gambogic-acid and Uterine-Cervical-Neoplasms

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.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Electrophoresis, Gel, Two-Dimensional; Female; G2 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; MCF-7 Cells; Mice, Nude; Microscopy, Confocal; Proteome; Proteomics; RNA Interference; Tandem Mass Spectrometry; Time Factors; Tumor Burden; Uterine Cervical Neoplasms; Vimentin; Xanthones; Xenograft Model Antitumor Assays

Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells.. The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique.. Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA.. These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Survival; Down-Regulation; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; Female; Garcinia; Heat-Shock Proteins; HeLa Cells; Humans; JNK Mitogen-Activated Protein Kinases; Phytotherapy; Plant Extracts; Transcriptional Activation; Up-Regulation; Uterine Cervical Neoplasms; Xanthones