摘要
目的:探讨没药甾酮增强化疗药物替莫唑胺抗脑胶质瘤的作用及其分子机制。方法:用没药甾酮、替莫唑胺单药及其联合处理U251细胞后,采用平板克隆形成实验、5-乙炔基-2′-脱氧尿苷(EdU)实验检测各组细胞增殖能力;采用Transwell小室实验、划痕愈合实验检测各组细胞侵袭、迁移能力;采用Western Blot检测细胞中mTOR/自噬通路关键蛋白的表达水平。结果:低氧条件下(200μmol/L的CoCl2诱导48 h),没药甾酮单药(10、30、100μmol/L)和替莫唑胺单药(6.25、12.5、25μmol/L)能显著抑制U251细胞克隆形成数(P<0.001);与替莫唑胺单药组(6.25、12.5μmol/L)比较,没药甾酮(10μmol/L)联合替莫唑胺(6.25、12.5μmol/L)抑制U251细胞克隆形成的作用增强,但差异无统计学意义(P>0.05)。没药甾酮单药(10、30、100μmol/L)和替莫唑胺单药(100、200、400μmol/L)显著下调了U251细胞EdU阳性细胞率;与替莫唑胺单药组(200、400μmol/L)比较,没药甾酮(30、100μmol/L)联合替莫唑胺(200、400μmol/L)显著下调了U251细胞EdU阳性细胞率(P<0.05或P<0.01)。与替莫唑胺单药(400μmol/L)比较,联用没药甾酮(30μmol/L)显著抑制U251细胞迁移、侵袭能力(P<0.001)。与替莫唑胺单药(400μmol/L)比较,联用没药甾酮(30μmol/L)显著下调U251细胞中Phospho-mTOR(Ser2448)、Phospho-4E-BP1(Ser65)蛋白表达,显著上调MAPKAP1、p-P70(S6K)蛋白表达(P<0.05或P<0.01),对mTOR、Phospho-mTOR(Ser2481)、Rictor、Raptor、GβL表达的下调也有一定的增强作用,但差异无统计学意义。与替莫唑胺单药(400μmol/L)比较,联用没药甾酮(30μmol/L)显著上调U251细胞中自噬相关蛋白Beclin-1、LC3-Ⅱ/LC3-Ⅰ、P62的表达(P<0.05~P<0.001),对Atg12-5/free-Atg12也有上调作用,但差异无统计学意义。结论:没药甾酮可通过调控mTOR/自噬信号通路增强替莫唑胺抗脑胶质瘤作用。
Objective:To investigate the effect and molecular mechanism of guggulsterone combined with chemotherapeutic drug temozolomide on brain glioma enhanced by myrrh steroid.Methods:After U251 cells were treated with guggulsterone,temozolomide and their combination,cell proliferation capacity of each group was detected by plate cloning assay and 5-acetyne-2'-deoxyuridine(EdU)assay.Transwell assay and scratch healing assay were used to detect cell invasion and migration ability of each group.The expression levels of key proteins in the mTOR/autophagy pathway were detected by Western Blot.Results:Under hypoxia conditions(200μmol/L CoCl2 induction for 48 hours),the number of clonal formation of U251 cells was significantly inhibited by guggulsterone monotherapy(10,30,100μmol/L)and temozolomide monotherapy(6.25,12.5,25μmol/L)(P<0.001).Compared with temozolomide single treatment group(6.25,12.5μmol/L),the combination of guggulsterone(10μmol/L)and temozolomide(6.25,12.5μmol/L)showed enhanced inhibitory effect on the clonal formation of U251 cells,but there was no significant difference.The EdU positive cell rate of U251 cells was significantly decreased by guggulsterone monotherapy(10,30,100μmol/L)and temozolomide monotherapy(100,200,400μmol/L).Compared with temozolomide monotherapy group(200,400μmol/L),the EdU positive cell rate of U251 cells was significantly decreased by guggulsterone(30,100μmol/L)combined with temozolomide(200,400μmol/L)(P<0.05或P<0.01).Compared with temozolomide monotherapy(400μmol/L),combined treatment with guggulsterone(30μmol/L)significantly inhibited the migration and invasion ability of U251 cells(P<0.001).Compared with temozolomide monotherapy(400μmol/L),combined treatment with guggulsterone(30μmol/L)significantly decreased Phospho-mTOR(Ser2448)and Phospho-4E-BP1(Ser65)expression in U251 cells,and significantly increased MAPKAP1,p-P70(S6K)expression in U251 cells(P<0.05 or P<0.01).The down-regulation of mTOR,Phospho-mTOR(Ser2481),Rictor,Raptor,GβL was enhanced,but there was no significant difference.Compared with temozolomide monotherapy(400μmol/L),the expressions of autophagy related proteins Beclin-1,LC3-Ⅱ/LC3-Ⅰand P62 in U251 cells were significantly up-regulated when combined with guggulsterone(30μmol/L)(P<0.05~P<0.001),and Atg12-5/free-Atg12 were also up-regulated,but there was no significant difference.Conclusion:Guggulsterone can enhance the antiglioma effect of temozolomide by regulating the mTOR/autophagy signaling pathway.
作者
余卓伦
张其海
樊昊雯
潘晶
徐宏彬
YU Zhuo-lun;ZHANG Qi-hai;FAN Hao-wen;PAN Jing;XU Hong-bin(Department of Pharmacy,The Second Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210017,China)
出处
《中药材》
CAS
北大核心
2023年第2期462-468,共7页
Journal of Chinese Medicinal Materials
基金
国家自然科学基金资助项目(81774040)
南京中医药大学自然科学基金项目(XZR2020049)
作者简介
余卓伦(1997-),女,在读硕士研究生,专业方向:中药抗肿瘤药理机制,E-mail:765252074@qq.com;通讯作者:徐宏彬,E-mail:xuhongbin@tongji.edu.cn
