title: Clodronate-induced cell apoptosis in human thyroid carcinoma is mediated via the P2 receptor signaling pathway.
abstract: Clodronate, a halogenated bisphosphonate, can inhibit the growth of human thyroid carcinoma (TC) cells. Previously, we found that a clodronate-induced Ca(2+) 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 Ca(2+) transients induced by clodronate and ATP. The release of Ca(2+) from the endoplasmic reticulum and membrane depolarization of mitochondria was observed during the clodronate-induced Ca(2+) 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-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] or store-operated Ca(2+) 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.
authors.affiliation: Departments of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China.
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Yang
authors.forename: Hsiao-Chuan
authors.initials: HC
authors.lastname: Teng
authors.forename: Kuan-Hsuan
authors.initials: KH
authors.lastname: Chen
authors.forename: Ming-Long
authors.initials: ML
authors.lastname: Tsai
authors.forename: Ting-Kuei
authors.initials: TK
authors.lastname: Lee
authors.forename: Yueh-Ching
authors.initials: YC
authors.lastname: Chou
authors.forename: Chin-Wen
authors.initials: CW
authors.lastname: Chi
authors.forename: Shi-Hwa
authors.initials: SH
authors.lastname: Chiou
authors.forename: Chen-Hsen
authors.initials: CH
authors.lastname: Lee
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D002291:Carcinoma, Papillary / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004002:Clodronic Acid / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*
chemical_list: D050071:Bone Density Conservation Agents; D018048:Receptors, Purinergic P2; D004002:Clodronic Acid