Data Sheet 3_Saikosaponin A induces cellular senescence in triple-negative breast cancer by inhibiting the PI3K/Akt signalling pathway.docx

BackgroundBreast cancer has now become the most prevalent cancer worldwide. Existing therapeutic agents are generally accompanied by significant side effects. Here, we highlight Saikosaponin A (SSA), a promising natural metabolite characterized by low toxicity, demonstrating significant efficacy against breast cancer through the induction of cellular senescence.
MethodsThe antitumor property of SSA was determined via MTT colorimetric assay, 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, colony formation, and propidium iodide (PI) staining in vitro, as well as xenograft in vivo model. A network approach was used to predict potential targets of SSA reevant for a potential anti-tumor effect and verified through senescence-associated β-galactosidase (SA-β-gal), flow-cytometry analysis, RT-PCR, Western blotting, and immuno-histochemistry assay.
ResultsSSA significantly suppressed proliferation and triggered cell cycle arrest of SUM159PT and MDA-MB-231 cells. Revealed by network analysis, cellular senescence, and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway were implemented in the anti-tumor effects of SSA. SSA-stimulated senescence was associated with increased ROS production, distinct senescence-associated secretory phenotype (SASP), and restricted PI3K/Akt signaling, as well as p21 and p53 accumulation. Furthermore, SSA displayed inhibitory effects on tumor growth with minimal toxicity in animal studies, accompanied by activated biomarkers of cellular senescence and decreased expression of p-Akt and p-PI3K.
ConclusionTaken together, based on the preliminary results of network analysis and further experimental validation, this study revealed that SSA significantly induced cell cycle arrest and senescence, and the inhibition of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

背景
乳腺癌现已成为全球范围内发病率最高的癌症。现有治疗药物普遍伴随显著的不良反应。本研究聚焦柴胡皂苷A（Saikosaponin A, SSA）——一种极具潜力的低毒性天然代谢产物，其通过诱导细胞衰老展现出显著的抗乳腺癌活性。

方法
本研究通过体外MTT比色法、5-乙炔基-2′-脱氧尿苷（5-ethynyl-2′-deoxyuridine, EdU）掺入实验、集落形成实验以及碘化丙啶（propidium iodide, PI）染色，结合体内异种移植瘤模型，评估了SSA的抗肿瘤特性。采用网络分析方法预测SSA潜在的抗肿瘤作用靶点，并通过衰老相关β-半乳糖苷酶（senescence-associated β-galactosidase, SA-β-gal）检测、流式细胞术分析、逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法（Western blotting）以及免疫组织化学实验进行验证。

结果
SSA可显著抑制SUM159PT与MDA-MB-231细胞的增殖并诱导其细胞周期阻滞。网络分析结果显示，细胞衰老及磷脂酰肌醇3-激酶（phosphatidylinositol-3-kinase, PI3K）/蛋白激酶B（Akt）信号通路参与了SSA的抗肿瘤作用过程。SSA诱导的细胞衰老与活性氧（reactive oxygen species, ROS）生成增加、典型的衰老相关分泌表型（senescence-associated secretory phenotype, SASP）以及PI3K/Akt信号通路抑制、p21和p53蛋白积累密切相关。此外，动物实验表明SSA可抑制肿瘤生长且毒性极低，同时伴随细胞衰老相关生物标志物的激活以及p-Akt和p-PI3K表达水平的下调。

结论
综上，基于网络分析的初步结果及后续实验验证，本研究证实SSA可显著诱导细胞周期阻滞与细胞衰老，而ROS介导的PI3K/Akt通路抑制可能是其发挥抗肿瘤作用的潜在分子机制。