Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, 121 Jiang Jia Yuan, Nanjing, 210011, Jiangsu Province, China.
Dig Dis Sci. 2013 Jun;58(6):1636-43. doi: 10.1007/s10620-012-2523-7. Epub 2013 Jan 12.
NK4, a competitive antagonist for hepatocyte growth factor (HGF) and the Met receptor, is a bifunctional molecule that acts as an HGF antagonist and an angiogenesis inhibitor. The objective of this study was to investigate the anti-tumor effects of NK4 on the cholangiocarcinoma (CCA) cell line HuCC-T1.
We assessed the effects of NK4 on proliferation, invasion, migration, and cell cycle progression in mock-transfected HuCC-T1 clones, empty-vector-transfected clones of HuCC-T1 (Hu-Em), and NK4-transfected clones of HuCC-T1 (Hu-NK4), with HuCC-T1 cells serving as the control cells. Correlated with these effects on cellular functions, the mRNA levels of cyclin D1 and cyclin A were monitored using reverse transcription (RT)-PCR and quantitative PCR, and the corresponding protein levels were monitored using Western blotting. In addition, Met phosphorylation and the activity of its important downstream signaling targets protein kinase B (Akt) and glycogen synthase kinase (GSK)-3β were evaluated by Western blotting.
Our data indicate that cell proliferation, invasion, and cell cycle progression of the three types of clones were essentially the same, while these processes were stimulated by HGF in HuCC-T1 and Hu-Em cells, but not in Hu-NK4 cells. Moreover, when stimulated with HGF, the increases in mRNA levels of cyclin D1 and cyclin A were accompanied by corresponding increases in protein levels, and the phosphorylation of Met, Akt, and GSK-3β was upregulated in HuCC-T1 and Hu-Em cells, compared to the levels in the Hu-NK4 cells.
These findings suggest that NK4 gene therapy inhibits HGF/Met-induced growth of human CCA cells by arresting cell cycle progression. It also interferes with Met activation and the downstream phosphatidylinositol-3-kinase/Akt/GSK-3β signaling pathway.
NK4 是肝细胞生长因子（HGF）和 Met 受体的竞争性拮抗剂，是一种双功能分子，既能充当 HGF 拮抗剂，又能抑制血管生成。本研究旨在探讨 NK4 对胆管癌细胞系 HuCC-T1 的抗肿瘤作用。
我们评估了 NK4 对 mock 转染 HuCC-T1 克隆、空载体转染 HuCC-T1 克隆（Hu-Em）和 NK4 转染 HuCC-T1 克隆（Hu-NK4）中增殖、侵袭、迁移和细胞周期进展的影响，HuCC-T1 细胞作为对照细胞。与这些细胞功能相关，使用逆转录（RT）-PCR 和定量 PCR 监测细胞周期蛋白 D1 和细胞周期蛋白 A 的 mRNA 水平，使用 Western blot 监测相应的蛋白水平。此外，通过 Western blot 评估 Met 磷酸化及其重要下游信号靶标蛋白激酶 B（Akt）和糖原合成酶激酶（GSK）-3β的活性。
我们的数据表明，三种类型克隆的细胞增殖、侵袭和细胞周期进展基本相同，而 HuCC-T1 和 Hu-Em 细胞中的这些过程受到 HGF 的刺激，但 Hu-NK4 细胞不受刺激。此外，当受到 HGF 刺激时，细胞周期蛋白 D1 和细胞周期蛋白 A 的 mRNA 水平增加，相应的蛋白水平也增加，HuCC-T1 和 Hu-Em 细胞中的 Met、Akt 和 GSK-3β 的磷酸化水平上调，而 Hu-NK4 细胞中的水平则下调。
这些发现表明，NK4 基因治疗通过阻止细胞周期进程抑制 HGF/Met 诱导的人胆管癌细胞生长。它还干扰了 Met 的激活及其下游磷酸肌醇-3-激酶/Akt/GSK-3β 信号通路。
