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

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相比, AE组和山奈苷组A431细胞迁移距离[(36.7±1.0) μm和(44.7 ± 3.5) μm]显著缩短(F = 52.34, P < 0.001), 而HaCaT细胞迁移距离差异无统计学意义(F = 1.73, P = 0.238)。Transwell小室实验表明, 与二甲基亚砜组A431细胞跨膜细胞数量(195.3 ± 5.7)相比, AE组和山奈苷组A431细胞显著抑制(145.0 ± 2.5和94.7 ± 4.1, F = 72.85, P < 0.001), 而对HaCaT细胞则无明显抑制作用(F = 3.91, P = 0.055)。动物实验表明, 与二甲基亚砜组裸鼠移植瘤体积(841.88 ± 84.63) mm3相比, AE组和山奈苷组显著下降[(407.42 ± 70.37) mm3与(368.77 ± 62.7) mm3, F = 73.78, P < 0.001]。HE染色证实, AE和山奈苷干预可改善其病理变化。qRT-PCR与Western印迹结果显示, AE和山奈苷明显上调瘤体组织中上皮钙黏着蛋白与p-GSK-3β mRNA和蛋白表达水平(均P < 0.001), 下调波形蛋白、β联蛋白及GSK-3β mRNA和蛋白表达水平(均P < 0.001)。结论分子对接筛选的小分子抑制剂与EphB2可形成稳定复合物, 并通过影响Wnt/β联蛋白通路诱导的上皮间质转化现象来抑制CSCC进程。

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 analyze the effect of AE and kaempferitrin on the mRNA and protein expression of E-cadherin,vimentin,glycogen synthase kinase 3β(GSK-3β),phosphorylated GSK-3β(p-GSK-3β)andβ-catenin respectively.One-way analysis of variance and t test were used for comparisons between groups.Results Two small-molecule compounds AA-504/20999031(kaempferitrin)and AA-466/21162055(AE)with high inhibitory activity against EphB2 were screened out.MTT assay showed that both AE and kaempferitrin exhibited strong cytotoxicity to SCL-1 and A431 cells compared with HaCaT cells,and their toxicity increased with the increase of their concentration(F=17.95,11.34,respectively,both P<0.001);after 48-hour treatment,the 50%inhibitory concentrations(IC50s)of AE against SCL-1 and A431 cells were 124.59 and 80.85μmol/L respectively,and the IC50s of kaempferitrin against SCL-1 and A431 cells were 119.64 and 64.96μmol/L respectively.Scratch assay showed that the migration distance of A431 cells was significantly shorter in the AE group and kaempferitrin group(36.7±1.0μm,44.7±3.5μm,respectively)than in the DMSO group(88.1±1.4μm,F=52.34,P<0.001),while there was no significant difference in the migration distance of HaCaT cells among the above groups(F=1.73,P=0.238).Transwell assay showed that the number of A431 cells crossing the Transwell membrane significantly decreased in the AE group and kaempferitrin group(145.0±2.5,94.7±4.1,respectively)compared with the DMSO group(195.3±5.7,F=72.85,P<0.001),while neither AE nor kaempferitrin showed significant inhibitory effects of on the number of HaCaT cells crossing the Transwell membrane(F=3.91,P=0.055).The animal experiment revealed significantly decreased volumes of transplanted tumors in nude mice in the AE group and kaempferitrin group(407.42±70.37 mm3,368.77±62.7 mm3,respectively)compared with the DMSO group(841.88±84.63 mm3,F=73.78,P<0.001).HE staining confirmed that AE and kaempferitrin could improve pathological changes of transplanted tumors.qRT-PCR and Western blot analysis showed that AE and kaempferitrin significantly up-regulated the mRNA and protein expression of E-cadherin and p-GSK-3βin tumor tissues(all P<0.001),and down-regulated the mRNA and protein expression of vimentin,β-catenin and GSK-3β(all P<0.001).Conclusion The small-molecule inhibitors screened by molecular docking can form a stable complex with EphB2,and inhibit the progression of CSCC by affecting the Wnt/β-catenin pathway-induced epithelial-mesenchymal transition.