摘要
目的:研究半枝莲石油醚提取物对EGF诱导的人三阴性乳腺癌细胞株(MDA-MB-231)Ezrin和RhoA蛋白表达的影响,探讨其抑制三阴性乳腺癌细胞增殖、分化的作用机制。方法:溶剂萃取法获得半枝莲石油醚提取物,通过Agilent HPLC-ESI-Q-TOF-MS MassHunter软件分析,于PubChem数据库中获取半枝莲石油醚提取物熊果酸的分子对接信息。采用分子对接技术对半枝莲石油醚提取物中熊果酸与RhoA蛋白受体的对接进行可视化分析。为探讨其抑制肿瘤细胞的分化、增殖机制,取对数生长期细胞分为7组,用含2%胎牛血清培养液进行饥饿培养24 h,给予30 ng/mL EGF分别干预30 min、24 h后,分别给予相应药物培养30 min,采用Western blotting法检测各组MDA-MB-231细胞中RhoA、p-RhoA及Ezrin蛋白表达情况。结果:质谱分析显示,半枝莲石油醚提取物中主要包括长链脂肪酸和萜类成分。生物信息分子对接发现,熊果酸是半枝莲调控RhoA的有效活性成分。Western blotting结果显示,EGF诱导MDA-MB-231细胞30 min及24 h后p-RhoA和Ezrin蛋白相对表达量均明显升高(P<0.05);经Fasudil和半枝莲石油醚提取物干预30 min后,MDA-MB-231细胞p-RhoA和Ezrin蛋白相对表达量均明显降低(P<0.05);7组MDA-MB-231细胞RhoA蛋白相对表达量比较,差异均无统计学意义(P>0.05)。EGF可能以RhoA蛋白磷酸化的形式参与下游蛋白信号调控,增强肿瘤癌细胞中Ezrin蛋白表达,进而促进癌细胞侵袭和转移。结论:熊果酸是半枝莲调控RhoA的有效活性成分。半枝莲石油醚提取物可通过RhoA信号通路抑制MDA-MB-231细胞的增殖、转移和分化,进而对人三阴性乳腺癌起到治疗作用。
Objective:To study the effect of petroleum ether extract of Banzhilian(Bearded Scutellaria)on the expression of Ezrin and RhoA proteins in human triple-negative breast cancer cell line(MDA-MB-231)induced by EGF,and explore its mechanism of inhibiting the proliferation and differentiation of triple negative breast cancer cells.Methods:The petroleum ether extract of Banzhilian was obtained by solvent extraction.The molecular docking information of ursolic acid from Banzhilian petroleum ether extract was obtained in PubChem database through Agilent HPLC-ESI-Q-TOF-MS MassHunter software analysis.The docking between ursolic acid and RhoA protein receptor in Banzhilian petroleum ether extract was visualized by molecular docking technology.In order to explore the mechanism of its inhibition on the differentiation and proliferation of tumor cells,cells in logarithmic growth phase were divided into 7 groups.The cells were starved for 24 hours in a culture medium containing 2%fetal bovine serum.After 30 ng/mL EGF was given for 30 min and 24 h,the cells were incubated with corresponding drugs for 30 min,respectively.Western blotting was used to detect the expression of RhoA,p-RhoA and Ezrin proteins in MDA-MB-231 cells in each group.Results:Mass spectrometry analysis showed that the petroleum ether extract of Banzhilian mainly contained long-chain fatty acids and terpenoids.Bioinformatics molecular docking found that ursolic acid was an effective active component of Banzhilian to regulate RhoA.Western blotting showed that the relative expression of p-RhoA and Ezrin protein in MDA-MB-231 cells induced by EGF increased significantly after 30 min and 24 h(P<0.05).After the intervention of Fasudil and Banzhilian petroleum ether extract for 30 min,the relative expression of p-RhoA and Ezrin protein in MDA-MB-231 cells decreased significantly(P<0.05).There was no significant difference in the relative expression of RhoA protein in MDA-MB-231 cells between the 7 groups(P>0.05).EGF may participate in downstream protein signal regulation in the form of RhoA protein phosphorylation,enhance Ezrin protein expression in tumor cells,and then promote cancer cell invasion and metastasis.Conclusion:Ursolic acid is an effective component of Banzhilian to regulate RhoA.The petroleum ether extract of Banzhilian can inhibit the proliferation,metastasis and differentiation of MDA-MB-231 cells through RhoA signaling pathway,and then play a therapeutic role in human triple negative breast cancer.
作者
牛俊杰
胡锦洋
王柱
NIU Junjie;HU Jinyang;WANG Zhu(Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha Hunan 410006,China)
出处
《中医药导报》
2022年第11期12-16,共5页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
湖南省中医药研究院课题一般项目(201810)。
