获得对阿霉素（DOX）的抗药性不可避免地限制了其在乳腺癌（BC）中的临床应用。异槲皮素（IS）是一种天然类黄酮，在蔬菜、水果和植物药中广泛存在，在先前的研究中被认为是可能的癌症化学预防剂，因为它表现出令人满意的抗肿瘤活性。到目前为止，使用IS作为增敏剂来缓解DOX对耐药性BC的疗效的策略尚未被涵盖。为了研究IS对增强耐药性BC细胞对DOX的体内外化疗敏感性的影响，并阐明可能的分子机制。使用MTS测定、群体形成测定、三维（3D）肿瘤球体模型和迁移试验来验证IS在有无DOX的情况下对耐药性BC细胞的抑制作用。通过流式细胞术确定细胞凋亡、细胞周期调节和内源性活性氧（ROS）。蛋白质水平则通过Western blotting进行监测。另外，则使用共聚焦激光扫描显微镜拍摄核染色和EdU增殖，以评估IS和DOX联合作用对异种移植实验中的肿瘤发生的影响，以进一步确认体外细胞毒性。IS显著抑制了细胞增殖和迁移，并增强了DOX对耐药性BC细胞的抗肿瘤能力，无论是在体内还是在体外。辅助IS（50 μM）通过抑制bcl 2的表达，增强cleaved caspase 3，最终导致DNA浓缩和断裂，有效地增强了DOX对耐药性BC细胞的促凋亡影响（35.38 ±3.18％， vs. DOX组的5.83 ±0.68％）。IS处理（20、30和50 μM）通过调节CDK1 / Cyclin B1复合物的表达，进而抑制BC增殖，显著增加了G2 / M人口（41.60 ±1.28％、44.60 ±1.14％和50.64 ±0.67％， vs.未经处理的MCF7 / ADR细胞的35.84 ±1.56％，p<0.01）。此外，IS（10、20、30和50 μM）刺激了MCF7 / ADR细胞内基质ROS的产生，分别为未经处理组的3.99倍、4.20倍、6.29倍和6.78倍（p<0.001），这些ROS涉及DNA损伤和AMPK引起的mTOR / p70S6K信号截断。我们的研究表明了IS作为DOX增敏剂的抗乳腺癌作用，并阐述了其潜在的分子机制，表明IS可以被认为是治疗耐药性BC的有力替代品。版权所有©2023 Elsevier GmbH。保留所有权利。

Acquired resistance to doxorubicin (DOX) inevitably limits its clinical use against breast cancer (BC). Isorhamnetin (IS), a native flavonoid which extensively available in vegetables, fruits, and phytomedicine, has been deemed to the probable cancer chemopreventive agent in preceding explorations since it exhibits satisfied antitumor activity. So far, the strategy for alleviating DOX resistance by using IS as a sensitizer against resistant BC has not yet been covered.To investigate the effect of IS on potentiating the chemoreceptivity of drug-resistant BC cells to DOX in vitro and in vivo and elucidate the possible molecular mechanisms.MTS assays, colony formation assays, three-dimensional (3D) tumor spheroid model, and migration assay were deployed to verify the inhibiting action of IS in the presence or absence of DOX on resistant BC cells in vitro. Apoptosis, cell cycle regulation, and endocellular reactive oxygen species (ROS) were determined by flow cytometry. Protein levels were monitored by western blotting. Nuclear staining and EdU proliferation were photographed with a confocal laser scanning microscope. The effects of the IS and DOX combination on the tumorigenesis in the xenograft experiments were evaluated for further confirming the in vitro cytotoxicity.IS significantly inhibited cell proliferation and migration and enhanced the antitumor competence of DOX against resistant BC cells both in vitro and in vivo. Adjuvant IS (50 μM) effectively enhanced the proapoptotic impacts of DOX in resistant BC cells (35.38 ± 3.18%, vs. 5.83 ± 0.68% in the DOX group) by suppressing the expression of bcl 2 in addition to enhancing cleaved caspase 3, ultimately leading to DNA condensation and fragmentation. IS (20, 30, and 50 μM) treatments induced significant increases in the G2/M populations (41.60 ± 1.28%, 44.60 ± 1.14%, and 50.64 ± 0.67%, vs. 35.84 ± 1.56% in the untreated control in MCF7/ADR cells, p < 0.01) via regulating CDK1/Cyclin B1 complex expression, subsequently triggering the inhibition of BC proliferation. In addition, IS (10, 20, 30, and 50 μM) stimulated the production of interstitial ROS in MCF7/ADR cells, by 3.99-, 4.20-, 6.29-, and 6.78-fold, respectively, versus the untreated group (p < 0.001), which were involved in DNA damage and AMPK-caused intercept of the mTOR/p70S6K signaling.Our study suggested the anti-breast cancer actions of IS as a DOX sensitizer and expounded the underlying molecular mechanisms, showing that IS could be deemed to a capable alternative for resistant BC cure.Copyright © 2023 Elsevier GmbH. All rights reserved.