The anthelmintic drug flubendazole induces cell apoptosis and inhibits NF-κB signaling in esophageal squamous cell carcinoma

Jiali Tao,1 Hongmei Zhao,1 Xiaochen Xie,2 Man Luo,1 Zhiwei Gao,1 Hong Sun,1 Ziming Huang3 1Department of Emergency, The Affiliated Huaian No 1 People’s Hospital of Nanjing Medical University, Huai’an 223300, Jiangsu, China; 2Department of Respiratory, The Affiliated Huaian No 1 People’s Hospital of Nanjing Medical University, Huai’an 223300, Jiangsu, China; 3Department of Emergency Surgery, The Affiliated Huaian No 1 People’s Hospital of Nanjing Medical University, Huai’an 223300, Jiangsu, China Background and objectives: The nuclear factor kappa B (NF-κB) signaling is activated in esophageal squamous cell carcinoma (ESCC) and can be used as a potential target for anti-ESCC drug discovery. In this study, we aimed to investigate the function of flubendazole as a novel NF-κB inhibitor in ESCC cells. Materials and methods: Cell Counting Kit-8 assay was carried out to assess cell viability of ESCC cells. Flow cytometry and immunoblotting were performed to examine cell apoptosis. Immunoblotting assay was used to analyze the protein expression of NF-κB signaling. Luciferase assay was performed to explore the activation of NF-κB. Plasmids were transfected into ESCC cells using Lipofectamine® 2000. Results: In this study, the anthelmintic drug flubendazole was found to inhibit the activation of IκBα kinases (IKKs), block the activation of IκBα, and decrease the phosphorylation of NF-κB p65, which could be a novel NF-κB inhibitor in ESCC cells. We also found that flubendazole inhibited the cell survival of different ESCC cells and induced cell apoptosis in both EC9706 and TE1 cells. Moreover, overexpression of constitutively activated IKKβ markedly decreased the cytotoxic effect of flubendazole on EC9706 and TE1 cells. In addition, flubendazole also showed a synergistic effect on ESCC cells when combined with doxorubicin. Conclusion: The results above demonstrated that flubendazole showed its anti-tumor action by suppressing the NF-κB signaling pathway and suggested that flubendazole might be re-purposed for anti-ESCC therapy in clinic as a single agent or in combination with other anti-tumor drugs. Keywords: flubendazole, cell apoptosis, NF-κB, esophageal squamous cell carcinoma, re-purpos