地榆皂苷Ⅱ抑制结肠癌细胞增殖、迁移、侵袭和诱导凋亡的体外实验研究

Experimental study in vitro of ziyuglycoside Ⅱ in inhibition of proliferation,migration,invasion and induction of apoptosis of colon cancer cells

目的探讨地榆皂苷Ⅱ对结肠癌细胞HT-29增殖、迁移、侵袭和凋亡的作用并探讨其作用机制。方法采用CCK-8法检测地榆皂苷Ⅱ对细胞增殖的影响,采用划痕试验检测地榆皂苷Ⅱ对细胞迁移能力的影响,采用Transwell小室实验检测地榆皂苷Ⅱ对细胞侵袭能力的影响,采用流式细胞术测定地榆皂苷Ⅱ对细胞凋亡的影响,分别采用实时定量反转录聚合酶链反应(qRT-PCR)和Western blot法测定地榆皂苷Ⅱ对细胞中蛋白激酶B(AKT)/磷脂酰肌醇-3-激酶(PI3K)信号通路mRNA和蛋白表达的影响。结果地榆皂苷Ⅱ(0、1、5、10、20、40、60和80μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的增殖;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的迁移能力;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性抑制结肠癌细胞HT-29的侵袭能力;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性促进结肠癌细胞HT-29的凋亡;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性降低结肠癌细胞HT-29中AKT和PI3K mRNA的表达,增加Caspase-3和Caspase-9 mRNA的表达;地榆皂苷Ⅱ(5、10和20μmol/mL)可剂量依赖性地降低结肠癌细胞HT-29中磷酸化蛋白激酶B(p-AKT)和磷酸化磷脂酰肌醇-3-激酶(p-PI3K)蛋白的表达,增加Cleaved-Caspase-3和Cleaved-Caspase-9蛋白的表达,差异有统计学意义(P<0.05)。结论地榆皂苷Ⅱ具有抑制结肠癌细胞HT-29的增殖、迁移、侵袭,促进细胞凋亡作用,可能与其促进AKT和PI3K蛋白磷酸化、Caspase-3和Caspase-9蛋白活化而调节AKT/PI3K信号通路有关。

Objective To investigate the effect and its mechanism of ziyuglycoside Ⅱ on proliferation,migration,invasion and apoptosis of colon cancer cells HT-29.Methods The effect of ziyuglycoside Ⅱ on cell proliferation of colon cancer cells HT-29 was determined by CCK-8 method;the effect of ziyuglycoside Ⅱ on cell migrative capacity of colon cancer cells HT-29 was determined by scratch assay;the effect of ziyuglycoside Ⅱ on cell invasive capacity of colon cancer cells HT-29 was determined by transwell assay;the effects of ziyuglycoside Ⅱ on cell apoptosis of colon cancer cells HT-29 was determined by flow cytometry;the effects of ziyuglycoside Ⅱ on mRNA and protein expression of protein kinase B(AKT)/phosphatidylinositol-3-kinase(PI3K)signal pathway were determined by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and western-blot,respectively.Results Ziyuglycoside Ⅱ(0,1,5,10,20,40,60 and 80μmol/mL)inhibited proliferation of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20μmol/mL)inhibited migration of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20μmol/mL)inhibited invasion of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20μmol/mL)promoted apoptosis of colon cancer cells HT-29 in a dose-dependent manner.Ziyuglycoside Ⅱ(5,10 and 20μmol/mL)increased mRNA expression of AKT and PI3K,decreased mRNA expression of Caspase-3 and Caspase-9.Ziyuglycoside Ⅱ(5,10 and 20μmol/mL)increased protein expression of phosphorylated protein kinase B(p-AKT)and phosphorylphosphatidylinositol-3-kinase(p-PI3K),increased the expression of Cleaved-Caspase-3 and Cleaved-Caspase-9 protein.Conclusion Ziyuglycoside Ⅱ can inhibit proliferation,migration and invasion of colon cancer cells HT-29,and promote the apoptosis of colon cancer cells HT-29.Its mechanism may be related to regulating the signal pathway of AKT/PI3K via promoting phosphorylation of AKT and PI3K protein,activation of Caspase-3 and Caspase-9 protein.