药学学报, 2022, 57(4): 1063-1072
引用本文:
郑凯露, 唐梅, 邓海东, 刘思齐, 刘晓宇, 李燕. 咖啡酸苯乙酯衍生物PEC01抗小鼠G422神经胶质瘤药效学作用及其机制[J]. 药学学报, 2022, 57(4): 1063-1072.
ZHENG Kai-lu, TANG Mei, DENG Hai-dong, LIU Si-qi, LIU Xiao-yu, LI Yan. The antitumor activity and mechanisms of action caffeic acid phenethyl ester derivative PEC01 in mouse G422 glioma[J]. Acta Pharmaceutica Sinica, 2022, 57(4): 1063-1072.

咖啡酸苯乙酯衍生物PEC01抗小鼠G422神经胶质瘤药效学作用及其机制
郑凯露1, 唐梅1, 邓海东1, 刘思齐1, 刘晓宇2*, 李燕1*
1. 中国医学科学院、北京协和医学院药物研究所, 新药作用机制研究与药效评价北京市重点实验室, 北京 100050;
2. 中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 活性物质发现与适药化研究北京市重点实验室, 北京 100050
摘要:
本研究旨在初步探索PEC01抑制小鼠G422神经胶质瘤的活性及其作用机制。采用MTT法、流式细胞术(FCM)、细胞划痕实验和Transwell细胞穿膜实验分别检测PEC01对G422细胞增殖、凋亡、迁移能力的影响。采用皮下移植的小鼠G422胶质瘤模型评价PEC01的体内药效学。动物福利和实验过程均遵循中国医学科学院北京协和医学院药物研究所动物伦理委员会的规定。Western blot检测PEC01作用2、96 h的G422细胞及30.0 mg·kg-1 PEC01给药后的小鼠G422肿瘤组织中EGFR、Src及下游MAPK/ERK、PI3K/Akt/mTOR通路蛋白水平。结果显示,PEC01以时间和剂量依赖性方式明显抑制体外G422细胞增殖,96 h作用时的IC50为(9.02 ±0.36)μmol·L-1;PEC01(10.0和20.0 μmol·L-1)作用96 h后,可明显诱导G422细胞发生早期和晚期凋亡;PEC01[(0.625~5.0)μmol·L-1]在12~48 h剂量依赖地显著抑制G422细胞划痕愈合能力;与DMSO组相比,不同浓度PEC01(5.0、10.0和20.0 μmol·L-1)作用8 h后Transwell穿膜的G422细胞数量明显减少。体内药效学实验中,PEC01(30.0和60.0 mg·kg-1)作用14天后的瘤重抑制率分别为72.29%和59.44%。Western blot结果显示,与DMSO组相比,PEC01在2和96 h作用时,G422细胞中p-EGFR、p-Src蛋白表达明显降低;PEC01作用96 h后G422细胞中MAPK/ERK、PI3K/Akt/mTOR通路相关蛋白以及c-myc和HIF-1α表达下调;侵袭转移相关蛋白Snail、N-cadherin和MMP-9表达明显降低;cyclin E1、cyclin B1/CDK1蛋白水平明显下调。研究结果表明,咖啡酸苯乙酯衍生物PEC01具有较好的体内外抗小鼠G422胶质瘤活性,其抗肿瘤作用可能是通过抑制EGFR、Src激酶活性和表达水平,调控下游MAPK/ERK、PI3K/Akt/mTOR信号通路,进而通过抑制肿瘤细胞增殖,促进肿瘤细胞凋亡、抑制侵袭转移等作用实现的。
关键词:    神经胶质瘤      咖啡酸苯乙酯      表皮生长因子受体      Src      MAPK/ERK信号通路      PI3K/Akt/mTOR信号通路     
The antitumor activity and mechanisms of action caffeic acid phenethyl ester derivative PEC01 in mouse G422 glioma
ZHENG Kai-lu1, TANG Mei1, DENG Hai-dong1, LIU Si-qi1, LIU Xiao-yu2*, LI Yan1*
1. Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
2. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
This study aimed to research the antitumor activity and mechanisms of caffeic acid phenethyl ester derivative PEC01 in mouse G422 glioma. MTT assay, flow cytometry (FCM) and Transwell migration assay were used to detect the effects of PEC01 on proliferation, apoptosis, and migration of G422 cells respectively. Mouse subcutaneously transplanted G422 tumor model was used to analyse the effect of PEC01 on the growth of glioma in vivo. Animal welfare and experimental procedure are in accordance with the regulations of the Animal Ethics Committee of institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. Western blot was used to detect the protein levels of epidermal growth factor receptor (EGFR), Src and their downstream signaling pathways in G422 cells and tumor issue. The results showed that PEC01 inhibited proliferation of G422 cells in a time- and dose-dependent manner, with IC50 of (9.02 ±0.36) μmol·L-1 at 96 h. PEC01 significantly induced early apoptosis and late apoptosis of G422 cells at 10.0 and 20.0 μmol·L-1 concentrations for 96 h. Scratch healing rate of G422 cells reduced after treated with different concentrations (0.625-5.0 μmol·L-1) of PEC01 for 12-48 h in scratch healing assay. The number of transmembrane G422 cells decreased in groups treated with PEC01 for 8 h compared with DMSO group. The average tumor weight of groups treated with 30.0 and 60.0 mg·kg-1 PEC01 was significantly reduced in G422 insubcutaneously transplanted tumor model, and the inhibition rate of tumor weight was 72.29% and 59.44%, respectively. Protein levels of EGFR, Src, c-myc and hypoxia-inducible factor 1-alpha (HIF-1α) decreased significantly in G422 cells and tumor tissue. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway related proteins were down-regulated. Related proteins of invasion, metastasis and cell cyclin were significantly down-regulated. PEC01 can suppress the growth of G422 glioma in vitro and in vivo. The antitumor activity of PEC01 in mouse subcutaneously transplanted G422 tumor model might be related to the blockcade of PI3K/Akt/mTOR and MAPK/ERK signaling pathways.
Key words:    glioma    caffeic acid phenethyl ester    epidermal growth factor receptor    Src    MAPK/ERK pathway    PI3K/Akt/mTOR pathway   
收稿日期: 2022-02-25
DOI: 10.16438/j.0513-4870.2022-0248
通讯作者: 刘晓宇,Tel:86-10-63169181,E-mail:liyanxiao@imm.ac.cn;李燕,E-mail:lxya@imm.ac.cn
Email: liyanxiao@imm.ac.cn;lxya@imm.ac.cn
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