鸦胆子醇提物抗三阴性乳腺癌机制研究

Study on the anti-triple-negative breast cancer mechanism of Brucea javanica ethanol extract

关键词:  三阴性乳腺癌  鸦胆子  凋亡  转录组测序

Key words:  triple-negative breast cancer  Brucea javanica  apoptosis  transcriptome sequencing

摘要:

[目的] 探究鸦胆子醇提物对于三阴性乳腺癌（TNBC）的治疗作用，验证其可能的作用机制。[方法] 通过细胞计数试剂盒-8（CCK-8）实验检测鸦胆子醇提物24 h抑制TNBC细胞增殖的能力；克隆形成实验检测鸦胆子醇提物长期对TNBC细胞增殖的影响；倒置显微镜观察鸦胆子醇提物处理后细胞形态变化；基于4T1荷瘤小鼠模型评价鸦胆子醇提物对TNBC的体内药效；对4T1荷瘤小鼠肿瘤组织进行转录组学测序分析，探究鸦胆子醇提物在TNBC治疗领域的潜在机制；利用流式细胞术检测鸦胆子醇提物对TNBC细胞凋亡的影响；实时荧光定量聚合酶链式反应（RT-qPCR）和蛋白免疫印迹（Western blot）法验证凋亡相关基因与蛋白变化。[结果] CCK-8结果显示，与对照组相比，鸦胆子醇提物显著抑制了4T1细胞和MDA-MB-231细胞活力（P<0.05或P<0.01），且具有剂量依赖性；克隆形成实验表明鸦胆子醇提物可以抑制4T1细胞和MDA-MB-231细胞的长期增殖（P<0.01）；鸦胆子处理后的细胞形态发生明显变化；在体内，鸦胆子醇提物同样可以抑制TNBC肿瘤生长；转录组测序提示鸦胆子醇提物抗TNBC的作用机制可能是诱导凋亡，流式细胞术结果证实鸦胆子醇提物可以诱导TNBC细胞凋亡，RT-qPCR检测到凋亡相关基因Bcl-2相关X蛋白（Bax）和半胱氨酸天冬氨酸蛋白酶-3（caspase-3）上调，B淋巴细胞瘤-2基因（Bcl-2）下调（P<0.05）；Western blot结果显示促凋亡蛋白Bax和核转录因子-κB（NF-κB）表达上调，抗凋亡蛋白Bcl-2表达下调（P<0.05）。[结论] 鸦胆子醇提物可以通过诱导凋亡抑制TNBC。

Abstract:

[Objective] To explore the therapeutic effect of Brucea javanica ethanol extract（BJ-EE） on triple-negative breast cancer（TNBC） and verify its possible mechanism of action. [Methods] The ability of BJ-EE to inhibit TNBC cell proliferation after 24 h was detected by CCK-8 assay. The long-term effect of BJ-EE on TNBC cell proliferation was evaluated by colony formation assay. Morphological changes of cells after BJ-EE treatment were observed using an inverted microscope. The in vivo efficacy of BJ-EE against TNBC was evaluated based on a 4T1 tumor-bearing mouse model. Transcriptomic sequencing analysis was performed on tumor tissues from 4T1 tumor-bearing mice to explore the potential mechanism of BJ-EE in TNBC treatment. The effect of BJ-EE on TNBC cell apoptosis was detected by flow cytometry. Apoptosis-related gene and protein changes were verified by quantitative real-time polymerase chain reaction（RT-qPCR） and Western blot. [Results] CCK-8 results showed that，compared with the control group，BJ-EE significantly inhibited the viability of 4T1 and MDA-MB-231 cells（P<0.05 or P<0.01） in a dose-dependent manner. Colony formation assays showed that BJ-EE could inhibit the long-term proliferation of 4T1 and MDA-MB-231 cells（P<0.01）. Cell morphology changed significantly after BJ-EE treatment. In vivo，BJ-EE could also inhibit TNBC tumor growth. Transcriptomic sequencing suggested that the mechanism of action of BJ-EE against TNBC might be inducing apoptosis. Flow cytometry results confirmed that BJ-EE could induce apoptosis in TNBC cells. RT-qPCR detected upregulation of the apoptosis-related genes Bax and caspase-3，and downregulation of Bcl-2（P<0.05）. Western blot results showed upregulation of the pro-apoptotic protein Bax and the nuclear transcription factor-κB（NF-κB），and downregulation of the anti-apoptotic protein Bcl-2（P<0.05）. [Conclusion] Brucea javanica ethanol extract can inhibit TNBC by inducing apoptosis.