基于Wnt/β联蛋白信号通路探讨EphB2抑制剂对皮肤鳞状细胞癌的影响及作用机制  

Exploration of the effect of EphB2 inhibitors on cutaneous squamous cell carcinoma and their mechanisms of action based on Wnt/β-catenin signaling pathway

作　　者：李艳[1] 张选奋[2] 张文芳[1] Li Yan;Zhang Xuanfen;Zhang Wenfang(Department of Ophthalmology,The Second Hospital of Lanzhou University,Lanzhou 730030,China;Department of Plastic Surgery,The Second Hospital of Lanzhou University,Lanzhou 730030,China)

机构地区：[1]兰州大学第二医院眼科,兰州730030 [2]兰州大学第二医院整形外科,兰州730030

出　　处：《中华皮肤科杂志》2022年第4期321-328,共8页Chinese Journal of Dermatology

基　　金：甘肃省自然科学基金(1506RJZA250)。

摘　　要：目的采用分子对接法筛选酪氨酸蛋白激酶受体B2(EphB2)小分子抑制剂, 研究其对皮肤鳞状细胞癌(CSCC)的影响及可能机制。方法利用Schrodinger对接工具预测EphB2蛋白的三维结构及其配体结合位点, 通过分子对接进行高通量虚拟筛选EphB2抑制剂, 通过体内外实验验证筛出的EphB2抑制剂山奈苷与芦荟大黄素(AE)抗CSCC的作用及机制。体外实验中, 将人CSCC细胞系A431、SCL^(-1)及人永生化表皮细胞HaCaT分别分为空白对照组、二甲基亚砜组、AE组与山奈苷组, 通过MTT实验(AE浓度:20、40、80、160 μmol/L;山奈苷浓度:12.5、25、50、100 μmol/L)、划痕实验及Transwell小室实验(AE浓度:80 μmol/L, 山奈苷浓度:50 μmol/L)分析EphB2抑制剂对CSCC细胞增殖、迁移、侵袭的影响。体内实验中, SPF级BALB/c雌性裸鼠皮下注射0.2 ml A431细胞悬液, 待成功长出瘤体以后, 随机分为4组(n = 6), 空白对照组、二甲基亚砜组、AE组(腹腔注射AE 20 mg·kg^(-1)·d^(-1) AE)与山奈苷组(腹腔注射山奈苷25 mg·kg^(-1)·d^(-1));每周测量裸鼠的肿瘤大小和体重;连续给药28 d后, 剥取裸鼠移植瘤进行HE染色, qRT-PCR与Western blot分析AE和山奈苷对裸鼠移植瘤中上皮钙黏着蛋白、波形蛋白、磷酸化葡萄糖合成激酶3β(p-GSK-3β)、β联蛋白及GSK-3β表达的影响。组间比较采用单因素方差分析及t检验。结果筛选出对EphB2具有较高抑制活性的两个小分子化合物AA-504/20999031(山奈苷)和AA-466/21162055(AE)。MTT实验结果表明, 与HaCaT细胞相比, AE对SCL^(-1)和A431细胞具有强烈的细胞毒性, 且随AE浓度升高毒性变强(F = 17.95, P<0.001), 作用48 h时, IC50分别为124.59 μmol/L和80.85 μmol/L;山奈苷对SCL^(-1)和A431细胞具有强烈的细胞毒性, 且随山奈苷浓度升高毒性变强(F = 11.34, P<0.001), 作用48 h时, IC50分别为119.64 μmol/L和64.96 μmol/L。划痕实验显示, 与二甲基亚砜组细胞迁移距离(88.1±1.4) μm相�Objective To screen small-molecule inhibitors of tyrosine kinase receptor B2(EphB2)by using a molecular docking method,and to investigate their effect on cutaneous squamous cell carcinoma(CSCC)and possible mechanisms of action.Methods The three-dimensional structure of EphB2 protein and its ligand binding sites were predicted by using the docking tool Schrodinger,and high-throughput virtual screening of EphB2 inhibitors was carried out by molecular docking.The anti-CSCC effect and mechanism of action of the screened EphB2 inhibitors kaempferitrin and aloe-emodin(AE)were verified in in vitro and in vivo experiments.In the in vitro experiments,human CSCC cell lines A431 and SCL-1,as well as the human immortalized keratinocyte HaCaT,were all divided into blank control group,dimethyl sulfoxide(DMSO)group,AE group and kaempferitrin group.Methyl thiazol tetrazolium(MTT)assay(AE at concentrations of 20,40,80,160μmol/L,kaempferitrin at concentrations of 12.5,25,50,100μmol/L),scratch and Transwell assays(AE at a fixed concentration of 80μmol/L,kaempferitrin at a fixed concentration of 50μmol/L)were performed to analyze the effect of EphB2 inhibitors on the proliferation,migration and invasion of CSCC cells.In the in vivo experiments,specific pathogen-free BALB/c female nude mice were subcutaneously injected with 0.2 ml of A431 cell suspension.After tumor growth,24 tumor-bearing mice were randomly and equally divided into 4 groups:AE group and kaempferitrin group intraperitoneally injected with 20 mg·kg^(−1)·d^(−1)AE and 25 mg·kg^(−1)·d^(−1)kaempferitrin respectively,blank control group and DMSO group intraperitoneally injected with the same volume of sodium chloride physiological solution and DMSO respectively;the tumor size and body weight of nude mice were measured weekly;after consecutive treatment for 28 days,transplanted tumors were resected from the nude mice for hematoxylin and eosin(HE)staining,and real-time fluorescence-based quantitative PCR(qRT-PCR)and Western blot analysis were performed to ana

关 键 词：癌 鳞状细胞 受体 EPHB2 上皮-间质转化 Wnt信号通路 Β连环素 肿瘤 实验性 芦荟大黄素 山奈苷 分子对接 虚拟筛选 

分 类 号：R739.5[医药卫生—肿瘤]