依维莫司诱导自噬抗肝母细胞瘤作用的研究  

Study of everolimus-induced autophagy and its antitumor effects in hepatoblastoma

作　　者：黄海金 谢玉娟 张守华[3] 冯燕平 曾勇 钟小娟 刘潜 Huang Haijin;Xie Yujuan;Zhang Shouhua;Feng Yanping;Zeng Yong;Zhong Xiaojuan;Liu Qian(First Affiliated Hospital,Gannan Medical University and Clinical Research Center for Vascular Diseases of Jiangxi Province,Ganzhou 341000,China;Department of Neonatology,Fengcheng People's Hospital,Yichu 331199,China;Department of Surgical Oncology,Jiangxi Children's Hospital,Nanchang Medical College,Nanchang 330006,China;Institute of Integrative Chinese and Western Medicine for Children's Health&Drug Innovation,Jiangxi University of Traditional Chinese Medicine/Jiangxi Key Laboratory of Traditional Chinese Medicine Prevention&Treatment of Hemangioma,Nanchang 330004,China)

机构地区：[1]赣南医科大学第一附属医院,江西省脉管性疾病临床研究中心,赣州341000 [2]丰城市人民医院新生儿科,宜春331199 [3]江西省儿童医院(南昌医学院附属儿童医院)肿瘤外科,南昌330006 [4]江西中医药大学儿童健康与药物创新中西医结合研究所,江西省血管瘤中医药防治重点实验室,南昌330004

出　　处：《中华小儿外科杂志》2024年第11期1043-1049,共7页Chinese Journal of Pediatric Surgery

基　　金：江西省卫健委科技计划项目(202410049;202410336);赣州市指导性科技计划项目(GZ2023ZSF112);江西省教育厅科技项目(GJJ161001)。

摘　　要：目的探讨依维莫司诱导自噬抗肝母细胞瘤的作用。方法构建肝母细胞瘤人源肿瘤异种移植(patient-derived tumor xenograft,PDX)模型评价依维莫司抗肝母细胞瘤效果,采用MTT实验、蛋白质印迹法、流式细胞技术、Transwell等方法体外评价依维莫司对HepG2及HuH-6细胞增殖、凋亡、侵袭及诱导自噬能力等情况。结果肝母细胞瘤PDX模型中,和对照组相比,依维莫司组与顺铂组肿瘤体积差异均存在统计学意义(P<0.05),和顺铂组相比,依维莫司组肿瘤抑瘤率更高(P<0.05),说明依维莫司单药能显著抑制肝母细胞瘤PDX模型肿瘤的生长,且效果优于经典化疗药物顺铂单药。依维莫司对HepG2及HuH-6细胞的IC50值分别为(39.01±1.10)μmol/L及(25.56±1.00)μmol/L,相较于对照组,经依维莫司处理HepG2及HuH-6细胞PNCA表达上调(P<0.05),提示依维莫司体外能有效地抑制肝母细胞瘤的增殖。Transwell实验显示,依维莫司对肝母细胞瘤细胞迁移能力呈现剂量依赖性抑制效应(P<0.001)。依维莫司体外可诱导肝母细胞瘤的凋亡[HepG2及HuH-6细胞凋亡比例皆呈现剂量依赖性升高(P<0.001);与对照组相比,HepG2及HuH-6高浓度组Bax/Bcl-2表达比值显著增高(P<0.001)]。依维莫司可促进肝母细胞瘤自噬相关蛋白LC3B和ATG16L1表达上调(P<0.05),并且明显上调自噬非经典通路相关EVA1A蛋白的表达(P<0.05)。结论依维莫司能诱导肝母细胞瘤自噬抑制肝母细胞瘤,可作为治疗肝母细胞瘤的潜在靶向药物,EVA1A可能参与了依维莫司调控肝母细胞瘤自噬。Objective:To explore the role of everolimus in inducing autophagy and to examine its anti-hepatoblastoma(anti-HB)effects.Methods:HB PDX model was established for assessing the anti-HB efficacy of everolimus.In vitro experiments utilizing methyl thiazolyl tetrazolium(MTT),Western blot,flow cytometry,Transwell and other techniques were employed for evaluating the proliferation,apoptosis,invasion and autophagy induction capabilities of everolimus on HuH-6/HepG2 cells.Results:In HB PDX model,both everolimus and cisplatin groups exhibited statistically significant differences in tumor volume as compared with control group(P<0.05).Additionally,everolimus group demonstrated a superior tumor inhibition rate as compared with cisplatin group(P<0.05).It hinted that everolimus as a monotherapy could effectively suppress tumor growth in HB PDX models with greater efficacy than classical chemotherapeutic agent cisplatin alone.IC50 values of everolimus for HepG2/HuH-6 cells were(39.01±1.10)and(25.56±1.00)μmol/L.Dosing of everolimus resulted in an up-regulation of PNCA expression in HepG2/HuH-6 cells as compared with control group(P<0.05).It implied that everolimus could efficiently suppress the proliferation of HB cells in vitro.Transwell assay revealed a dose-dependent inhibition of migratory capability of HB cells by everolimus(P<0.001).Everolimus induced apoptosis in HB cells in a dose-dependent manner.In HepG2 and HuH-6 cells,apoptotic rates spiked markedly as compared with control group(P<0.001).Moreover,a high concentration of everolimus led to a substantial elevation in Bax/Bcl-2 expression ratio in HepG2/HuH-6 cells(P<0.001).Everolimus promoted an up-regulation of autophagy-related proteins LC3B and ATG16L1 expression(P<0.05),as well as significant up-regulation of non-classical autophagy pathway-related EVA1A protein(P<0.05).Conclusion:Everolimus offers the potential of inducing autophagy and arresting HB,thus emerging as a promising targeted therapeutic agent for HB.Furthermore,EVA1A may play a role in everolimus-m

关 键 词：依维莫司 肝母细胞瘤 自噬 哺乳动物雷帕霉素靶蛋白 靶向药物 

分 类 号：R735.7[医药卫生—肿瘤]