Is Vascular Endothelial Growth Factor Modulation a Predictor of the Therapeutic Efficacy of Gefitinib for Bladder Cancer?

Purpose

The epidermal growth factor receptor inhibitor gefitinib (Iressa®) is currently being studied in patients with bladder cancer and it has significant anti-angiogenic activity. We investigated the relationship between the modulation of vascular endothelial growth factor (Santa Cruz Biotechnology, Santa Cruz, California) expression and the biological efficacy of gefitinib for bladder cancer.

Materials and Methods

In vitro the 4 bladder cancer cell lines 253JB-V, UMUC-3, KU-7 and UMUC-13 were treated with gefitinib and vascular endothelial growth factor secretion was measured. The effects of gefitinib on vascular endothelial growth factor promoter, proliferation, cell cycle and downstream signals were evaluated. In vivo 253JB-V and UMUC-13 were injected into nude mice and tumors were treated with 2 mg gefitinib per day. Tumor kinetics were determined and the levels of phospho-epidermal growth factor receptor (Biosource^™), vascular endothelial growth factor, phospho-vascular endothelial growth factor (Cell Signaling Technology®), angiogenesis and apoptosis were measured.

Results

Epidermal growth factor receptor (Neomarkers, Fremont, California) phosphorylation was blocked efficiently in all cell lines at concentrations of 0.5 μM or greater. Gefitinib (1 μM) induced an accumulation of cells in G0/G1 without apoptosis in 253J B-V cells, whereas it had no effect in other cell lines. Gefitinib inhibited vascular endothelial growth factor secretion in 253JB-V and UMUC-13 (concentration inhibiting a 50% response 0.5 and 0.1 μM, respectively) but not in UMUC-3 or KU-7. Gefitinib decreased vascular endothelial growth factor promoter activity in 253JB-V and UMUC-13 by 40% to 60%. In vivo the growth of 253JB-V tumors was significantly inhibited by gefitinib, whereas no effect was demonstrated in UMUC-13 tumors. Vascular endothelial growth factor expression and vascular endothelial growth factor receptor activation were significantly decreased in 253JB-V tumors and to a greater extent in resistant UMUC-13 tumors. Gefitinib inhibited angiogenesis and induced apoptosis in sensitive 253JB-V tumors only.

Conclusions

Epidermal growth factor receptor blockade exerts an anti-angiogenic effect on bladder cancer cells, in part by modulating vascular endothelial growth factor expression. However, down-regulation of vascular endothelial growth factor expression is not sufficient to inhibit bladder cancer growth and it should not be used as a predictor of the therapeutic efficacy of gefitinib.

Key Words: urinary bladder, carcinoma, transitional cell, gefitinib, vascular endothelial growth factor, angiogenesis inhibitors

Abbreviations and Acronyms: EGFR, epidermal growth factor receptor, ELISA, enzyme-linked immunosorbent assay, ERK, extracellular signal-regulated kinase, GSK, glycogen synthase kinase, Jnk, c-Jun N-terminal kinase, p, phospho, PBS, phosphate buffered saline, PI3, phosphatidylinositol 3, VEGF, vascular endothelial growth factor, VEGFR, VEGF receptor