Modulation of Tumor Growth and Tumor Induced Angiogenesis After Epidermal Growth Factor Receptor Inhibition by ZD1839 in Renal Cell Carcinoma

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Abstract
ZD1839 or Iressa (AstraZeneca, Macclesfield, United Kingdom) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in human renal cell carcinomas (RCC). Six established human RCC lines and surgical specimens from 10 patients with RCC were used. Protein expression was detected by Western blotting and/or immunohistochemistry. The concentrations of vascular endothelial growth factor and interleukin-8 in the supernatants were measured by enzyme-linked immunosorbent assay. Cell cycle progression and the apoptotic ratio were evaluated by flow cytometry. In vitro angiogenesis was measured using human umbilical vascular endothelial cells by capillary-like network formation analysis. In vivo SKRC-49 cells injected subcutaneously into athymic nude mice were treated with various doses of ZD1839 orally. The effect of ZD1839 on tumor xenografts angiogenesis was evaluated by immunohistochemistry using CD34 Ab. Epidermal growth factor receptor was activated in all RCC lines and over expressed in 7 of 10 RCC specimens (70%) compared with adjacent normal renal tissues. Treatment of SKRC-49 cells with ZD1839 (0.1 to 10 μM) for 48 hours resulted in the accumulation of cells in the G1 phase and a 30% to 50% decrease in cellular proliferation compared with untreated controls (p <0.01). The tumor xenograft study confirmed that ZD1839 (50 to 100 mg/kg daily) significantly inhibited SKRC-49 tumor growth compared with controls within 3 weeks after treatment (p <0.01). Vascular endothelial growth factor and interleukin-8 production was significantly decreased in ZD1839 treated SKRC-49 cells compared with untreated controls (p <0.01). Treatment of human umbilical vascular endothelial cells with SKRC-49 supernatants treated with ZD1839 for 11 days resulted in an approximate 80% decrease in tubule formation compared with untreated controls (p <0.01). Immunohistochemical assays showed that ZD1839 treatment resulted in a significant decrease in CD34 positive neovessels compared with controls in SKRC-49 xenografts. These results suggest that the antitumor activity of ZD1839 appears to derive not only from direct inhibition of cell proliferation, but also from the inhibition of tumor angiogenesis in RCC.