Yang De-Ming, Teng Hsiao-Chuan, Chen Kuan-Hsuan, Tsai Ming-Long, Lee Ting-Kuei, Chou Yueh-Ching, Chi Chin-Wen, Chiou Shi-Hwa, Lee Chen-Hsen
Departments of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China.
J Pharmacol Exp Ther. 2009 Aug;330(2):613-23. doi: 10.1124/jpet.109.152447. Epub 2009 May 15.
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.
氯膦酸盐是一种卤代双膦酸盐，可抑制人甲状腺癌细胞（TC）的生长。此前，我们发现氯膦酸盐诱导的Ca(2+)瞬变与氯膦酸盐诱导的TC细胞生长抑制相关。然而，氯膦酸盐对TC细胞抗增殖作用的信号传导过程细节尚不清楚。在本研究中，我们使用多种药理药物组合研究了氯膦酸盐对乳头状TC（PTC）细胞和异种移植动物的抗增殖机制。逆转录-聚合酶链反应分析证实了PTC细胞中P2Y受体亚型的内源性表达。P2拮抗剂苏拉明不仅抑制氯膦酸盐和ATP对TC细胞的抗增殖作用，还阻断了氯膦酸盐和ATP诱导的所有Ca(2+)瞬变。在氯膦酸盐诱导的Ca(2+)瞬变过程中，观察到内质网Ca(2+)释放和线粒体膜去极化。末端脱氧核苷酸转移酶dUTP缺口末端标记试验以及用膜联蛋白V和半胱天冬酶-3染色的流式细胞术结果表明，ATP和氯膦酸盐均诱导细胞凋亡。在氯膦酸盐处理的PTC细胞中也观察到肿瘤侵袭和集落形成受到显著抑制。我们进一步证明，只有cAMP抑制剂9-(四氢-2-呋喃基)-9H-嘌呤-6-胺（SQ22536），而不是磷脂酶C抑制剂[1-[6-[[17β-甲氧基雌甾-1,3,5(10)-三烯-17-基]氨基]己基]-1H-吡咯-2,5-二酮（U73122）]或储存操纵性Ca(2+)内流抑制剂（2-氨基乙基二苯基硼酸盐），能显著逆转氯膦酸盐的作用。最后，体内动物和绿色荧光蛋白成像研究进一步证明，苏拉明处理可逆转氯膦酸盐对异种移植CG3细胞的肿瘤抑制作用。总之，我们证明氯膦酸盐诱导的PTC细胞凋亡和肿瘤抑制部分由P2Y受体-cAMP级联介导。
