NVPAST inhibits the growth of NIHTRETCW xenografts in athymic nude mice.Mice were treated with the indicated dose of drug or weekly.Tumor volumes were determined according to the formula: length vehicle daily by Flunisolide gavage.In our hands, TPC cells did not grow as readily as xenografts.Hence, in vivo experiments to test the effects of NVPAST were done exclusively on TT cells.These data were replicated in two additional efficacy experiments.Treatment of athymic mice was not associated with significant body weight change of the animals at any of the concentrations tested.A notable finding from these experiments was that the plasma levels of human calcitonin, which were measured as a biomarker of tumor responsiveness to NVPAST, seemed to decrease prior to any changes in tumor Thujone volume.This difference was statistically significant when the calcitonin concentrations were normalized to the tumor weight. NVPAST directly inhibits calcitonin gene expression, independent of effects on TT cell growth.We next examined the effects of NVPAST on calcitonin gene expression in vitro. The quantification of this effect in three independent biological replicates is shown as supplemental data.The delayed response to the compound is likely due to the prolonged halflife of calcitonin mRNA, as inhibition of nascent transcription by treatment with actinomycin D resulted in a very modest decay of mature calcitonin mRNA over a hincubation, which is consistent with the previously reportedt of calcitonin mRNA in TT cells. Calcitonin secretion into conditioned media was markedly inhibited by NVPAST in a concentrationdependent fashion, with maximal effects observed at to nmolL. Treatment with nmolL of NVPAST resulted in a fold inhibition of promoter activity after h.The pCT promoter fragment that lacks the domain conferring responsiveness to RAS also seemed to be inhibited by the kinase inhibitor. The effects of NVPAST were unlikely to be due to offtarget actions because PP and ZD, compounds previously shown to inhibit RET kinase activity. All three compounds inhibited CGRP mRNA to a comparable level.This is consistent with an effect on the abundance of the primary CTNCGRP transcript rather than through regulation of alternative splicing of the gene.Persephin and GDNF induce calcitonin gene expression in MTCM cells.To this end, we used the mouse medullary thyroid cancer cell line, MTCM, which was shown to have a wildtype sequence. By contrast to GDNF, treatment of cells with persephin alone induced calcitonin mRNA. There was no corresponding decrease in thyroid tissue calcitonin mRNA at these time points. NVPAST decreases TT cell calcitonin gene transcription, mRNA abundance, and secretion in vitro.Quantitation of effects of NVPAST on calcitonin mRNA is presented in supplemental data.B, calcitonin concentration in conditioned media of TT cells incubated with the indicated concentration of NVPAST for h, with media exchange at h.Columns, means of three independent experiments expressed relative to DNA content of cells harvested from the wells after removal of the conditioned media; bars, SE.C, treatment of TT cells with NVPAST inhibits calcitonin gene transcription.Similar findings were obtained in three additional biological replicates.GDNF and persephin induce calcitonin mRNA in MTCM cells.A, MTCM cells were placed in serumfree media for hand then treated with GDNF for the indicated times.