Figure 1.

Synergistic cytotoxic effects of combinations of eeyarestatin with sunitinib or vandetanib in MTC cells. A, Cytotoxic activity of eeyarestatin in TT and MZCRC1 cells. Cells were treated as indicated for 48 h, and cell viability was measured by an MTT assay. Data presented as the mean of three independent experiments. Error bars, ±SD (n=3). B, Eeyarestatin induces cell death. TT or MZCRC1 cells were treated as indicated for 48 h, dead cells were quantified using an annexin V/propidium iodide (PI) assay by flow cytometry. Data presented as the mean of three independent experiments. Error bars, ±SD (n=3). *p<0.05, **p<0.01 (unpaired two-tailed t-test). C, Eeyarestatin activates ATF4 and its target genes in TT and MZCRC1 cells. Quantitative real-time PCR showing mRNA levels of indicated genes using HPRT mRNA as an internal control. Error bars, ±SD (n=3). D, E, TT or MZCRC1 cells were treated with eeyarestatin for 48 h at indicated doses; immunoblot depicts ATF4 and RET expression. Vinculin served as a loading control. F, G. MTT assay of TT and MZCRC1 cells treated with eeyarestatin, sunitinib, vandetanib or their combination as indicated for 48 h. Percentage inhibition at each concentration of the drugs in TT and MZCRC1 cells is shown. Data presented as the mean of three independent experiments. Error bars, ±SD (n=3). H, I, normalized Isobologram for non-constant ratios for eeyarestatin combination with sunitinib or vandetanib in TT cells were obtained using CompuSyn software, which performs drug dose-effect calculation using the median effect method.