和厚朴酚通过SMAD2/3通路抑制胰腺癌KPC小鼠肿瘤生长及其原代细胞的侵袭和迁移  

Honokiol suppresses the tumor growth and invasion and migration of KPC mice and primary cells via inhibiting SMAD2/3 pathway

作　　者：李杰[1] 秦涛[1] 武帅[1] 马清涌[1] 仵正[1] LI Jie;QIN Tao;WU Shuai;MA Qingyong;WU Zheng(Department of Hepatobiliary Surgery,the First Affiliated Hospital of Xi'an Jiaotong University,Shaanxi Xi'an 710061,China)

机构地区：[1]西安交通大学第一附属医院肝胆外科,陕西西安710061

出　　处：《现代肿瘤医学》2022年第13期2326-2331,共6页Journal of Modern Oncology

基　　金：陕西省创新能力支撑计划(编号:2022PT-35)。

摘　　要：目的:探究和厚朴酚(honokiol,HNK)在胰腺癌KPC小鼠(LSL-Kras^(G12D/+);Trp53^(fl/+);Pdx1-Cre)肿瘤生长及其原代肿瘤细胞侵袭、迁移及上皮间质转化(epithelial mesenchymal transition,EMT)中的作用和机制。方法:自发性胰腺癌转基因小鼠KPC成瘤后灌胃给药,1个月后剖杀观察肿瘤大小和周围浸润情况,免疫组化染色检测EMT相关指标;提取KPC小鼠原代肿瘤细胞培养,以不同浓度和厚朴酚干预,Transwell小室检测其侵袭、迁移能力,Westren blot检测E-cadherin、N-cadherin、Vimentin、p-SMAD2/3、t-SMAD2/3蛋白的表达;利用分子对接模型,预测和厚朴酚和SMAD2的最小距离氢键结合位置;使用外源性TGF-β1作为SMAD2/3的激动剂,联合和厚朴酚,干预肿瘤细胞,Transwell小室和Western blot检测细胞侵袭、迁移和EMT相关蛋白表达情况。结果:和厚朴酚能抑制KPC小鼠自发胰腺肿瘤的大小和EMT,细胞实验也证实和厚朴酚能抑制KPC小鼠原代肿瘤细胞的侵袭、迁移,抑制N-cadherin、Vimentin蛋白的表达,促进E-cadherin蛋白的表达,同时抑制SMAD2/3的磷酸化。软件预测SMAD2的459号脯氨酸残基与和厚朴酚羟基形成0.24 nm的氢键。回复实验证实和厚朴酚能够抑制TGF-β1激活SMAD2/3促进的胰腺癌细胞的侵袭、迁移和EMT。结论:动物和细胞实验都证实和厚朴酚可能封闭SMAD2蛋白质口袋,抑制SMAD2/3的磷酸化,进而抑制KPC小鼠肿瘤的侵袭、迁移和EMT。Objective:To investigate the role and mechanism of honokiol(HNK)in the invasion,migration and EMT of pancreatic cancer cells in KPC mice(LSL-Kras^(G12D/+),Trp53^(fl/+),Pdx1-Cre).Methods:Gene editing engineering mouse KPC was treated with gavage.To harveste and observe the tumor size and infiltration after one month.The related indexes of EMT were detected by immunohistochemistry.Primary tumor cells were extracted from KPC mice and cultured.Transwell assay was applied to analyze the invasion and migration ability of tumor cells after different levels of HNK intervention.The expression of E-cadherin,N-cadherin,Vimentin,p-SMAD2/3 and t-SMAD2/3 were detected by Western blot.AutoDock was used to predict the minimum distance of hydrogen bond position between HNK and SMAD2.TGF-β1,as an agonist for SMAD2/3,was used in combination with HNK to treat tumor cells,Transwell assay and Western blot were applied to detect cell invasion,migration and EMT.Results:HNK could inhibit tumor size and the related indexes of EMT in KPC.HNK could significantly inhibit the invasion and migration of KPC cells,down-regulate the expression of N-cadherin and Vimentin,up-regulate the expression of E-cadherin protein,and inhibit the phosphorylation of SMAD2/3.AutoDock predicted that the 459 proline residue of SMAD2 formed a 0.24 nm hydrogen bond with the HNK hydroxyl group.HNK inhibited TGF-β1-induced invasion,migration and EMT of pancreatic cancer cells.Conclusion:HNK could block the SMAD2 protein pocket,inhibit phosphorylation of SMAD2/3,thus prevent invasion,migration and EMT of pancreatic cancer in vitro and in vivo.

关 键 词：和厚朴酚 SMAD2/3通路 KPC 侵袭 上皮间质转化 

分 类 号：R735.9[医药卫生—肿瘤]