Xing Zhao-Yu, Wang Yin, Cheng Long, Chen Jie, He Xiao-Zhou, Xing Wei
The Department of Urology, the Third Affiliated Hospital, Soochow University, Changzhou, China.
Institute of Neuroscience, Soochow University, Suzhou, China.
Cell Physiol Biochem. 2018;50(2):640-653. doi: 10.1159/000494185. Epub 2018 Oct 11.
BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) is a valuable treatment target of renal cell carcinoma (RCC). Palomid 529 is a novel mTORC1/2 dual inhibitor.
RCC cells were treated with different concentrations of Palomid 529. Cell survival was tested by MTT assay and clonogenicity assay. Cell proliferation was tested by BrdU ELISA assay. Cell apoptosis was tested by the Hoechst-33342 nuclei staining assay and Histone DNA ELISA assay. mTOR signaling was tested by Western blotting assay and co-immunoprecipitation (IP) assay. The SCID mouse 786-O xenograft model was established to test RCC cell growth in vivo.
Palomid 529 exerted cytotoxic, anti-proliferative and pro-apoptotic activities in 786-O RCC cells. Palomid 529 disassembled mTORC1/2, causing de-phosphorylation of mTORC1/2 substrates. Bromodomain-containing protein 4 (BRD4) is a primary resistant factor of Palomid 529. Palomid 529-induced 786-O cell apoptosis was sensitized by BRD4 inhibitors or BRD4 silencing, but inhibited with BRD4 over-expression. Palomid 529-induced cytotoxicity in the primary human RCC cells was negatively correlated with BRD4 expression level. In vivo, Palomid 529 i.p. administration inhibited 786-O xenograft tumor growth in SCID mice. Its anti-tumor activity was further sensitized by co-administration of the BRD4 inhibitor JQ1. Cconclusion: Palomid 529 inhibits RCC cell growth in vitro and in vivo. BRD4 inhibition could further sensitize Palomid 529 against RCC cells.
背景/目的：雷帕霉素哺乳动物靶点（mTOR）是肾细胞癌（RCC）的一个重要治疗靶点。Palomid 529是一种新型的mTORC1/2双重抑制剂。
用不同浓度的Palomid 529处理肾癌细胞。通过MTT法和克隆形成试验检测细胞存活率。通过BrdU ELISA试验检测细胞增殖。通过Hoechst-33342细胞核染色试验和组蛋白DNA ELISA试验检测细胞凋亡。通过蛋白质免疫印迹法和免疫共沉淀（IP）试验检测mTOR信号通路。建立SCID小鼠786-O异种移植模型以检测肾癌细胞在体内的生长情况。
Palomid 529对786-O肾癌细胞具有细胞毒性、抗增殖和促凋亡活性。Palomid 529使mTORC1/2解聚，导致mTORC1/2底物去磷酸化。含溴结构域蛋白4（BRD4）是Palomid 529的主要耐药因子。BRD4抑制剂或BRD4沉默可使Palomid 529诱导的786-O细胞凋亡敏感化，但BRD4过表达则抑制这种凋亡。Palomid 529对原代人肾癌细胞诱导的细胞毒性与BRD4表达水平呈负相关。在体内，腹腔注射Palomid 529可抑制SCID小鼠体内786-O异种移植瘤的生长。联合使用BRD4抑制剂JQ1可进一步增强其抗肿瘤活性。结论：Palomid 529在体外和体内均能抑制肾癌细胞生长。抑制BRD4可进一步增强Palomid 529对肾癌细胞的敏感性。
