Medullary thyroid carcinoma (MTC) accounts for ~510% of thyroid cancers. Intense efforts are currently directed toward the identification of new druggable targets for the treatment of MTC. The aim of this study was to investigate whether drugs acting at voltage-gated T-type calcium channels could affect hormone release and/or cell proliferation in a MTC cell line, the TT cells. The expression of the three isoforms of T-type calcium channels (CaV3.1, CaV3.2 and CaV3.3) was evaluated by RT-PCR whereas T-type currents were recorded by whole cell patch clamp electrophysiology. The effect of T-type calcium channel blockers (Ni2+, mibefradil and NNC 55-0396) on calcitonin secretion and cell proliferation and viability was evaluated respectively by measuring calcitonin levels, and the percentage of cell cycle arrest and/or apoptosis by WST assay, flow cytometry and FLICA assay. At RT-PCR analysis, all three isoforms of T-type channels were found. Whole cell patch clamp experiments showed that T-type currents represent the only form of Ca2+ current elicited by depolarization in these cells. The amplitude of T-type currents was reduced to <30% of their basal values when TT cells were perfused with an extracellular solution containing either the transition metal Ni2+(150 M) or mibefradil (10 M) or NNC 55-0396 (10 M). Basal calcitonin release from cultured TT cells was dose-dependently inhibited in the presence of T-type blockers (IC50: ~15 M). The viability of TT cells was inhibited by Ni2+(32%), NNC 55-0396 (76%) and Mibefradil (70%) at the concentration 1 mM, 1030 μM and 30 μM respectively. Moreover, NNC 55-0396 dramatically increased the percentage of cells undergoing apoptosis whereas only limited effects were observed on cell cycle. At FLICA assay a significant apoptotic effect was observed by binding of red fluorochrome to caspases in cells treated with NNC 55-0396, confirming the evidence that upon NNC 55-0396 exposure TT cells underwent apoptosis. In conclusion, T-type channels seem to play a relevant role in controlling both hormone secretion and cell viability in MTC cells. These data prompt further studies aimed to investigate the potential of T-type channel blockers in the pharmacological treatment of MTC.
30 Apr - 04 May 2011
European Society of Endocrinology