Abstract
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.
Highlights
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; ʈCollege of Pharmaceutical Sciences, Soochow University, Suzhou, China
Statistical analysis results indicated that, in cells treated with Gambogic acid (GA) at 2 M or 4 M for 24 h, the early apoptosis rate was 6.37 Ϯ 1.05% and 11.47 Ϯ 1.09%, respectively, which were statistically different from the control (2.64 Ϯ 0.61%) with p value of 0.037 and 0.006, respectively
These results demonstrated that GA induced apoptosis of HeLa cells in a dose-dependent manner
Summary
Chemicals—GA with a purity of more than 97% was purchased from Sigma-Aldrich Chemical Co. Cells were washed three times with cold TBS, harvested using a cell scraper, and lysed in 10 volume of cold lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% Nonidet P-40, 2 mM EDTA, 10% glycerol, 1 mM PMSF, 5 g/ml Aprotinin, 5 g/ml Leupeptin) on ice. The concentration of protein samples was quantified using the Bio-Rad protein assay. Protein samples from three independent experiments were used for quantification of vimentin phosphopeptides in control and GA-treated group. The cytotoxicity of GA on cells with overexpression of vimentin was checked and compared with that of negative control cells using MTT assay and flow cytometric analysis as described above. The cytotoxicity of GA on cells treated with siRNA against p38, HSP27 or vimentin was checked using flow cytometric analysis as described above. For each variable three independent experiments were carried out unless otherwise indicated
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