摘要
目的:探讨黄芩苷在体内外对结肠癌细胞凋亡和细胞周期的影响,以及其可能的分子作用机制。方法:不同质量浓度(0、50、100、200、400和800μg/mL)的黄芩苷分别作用人正常结直肠黏膜FHC细胞和人结肠癌HCT116细胞48 h后,倒置显微镜下观察细胞形态变化,并采用MTT法检测各组细胞活力变化。FCM法检测黄芩苷干预后结肠癌HCT116细胞凋亡率和细胞周期分布的变化;同时,采用蛋白质印迹法检测凋亡相关蛋白聚腺苷二磷酸核糖聚合酶-1(poly ADP-ribose polymerase-1,Parp-1)、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)、核因子-κB(nuclear factor-κB,NF-κB)、caspase 3、p53、Bcl-2和Bax,以及细胞周期相关蛋白cyclin D1和cyclin B1表达水平的变化。此外,通过构建结直肠癌HCT116细胞的原位移植瘤小鼠模型,观察黄芩苷灌胃治疗后小鼠体质量以及体内肿瘤生长体积的变化,并应用TUNEL法检测黄芩苷对移植瘤小鼠体内肿瘤细胞凋亡的影响。结果:与黄芩苷未处理的对照组相比,50~800μg/mL黄芩苷对结肠癌HCT116细胞活力有明显的抑制作用(P值均<0.05)。而且黄芩苷对正常结直肠黏膜细胞FHC的半数抑制浓度(half maximal inhibitory concentration,IC50)值明显高于HCT116细胞(P<0.01)。200和400μg/mL黄芩苷处理后,结肠癌HCT116细胞的凋亡率明显升高(P值均<0.01);50和100μg/mL黄芩苷作用48 h后,Parp-1和caspase 3剪切体蛋白的表达水平比对照组均明显升高(P值均<0.01),而XIAP、NF-κB和Bcl-2蛋白的表达水平均明显降低(P值均<0.05)。100和200μg/mL黄芩苷处理后,HCT116细胞周期阻滞在G1期(P值均<0.01);50和100μg/mL黄芩苷作用48 h后,cyclin D1和cyclin B1蛋白的表达水平均明显降低(P值均<0.01)。黄芩苷可以抑制小鼠原位移植瘤的生长(P<0.01),并促进肿瘤细胞凋亡,但是对小鼠体质量无明显影响(P>0.05)。结论:黄芩苷在体内外均可以明显抑制结肠癌HCT116细胞的生长活性,诱导细胞凋亡,并使细胞周期阻滞于G1期。
Objective： To investigate the effects of baicalin on the cell cycle and apoptosis of human colon cancer in vitro and in vivo, and to further clarify its possible molecular mechanism. Methods： After treatment with different concentrations （0, 50, 100, 200, 400 and 800μg/mL） of baicalin for 48 h, the morphology and viability of human normal colorectal mucosa FHC cells and human colon cancer HCT116 cells were detected by invert microscopy and MTT method, respectively. The changes of apoptosis rate and cell cycle distribution of HCT116 cells after baicalin treatment were detected by flow cytometry. The expression levels of apoptosis-related proteins [poly ADP-ribose polymerase-1 （Parp-1）, caspase 3, X-linked inhibitor of apoptosis protein （XlAP）, nuclear factor-κB （NF-κB）, p53, Bcl-2 and Bax] and cell cycle-related proteins （cyclin D1 and cyclin B1） in HCT116 cells treated with baicalin were measured by Western blotting. After the orthotopic xenograft tumor model of colon cancer HCT116 cells in nude mice were constructed and treated with baicalin by gavage, the body weight of mice and the tumor size were checked, and the baicalin-induced apoptosis in xenograft tumors was also assayed using TUNEL methods. Results： As compared with baicalin-untreated control group, 50-800 μg/mL baicalin significantly suppressed the viability of colon cancer HCT116 cells （all P 〈 0.05）. The half maximal inhibitory concentration （IC50） of baicalin in the normal colorectal mucosa FHC cells was significantly higher than that in colon cancer HCT116 cells （P 〈 0.01）. After treatment with 200 and 400 μg/mL baicalin, the apoptosis rate of HCT116 cells was significantly increased （both P 〈 0.01 ）. After treatment with 50 and 1 00 μg/mL baicalin for 48 h, the expression levels of cleaved-Parp-1 and cleaved-caspase 3 proteins were significantly up-regulated （all P 〈 0.01）, while the expression levels of XlAP, NF-κB and Bcl-2 protein were significantly down-regulated （all P 〈 0.05）. Furthermore, after treatment with 100 and 200 ~g/mL baicalin, HCT116 cell cycle arrested at G1 phase （both P 〈 0.01）. After treatment with 50 and 1 00 μg/mL baicalin for 48 h, the levels of cyclin D1 and cyclin B1 proteins in HCT116 cells were significantly down- regulated （all P 〈 0.01）. Additionally, baicalin significantly inhibited the growth of xenograft tumors in mice （P 〈 0.01）, and promoted the apoptosis of tumor cells in vivo, but had no obvious effect on the body weight of mice （P 〉 0.05）. Conclusion： Baicalin can inhibit the growth of colon cancer HCT116 cells in vivo and in vitro through inducing apoptosis and cell cycle arrest at G1 phase.
出处
《肿瘤》
CAS
CSCD
北大核心
2017年第3期208-217,共10页
Tumor
