Monin Malte B, Krause Petra, Stelling Robin, Bocuk Derya, Niebert Sabine, Klemm Florian, Pukrop Tobias, Koenig Sarah
Department of General, Visceral and Paediatric Surgery, University Medical Centre, Georg-August-University Goettingen, Göttingen, Germany.
Department of Haematology and Oncology, University Medical Centre, Georg-August-University Goettingen, Göttingen, Germany.
J Surg Res. 2016 Jun 1;203(1):193-205. doi: 10.1016/j.jss.2016.03.051. Epub 2016 Mar 30.
Wnt/β-catenin signaling is known to play an important role in colorectal cancer (CRC). Niclosamide, a salicylamide derivative used in the treatment of tapeworm infections, targets the Wnt/β-catenin pathway. The objective of this study was to investigate niclosamide as a therapeutic agent against CRC.
The antiproliferative effects of 1, 3, 10, and 50 μM concentrations of niclosamide on human (SW480 and SW620) and rodent (CC531) CRC cell lines were determined by MTS assay and direct cell count. The lymphoid enhancer-binding factor 1/transcription factor (LEF/TCF) reporter assay monitored the activity of Wnt signaling. Immunofluorescence staining demonstrated the expression pattern of active β-catenin. Gene expression of canonical and noncanonical Wnt signaling components was analyzed using qRT-PCR. Western blot analysis was performed with antibodies detecting nuclear localization of β-catenin and c-jun.
Cell proliferation in CRC cell lines was blocked dose dependently after 12 and 24 h of incubation. The Wnt promoter activity of LEF/TCF significantly decreased with niclosamide concentrations of 10 and 50 μM after 12 h of incubation. Active β-catenin did not shift from the nuclear to the cytosolic pool. However, canonical target genes (met, MMP7, and cyclin D1) as well as the coactivating factor Bcl9 were downregulated, whereas the noncanonical key player c-jun was clearly activated.
Niclosamide treatment is associated with an inhibitory effect on CRC development and reduced Wnt activity. It may exert its effect by interfering with the nuclear β-catenin-Bcl9-LEF/TCF triple-complex and by upregulation of c-jun representing noncanonical Wnt/JNK signaling. Thus, our findings warrant further research into this substance as a treatment option for patients with advanced CRC.
已知Wnt/β-连环蛋白信号通路在结直肠癌（CRC）中发挥重要作用。氯硝柳胺是一种用于治疗绦虫感染的水杨酰胺衍生物，作用于Wnt/β-连环蛋白通路。本研究的目的是探究氯硝柳胺作为一种抗结直肠癌的治疗药物的效果。
通过MTS法和直接细胞计数法测定1、3、10和50 μM浓度的氯硝柳胺对人（SW480和SW620）和啮齿动物（CC531）结直肠癌细胞系的抗增殖作用。淋巴样增强因子1/转录因子（LEF/TCF）报告基因检测法监测Wnt信号的活性。免疫荧光染色显示活性β-连环蛋白的表达模式。使用qRT-PCR分析经典和非经典Wnt信号通路成分的基因表达。用检测β-连环蛋白和c-jun核定位的抗体进行蛋白质印迹分析。
孵育12和24小时后，结直肠癌细胞系中的细胞增殖呈剂量依赖性受阻。孵育12小时后，10和50 μM浓度的氯硝柳胺使LEF/TCF的Wnt启动子活性显著降低。活性β-连环蛋白没有从细胞核转移到细胞质中。然而，经典靶基因（met、MMP7和细胞周期蛋白D1）以及共激活因子Bcl9被下调，而非经典关键因子c-jun被明显激活。
氯硝柳胺治疗与对结直肠癌发展的抑制作用和Wnt活性降低有关。它可能通过干扰细胞核β-连环蛋白-Bcl9-LEF/TCF三聚体复合物以及上调代表非经典Wnt/JNK信号通路的c-jun发挥作用。因此，我们的研究结果值得进一步研究这种物质作为晚期结直肠癌患者的一种治疗选择。
