments began when tumors reached ∼300 mg. P1 shows the response of tumors where mice received daily treatments of SU6668 (100 mg/kg orally, Q1 × 10), paclitaxel (6 mg/kg i.v., Q1 × 10), or the combination. P2 shows the response of xenografts to SU6668 (100 mg/kg orally, Q1 × 10), paclitaxel (20 mg/kg i.v., Q4 × 3), or the combination. No significant body weight loss was observed in the treated groups. PTX, paclitaxel.
↵* n = number of mice evaluated.
↵† %T/C ≤ 40%.
↵‡ P < 0.01.
Volume 12, Issue 6
