Chen Min-Bin, Zhou Zhen-Tao, Yang Lan, Wei Mu-Xin, Tang Min, Ruan Ting-Yan, Xu Jun-Ying, Zhou Xiao-Zhong, Chen Gang, Lu Pei-Hua
Department of Oncology, Kunshan First People's Hospital Affiliated to Jiangsu University, Kunshan 215300, China.
Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou 215000, China.
Oncotarget. 2016 Mar 29;7(13):17047-59. doi: 10.18632/oncotarget.7742.
Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.
在此，我们在临床前肝细胞癌（HCC）模型中测试了KU-0060648的抗肿瘤活性。我们的结果表明，KU-0060648在已建立的（HepG2、Huh-7和KYN-2细胞系）及原代人HCC细胞中具有抗增殖和促凋亡作用，但对非癌性HL-7702肝细胞无细胞毒性。DNA-PKcs（DNA激活蛋白激酶催化亚基）是KU-0060648的一个重要但非唯一靶点。DNA-PKcs基因敲低或显性负突变抑制HCC细胞增殖。另一方面，野生型DNA-PKcs的过表达增强了HepG2细胞的增殖。重要的是，KU-0060648对DNA-PKcs沉默或突变的HepG2细胞仍具有细胞毒性，尽管其在这些细胞中的活性相对较弱。进一步研究表明，KU-0060648抑制PI3K-AKT-mTOR激活，且不依赖于DNA-PKcs。在HepG2细胞中，用组成型激活的AKT1（CA-AKT1）处理可恢复KU-0060648处理后AKT-mTOR的激活，并减轻随后的细胞毒性。在体内，腹腔注射KU-0060648可显著抑制裸鼠体内HepG2异种移植瘤的生长。异种移植瘤中的AKT-mTOR激活也受到抑制。最后，我们发现DNA-PKcs在人HCC组织中的表达显著上调。然而，一种抗DNA-PKcs的miRNA即miRNA-101表达下调。在HepG2细胞中过表达miR-101可抑制DNA-PKcs表达和细胞增殖。总之，这些结果表明KU-0060648通过依赖和不依赖DNA-PKcs的机制抑制HCC细胞。
