The Department of Orthopaedics, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, PR China.
The Department of Rehabilitation, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, PR China.
Tissue Cell. 2019 Aug;59:51-61. doi: 10.1016/j.tice.2019.07.002. Epub 2019 Jul 2.
We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.
我们使用具有高转移潜能的小鼠自发性骨肉瘤细胞系 K7M2 细胞系来研究 Notch 信号通路在骨肉瘤生物学表现中的作用，以了解其在调节细胞增殖和迁移中的潜在机制，并通过发现新的治疗靶点来改善骨肉瘤患者的预后。首先，通过免疫染色确定 K7M2 中的 Notch 表达，并用 γ-分泌酶抑制剂 N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-S-苯基甘氨酸叔丁酯（DAPT）抑制 Notch 细胞内结构域（NICD）的蛋白水解裂解，从而抑制 Notch 激活。通过使用 Sulforhodamine B 测定法、集落形成单位测定法、Brdu 和 Ki67 染色以及细胞凋亡和细胞周期阶段的流式细胞术检测，发现 DAPT 以剂量依赖性方式抑制 K7M2 的增殖。通过使用划痕愈合和 Transwell 迁移测定法，发现 DAPT 也以剂量依赖性方式抑制 K7M2 的迁移。通过结合微拉曼光谱和 K-均值聚类分析，我们发现 DAPT 抑制大多数 K7M2 细胞结构中多种重要的细胞代谢相关成分。然后，通过 Western blot 发现 DAPT 以浓度依赖性方式抑制 Notch1ICD 表达，并且这种表达与磷酸化-Erk1/2（p-Erk）直接相关。为了证实这一发现，我们使用 Notch 信号配体 Jagged1 激活 Notch 信号通路，从而上调 p-Erk，导致 K7M2 的增殖和迁移增加。使用 Erk 通路抑制剂 U0126，我们表明 p-Erk 下调，K7M2 的增殖和迁移随之减少。最后，我们构建了 K7M2 小鼠胫骨旁肿瘤模型和肺转移模型。我们发现 DAPT 在体内抑制 p-Erk，有效控制肿瘤生长，减少血管生成，减少向肺部转移，并提高整体生存率。总之，Notch 信号通路通过 p-Erk 发挥癌基因作用并促进骨肉瘤转移。DAPT 通过抑制 Erk 磷酸化，有效抑制骨肉瘤在体内和体外的增殖和转移。因此，抑制 Notch 激活导致 Erk 通路磷酸化的下调可作为临床治疗中改善骨肉瘤预后的潜在治疗靶点。
