非同源末端连接是一种重要的 DNA 双链断裂修复途径，在赋予放射治疗药物耐药性、导致癌症进展和复发方面发挥着重要作用。胡椒碱和许多其他药物的放射增敏作用涉及的分子参与者植物化合物在很大程度上仍然难以捉摸。该研究旨在评估胡椒碱（一种植物生物碱）是否可以通过调节非同源末端连接机制的表达来改变放射抗性。培养雌激素受体阳性/阴性的乳腺癌细胞，以了解胡椒碱与放射治疗的协同效应。顺铂和巴多昔芬用作阳性对照。使用 Low Dose gamma Irradiator-2000 将细胞暴露于 γ-辐射。通过蛋白质印迹、免疫荧光、酵母雌激素受体 LacZ 报告基因测定和核易位分析，研究胡椒碱对雌激素受体调节、DNA 损伤、DNA 损伤反应和细胞凋亡的影响。对 DNA 损伤和基因毒性进行微核测定，并使用共聚焦显微镜通过 γH2AX 焦点染色对 DSB 进行定量。进行流式细胞术分析以确定细胞周期、线粒体膜去极化和活性氧的产生。使用薛定谔软件进行药效团分析和蛋白质-配体相互作用研究。通过计算机统计分析计算协同作用。使用 GraphPad 棱镜计算标准误差/偏差/显着性。使用胡椒碱，我们提出了一种通过雌激素受体介导的 NHEJ 途径调节来克服获得性放射抗性的新策略。这是第一个阐明胡椒碱放射增敏作用机制的综合研究。胡椒碱增强辐射诱导的细胞死亡并增强雌激素受体β的表达和激活，而雌激素受体α的表达和激活减少。此外，胡椒碱与大多数已知的雌激素激动剂和拮抗剂具有共同的药效团特征。它改变了雌激素受体α/β比例以及DDR和NHEJ途径雌激素反应蛋白的表达。 DDR 蛋白、ATM、p53 和 P-p53 的表达增强，具有低 DNA-PK 修复复合物（由 DNA-PKcs/Ku70/Ku80 组成），导致辐射诱导的 DNA 双链断裂的积累（如MNi 和 γH2AX-foci）最终导致细胞周期停滞和线粒体凋亡途径。总之，我们的研究首次报道胡椒碱通过积累 DNA 断裂、改变 DNA-PK 复合物的表达来使乳腺癌细胞对辐射敏感。和 DDR 蛋白，通过选择性雌激素受体调节，提供一种对抗放射抗性的新策略。版权所有 © 2023 Elsevier GmbH。版权所有。

Non-homologous end joining, an important DNA-double-stranded break repair pathway, plays a prominent role in conferring resistance to radiotherapeutic agents, resulting in cancer progression and relapse.The molecular players involved in the radio-sensitizing effects of piperine and many other phytocompounds remain evasive to a great extent. The study is designed to assess if piperine, a plant alkaloid can alter the radioresistance by modulating the expression of non-homologous end-joining machinery.Estrogen receptor-positive/negative, breast cancer cells were cultured to understand the synergetic effects of piperine with radiotherapy. Cisplatin and Bazedoxifene were used as positive controls. Cells were exposed to γ- radiation using Low Dose gamma Irradiator-2000. The piperine effect on Estrogen receptor modulation, DNA-Damage, DNA-Damage-Response, and apoptosis was done by western blotting, immunofluorescence, yeast-based-estrogen-receptor-LacZ-reporter assay, and nuclear translocation analysis. Micronuclei assay was done for DNA damage and genotoxicity, and DSBs were quantified by γH2AX-foci-staining using confocal microscopy. Flow cytometry analysis was done to determine the cell cycle, mitochondrial membrane depolarization, and Reactive oxygen species generation. Pharmacophore analysis and protein-ligand interaction studies were done using Schrodinger software. Synergy was computed by compusyn-statistical analysis. Standard errors/deviation/significance were computed with GraphPad prism.Using piperine, we propose a new strategy for overcoming acquired radioresistance through estrogen receptor-mediated modulation of the NHEJ pathway. This is the first comprehensive study elucidating the mechanism of radio sensitizing potential of piperine. Piperine enhanced the radiation-induced cell death and enhanced the expression and activation of Estrogen receptor β, while Estrogen receptor α expression and activation were reduced. In addition, piperine shares common pharmacophore features with most of the known estrogen agonists and antagonists. It altered the estrogen receptor α/β ratio and the expression of estrogen-responsive proteins of DDR and NHEJ pathway. Enhanced expression of DDR proteins, ATM, p53, and P-p53 with low DNA-PK repair complex (comprising of DNA-PKcs/Ku70/Ku80), resulted in the accumulation of radiation-induced DNA double-stranded breaks (as evidenced by MNi and γH2AX-foci) culminating in cell cycle arrest and mitochondrial-pathway of apoptosis.In conclusion, our study for the first time reported that piperine sensitizes breast cancer cells to radiation by accumulating DNA breaks, through altering the expression of DNA-PK Complex, and DDR proteins, via selective estrogen receptor modulation, offering a novel strategy for combating radioresistance.Copyright © 2023 Elsevier GmbH. All rights reserved.