A39
The effects of vandetanib on calcitonin (CTN) mRNA levels in cultured TT human medullary thyroid carcinoma cells
Susie L Weston, Paula M McArdle, Neil H James, Susan C Lovick, Anderson J Ryan
AstraZeneca, Macclesfield, Cheshire, UK
Medullary thyroid cancer (MTC) arises from CTN-producing C-cells in the thyroid. Measuring blood CTN levels in MTC patients is considered a sensitive method to monitor disease progression and response to therapy. Vandetanib (ZACTIMA) has shown clinical activity in MTC, producing marked decreases in serum CTN in some patients. The present study was undertaken to determine the effect of vandetanib treatment on CTN mRNA levels in human MTC cells (TT) in vitro. TT cells were cultured in RPMI 1640 media containing 10% FCS. Cell growth was analysed as confluence (%) following 48, 72 and 160h exposure to vandetanib (03.0M) using an Incucyte image acquisition system (Essen Instruments). mRNA levels were determined by RT-PCR following 24, 48 and 72h exposure to vandetanib (0-2.5μM) using TaqMan Gene Expression Assays (Applied Biosystems). CTN and Ki-67 mRNA expression were determined by the comparative Ct method with PPIA as an endogenous control. CTN protein was measured in cell culture supernatants by ELISA (MD Biosciences). Vandetanib potently inhibited TT cell growth (IC50 0.07M) in cell growth assays. Vandetanib treatment (0.10μM) for 24h did not affect CTN mRNA levels. However, there was a modest reduction in CTN mRNA at 48h (1.5x) and 72h (2.3x) after treatment. Vandetanib (0.10uM) did not reduce CTN protein levels in cell culture medium. Ki67 mRNA was little changed 24h after vandetanib treatment (1.2x reduction), but was markedly reduced after 48h (16.7x) and 72h (74.2x) of treatment. Analogous results were observed at higher concentrations of vandetanib (0.502.50uM). These data suggest that the marked decreases in serum CTN observed in MTC patients treated with vandetanib are unlikely to be due to effects on CTN mRNA levels. In contrast, vandetanib treatment led to a marked reduction in Ki67 mRNA, a gene product known to be associated with cell cycle progression and proliferation.
