title: Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.
abstract: Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.
authors.affiliation: Institute of Oncology, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 201999, P.R. China.|Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Qingxi
authors.identifier: http://orcid.org/0000-0003-1766-5298
authors.initials: Q
authors.lastname: Yue
authors.affiliation: Institute of Oncology, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 201999, P.R. China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhen
authors.affiliation: Center for Integrative Proteomics Research, BioMaPS Institute and Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854-8076, United States of America.
authors.forename: Ming
authors.initials: M
authors.lastname: Huang
authors.affiliation: Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854-8020, United States of America.
authors.forename: Xi
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Lixing
authors.initials: L
authors.lastname: Feng
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Baohong
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Wanying
authors.initials: W
authors.lastname: Wu
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 201203, P.R. China.
authors.forename: Dean
authors.initials: D
authors.lastname: Guo
mesh_terms: D000664:Amphibian Venoms / Q000737:chemistry; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D002018:Bufanolides / Q000737:chemistry / Q000494:pharmacology*; D002584:Cervix Uteri / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D062105:Molecular Docking Simulation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D060066:Protein Interaction Maps / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology
chemical_list: D000664:Amphibian Venoms; D000970:Antineoplastic Agents; D002018:Bufanolides; D061988:Proteasome Inhibitors; C055393:arenobufagin; D046988:Proteasome Endopeptidase Complex; D000254:Sodium-Potassium-Exchanging ATPase