胡桃楸树皮提取物对SMMC-7721、MCF-7和A549肿瘤细胞的抑制作用及其机制

Inhibitory effects of Juglans Mandshurica Maxim stem-bark extract on SMMC-7721,MCF-7 and A549 in vitro and mechanisms

目的:观察胡桃楸树皮提取物(JMME)对SMMC-7721、MCF-7和A549 3种肿瘤细胞的生长抑制作用,确定其抑瘤的药用价值,通过检测JMME诱导肿瘤细胞凋亡、抑制端粒酶活性变化阐述其抑瘤机制。方法:分别以25、50、100、200、400和800 mg.L-1JMME作用于体外培养的SMMC-7721、MCF-7和A549肿瘤细胞72 h,采用MTT法检测生长抑制率;以200 mg.L-1JMME作用于体外培养的SMMC-7721、MCF-7和A549,于倒置显微镜下观察细胞形态学变化;琼脂糖凝胶电泳法检测细胞凋亡;PCR-ELISA法检测端粒酶活性。结果:不同浓度JMME(25、50、100、200、400和800 mg.L-1)对SMMC-7721的细胞生长抑制率(%)分别为13.06、21.77、29.88、41.74、69.82和78.08;对MCF-7的抑制率(%)为24.91、38.63、49.72、61.84、65.69和81.28;对A549的抑制率(%)为12.32、26.21、41.65、55.38、62.87和80.13;与对照组比较,JMME各浓度作用的3种肿瘤细胞的生长抑制率均升高(P<0.05),抑制率随JMME对SMMC、MCF-7和A549细胞的浓度的增加而增高(P<0.05)。JMME对SMMC、MCF-7和A549细胞的半数抑制浓度(IC50)分别为211.21、111.07和176.20 mg.L-1,对MCF-7的IC50小于其他两种瘤细胞株。经不同浓度JMME作用单位视野内细胞数量明显减少,细胞间隙增大,细胞体积明显缩小,细胞失去原有的形态,胞体皱缩变圆,出现膜发泡现象,对照组无此变化。提取DNA进行琼脂糖凝胶电泳,见DNA Ladder,表明3种肿瘤细胞经不同浓度JMME作用后出现不同程度凋亡。3种肿瘤细胞与阴性对照组比较端粒酶活性升高,而经过JMME作用后端粒酶活性均受到了抑制,抑制率分别为42.86%、73.30%和58.78%。结论:胡桃楸树皮提取物在体外有抗肿瘤活性,其机制可能与其诱导肿瘤细胞凋亡?抑制端粒酶活性有关。

Objective To investigate the inhibitory effects of Juglans Mandshuriea Maxim stem bark extract （JMME） on SMMC 7721, MCF-7 and A549 cells in vitro, and to confirm the antitumor medicative value of JMME, and to verify antitumor mechanisms of JMME by examining the apoptosis and telomerase activitiy change induced by JMME. Methods SMMC-7721, MCF-7 and A549 cells in culture medium were treated with 25, 50, 100, 200, 400 and 800 mg· L^-1 JMME respectively. The inhibitory rates of these cells were measured by MTT assay. After SMMC-7721, MCF-7 and A549 cells in culture medium were respectively treated with 200 mg · L^-1 JMME, the morphological changes of cells were observed under inverted microscope and the apoptosis was detected by agarose gel eIeetrophoresis assay. Then PCR-enzyme-linked immunosorbent assay （ELISA） was used to detect telomerase activities of SMMC-7721, MCF-7 and A549 cells. Results The inhibitory rates （%） of JMME with different concentrations （25, 50, 100, 200, 400 and800mg· L^-1） on SMMC-7721 were 13.06, 21.77, 29.88, 41.74, 69.82 and 78.08, respectively; 24.90, 35.61, 45.42, 65.65, 69.69 and 81.39 on MCF-7; 12.32, 26.21, 41.65, 55.38, 62.87 and 80.13 on A549. The results showed that JMME had cytolytie action on tumor cells compared with control group （P 〈0.05）. The inhibitory rates increased with the increasing of the concentration of JMME （P〈0.05）. IC50 of SMMC-7721, MCF-7 and A549 after treated with JMME were 211.21, 111.07 and 176.20 mg· L^-1 respectively. The ICs0 of MCF-7 cells were lower than those of SMMC- 7721 and A549 cells. The typical apoptosis morphology was identified under inverted microscope. The typical ladder bar was observed. These results demonstrated that the apoptosis of tumor cells were induced by JMME. The telomerase activities of SMMC-7721, MCF-7 and A549 were stronger than that in negative control , but the telomerase activities of the tumor cells treated with extracts were much weaker than that of the tumor cells untreated. The inhibitory rates were 42.86%, 73.30% and 58.78%, respectively. Conclusion JMME has antitumor effect in vitro. It＇s mechanism is associated with inducing tumor cells apoptosis and inhibiting telomerase activity.