石蒜碱通过激活STMN1阻滞人乳腺癌MCF-7细胞周期分子机制研究  

Mechanism of lycorianine on inhibiting MCF-7 cell cycle by activating STMN1

作　　者：冯洁 綦峥 薛沁冰[1] 李超宇 赵梓桐 王昊 刘婷 付叶珊 王冰 于淼[1,3,4] FENG Jie;QI Zheng;XUE Qinbing;LI Chaoyu;ZHAO Zitong;WANG Hao;LIU Ting;FU Yeshan;WANG Bing;YU Miao(Engineering Research Center for Medicine,Harbin University of Commerce,Harbin 150076,China;School of Food Engineering,Harbin University of Commerce,Harbin 150076,China;Engineering Research Center of Natural Antitumor Drugs,Ministry of Education,Harbin 150076,China;Heilongjiang Key Laboratory of Cancer Prophylaxis and Anticancer Drugs Research,Harbin 150076,China;Wisdom Lake Academy of Pharmacy,Xi’an Jiaotong Liverpool University,Suzhou 215123,China)

机构地区：[1]哈尔滨商业大学药物工程技术研究中心,黑龙江哈尔滨150076 [2]哈尔滨商业大学食品工程学院,黑龙江哈尔滨150076 [3]国家教育部抗肿瘤天然药物工程研究中心,黑龙江哈尔滨150076 [4]黑龙江省肿瘤预防与抗肿瘤药物研究重点实验室,黑龙江哈尔滨150076 [5]西交利物浦大学慧湖药学院,江苏苏州215123

出　　处：《中草药》2024年第15期5125-5134,共10页Chinese Traditional and Herbal Drugs

基　　金：黑龙江省重点研发项目(2022ZX02C07);黑龙江省普通高等学校青年创新人才培养计划项目(UNPYSCT-2020218);哈尔滨商业大学2023年研究生创新科研基金项目(YJSCX2023-785HSD);2023年度哈尔滨商业大学镜湖学者支持计划项目(JHQNRC06);哈尔滨商业大学博士科研支持计划项目(24BQ31)。

摘　　要：目的探讨石蒜碱通过激活微管相关基因STMN1阻滞人乳腺癌MCF-7细胞周期相关分子机制。方法MCF-7细胞给予石蒜碱干预后,采用CCK-8法检测细胞增殖抑制率;流式细胞术检测G_(2)/M期细胞比例、磷酸化组蛋白H3(phosphorylated histone H3,p-H3)表达;荧光显微镜观察石蒜碱对MCF-7细胞微管形态的影响;qRT-PCR检测MCF-7细胞周期、骨架及微管相关基因的表达;Western blotting检测MCF-7细胞内5个周期阻滞相关蛋白的表达。结果石蒜碱对MCF-7细胞的半数抑制浓度(half inhibitory concentration,IC_(50))为13.98μmol/L。石蒜碱呈剂量相关性地将MCF-7细胞阻滞于G_(2)/M期(P<0.01),并增加p-H3表达(P<0.05、0.01)。当石蒜碱浓度为28μmol/L时,细胞微管形态接近于阳性对照药物长春新碱。石蒜碱可下调5个信号传导相关基因,上调2个微管相关基因,下调2个M期相关基因,进而下调5个周期阻滞相关蛋白。相关性分析表明石蒜碱通过激活微管相关基因STMN1,导致微管动态平衡被破坏,最终导致M期阻滞。抑制STMN1基因后,石蒜碱无法将MCF-7细胞周期阻滞在M期。结论石蒜碱抑制MCF-7细胞增殖并通过促进微管解聚将其阻滞于M期,且阻滞M期的关键位点是基因STMN1,为未来药物设计和癌症治疗策略的探索提供了有价值的信息。Objective To investigate the molecular mechanism of lycorine blocking MCF-7 cell cycle through activating microtubule-associated gene STMN1.Methods After intervention with lycorine on MCF-7 cells,cell proliferation inhibition rate was measured by CCK-8 assay;The proportion of cells in G_(2)/M phase and phosphorylated histone H3(p-H3)expression were detected by flow cytometry;The effect of lycorine on microtubule morphology of MCF-7 cells was observed by fluorescence microscopy;The expressions of MCF-7 cell cycle,skeleton and microtubule related genes were detected by qRT-PCR;Western blotting was employed to detect the expressions of five cycle arrest related proteins in MCF-7 cells.Results The half inhibitory concentration(IC_(50))of lycorine in MCF-7 cells was 13.98μmol/L.Lycorianine inhibited MCF-7 cells in G_(2)/M phase(P<0.01)and increased p-H3 expression(P<0.05,0.01)in a dose-dependent manner.When the concentration of lycorine was 28μmol/L,the microtubule morphology of cells was similar to that of the positive control drug vincristine.Lycorine down-regulated five signal transduction-related genes,up-regulated two microtubule-related genes,down-regulated two M phase-related genes,and subsequently down-regulated five cell cycle arrest-related proteins.Correlation analysis indicated that lycorine could disrupt microtubule homeostasis by activating microtubule related gene STMN1,and eventually lead to M-phase arrest.After inhibiting STMN1 gene,lycorine could not block MCF-7 cell cycle in M phase.Conclusion Lycorine effectively inhibits the proliferation of MCF-7 cells and induces M-phase arrest by promoting microtubule depolymerization,with the STMN1 gene being key point of M-phase blockade.This study provides valuable basic information for future drug design and exploration of cancer treatment strategies.

关 键 词：石蒜碱 MCF-7细胞 乳腺癌 M期阻滞 STMN1 微管蛋白 

分 类 号：R285.5[医药卫生—中药学]