Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) and Institute of Biomedicine, University of León, León, Spain.
Br J Cancer. 2013 Feb 5;108(2):442-9. doi: 10.1038/bjc.2012.563. Epub 2012 Dec 20.
Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a.
Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase-polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells.
Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr(32) and Ser(253), and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation.
This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.
褪黑素可诱导多种不同的癌细胞系凋亡，包括肝癌细胞。然而，负责的途径尚未明确阐明。叉头转录因子家族的一个成员 FoxO3a 已被牵涉到促凋亡蛋白 Bim（一种 Bcl-2 相互作用的细胞死亡介质）的表达中。在这项研究中，我们使用人 HepG2 肝癌细胞作为体外模型，研究褪黑素处理是否通过转录因子 FoxO3a 的调节诱导 Bim。
比较褪黑素在 HepG2 肝母细胞瘤细胞和原代人肝细胞中的细胞毒性。通过逆转录聚合酶链反应和 Western blot 分析促凋亡 Bim 的表达。进行报告基因分析和染色质免疫沉淀分析，以分析 FoxO3a 是否转录激活 Bim 启动子。使用小干扰 RNA（siRNA）研究 FoxO3a 在 Bim 表达中的作用。进行免疫荧光分析以研究 HepG2 细胞中 FoxO3a 的定位。
褪黑素处理可诱导 HepG2 细胞凋亡，但不能诱导原代人肝细胞凋亡。促凋亡作用是通过增加 BH3 仅蛋白 Bim 的表达介导的。在褪黑素处理期间，我们观察到叉头反应元件的转录活性增加，并能证明 FoxO3a 结合到 Bim 启动子内的特定序列。此外，褪黑素降低了 FoxO3a 在 Thr(32)和 Ser(253)上的磷酸化，并诱导其核内定位增加。此外，用 FoxO3a siRNA 进行的沉默实验可防止 Bim 的上调。
本研究表明，褪黑素可通过核转位和转录因子 FoxO3a 的激活来上调促凋亡的 Bim，从而诱导 HepG2 肝癌细胞凋亡。
