Scutebarbatine A（SBT-A）是一种二萜生物碱，我们之前的研究表明其对肝细胞癌细胞具有细胞毒作用。本研究探讨了SBT-A在乳腺癌细胞中的抗肿瘤活性和作用机制。通过尝试蓝染色，5-乙炔基-2'-脱氧尿嘧啶（EdU）合成和克隆形成实验分析SBT-A的抗增殖作用。γ-H2AX核聚焦形成评估DNA双链断裂（DSBs）。细胞周期分布通过流式细胞术评估。细胞凋亡通过TUNEL试验确定。2'，7'-二氯荧光素乙酯（DCFH-DA）和二氢乙啶（DHE）染色分别测定胞内活性氧（ROS）产生和超氧产生。结果表明，SBT-A表现出对乳腺癌细胞的剂量依赖性细胞毒作用，同时对MCF-10A乳腺上皮细胞的毒性较小。此外，SBT-A显著诱导MDA-MB-231和MCF-7细胞的DNA损伤、细胞周期阻滞和细胞凋亡。SBT-A处理增加了ROS水平和胞质超氧产生。使用ROS清除剂N-乙酰半胱氨酸（NAC）处理足以阻止SBT-A引起的存活率降低、DNA损伤、凋亡和内质网应激。SBT-A暴露后，c-Jun N端激酶（JNK）和p38丝裂原活化蛋白激酶（p38MAPK）的磷酸化上调，而细胞外信号调节激酶（ERK）的磷酸化下调。此外，SBT-A通过降低EGFR表达和Akt和p70S6K的磷酸化来抑制EGFR信号通路。总之，通过诱导DNA损伤、细胞凋亡和内质网应激、生成ROS及调节MAPK和EGFR/Akt信号通路，SBT-A对乳腺癌细胞具有强大的抑制作用。Copyright © 2023. Elsevier B.V.出版。

Scutebarbatine A (SBT-A), a diterpenoid alkaloid, has exerted cytotoxicity on hepatocellular carcinoma cells in our previous works. Here, the antitumor activity of SBT-A in breast cancer cells and the underlying mechanism were explored. The anti-proliferative effect of SBT-A was measured by trypan blue staining, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and colony formation assay. DNA double-strand breaks (DSBs) were evaluated by observing the nuclear focus formation of γ-H2AX. Cell cycle distribution was assessed by flow cytometry. Apoptosis was determined by a TUNEL assay. Intracellular reactive oxygen species (ROS) generation and superoxide production were measured with 2', 7'-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) staining, respectively. The results indicated that SBT-A showed a dose-dependent cytotoxic effect against breast cancer cells while revealing less toxicity toward MCF-10A breast epithelial cells. Moreover, SBT-A remarkably induced DNA damage, cell cycle arrest and apoptosis in both MDA-MB-231 and MCF-7 cells. SBT-A treatment increased the levels of ROS and cytosolic superoxide production. Pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, was sufficient to block viability reduction, DNA damage, apoptosis and endoplasmic reticulum (ER) stress caused by SBT-A. By exposure to SBT-A, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) was upregulated, while the phosphorylation of extracellular signal-regulated kinase (ERK) was downregulated. In addition, SBT-A inhibited the EGFR signaling pathway by decreasing EGFR expression and phosphorylation of Akt and p70S6K. As mentioned above, SBT-A has a potent inhibitory effect on breast cancer cells through induction of DNA damage, apoptosis and ER stress via ROS generation and modulation of MAPK and EGFR/Akt signaling pathway.Copyright © 2023. Published by Elsevier B.V.