Dual epidermal growth factor receptor and vascular endothelial growth factor receptor inhibition with vandetanib sensitizes bladder cancer cells to cisplatin in a dose- and sequence-dependent manner

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Abstract

OBJECTIVE

To investigate the activity of the combination of vandetanib and cytotoxic agents using in vitro models of bladder cancer, as modern chemotherapy regimens are built around cisplatin, with gemcitabine or a taxane such as docetaxel also commonly added in combination for the treatment of advanced bladder cancer.

MATERIALS AND METHODS

Human bladder cancer cells HTB3, HT1376, J82, RT4, CRL1749, T24, SUP and HTB9 were cultured. The activity of gefitinib (ZD1839) and vandetanib (ZD6474) was assessed in these eight bladder cancer cell lines with a tetrazolium-based assay of cell viability. RT4 bladder cancer cells, determined to have moderate cisplatin resistance and also moderate sensitivity to vandetanib, were treated with vandetanib and cisplatin. RT4 and T24 cells were treated with six different regimens. The apoptosis and cell-cycle analysis were studied by flow cytometry. Expression of p21 and p27 was detected by Western blotting. Fluorescence in situ hybridization (FISH) analysis of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 was performed for all cell lines.

RESULTS

At equal concentrations, vandetanib was a more potent inhibitor of cell viability, compared to gefitinib. At vandetanib concentrations of ≤2 µM, the combination with cisplatin was synergistic, especially in the treatment sequence of cisplatin followed by vandetanib, and additive with vandetanib followed by cisplatin. An analysis of the cell-cycle distribution showed that vandetanib treatment induced G1 arrest at high concentrations, but not at lower concentrations. High-concentration treatment was associated with increased levels of the cyclin-dependent kinase p27. FISH analysis showed that there was a low level of genomic gain, and no gene amplification. Mutational analysis of exons 18, 19, and 21 of EGFR in each cell line revealed no mutation.

CONCLUSION

Vandetanib has synergistic activity when given at low concentration with cytotoxic chemotherapy. The addition of vandetanib to cisplatin-based chemotherapy regimens merits further study.

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