华蟾素通过调控Wnt/β-catenin信号通路和EMT抑制结肠癌侵袭和转移的实验研究  

Experimental study on the inhibition of colon cancer invasion and metastasis by cinobufacini through the regulation of Wnt/β-catenin signaling pathway and EMT

作　　者：崔旭旭 刘璐 张洁 Cui Xuxu;Liu Lu;Zhang Jie(Department of Oncology,Second Affiliated Hospital of Air Force Medical University,Shaanxi Province,Xi'an 710038,China;不详)

机构地区：[1]空军军医大学第二附属医院肿瘤科,西安710038 [2]西安大兴医院肿瘤科,西安710016 [3]陕西中医药大学附属医院肿瘤科,陕西咸阳712000

出　　处：《疑难病杂志》2024年第12期1507-1512,共6页Chinese Journal of Difficult and Complicated Cases

基　　金：国家自然科学基金(81702847);陕西省中医药管理局中医药科研课题(LCPT047)。

摘　　要：目的探索华蟾素在结肠癌侵袭和转移中的作用及其机制。方法于2022年9月—2023年3月在空军军医大学第二附属医院实验室进行实验。使用不同浓度的华蟾素(0、2.5、5、10、20、40、80、160 mg/L)对人结肠癌细胞系HCT 116和人正常结肠上皮细胞系NCM 460进行处理,CCK-8法检测华蟾素对结肠癌细胞和正常结肠上皮细胞增殖的影响。将HCT 116细胞随机分为对照组和华蟾素(80 mg/L)组,干预24 h后光学显微镜下观察细胞形态变化;Annexin V-FITC/PI双染检测细胞凋亡;Transwell实验检测细胞迁移和侵袭;Western blot检测癌细胞转移、上皮—间充质转化(EMT)和Wnt/β-catenin相关蛋白水平。结果CCK-8检测结果显示,不同浓度的华蟾素处理后,华蟾素对HCT 116细胞的增殖抑制率以剂量和时间依赖的方式逐渐升高(P<0.001),24 h半抑制浓度(IC50)为80 mg/L,而对正常结肠上皮细胞NCM 460的增殖能力无影响。对照组HCT 116细胞状态良好,而华蟾素组细胞贴壁能力下降,分泌物增多,形态皱缩呈圆形上皮细胞样表型,伴随不同程度细胞膜破裂;流式细胞术结果显示,华蟾素处理24、48和72 h后,HCT 116细胞凋亡率分别较处理0 h时显著增加(t/P=17.222/<0.001、17.041/<0.001、23.651/<0.001);Transwell实验结果显示,华蟾素组HCT 116细胞的迁移和侵袭细胞数较对照组明显降低(t/P=52.754/<0.001、7.584/0.002);Western blot结果显示,与对照组比较,华蟾素组细胞中MMP-2和MMP-9表达降低,上皮标志物E-cadherin表达增加,而间充质标志物N-cadherin和Snail表达减少,Wnt3a、β-catenin、c-myc、cyclin D1和MMP-7蛋白水平均下调,而APC蛋白水平上调(t/P=8.908/0.001、29.394/0.001、36.406/0.001、12.860/0.001、14.203/0.001、27.862/0.001、20.176/0.001、24.012/0.001、37.245/0.001、33.627/0.001、6.971/0.002)。结论华蟾素能够抑制结肠癌细胞侵袭和转移,其机制可能与抑制EMT和阻断Wnt/β-catenin信号通路有关。Objective To explore the role of cinobufacini in colon cancer invasion and metastasis and its mechanism.Methods The experiment was conducted in the laboratory of the Second Affiliated Hospital of the Air Force Military Medical University from September 2022 to March 2023.Human colon cancer cell line HCT 116 and human normal colon epithelial cell line NCM 460 were treated with different concentrations of cinobufacini(0,2.5,5,10,20,40,80,160 mg/L)for treatment,and the effects of cinobufacini on the proliferation of colon cancer cells and normal cells were detected by CCK-8 method.HCT 116 cells were randomly divided into the control group and the cinobufacini(80 mg/L)group,and the morphological changes of the cells were observed under the light microscope;apoptosis was detected by Annexin V-FITC/PI double staining;cell migration and invasion were detected by Transwell assay;and metastasis of the cancer cells,EMT,and Wnt/β-catenin related protein expression.Results CCK-8 results showed that the proliferation inhibition rate of HCT 116 cells was gradually increased in a dose and time dependent manner(P<0.001)after treatment with different concentrations of cinobufacini,and the 24 h semi-inhibitory concentration(IC50)was about 80 mg/L,whereas it did not affect the proliferative ability of normal colonic epithelial cells NCM460.The HCT 116 cells in the control group were in good condition,while the cells in the cinobufacini group showed decreased wall adhesion ability,increased secretion,and wrinkled morphology with a round epithelial cell like phenotype,accompanied by cell membrane rupture of varying degrees;the results of flow cytometry showed that the apoptosis rate of the HCT 116 cells was significantly increased compared with that of the treatment at 0 h after cinobufacini treatment for 24,48,and 72 h,respectively(t=17.222,17.041,23.651,P<0.001);and the results of the Transwell experiments showed that the apoptotic rate of HCT 116 cells increased significantly compared with that of the control group.Transwell assa

关 键 词：结肠癌 华蟾素 上皮—间充质转化 WNT/Β-CATENIN信号通路 侵袭 转移 

分 类 号：R735.35[医药卫生—肿瘤]