Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：非同源末端连接，一个重要的DNA双链断裂修复途径，在赋予放射治疗剂抗性方面发挥着重要作用，导致癌症进展和复发。
目的：参与胡椒碱和许多其他植物化合物的放射增敏作用的分子分子在很大程度上仍然回避。这项研究旨在评估胡椒碱，植物生物碱可以通过调节非同源末端连接机制的表达来改变辐射抗性。
方法：雌激素受体阳性/阴性，培养乳腺癌细胞以了解胡椒碱与放疗的协同作用。顺铂和巴泽多昔芬用作阳性对照。使用低剂量γ辐射器-2000将细胞暴露于γ-辐射。胡椒碱对雌激素受体调节的作用，DNA损伤,DNA损伤反应,细胞凋亡是通过蛋白质印迹完成的，免疫荧光，基于酵母的雌激素-受体-LacZ-报告基因测定，和核易位分析。对DNA损伤和遗传毒性进行了微核分析，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Schrodinger软件进行药效团分析和蛋白质-配体相互作用研究。协同作用是通过复合统计分析计算的。用GraphPad棱镜计算标准误差/偏差/显著性。
结果：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。这是第一个全面的研究阐明胡椒碱的放射增敏潜力的机制。胡椒碱增强辐射诱导的细胞死亡，增强雌激素受体β的表达和激活，雌激素受体α的表达和激活降低。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DDR和NHEJ途径的雌激素受体α/β比例以及雌激素反应蛋白的表达。DDR蛋白表达增强，ATM,p53和具有低DNA-PK修复复合物的P-p53（包括DNA-PKcs/Ku70/Ku80），导致辐射诱导的DNA双链断裂的积累（如MNi和γH2AX-foci所证明的），最终导致细胞周期停滞和线粒体凋亡途径。
结论：结论：我们的研究首次报道，胡椒碱通过积累DNA断裂使乳腺癌细胞对辐射敏感，通过改变DNA-PK复合物的表达，和DDR蛋白，通过选择性雌激素受体调节，提供了一种新的对抗辐射抗性的策略。
目的：参与胡椒碱和许多其他植物化合物的放射增敏作用的分子分子在很大程度上仍然回避。这项研究旨在评估胡椒碱，植物生物碱可以通过调节非同源末端连接机制的表达来改变辐射抗性。
方法：雌激素受体阳性/阴性，培养乳腺癌细胞以了解胡椒碱与放疗的协同作用。顺铂和巴泽多昔芬用作阳性对照。使用低剂量γ辐射器-2000将细胞暴露于γ-辐射。胡椒碱对雌激素受体调节的作用，DNA损伤,DNA损伤反应,细胞凋亡是通过蛋白质印迹完成的，免疫荧光，基于酵母的雌激素-受体-LacZ-报告基因测定，和核易位分析。对DNA损伤和遗传毒性进行了微核分析，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Schrodinger软件进行药效团分析和蛋白质-配体相互作用研究。协同作用是通过复合统计分析计算的。用GraphPad棱镜计算标准误差/偏差/显著性。
结果：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。这是第一个全面的研究阐明胡椒碱的放射增敏潜力的机制。胡椒碱增强辐射诱导的细胞死亡，增强雌激素受体β的表达和激活，雌激素受体α的表达和激活降低。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DDR和NHEJ途径的雌激素受体α/β比例以及雌激素反应蛋白的表达。DDR蛋白表达增强，ATM,p53和具有低DNA-PK修复复合物的P-p53（包括DNA-PKcs/Ku70/Ku80），导致辐射诱导的DNA双链断裂的积累（如MNi和γH2AX-foci所证明的），最终导致细胞周期停滞和线粒体凋亡途径。
结论：结论：我们的研究首次报道，胡椒碱通过积累DNA断裂使乳腺癌细胞对辐射敏感，通过改变DNA-PK复合物的表达，和DDR蛋白，通过选择性雌激素受体调节，提供了一种新的对抗辐射抗性的策略。
