多激酶抑制剂对特定突变甲状腺癌碘和葡萄糖代谢的影响及其机制

Dedifferentiation of differentiated thyroid carcinoma (DTC) represents a main cause of radioiodine refraction. Radioiodine-refractory DTC is currently incurable, leading to a much higher mortality than that of classic DTC. The constitutive activation of Ras-Raf-MAPK/ERK signaling pathway plays an important role in tumorigenesis and is associated with silencing of various thyroid iodide-metablolizing genes and other malignant clinicopathologic features. Sponsored by the National Natural Science Foundation of China (No.30700187 and 30600155), Shanghai Rising-Star Program (No.08QA14040 and 06QA14040), and the Public Health Bureau of Shanghai Municipality (2006Y34), our group has recently achieved success in many basic and clinical studies, including expression and modulation of iodide-metabolizing gene (sodium iodide symporter, NIS), development of radionuclide-labled agents for detection of apoptosis in vivo, and multidisciplinary treatment of thyroid carcinoma. Given the encouraging results of our preliminary study more recently, which showed up-regulation of NIS expression via supression of the Ras-Raf-MAPK/ERK signaling pathway by sorafenib, the present study is designed to investigate the feasibility of this multikinase inhibitor to enhance radioiodine uptake and decrease the accumulation of FDG in thyroid carinoma cell lines harboring specific gene alterations in vitro and in vivo. The effects and the underling mechanisms of sorafeninb on inhibition of proliferation, inducton of differentiation, and acceleration of apoptosis are also evaluated using RT-PCT, Western blotting, immunofluorescence, flow cytometry, micro-PET, and SPCET. Aiming at exploring potential of novel redifferentiation therapy and combined therapy of radioiodine and tyrosin kinase inhibitor, our project also lays a foundation for molecular imaging and gene detection on individualized treatment of thyroid carcinoma.

分化型甲状腺癌(DTC)的失分化是导致131I治疗无效和病死率升高的主要原因。研究发现，RAS-RAF-MAPK/ERK信号通路的组成性激活和DTC的发生、碘代谢相关基因沉默及其恶性临床病理特征密切相关。我们继碘代谢相关基因的表达与调控、核素标记凋亡显像剂的研制、DTC多学科诊治等研究取得成功后，通过预实验证实了多激酶抑制剂索拉非尼可上调甲状腺癌细胞钠/碘转运体（NIS）的表达。在此基础上，我们选取特定基因突变甲状腺癌细胞系，通过RT-PCR、Western blot、免疫荧光、FCM等体外实验和小动物PET、SPECT等体内实验，深入研究索拉非尼能否提高DTC细胞摄碘能力并降低葡萄糖代谢水平，探索其抑制增殖、诱导分化和促进凋亡的潜能和机制。本项目在探索131I难治性DTC治疗新模式（再分化治疗和131I/激酶抑制剂联合治疗）的同时，为分子影像和基因检测指导甲状腺癌的个体化治疗奠定基础。

目的：碘代谢相关基因的沉默和葡萄糖代谢相关基因表达水平的增高造成了治疗碘-131难治性甲状腺乳头状癌的一大难题。本研究旨在研究索拉非尼和卡博替尼对 RET/PTC1重排的甲状腺乳头状癌的碘和葡萄糖代谢相关的基因表达的影响，并对影响碘和葡萄糖代谢相关基因表达的潜在的机制也做了探讨研究。方法:索拉非尼和卡博替尼治疗对细胞生长、细胞周期和细胞凋亡的影响分别用CCK8，细胞周期分析和Annexin V-FITC细胞凋亡分析进行检测。索拉非尼和卡博替尼对信号传导通路的影响用Western blot进行检测。实时定量PCR，Westernblot,和免疫荧光被用来检测碘和葡萄糖代谢相关基因的表达。结果：索拉非尼和卡博替尼随着时间和剂量的增加抑制细胞增殖的效应越明显。用索拉非尼和卡博替尼处理细胞48小时后的IC50分别是2.341μM和 0.441 μM。索拉非尼和卡博替尼引起细胞周期阻滞在G0/G1期，而不引起凋亡。索拉非尼降低ERK1/2, SAPK/JNK 和AKT的磷酸化，而卡博替尼降低了P38, SAPK/JNK 和AKT的磷酸化。同时，索拉非尼或卡博替尼可抑制碘代谢相关基因和葡萄糖代谢相关基因（GLUT1和GLUT3）的表达。结论：通过抑制RET/PTC1重排的甲状腺乳头状癌细胞的MAPK通路和PI3K/AKT通路的表达，索拉非尼和卡博替尼对提高碘代谢相关基因表达和降低葡萄糖代谢相关基因的表达有潜在价值。