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T-TYPE CALCIUM CHANNEL BLOCKERS INHIBIT HORMONE SECRETION AND INDUCE APOPTOTIC CELL DEATH IN A MEDULLARY THYROID CANCER CELL LINE. R.Pivonello, C.Pivonello, A.Cavaccini, G. Petrillo,P.Rousaki, A.Faggiano, G.Lombardi, A.Colao, M.Cataldi. Department of Molecular and Clinical Endocrinology and Oncology, Department of Neurosciences, Division of Pharmacology, “Federico II” University, Naples, Italy Background Medullary thyroid carcinoma (MTC) accounts for approximately 5-10% of thyroid cancers. Intense efforts are currently directed toward the identification of new druggable targets for the treatment of MTC. Calcium channels play a pivotal role in the physiology of neuroendocrine cells, being involved in the mechanisms of control of hormone secretion and cell proliferation. The T-type channels was found to represent the only voltage gated calcium current in the medullary thyroid cancer (MTC) cell line, named TT, where their block obtained by specific pharmacological agents were found to inhibit calcium currents. Aim The aim of this study was to investigate whether drugs acting at voltage-gated T-type calcium channels could affect hormone release, cell proliferation and apoptosis in a MTC cell line, the TT cells. Materials and Methods The expression of T-type calcium channels (Cav α1G, Cav α1H, and Cav α1I) was evaluated by RT-PCR, and by the measurements of calcium currents, performed by whole cell patch clamp technique. Hormone secretion was evaluated by measuring calcitonin concentrations by CLIA, cell vitality was evaluated by WST-1 assay, by FACS analysis was evalueted the cell proliferation and apoptosis that was confirmed by FLICA assay, before and after the administration of the T-type channel blockers, including nichel (150 µM), Mibefradil (30 µM) and or NNC 55-0396 (10 µM). Results The results of RT-PCR demonstrated that all three isoforms of the T-type calcium (Cav 3.1, Cav 3.2, and Cav 3.3) channels were expressed in TT cells (Fig.1). The electrophysiologic experiments showed that the calcium currents elicited in TT cells are typical of T-type calcium channels and that the three T-type calcium channel blockers induced a significant inhibition of calcium currents (Data not showed). A significant inhibition of calcitonin secretion was found after adding of Nichel (36,4%), NNC 55-0396 ( 50%) and Mibefradil (54%) (Fig. 2). The viability of TT cells was inhibited by Nichel (32%), NNC 55-0396 (76%) and Mibefradil (70%) rispectively at the concentration 1mM, 10-30 µM and 30 µM (Fig. 3). In Fig. 4B and 4C an increase of apoptotic cells was showed in G1 phase under the effect of Mibefradil more than NNC 55-0396, moreover an arrest in S phase was detected after both treatments. The apoptotic effect of NNC 55-0396 was confirmed by a FLICA assay using inhibitors of Caspase 3 (ICT) binding red fluorocrome (Fig. 5C and 5D). Conclusions In conclusion, T-type channels seem to play a relevant role in controlling both hormone secretion, cell viability and apoptosis in MTC cells. These data prompt further studies aimed to investigate the potential of T-type channel blockers in the pharmacological treatment of MTC. FIG.1. A) Expression of T-type calcium channels (Cav 3.1, Cav 3.2 and Cav 3.3) B) Minus RT-PCR in TT cells FIG. 2. Inhibition of calcitonin secretion induced by Nichel, NNC 55-0396 and Mibefradil FIG. 4. Effect of the T-type calcium blockers Mibefradil and NNC 55-0396 on cell cicle in cultured TT cells. FIG. 3. Inhibition of cell viability induced by Nichel, NNC 55-0396 and Mibefradil FIG. 5. FLICA assay. A) TT cells control with Hoechst B): TT cells control with ICT C): TT cells treated with NNC 55-0396 labelled with Hoechst D): TT cells treated with NNC 55-0396 labelled with ICT