基于鼠类肉瘤病毒癌基因信号通路探究α-番茄碱对肝癌细胞糖酵解的机制  

Exploring the mechanism ofα-tomatine on glycolysis in hepatocellular carcinoma cells based on KRAS signaling pathway

作　　者：张金忠 朱应超 李富永 郭晓青 亓久德 张磊 ZHANG Jinzhong;ZHU Yingchao;LI Fuyong;GUO Xiaoqing;QI Jiude;ZHANG Lei(Department of Oncology,People’s Hospital Affiliated to Shandong First Medical University,Ji’nan 271199,China;Department of Cardiothoracic Surgery,People’s Hospital Affiliated to Shandong First Medical University,Ji’nan 271199,China)

机构地区：[1]山东第一医科大学附属人民医院肿瘤科,济南271199 [2]山东第一医科大学附属人民医院胸心外科,济南271199

出　　处：《西北药学杂志》2025年第2期95-102,共8页Northwest Pharmaceutical Journal

基　　金：山东省老年医学学会2021年科技攻关计划项目(编号:LKJGG2021W057);济南市卫生健康委员会科技计划项目(编号:2021-1-10)。

摘　　要：目的探讨α-番茄碱基于鼠类肉瘤病毒癌基因(kirsten rat sarcoma viral oncogene,KRAS)信号通路作用于肝癌细胞糖酵解的机制。方法将肝癌细胞系随机均分为对照组、α-番茄碱低剂量(1μmol·L^(−1))组、α-番茄碱中剂量(2μmol·L^(−1))组、α-番茄碱高剂量(5μmol·L^(−1))组,经过多实验筛选,最终确定5μmol·L^(−1)的α-番茄碱作为人肝癌细胞干预浓度。将Huh7细胞随机均分为对照组、α-番茄碱组、LY294002抑制剂组(25μmol·L^(−1))、α-番茄碱(5μmol·L^(−1))联合LY294002(25μmol·L^(−1))抑制剂组。用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴化物[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]法测定细胞活力,用穿越小室(Transwell)实验测定细胞的侵袭能力,用划痕实验测定细胞的迁移能力;用蛋白质印迹法(Western blotting)分析B淋巴细胞瘤-2(Bcell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2-associated X,Bax),KRAS和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)蛋白的表达情况;用邻甲苯胺法检测葡萄糖含量,用对羟基联苯比色法检测乳酸释放量,用比色法检测细胞内三磷酸腺苷(adenosine triphosphate,ATP)含量。结果MTT实验结果表明,α-番茄碱对Huh7细胞增殖有明显的抑制作用。同时,α-番茄碱能抑制细胞侵袭和迁移,抑制葡萄糖消耗、乳酸释放和降低ATP含量,促进细胞凋亡。α-番茄碱在Huh7细胞中激活mTOR/PI3K信号通路,并通过mTOR/PI3K信号通路促进细胞凋亡,抑制细胞的侵袭和迁移能力,进一步抑制糖酵解。结论α-番茄碱通过mTOR/PI3K信号通路抑制糖酵解,降低肝癌细胞的活性及侵袭和迁移能力。Objective To investigate the mechanism ofα-tomatine based on the kirsten rat sarcoma viral oncogene(KRAS)signaling pathway in the glycolysis of hepatoma cells.Method Hepatocellular carcinoma cell lines were randomly divided into 4 groups:control,low(1μmol·L^(−1)),medium(2μmol·L^(−1)),and high(5μmol·L^(−1))dose groups.5μmol·L^(−1)ofα-tomatine was finally chosen to treat Huh7 cells basing on several experiments.Huh7 cells were also randomly assigned to a control group,anα-tomatine group,a LY294002 inhibitor(25μmol·L^(−1))group,and anα-tomatine(5μmol·L^(−1))combined with LY294002 inhibitor(25μmol·L^(−1))group.Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,invasion ability was evaluated through the Transwell assay,and migration ability was measured using the scratch assay.Western blot analysis was conducted to assess the protein expressions of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax),kirsten rat sarcoma viral oncogene(KRAS),mammalian target of rapamycin(mTOR),and phosphatidylinositol 3-kinase(PI3K).Glucose content was measured using the o-toluidine method,lactic acid release was quantified via p-hydroxy biphenyl colorimetry,and adenosine triphosphate(ATP)content was determined through colorimetric analysis.Results The MTT assay results indicated thatα-tomatine significantly inhibited the proliferation of Huh7 cells.Additionally,α-tomatine reduced cell invasion and migration,glucose consumption,lactic acid release,and ATP production,but promoted apoptosis.It was found thatα-tomatine activated the mTOR/PI3K signaling pathway in Huh7 cells.The pro-apoptotic capacity,anti-invasive and anti-migratory abilities,and thereby antiglycolysis ability ofα-tomatine were dependent on the activation of mTOR/PI3K signaling pathway.Conclusionα-tomatine inhibited glycolysis,and the proliferation,invasion and migration of hepatoma cells through activating mTOR/PI3K signaling pathway.

关 键 词：α-番茄碱 肝癌 肉瘤病毒癌基因 糖酵解 

分 类 号：R96[医药卫生—药理学]