Antitumor Effects of ZD6474, a Small Molecule Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, with Additional Activity against Epidermal Growth Factor Receptor Tyrosine Kinase

Dose-dependent inhibition of EGF-induced EGFR autophosphorylation by ZD6474 or by ZD1839 in human MCF-10A ras cells. Serum-starved MCF-10A ras cells were treated for 3 h with ZD6474 or with ZD1839 (0.1, 0.5, or 1 μm), followed by addition of complete medium containing EGF (20 ng/ml) for 15 min. Protein extracts were immunoprecipitated with the MAb 528 anti-EGFR MAb, resolved by a 7.5% SDS-PAGE, and probed with either the PY20 anti-P-tyr MAb (A) or an antihuman EGFR MAb (B). Immunoreactive proteins were visualized by enhanced chemiluminescence. C, Control untreated cells: Lane 1, cells treated with ZD1839 (0.1 μm); Lane 2, cells treated with ZD6474 (0.1 μm); Lane 3, cells treated with ZD1839 (0.5 μm); Lane 4, cells treated with ZD6474 (0.5 μm); Lane 5, cells treated with ZD1839 (1 μm); and Lane 6, cells treated with ZD6474 (1 μm).

A, dose-dependent growth inhibitory effects of ZD6474 on the soft agar growth of human ZR-75-1, MCF-10A ras, OVCAR-3, CALU-6, AGS, MNK-28, and GEO cells. Cells were treated with the indicated concentrations of ZD6474 each day for 5 consecutive days. Colonies were counted after 10–14 days. Data represent the average (±SD) of three different experiments, each performed in triplicate. B–E, dose-dependent induction of programmed cell death by treatment with ZD6474 in human OVCAR-3, ZR-75-1, MCF-10A ras, and GEO cells, respectively. Cells were treated each day for 3 days with the indicated doses of ZD6474. Data represent the average (±SD) of quadruplicate determinations.

Flow cytometric analysis of induction of programmed cell death by treatment with ZD6474 in human OVCAR-3 (A) and GEO (B) cells, respectively. Cells were treated each day for 3 days with the indicated doses of ZD6474. Data represent the average (±SD) of triplicate determinations.

Growth inhibitory effects of treatment with ZD6474 (0.01, 0.05, 0.1 μm) in combination with docetaxel (A) or with paclitaxel (B) on the soft agar growth of human GEO cells. Cells were treated with the indicated concentrations of cytotoxic drug on day 1 followed by the indicated concentrations of ZD6474 on each day from day 2 to day 6. Colonies were counted after 10–14 days. Data represent the average (±SD) of three different experiments, each performed in triplicate.

Growth inhibitory effects of the combined treatment with ZD6474 and docetaxel (A) or paclitaxel (B) on the soft agar growth of GEO, MCF-10A ras, ZR-75-1, OVCAR-3, CALU-6, MNK-28, and AGS cancer cell lines. Cells were plated in soft agar and treated with the indicated concentration of cytotoxic drug on day 1 and with ZD6474 (0.05 μm) on days 2–6 or with each agent alone. Colonies were counted after 10–14 days. Data are expressed as percentage growth inhibition as compared with the growth of untreated control cells. For each cell line, Lane 1, docetaxel (or paclitaxel); Lane 2, ZD6474; Lane 3, docetaxel (or paclitaxel) plus ZD6474. Data represent the average (±SD) of three different experiments, each performed in duplicate.

Induction of apoptosis by treatment with ZD6474 in combination with taxanes in OVCAR-3 (A), ZR-75-1 (B), MCF-10A ras (C), and GEO (D). Cells were treated with ZD6474 (0.5 μm) each day for 3 days; docetaxel (0.25 nm) on day 1; paclitaxel (1 nm) on day 1; or with the combination of ZD6474 and each taxane. On day 4, the cells were collected. Data are presented as relative increase in apoptosis of treated cells compared with untreated control cells and represent the average (SD) of quadruplicate determinations.

A, antitumor activity of ZD6474 treatment on established GEO human colon carcinoma xenografts. Mice were injected s.c. into the dorsal flank with 10⁷ GEO cells. After 7 days (average tumor size, 0.25 cm³), the mice were treated i.p. on days 1–5 of each week for 2 weeks with ZD6474 at the indicated daily doses. Each group consisted of 10 mice. Data represent the average (± SD). Student’s t test was used to compare tumor sizes among different treatment groups at day 21 after GEO cell injection. ZD6474 (25 mg/kg/dose) versus control (two-sided P < 0.01); ZD6474 (50 mg/kg/dose) versus control (two-sided P < 0.01); ZD6474 (100 mg/kg/dose) versus control (two-sided P < 0.01); and ZD6474 (150 mg/kg/dose) versus control (two-sided P < 0.01). B, antitumor activity of ZD6474 treatment in combination with paclitaxel on established GEO human colon carcinoma xenografts. Mice were injected s.c. in the dorsal flank with 10⁷ GEO cells. Data represent the average (±SD). After 7 days (average tumor size, 0.2–0.3 cm³), mice were treated i.p. on days 1–5 of each week for 4 weeks with ZD6474 (100 or 150 mg/kg/dose), alone or in combination with paclitaxel (20 mg/kg/dose) on day 1 of each week for 4 weeks.