All-trans retinoic acid suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and metastasis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis. Adjuvant therapies, chemotherapy, radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma. All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development, growth, neural function, immune function, reproduction, and vision. It is one of the most potent therapeutic agents used for treating cancers, especially lung adenocarcinomas. ATRA inhibits metastatic potential and angiogenesis in several tumor models. We investigated the effects of ATRA on the expression of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and receptor Tie-2 in EC1 cells in vitro. We also assessed the growth and migration of EC1 cells in vitro. ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours, relative to the control. ATRA plus fluorouracil treatment reduced the viability more strongly than either drug alone, indicating an additive effect. Moreover, ATRA decreased EC1 migration by 87%. Furthermore, ATRA treatment led to a marked decrease of the transcript levels of Ang-1, Ang-2, Tie-2, VEGF, and VEGF receptors, as assessed by real-time RT-PCR. Importantly, the protein levels of Ang-1, Ang-2 and Tie-2 were reduced by ATRA treatment. In vivo, we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice. Importantly, ATRA treatment decreased the expression of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells. Collectively, our findings demonstrate that ATRA exhibits a dose- and temporal-dependent effect on the metastatic behavior, suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and the progression of xenograft tumors of EC1 cells.食管鳞状细胞癌（Esophageal squamous cell carcinoma, ESCC）是河南省第二高发的恶性肿瘤，以侵袭性强、预后极差著称。辅助治疗、化疗、放疗及内镜治疗均未能改善晚期食管癌患者的生存率。全反式维甲酸（All-trans retinoic acid, ATRA）是维生素A的活性成分，可调控广泛的生物学过程，包括发育、生长、神经功能、免疫功能、生殖与视觉功能。它是目前最有效的抗癌治疗药物之一，尤其对肺腺癌疗效显著。ATRA可在多种肿瘤模型中抑制肿瘤转移潜能与血管生成。本研究体外探究了ATRA对EC1细胞中血管生成素1（Angiopoietin 1, Ang-1）、血管生成素2（Angiopoietin 2, Ang-2）及其受体Tie-2表达的影响，同时评估了EC1细胞的增殖与迁移能力。与对照组相比，ATRA处理24小时、48小时后，EC1细胞的增殖能力分别下降29.5%与40.3%。ATRA联合氟尿嘧啶（fluorouracil）处理较单一药物处理更显著地降低了细胞活力，提示二者存在叠加效应。此外，ATRA可使EC1细胞的迁移能力降低87%。经实时荧光定量逆转录聚合酶链反应（real-time RT-PCR）检测，ATRA处理可显著降低Ang-1、Ang-2、Tie-2、血管内皮生长因子（Vascular Endothelial Growth Factor, VEGF）及其受体的转录水平。更为重要的是，ATRA处理还可降低Ang-1、Ang-2及Tie-2的蛋白表达水平。体内实验显示，ATRA处理可抑制小鼠肿瘤生长，并改善小鼠恶病质状态。值得注意的是，ATRA处理可降低EC1细胞皮下移植瘤中CD31、Ang-1、Ang-2及Tie-2的表达水平。综上，本研究结果表明，ATRA对EC1细胞的转移行为具有剂量与时间依赖性调控作用，可抑制血管生成素-Tie2通路，进而阻断血管生成并延缓EC1细胞异种移植瘤的进展。