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 2ϩ 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 2ϩ transients induced by clodronate and ATP. The release of Ca 2ϩ from the endoplasmic reticulum and membrane depolarization of mitochondria was observed during the clodronate-induced Ca 2ϩ 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 phospho-or store-operated Ca 2ϩ 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 receptorcAMP cascade.Papillary thyroid carcinoma (PTC) is one of the most commonly differentiated thyroid carcinomas (TCs) (Kondo et al., 2006;Kung et al., 2006). Genomic research has revealed that RET rearrangement and BRAF mutation are important in the carcinogenesis of TC/PTC (Lee et al., 1998;Kondo et al., 2006). Thus far, the treatment choice for TC is surgery in combination with postoperative radioiodine therapy (Huang et al., 2005). However, in patients with postoperative recurrent or lymphoid metastatic lesions without radioiodative uptake, the success of TC therapy is limited (Huang et al., 2005;Muresan et al., 2008).Anticancer or chemotherapeutic drugs are limited in effect and duration of response for the treatment of TC (Muresan et al., 2008). Recently, bisphosphonates (BPs) were found to have a broad range of in vivo and in vitro antitumor effects

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

“…A control group received an equivalent volume of the vehicle daily (sterile saline) by intraperitoneally injection. 25, 26 In some experiments, 10 mg/kg suramin 37 and 3  μ g/kg xanthine 38 were applied 1 h before to ATP injection.…”

Bisphosphonamidate Clodronate Prodrug Exhibits Selective Cytotoxic Activity against Melanoma Cell Lines

“…Relevant to the present study, the NNBP clodronate is minimally active against breast, ovarian and thyroid cancer cell lines (high µM to low mM IC 50 s) (12,21,22), and is inactive against melanoma (15), although it is reported to inhibit MMPs important for tumor invasion in melanoma cells (23, 24). In contrast to the limited preclinical activity observed, results from clinical trials suggest potential benefits of adjuvant therapy with clodronate for breast cancer in older patients.…”

“…Interestingly, although clodronate exhibits minimal activity against cancer cell lines, studies by Yang, et al highlight induction of apoptosis in human thyroid cancer cell lines correlating with calcium release from mitochondrial and endoplasmic reticulum that leads to membrane depolarization of mitochondria (21, 22). Nevertheless, CLO exhibits only weak growth inhibitory activity in these cells lines, presumably as a result of poor cellular uptake.…”

Macrophages are required for host survival in experimental urogenital schistosomiasis

“…Exposure of phagocytes to clodronate liposomes results in apoptosis (18). Effective clearance of apoptotic cells by professional phagocytes ( e.g.…”

“…Exposure of phagocytes to clodronate liposomes results in apoptosis (18). Effective clearance of apoptotic cells by professional phagocytes (e.g., macrophages) requires Fas/CD95-induced chemokines to serve as "findme" signals for these dying cells (19).…”