datasheet1_Sanguisorba officinalis L. Suppresses Triple-Negative Breast Cancer Metastasis by Inhibiting Late-Phase Autophagy via Hif-1α/Caveolin-1 Signaling.pdf

Sanguisorba officinalis L. (SA) is a common herb for cancer treatment in the clinic, particularly during the consolidation phase to prevent occurrence or metastasis. Nevertheless, there are limited studies reporting the molecular mechanisms about its anti-metastatic function. It is well demonstrated that autophagy is one of the critical mechanisms accounting for metastasis and anti-cancer pharmacological actions of Chinese herbs. On the threshold, the regulatory effects and molecular mechanisms of SA in suppressing autophagy-related breast cancer metastasis were investigated in this study. In vitro findings demonstrated that SA potently suppressed the proliferation, colony formations well as metastasis process in triple-negative breast cancer. Network and biological analyses predicted that SA mainly targeted caveolin-1 (Cav-1) to induce anti-metastatic effects, and one of the core mechanisms was via regulation of autophagy. Further experiments—including western blotting, transmission electron microscopy, GFP-mRFP-LC3 immunofluorescence, and lysosomal-activity detection—validated SA as a potent late-stage autophagic inhibitor by increasing microtubule-associated light chain 3-II (LC3-II) conversion, decreasing acidic vesicular-organelle formation, and inducing lysosomal dysfunction even under conditions of either starvation or hypoxia. Furthermore, the anti-autophagic and anti-metastatic activity of SA was Cav-1-dependent. Specifically, Cav-1 knockdown significantly facilitated SA-mediated inhibition of autophagy and metastasis. Furthermore, hypoxia inducible factor-1α (Hif-1α) overexpression attenuated the SA-induced inhibitory activities on Cav-1, autophagy, and metastasis, indicating that SA may have inhibited autophagy-related metastasis via Hif-1α/Cav-1 signaling. In both mouse breast cancer xenograft and zebrafish xenotransplantation models, SA inhibited breast cancer growth and inhibited late-phase autophagy in vivo, which was accompanied by suppression of Hif-1α/Cav-1 signaling and the epithelial-mesenchymal transition. Overall, our findings not only indicate that SA acts as a novel late-phase autophagic inhibitor with anti-metastatic activities in triple-negative breast cancer, but also highlight Cav-1 as a regulator in controlling late-phase autophagic activity.

地榆（Sanguisorba officinalis L., SA）是一种临床常用的抗癌中草药，尤其多用于肿瘤巩固治疗阶段，以预防肿瘤发生或转移。然而，目前关于其抗转移功能的分子机制研究较为匮乏。已有研究证实，自噬（autophagy）是中草药发挥抗癌作用及调控肿瘤转移的关键机制之一。基于此，本研究探讨了SA在抑制自噬相关乳腺癌转移中的调控作用及分子机制。体外实验结果显示，SA可有效抑制三阴性乳腺癌的细胞增殖、集落形成及转移进程。通过网络药理学与生物学分析预测，SA主要通过靶向窖蛋白-1（Caveolin-1, Cav-1）发挥抗转移作用，其核心机制之一涉及自噬调控。后续实验包括蛋白质印迹法（Western blotting）、透射电子显微镜、GFP-mRFP-LC3免疫荧光及溶酶体活性检测，结果证实SA是一种强效晚期自噬抑制剂：在饥饿或缺氧条件下，SA均可通过提升微管相关蛋白轻链3-II（LC3-II）的转化水平、减少酸性囊泡细胞器的形成、诱导溶酶体功能紊乱，发挥抑制作用。此外，SA的抗自噬与抗转移活性呈Cav-1依赖性。具体而言，敲低Cav-1可显著增强SA介导的自噬与转移抑制效果。进一步研究发现，缺氧诱导因子-1α（Hypoxia-inducible factor-1α, Hif-1α）过表达会削弱SA对Cav-1、自噬及转移的抑制活性，这表明SA可能通过Hif-1α/Cav-1信号通路抑制自噬相关的肿瘤转移。在小鼠乳腺癌异种移植模型与斑马鱼异种移植模型中，SA均可在体内抑制乳腺癌生长与晚期自噬，同时伴随Hif-1α/Cav-1信号通路及上皮间质转化（epithelial-mesenchymal transition, EMT）的抑制。综上，本研究结果不仅表明SA可作为一种新型晚期自噬抑制剂，在三阴性乳腺癌中发挥抗转移活性，同时也揭示了Cav-1作为晚期自噬活性调控因子的潜在作用。