Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid.
Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51-64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic.
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China; §Institute of Oncology, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; ¶College of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China; qxyue@sibs.ac.cn.
Qingxi
Q
Yue
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Lixing
L
Feng
‖College of Pharmaceutical Sciences, Soochow University, Suzhou, China.
Biyin
B
Cao
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Miao
M
Liu
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Dongmei
D
Zhang
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Wanying
W
Wu
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Baohong
B
Jiang
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Min
M
Yang
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;
Xuan
X
Liu
From the ‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China; ¶College of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China;
Dean
D
Guo
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D008819:Mice, Nude; D018613:Microscopy, Confocal; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D047368:Tumor Burden / Q000187:drug effects / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*; D044004:Xanthones / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
D020543:Proteome; D014746:Vimentin; D044004:Xanthones; C052659:gambogic acid