Abstract: 【Abstract】 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, 160 μmol/L, kaempferitrin at concentrations of 12.5, 25, 50, 100 μmol/L）, scratch and Transwell assays （AE at a fixed concentration of 80 μmol/L, kaempferitrin at a fixed concentration of 50 μmol/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） and β-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 80.85 μmol/L respectively, and the IC50s of kaempferitrin against SCL-1 and A431 cells were 119.64 and 64.96 μmol/L respectively. Scratch assay showed that the migration distance of A431 cells was significantly shorter in the AE group and kaempferitrin group （36.7 ± 1.0 μm, 44.7 ± 3.5 μm, respectively） than in the DMSO group （88.1 ± 1.4 μm, 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-3β in 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.

Key words: Carcinoma, squamous cell, Receptor, EphB2, Epithelial-mesenchymal transition, Wnt signaling pathway, beta Catenin, Neoplasms, experimental, Aloe-emodin, Kaempferitrin, Molecular docking, Virtual screening