Raw western blot scans.

<div><p>Purpose</p><p>Approximately 20% of all breast cancer cases are classified as triple-negative breast cancer (TNBC), which represents the most challenging subtype due to its poor prognosis and high metastatic rate. Caffeic acid phenethyl ester (CAPE), the main component extracted from propolis, has been reported to exhibit anticancer activity across various tumor cell types. This study aimed to investigate the effects and mechanisms of CAPE on TNBC.</p><p>Methods</p><p>MDA-MB-231 and MDA-MB-468 cells were treated with CAPE. CCK8 and colony formation assays were performed to analyze cell proliferation. Western blot, TUNEL and Annexin V-FITC/PI staining methods were employed to assess cell apoptosis. ROS, MDA, SOD, GSH, C11-bodipy staining, along with measurements of GPX4 and Ferritin levels, were utilized for ferroptosis detection. Western blot and immunofluorescence analysis were used to assess key regulatory molecules. The cells were subjected to treatments involving ferroptosis inhibition, AMPK inhibition, or Foxo3 inhibition, followed by CAPE administration to assess cell proliferation, apoptosis, and ferroptosis. Tumor xenografts were used to evaluate the antitumor efficacy of CAPE.</p><p>Results</p><p>CAPE not only suppressed cell proliferation but also promoted apoptosis followed by ferroptosis. Co-incubation with Fer-1 (a ferroptosis inhibitor) diminished CAPE’s suppressive effects on proliferation and apoptosis induction. CAPE treatment enhanced the phosphorylation of AMPK and promoted the nuclear translocation of Foxo3. Inhibition of both AMPK and Foxo3 by siRNAs or inhibitors (Compc, TIC10) reversed the growth retardation induced by CAPE as well as its pro-apoptotic effects leading to ferroptosis. Specifically, AMPK inhibition abrogated the CAPE-induced nuclear translocation of Foxo3. CAPE significantly inhibited tumor growth in nude mice bearing TNBC xenografts.</p><p>Conclusion</p><p>CAPE possesses a resistance effect on TNBC via activation of AMPK and Foxo3 signaling pathways.</p></div>