Crosstalk between Beclin-1-dependent autophagy and caspase-dependent apoptosis induced by tanshinone IIA in human osteosarcoma MG-63 cells
| Autor: | Yan-Xing Guo, Man-Yu Huang, Kun Ma, Guo-Qiang Hu, Chuan Zhang |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
autophagy Cancer Research Programmed cell death Blotting Western Bone Neoplasms Biology Caspase-Dependent Apoptosis 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Microscopy Electron Transmission Cell Line Tumor Humans MTT assay Propidium iodide Cell Proliferation chemistry.chemical_classification Osteosarcoma Reactive oxygen species Cell growth Autophagy apoptosis ROS Receptor Cross-Talk Articles General Medicine Flow Cytometry Antineoplastic Agents Phytogenic tanshinone IIA Molecular biology 030104 developmental biology Oncology chemistry Apoptosis Caspases 030220 oncology & carcinogenesis Abietanes Beclin-1 Reactive Oxygen Species |
| Zdroj: | Oncology Reports |
| ISSN: | 1791-2431 1021-335X |
| Popis: | The aim of the present study was to ascertain whether or not autophagy is induced by tanshinone IIA (TanIIA), and to explore the crosstalk between autophagy and apoptosis in regards to the antitumor effects of TanIIA on MG-63 cells and the potential mechanism. MG-63 cells were cultured in vitro with various concentrations of TanIIA (0, 2.5, 5, 10 and 20 mg/l) for 0, 24, 48 and 72 h, respec- tively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to evaluate the inhibition of the proliferation of osteosarcoma MG-63 cells by TanIIA or in the presence/absence of chloroquine (CQ). Autophagic vacuoles and characteristic autophagosomes were observed by transmission electron microscopy (TEM). TanIIA-induced autophagy in MG-63 cells was confirmed by GFP-LC3 punc - tate fluorescence. The expression levels of apoptosis-related proteins caspase-3, caspase-8, caspase-9 and cleaved-PARP and autophagy-related proteins LC3II/LC3I and Beclin-1 were detected by western blotting. FITC-Annexin V/propidium iodide (PI) staining, flow cytometry and Hoechst 33258 staining were used to analyze the apoptotic rate. Fluorescence intensity of reactive oxygen species (ROS) was examined under a fluorescence microscope using an analysis software system. Cell proliferation was obviously inhibited by TanIIA in a dose- and time-dependent manner. Generation of autophagy was triggered by TanIIA (0-20 mg/l) treatment, and in a Beclin-1-dependent manner. Compared with the control group, the apoptosis ratio following treatment with 2.5 mg/l TanIIA failed to achieve statistical significance. Expression of caspase-3, -8 and -9, and cleaved-PARP in the other groups was gradually enhanced in dose-dependent manner. Our analysis also suggested that the influence of autophagy on TanIIA cyto - toxicity had a phase effect; with low -dose drugs and shorter treatment periods, autophagy functioned as a damage repair mechanism. In conrast, when the cells were treated with higher doses of TanIIA for longer treatment periods, autophagic cell death contributed to apoptosis. Furthermore, generation of ROS occurred in a dose-dependent manner and pretreatment with NAC, a selective ROS scavenger, blocked the coexistence of Beclin-1 autophagy and caspase-dependent apoptosis. In conclusion, our findings provide strong evidence that TanIIA may be a potential therapeutic drug against osteosarcoma. Moreover, its cytotoxity can be enhanced with ROS agonists. |
| Databáze: | OpenAIRE |
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