RESUMO
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.
RESUMO
Objective To investigate the relationship in clinical features of invasive pituitary adenoma patients and their aggressive,matrix metalloproteinase 9 (MMP-9),vascular endothelial growth factor (VEGF) expression and tumor invasiveness of pituitary adenomas.Methods 96 patients with pituitary adenoma were selected,46 cases of invasive pituitary adenomas,50 cases of non-invasive pituitary adenomas,MMP-9,VEGF expression were detected by immunohistochemical assay,their expression levels were analyzed with tumor invasiveness correlation.Results MMP-9,VEGF expression in invasive pituitary adenomas strongly positive rates were 54.35％,52.17％,the total positive rates were 89.13％,95.65％,which were significantly higher than the non-invasive pituitary adenomas MMP-9,VEGF strong positive expression rate of 16.00％,20.00％,the total positive rates were 44.00％,56.00％ (x2 =15.618,9.416,21.631,20.090,all P ＜ 0.05);MMP-9,VEGF for invasive pituitary volume under positive circumstances,the maximum diameter was significantly higher than non-invasive of the group (t =4.32,3.67,2.49,2.54,all P ＜ 0.05),and MMP-9,VEGF were under negative circumstances invasive and non-invasive pituitary adenoma group,the difference was not statistically significant(t =1.56,1.47,0.53,0.21,all P ＞ 0.05);By correlation analysis,invasive pituitary adenoma MMP-9 expression was positively correlated with VEGF levels (r =0.734,P ＜ 0.01).Conclusion Invasive pituitary adenomas of MMP-9 and VEGF expression increased,MMP-9 and VEGF can be used as auxiliary judge biological indicators of invasive pituitary adenomas.
RESUMO
AIM:To investigate matrine-induced apoptosis of human medulloblastoma D 341 cells and the ex-pression of Bax , Bcl-2, serine/threonine kinase Akt and phosphorylated Akt ( p-Akt) in vitro.METHODS: D341 cells were divided into experimental groups ( added with matrine at different concentrations ) and control group ( under the same conditions without matrine).The proliferation of D341 cells was analyzed by CCK-8 assay.Apoptosis was detected by An-nexin V-FITC/PI double staining and the expression of Bax , Bcl-2, Akt and p-Akt was detected by Western blotting .RE-SULTS:Matrine significantly inhibited the proliferation of D 341 cells and increased the apoptosis in a dose-and time-de-pendent manner .The cell apoptosis was characterized by chromatin condensation with margination of chromatin to the nu -clear membrane , increased when and larger cytoplasmic vacuoles , and formation of apoptotic body after treatment with ma-trine.The expression of Bax increased , while the expression of Bcl-2 and p-Akt decreased when the drug concentration gradually increased .CONCLUSION:Matrine induces the apoptosis of human medulloblastoma D 341 cells in vitro by acti-vation of Bax, down-regulation of Bcl-2 and reduction of p-Akt expression level in the PI3K/Akt signaling pathway.