Inhibition of medullary thyroid carcinoma cell proliferation and RET phosphorylation by tyrosine kinase inhibitors.

Abstract

Most medullary thyroid carcinomas (MTCs) result from gain-of-function mutations in the RET proto-oncogene, which encodes a transmembrane tyrosine kinase receptor. Systemic therapies have not been effective in treating this disease. We evaluated the effects of 3 tyrosine kinase inhibitors (TKIs) on MTC cell growth and RET tyrosine kinase activity by using an in vitro model. An MTC cell line (TT cells, RETc634 mutant) cultured in RPMI medium was exposed to varying concentrations of STI571, genistein, or allyl-geldanamycin with controls (no TKI) for 3 to 48 hours. Cellular protein was analyzed by immunoprecipitated Western blot analysis probing with a monoclonal antiphosphotyrosine antibody. Cell proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays. RET phosphorylation was inhibited at 24 hours of exposure to 5 to 20 micromol/L STI571 and 48 hours of exposure to genistein (200 micromol/L) and allyl-geldanamycin (6 micromol/L). RET protein was detected in equal concentrations in all experimental conditions. MTT and BrdU assays demonstrated a dose-dependent decrease in TT cell proliferation with exposure to the 3 TKIs. These TKIs selectively inhibit cell growth and RET tyrosine kinase activity of MTC cells in vitro in a dose manner. This study suggests the use of TKIs in human trials as a systemic therapy for MTC.