Institute of Biomedicine (IBIOMED), University of León, Campus Universitario, 24071 León, Spain.
Br J Cancer. 2013 Jul 9;109(1):83-91. doi: 10.1038/bjc.2013.285. Epub 2013 Jun 11.
Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin.
HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT-qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP).
Melatonin at a pharmacological concentration (1 mM) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mM decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression.
Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.
肝细胞癌（HCC）的生长依赖于血管内皮生长因子（VEGF）释放的血管生成。肿瘤微环境中的缺氧会导致缺氧诱导因子 1 阿尔法（Hif1α）和信号转导和转录激活因子（STAT3）的细胞内稳定。褪黑素可诱导 HCC 细胞凋亡，并在多种肿瘤中表现出抗血管生成特性。在这项研究中，我们使用人 HepG2 肝癌细胞作为体外模型，研究褪黑素的抗血管生成作用。
在常氧或 CoCl2 诱导的缺氧条件下，用褪黑素处理 HepG2 细胞。通过 RT-qPCR 和 Western blot 分析基因表达。通过体内人脐静脉内皮细胞（HUVECs）管形成试验证实褪黑素的抗血管生成活性。通过 ELISA 测量分泌的 VEGF。通过免疫荧光分析 Hif1α细胞内定位。通过免疫沉淀和染色质免疫沉淀（ChIP）分析 Hif1α与其共激活因子的物理相互作用。
药理浓度（1mM）的褪黑素降低细胞和分泌的 VEGF 水平，并防止缺氧下 HUVECs 管形成，与 Hif1α蛋白表达、核定位和转录活性降低相关。虽然缺氧会增加磷酸化 STAT3、Hif1α 和 CBP/p300 作为 VEGF 启动子中的转录复合物的募集，但 1mM 的褪黑素会降低它们的物理相互作用。褪黑素和选择性 STAT3 抑制剂 Stattic 对 Hif1α、STAT3 和 VEGF 表达表现出协同作用。
褪黑素通过干扰 Hif1α 和 STAT3 对 VEGF 的转录激活，在 HepG2 细胞中发挥抗血管生成活性。我们的研究结果为将这种吲哚考虑为治疗 HCC 的有效抗血管生成剂提供了证据。
