Li Xiaobin, Li Zongze, Song Yimin, Liu Wenjing, Liu Ziwen
Cell Physiol Biochem. 2018;46(2):579-590. doi: 10.1159/000488625. Epub 2018 Mar 28.
BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) plays an important role in papillary thyroid carcinoma (PTC) cell progression. CZ415 is a novel, highly-efficient and specific mTOR kinase inhibitor. The current study tested the potential anti-tumor activity of CZ415 in human PTC cells.
The established (TPC-1 cell line) and primary human PTC cells were treated with CZ415. Cell survival and growth were tested by Cell Counting Kit-8 assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by caspase-3/-9 activity assay, Hoechst-33342 staining assay and single-stranded DNA ELISA assay. Cell cycle progression was tested by propidium iodide-FACS assay. The mTOR signaling was tested by Western blotting assay and co-immunoprecipitation assay. The mouse xenograft tumor model was applied to study the effect of CZ415 in vivo.
In cultured human PTC cells, treatment with CZ415 at nM concentrations significantly inhibited cell survival and growth. CZ415 induced apoptosis activation and cell cycle arrest in human PTC cells. CZ415 disrupted assembling of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-GβL association) in TPC-1 cells, which led to de-phosphorylation of the mTORC1 substrates (S6K1 and 4E-BP1) and the mTORC2 substrate AKT (Ser-473). Further studies show that the autophagy inhibitor 3-methyladenine (3-MA) or Beclin-1 shRNA aggravated CZ415-induced cytotoxicity against PTC cells. In vivo, CZ415 oral administration inhibited TPC-1 xenograft tumor growth in mice.
Our results show that mTOR blockage by CZ415 inhibits PTC cell growth in vitro and in vivo.
背景/目的：雷帕霉素哺乳动物靶点（mTOR）在甲状腺乳头状癌（PTC）细胞进展中起重要作用。CZ415是一种新型、高效且特异性的mTOR激酶抑制剂。本研究测试了CZ415在人PTC细胞中的潜在抗肿瘤活性。
用CZ415处理已建立的（TPC-1细胞系）和原代人PTC细胞。分别通过细胞计数试剂盒-8法和BrdU ELISA法检测细胞存活和生长情况。通过caspase-3/-9活性测定、Hoechst-33342染色测定和单链DNA ELISA测定检测细胞凋亡。通过碘化丙啶-FACS测定检测细胞周期进程。通过蛋白质免疫印迹法和免疫共沉淀法检测mTOR信号传导。应用小鼠异种移植瘤模型研究CZ415在体内的作用。
在培养的人PTC细胞中，用纳摩尔浓度的CZ415处理可显著抑制细胞存活和生长。CZ415诱导人PTC细胞凋亡激活和细胞周期停滞。CZ415破坏了TPC-1细胞中mTORC1（mTOR-Raptor结合）和mTORC2（mTOR-Rictor-GβL结合）的组装，导致mTORC1底物（S6K1和4E-BP1）和mTORC2底物AKT（Ser-473）去磷酸化。进一步研究表明，自噬抑制剂3-甲基腺嘌呤（3-MA）或Beclin-1短发夹RNA加重了CZ415对PTC细胞的细胞毒性。在体内，口服CZ415可抑制小鼠TPC-1异种移植瘤的生长。
我们的结果表明，CZ415阻断mTOR可在体外和体内抑制PTC细胞生长。
