Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina , Londrina, Paraná, Brasil.
Departamento De Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do Maranhão , São Luís, Maranhão, Brasil.
J Toxicol Environ Health A. 2020 Aug 17;83(15-16):547-558. doi: 10.1080/15287394.2020.1784339. Epub 2020 Jun 26.
Brachydins (Br) A, B, and C are flavonoids extracted from (Cham.) L.G. Lohmann roots (synonym ), whose extract previously exhibited cytotoxic and antitumor activity. cell culture of human prostate tumor cell line (PC-3) was used to determine cell viability as evidenced by MTT, neutral red, and LDH release using nine concentrations (0.24 to 30.72 µM) of each brachydin. A triple-fluorescent staining assay assessed the mechanism resulting in cell death. Genomic instability and protein expression were evaluated using comet assay and western blot analysis, respectively. The pro-oxidant status was analyzed using the5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-HDCFDA) probe. The IC values for brachydins BrA, BrB, and BrC were 23.41, 4.28, and 4.44 µM, respectively, and all compounds induced apoptosis and necrosis. BrB and BrC increased p21 levels indicating a possible G1 cell cycle arrest. BrA (6 µM) and BrB (3.84 µM) decreased phospho-AKT (AKT serine/threonine kinase) expression, which also influenced cell cycle and proliferation. BrA, BrB, and BrC elevated cleaved PARP (poly (ADP-ribose) polymerase), a protein related to DNA repair and induction of apoptotic processes. Therefore, this study determined the IC values of brachydins in the PC-3 cell line as well as the influence on cell proliferation and cell death processes, such as apoptosis and necrosis, indicating the proteins involved in these processes.
ANOVA: Analysis of Variance; BrA: Brachydin A; BrB: Brachydin B; BrC: Brachydin C; CGEN: Genetic Heritage Management Council; CID: Compound identification number; CM-HDCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CO: Carbon dioxide; DMSO: Dimethyl sulfoxide; DNA: Deoxyribonucleic acid; DTT: Dithiothreitol; DXR: Doxorubicin; ECL: Chemiluminescence; EDTA: Ethylenediaminetetraacetic acid; FBS: Fetal bovine serum; HO: Hydrogen peroxide; HRMS: High-Resolution Mass Spectrometry; IC50: Half maximal inhibitory concentration; LDH: Lactate dehydrogenase; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Na3VO4: Sodium Orthovanadate; NaOH: Sodium hydroxide; NCBI: National Center for Biotechnology Information; NMR: Nuclear Magnetic Resonance; PBS: Phosphate buffer saline; PCR: Polymerase chain reaction; PSMF: Phenylmethylsulfonyl fluoride; RPMI: Roswell Park Memorial Institute Medium; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis; STR: Short tandem repeat; TBS-T: Tris-buffered saline and Polysorbate 20; UPHLC: Ultra-Performance Liquid Chromatography.
从 （Cham.）L.G. Lohmann 根（同义词 ）中提取的黄酮类化合物 BrA、BrB 和 BrC 之前表现出细胞毒性和抗肿瘤活性。用人前列腺癌细胞系（PC-3）的细胞培养来确定细胞活力，这是通过 MTT、中性红和 LDH 释放来证明的，每种 Br 分别使用 9 个浓度（0.24 至 30.72µM）。三重荧光染色分析评估导致细胞死亡的机制。使用彗星分析分别评估基因组不稳定性和蛋白质表达，使用 Western blot 分析。使用 5-（和-6）-氯甲基-2'，7'-二氯二氢荧光素二乙酸酯（CM-HDCFDA）探针分析促氧化剂状态。Brachydins BrA、BrB 和 BrC 的 IC 值分别为 23.41、4.28 和 4.44µM，所有化合物均诱导细胞凋亡和坏死。BrB 和 BrC 增加了 p21 水平，表明可能存在 G1 细胞周期阻滞。BrA（6µM）和 BrB（3.84µM）降低了磷酸化 AKT（丝氨酸/苏氨酸激酶 AKT）的表达，这也影响了细胞周期和增殖。BrA、BrB 和 BrC 升高了裂解的 PARP（多聚（ADP-核糖）聚合酶），PARP 与 DNA 修复和诱导凋亡过程有关。因此，本研究确定了 Br 在 PC-3 细胞系中的 IC 值，以及对细胞增殖和细胞死亡过程（如凋亡和坏死）的影响，表明这些过程涉及的蛋白质。
ANOVA：方差分析；BrA：Brachydin A；BrB：Brachydin B；BrC：Brachydin C；CGEN：遗传遗产管理委员会；CID：化合物识别号；CM-HDCFDA，5-（和-6）-氯甲基-2'，7'-二氯二氢荧光素二乙酸盐，乙酰酯；CO：二氧化碳；DMSO：二甲基亚砜；DNA：脱氧核糖核酸；DTT：二硫苏糖醇；DXR：多柔比星；ECL：化学发光；EDTA：乙二胺四乙酸；FBS：胎牛血清；HO：过氧化氢；HRMS：高分辨率质谱；IC50：半最大抑制浓度；LDH：乳酸脱氢酶；MTT，3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐；Na3VO4：正钒酸钠；NaOH：氢氧化钠；NCBI：国家生物技术信息中心；NMR：核磁共振；PBS：磷酸盐缓冲盐水；PCR：聚合酶链反应；PSMF：苯甲基磺酰氟；RPMI：罗格斯大学纪念医院培养基；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；STR：短串联重复；TBS-T：三（羟甲基）氨基甲烷缓冲盐水和聚山梨醇酯 20；UPHLC：超高效液相色谱法。
