Diosgenin Suppresses Cholangiocarcinoma Cells Via Inducing Cell Cycle Arrest And Mitochondria-Mediated Apoptosis

Xiao-Mei Mao,1,* Pan Zhou,1,* Si-Yang Li,2,* Xiao-Yun Zhang,2 Jin-Xing Shen,2 Qing-Xi Chen,1 Jiang-Xing Zhuang,3 Dong-Yan Shen2 1School of Life Sciences, Xiamen University, Xiamen 361102, People’s Republic of China; 2Biobank, The First Affiliated Hospital, School of Medicine, Xiamen University, Xiamen 361003, People’s Republic of China; 3Fujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, Institute of Neuroscience, School of Medicine, Xiamen University, Xiamen, Fujian 361102, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dong-Yan ShenBiobank, The First Affiliated Hospital, School of Medicine, Xiamen University, Xiamen 361003, People’s Republic of ChinaTel +86 592 213 7507Fax +86 592 213 7509Email shendongyan@163.comJiang-Xing ZhuangFujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, Institute of Neuroscience, School of Medicine, Xiamen University, Xiamen, Fujian 361102, People’s Republic of ChinaEmail jiangxingzhuang@xmu.edu.cnPurpose: Diosgenin (DSG) is the precursor of steroid hormones and plays a crucial part in the proliferation of various carcinomas including human colorectal cancer and gastric carcinoma. Nevertheless, its specific features and mechanisms in human cholangiocarcinoma (CCA) remain unknown.Methods: MTS assay, colony-forming assay, and EdU assay were performed to determine the role of DSG on the progression of human CCA cells. The distributions of cell cycle, the ratio of apoptosis, and the mitochondrial membrane potential (ΔΨm) were studied by flow cytometry (FCM). AO/EB and Hoechst 33258 staining were performed to observe the morphological features of cell apoptosis. TEM was performed to observe the ultrastructures of QBC939 and HuCCT1 cells. The mRNA and protein expression of mitochondrial apoptotic pathway and GSK3β/β-catenin pathway were further confirmed by qPCR and Western blotting. The xenograft tumor model of HuCCT1 cells was built. Immunohistochemistry of tumor tissues was performed.Results: Our results indicated that DSG inhibited the progression of six CCA cell lines. In vivo tumor studies also indicated that DSG significantly inhibited tumor growth in xenografts in nude mice. The expression of mitosis-promoting factor cyclinB1 was decreased along with the elevating level of cell cycle inhibitor p21, resulting in arresting CCA cell cycles at G2/M phase. Furthermore, DSG induced apoptosis with the increased expressions of cytosol cytochrome C, cleaved-caspase-3, cleaved-PARP1 and the Bax/Bcl-2 ratio. Mechanistically, our study showed that GSK3β/β-catenin pathway was involved in the apoptosis of CCA cells. Thus, DSG might provide a new clue for the drug therapy of CCA.Conclusion: In our data, DSG was found to have efficient antitumor potential of human CCA cells in vitro and in vivo.Keywords: diosgenin, cholangiocarcinoma, apoptosis, mitochondria, GSK3β/β-catenin pathway