RESUMO
Gambogic acid (GA) is an anticancer agent in phase âb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Assuntos
Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Biologia Computacional/métodos , Proteômica/métodos , Xantonas/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Ensaios de Migração Celular , Inibição de Migração Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Expressão Gênica , Humanos , Queratina-18/genética , Oxirredução , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico , Transcrição Gênica/efeitos dos fármacos , Proteases Específicas de Ubiquitina/farmacocinética , Vimentina/genéticaRESUMO
The cytotoxicty of 9,11-dehydroergosterol peroxide (DHEP) isolated from the fruiting bodies of Ganoderma lucidum on HeLa cells was studied. DHEP treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50-value of 8.58 +/- 0.98 microM. Morphological changes of DHEP-treated cells indicated that DHEP induced apoptosis in HeLa cells. To identify the cellular targets of DHEP, two-dimensional electrophoresis analysis was performed to compare the protein expression profiles of DHEP-treated cells with that of control cells. Proteins altered in expressional level after DHEP exposure were identified by MALDI-TOF MS/MS. The cytotoxic effect of DHEP was associated with regulated expression of 6 proteins. Stathmin 1 might be an important target-related protein of DHEP. The regulation of stathmin 1 by DHEP treatment was also confirmed by Western blotting.