Clodronate-Induced Cell Apoptosis in Human Thyroid Carcinoma Is Mediated via the P2 Receptor Signaling Pathway

Clodronate, a halogenated bisphosphonate, can inhibit the growth of human thyroid carcinoma (TC) cells. Previously, we found that a clodronate-induced Ca²⁺ transient was correlated with clodronate-induced growth inhibition in TC cells. However, the details of the signaling process underlying the antiproliferative effect of clodronate on TC cells are not clear. In this study, we investigated the antiproliferative mechanism of clodronate on papillary TC (PTC) cells and xenotransplanted animals using a combination of pharmacological drugs. Reverse transcription-polymerase chain reaction analysis confirmed the endogenous expression of P2Y receptor isoforms in PTC cells. The P2 antagonist suramin not only inhibited the antiproliferative effect of clodronate and ATP on TC cells but also blocked all the Ca²⁺ transients induced by clodronate and ATP. The release of Ca²⁺ from the endoplasmic reticulum and membrane depolarization of mitochondria was observed during the clodronate-induced Ca²⁺ transients. The results of terminal deoxynucleotidyltransferase dUTP nick-end labeling assays and flow cytometry with annexin V and caspase-3 staining suggest that both ATP and clodronate induce apoptosis. Significant inhibition of tumor invasion and colony formation was also observed in clodronate-treated PTC cells. We further demonstrated that only the cAMP inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), and not inhibitors of phospholipase C [1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] or store-operated Ca²⁺ entry (2-aminoethyl diphenylborinate), can significantly reverse the effect of clodronate. Finally, in vivo animal and green fluorescent protein imaging studies further proved that the tumor inhibitory effect of clodronate on xenotransplanted CG3 cells can be reversed by treatment with suramin. In conclusion, we demonstrated that clodronate-induced PTC cell apoptosis and tumor inhibition are partially mediated by the P2Y receptor-cAMP cascade.