汉黄芩素调控Ywhaz蛋白抑制结肠癌细胞增殖及侵袭转移  

Effect of Wogonin on Inhibiting Proliferation,Invasion and Migration of Colorectal Cancer Cells by Regulating Ywhaz Protein Expression

作　　者：李瑶瑶[1,2] 王继军[1] 刘静[2] 郝臻凤[2] 张瑜[1] 侯亚颖 李艳[1] 卜平[1] 孔桂美[1] 郁多男[1] 

机构地区：[1]扬州大学医学院,江苏省中西医结合老年病防治重点实验室,江苏扬州225009 [2]泰兴市人民医院,江苏泰兴225400

出　　处：《中国实验方剂学杂志》2016年第11期97-101,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81272537);江苏省研究生创新工程项目(CXLX-1439);2014年泰兴市人民医院院级基金课题(try1401)

摘　　要：目的:探讨汉黄芩素对人结肠癌细胞HCT116和HT29细胞系的增殖、侵袭能力的影响,并研究汉黄芩素对酪氨酸3/色氨酸5-单加氧酶激活蛋白(Ywhaz)蛋白水平的影响。方法:体外培养HCT116和HT29细胞系,设空白组、汉黄芩素不同浓度组(浓度分别为5,10,20,40μmol·L-1)作用不同时间后,采用细胞计数试剂盒(CCK-8)法检测汉黄芩素对结肠癌细胞增殖的影响,并用Annexin V-FITC/PI双标后流式细胞仪检测结肠癌细胞的凋亡率;穿透小室(Transwell)小室法检测处理24 h后细胞侵袭和迁移力的变化;实时荧光定量-聚合酶链式反应(q PCR)检测不同浓度汉黄芩素作用24 h后Ywhaz mRNA的水平,并运用蛋白质免疫印迹(Western blot)检测该蛋白及其磷酸化水平。结果:与空白组比较,汉黄芩素可明显抑制结肠癌细胞系的增殖,具有浓度和时间依赖性,并促进结肠癌细胞系的凋亡率,其中20和40μmol·L-1的汉黄芩素抑制细胞增殖作用显著(P<0.01),且不同浓度的汉黄芩素(10,20,40μmol·L-1)作用于结肠癌细胞后,明显降低肿瘤细胞的穿膜数(P<0.05,P<0.01),汉黄芩素可下调Ywhaz mRNA水平和蛋白水平的表达,并降低Ywhaz的磷酸化水平(P<0.05,P<0.01)。结论:汉黄芩素可显著抑制HCT116和HT29细胞系的增殖、侵袭和迁移能力,并诱导细胞凋亡,其抗肿瘤机制可能与下调Ywhaz的蛋白水平及其蛋白的磷酸化水平有关。Objective： To study the effect of wogonin on inhibiting the proliferation,migration and invasion of human colorectal cancer cells（ HCT116 and HT29 cell lines）,and investigate its effect on tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein（ Ywhaz） expression levels. Method： HCT116 and HT29 cell lines were cultured in vitro,and then treated with wogonin of different concentrations（ 5,10,20,40μmol·L^-1） for various periods. Another blank group was also set up. Then the cell counting kit（ CCK-8） was used to detect the effect of wogonin on proliferation of colorectal cancer cells; Annexin V-FITC / PI double standard flow cytometry was used to detect the apoptosis of colorectal cancer cells; Transwell method was used to detect the cells migration and invasioncapacities after 24 h treatment; Fluorescense real-time quantitative PCR method was used to detect mRNA expression levels of Ywhaz after 24 h treatment with different concentrations of wogonin,and their protein expression level and phosphorylation level were detected by Western blot assay. Result： As compared with blank group, wogonin significantly inhibited the proliferation of colorectal cancer cell lines in a dosedependent manner, and promoted apoptosis of colorectal cancer cell lines; the inhibitory effect on cells proliferation was significant by 20,40 μmol·L^-1 wogonin（ P〈0. 01）. Moreover, different concentrations of wogonin（ 10,20,40 μmol·L^-1） could significantly reduce the number of transmembrane tumor cells（ P〈0. 05,P〈0. 01）,down-regulate the protein and mRNA expression levels of Ywhaz and reduce the phosphorylation level of Ywhaz in HCT116 and HT29 cell lines（ P〈0. 05,P〈0. 01）. Conclusion： Wogonin can significantly inhibit the proliferation,migration and invasion of HCT116 and HT29 cell lines and induce apoptosis. Its anti-tumor mechanism may be associated with down-regulating the protein expression of Ywhaz and its phosphorylation level.

关 键 词：汉黄芩素 结肠癌 增殖 侵袭 凋亡 迁移 酪氨酸3/色氨酸5-单加氧酶激活蛋白 

分 类 号：R285.5[医药卫生—中药学]