Abstract
The aim of the present study was to observe whether autophagy was induced by matrine, and to investigate the role of autophagy in the antitumor effects of matrine on human osteosarcoma MG-63 cells and its underlying mechanism. MG-63 cells were cultured in vitro in matrine at a concentration of 0.6, 0.8, 1.0 and 1.2 g/l for 0, 24, 48 and 72 h. A MTT assay was used to evaluate the proliferation inhibition of MG-63 cells by matrine, and Annexin V-fluorescein isothiocyanate/propidum iodide (PI) staining flow cytometry was used to analyze the apoptotic rate. Alterations in cell morphology was assessed by PI and Hoechst 33258 cell staining. Matrine-induced autophagy in MG-63 cells was confirmed by green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) b transfection and fluorescence microscopy, and cell viability was investigated by MTT assay following inhibition of autophagy by chloroquine (CQ) pretreatment. The expression level of apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2) and Bcl-2-like protein 4 (Bax), autophagy-associated LC3II protein, and the activation of extracellular signal-regulated kinase (ERK) was detected by western blotting. Cell proliferation was clearly inhibited by matrine in a dose- and time-dependent manner. Flow cytometry and Hoechst 33258/PI staining verified that matrine induced apoptosis in a time-dependent manner when cells were exposed to 1.1 g/l matrine; fluorescence microscopy demonstrated that green fluorescence puncta were enhanced with prolonged time of matrine incubation. Western blotting confirmed that the expression of pro-apoptosis-associated proteins Bax and LC3II, and phosphorylated-ERK were upregulated, and anti-apoptosis protein Bcl-2 was downregulated in a time-dependent manner following treatment with matrine. The cell viability of the matrine + CQ group was increased compared with the matrine group alone, which revealed that matrine treatment alone induced protective autophagy in MG-63 cells. In addiiton, expression of LC3II/LC3I decreased and the expression of BAX/Bcl-2 increased in the matrine + U0126 group compared with the matrine alone group. The present study demonstrated, to the best of our knowledge, for the first time that matrine induced protective autophagy via ERK activation in MG-63 cells, and matrine combined treatment with CQ or U0126 led to an increase in apoptosis in osteosarcoma cells.