背景:对多柔比星(DOX)的耐药性显着限制了其在乳腺癌(BC)患者中的治疗效果。柴胡皂苷D(SSD),从传统的柴胡草本植物中提取的三萜皂苷,由于其显着的抗肿瘤活性,在临床前研究中已显示出作为化疗增敏剂的希望。然而,SSD在DOX抗性BC细胞中的作用和机制仍未被研究。
目的:本研究旨在研究SSD对DOX耐药BC的化学增敏作用及其体内外分子机制。
方法:体外试验,包括细胞活力,克隆形成,三维肿瘤球体生长,和细胞凋亡分析,进行评估SSD和DOX对耐药BC细胞的协同作用。活性氧(ROS),GSH/GSSG,NADPH/NADP+,和NADH/NAD+检测用于评估SSD对细胞氧化还原稳态的影响。西方印迹,细胞周期分布测定,并进行了DOX摄取测定,以进一步阐明SSD可能的抗肿瘤机制。最后,建立裸鼠皮下MCF7/DOX细胞异种移植模型,以鉴定SSD联合DOX的体内抗癌作用。
结果:SSD显著抑制细胞活力,扩散,和克隆形成,在体外和体内增强DOX的抗癌功效。机械上,SSD降低STAT1、NQO1和PGC-1α蛋白水平,导致细胞氧化还原失衡,过量的ROS生成,以及GSH的耗尽,NADPH,还有NADH.SSD通过破坏氧化还原稳态诱导DNA损伤,导致G0/G1期细胞周期停滞。此外,SSD通过抑制P-gp蛋白表达和外排活性增加了BC细胞中DOX的积累。
结论:我们首次证明SSD通过STAT1/NQO1/PGC-1α信号通路的失活破坏细胞的氧化还原稳态,增强了化学抗性BC细胞对DOX的敏感性。本研究为SSD作为耐药BC治疗的佐剂提供了证据。
BACKGROUND: Drug resistance to doxorubicin (DOX) significantly limits its therapeutic efficacy in breast cancer (BC) patients. Saikosaponin D (SSD), a triterpene saponin derived from the traditional herb Radix Bupleuri, has shown promise as a chemotherapeutic sensitizer in preclinical studies due to its notable antitumor activity. However, the role and mechanism of SSD in DOX-resistant BC cells remain largely unexplored.
OBJECTIVE: This study aimed to investigate the chemosensitizing effect of SSD on DOX-resistant BC and the underlying molecular mechanisms both in vitro and in vivo.
METHODS: In vitro assays, including cell viability, clone formation, three-dimensional tumor spheroid growth, and apoptosis analysis, were conducted to evaluate the synergistic effect of SSD and DOX on resistant BC cells. Reactive oxygen species (ROS), GSH/GSSG, NADPH/NADP+, and NADH/NAD+ detections were employed to assess the impact of SSD on cellular redox homeostasis. Western blotting, cell cycle distribution assay, and DOX uptake assay were performed to further elucidate the possible antineoplastic mechanism of SSD. Finally, a subcutaneous MCF7/DOX cell xenografted model in nude mice was established to identify the in vivo anticarcinogenic effect of SSD combined with DOX.
RESULTS: SSD significantly inhibited cell viability, proliferation, and clone formation, enhancing DOX\'s anticancer efficacy in vitro and in vivo. Mechanistically, SSD reduced STAT1, NQO1, and PGC-1α protein levels, leading to cellular redox imbalance, excessive ROS generation, and depletion of GSH, NADPH, and NADH. SSD induced DNA damage by disrupting redox homeostasis, resulting in G0/G1 phase cell cycle arrest. Additionally, SSD increased DOX accumulation in BC cells via inhibiting P-gp protein expression and efflux activity.
CONCLUSIONS: We demonstrated for the first time that SSD enhances the sensitivity of chemoresistant BC cells to DOX by disrupting cellular redox homeostasis through inactivation of the STAT1/NQO1/PGC-1α signaling pathway. This study provides evidence for SSD as an adjuvant agent in drug-resistant BC treatment.