Effect of matrine on cell proliferation, apoptosis and autophagy of human medullobIastoma D341 cells in vitro / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To explore the effect of matrine induced proliferation, apoptosis and auto-phagy on human medulloblastoma cell line D341 in vitro. METHODS D341 cells in vitro were incubated with matrine 0, 0.5, 1.0, 1.5 and 2.0 g.L-1 for 24, 48 and 72 h, respectively. The proliferation of D341 cells was analyzed using Cell Counting Kit-8 assay. Apoptosis was detected by flow cytometry. The mor-phologic change of cells was observed under a transmission electron microscope. The expression of Bax, Bcl-2, caspase 3, microtubule associated protein 1 light chain 3 (LC3) and beclin1 was detected by Western blotting, and the expression of LC3 and beclin1 was detected by Western blotting with or without the autophagy inhibitor 3-methyladenine(3-MA). 3-MA was added 1 h before matrine and the final concentration of 3-MA was 5 mmol.L-1 . RESULTS Matrine significantly inhibited the proliferation of D341 cells. There was a concentration-effect relationship ( r24 h = 0.994, r48 h = 0.992, r72 h = 0.996, P<0.01). Matrine could induce the cell apoptosis (r24 h = 0.937, r48 h = 0.947, r72 h = 0.987, P<0.01). When the concentration of matrine was 2.0 g.L-1 , the inhibitory effect on D341 cell proliferation (r=0.999, P<0.01) and the induction of cell apoptosis (r=0.990, P<0.01) had a time-dependence. When the concen-tration of matrine was 2.0 g.L-1 , the ultrastructure of the D341 cells had obvious change. Cells with acoustic cavitation bubble structure, chromatin condensation, and marginalization were observed after matrine treatment for 24 h. After 48 h treatment with matrine, nuclear chromatin condensation and more vacuoles in the cytoplasm were observed. After 72 h treatment with matrine, cells exhibited apoptotic characteristics with obvious nuclear chromatin condensation, and nuclear fragmentation, significantly increased the larger cytoplasmic vacuoles. Western blotting analysis showed that matrine could increase the expression of Bax (r24 h =0.981, r48 h =0.967, r72 h =0.998, P<0.01), and decrease the expression of Bcl-2 (r24 h = -0.977, r48 h = -0.989, r72 h = -0.968, P<0.01). Matrine could increase the expression of caspase 3 when the effect time was 48 h (r48 h =0.995, P<0.01). Matrine also increased the expression of beclin1 (r24 h =0.989, r48 h =0.986, r72 h =0.966, P<0.01). The autophagy inhibitor 3-MA could reduce this effect ( P < 0.05). Matrine decreased the expression of LC3-Ⅰ but increased the expression of LC3-Ⅱ and thus the ratio of LC3-Ⅰ/ LC-Ⅱ was decreased (r24 h = -0.795, r48 h = -0.886, r72 h = -0.901, P<0.05). 3-MA could reduce the effects of matrine on LC3-Ⅰ and LC3-Ⅱ expression of D341 cells (P<0.05). CONCLUSION Matrine can inhibit proliferation, induce apoptosis and promote autophagy of D341 cells in vitro.