Antitumorigenic mechanisms of the monoacylglycerol lipase inhibitor JZL184

单酰甘油脂肪酶抑制剂JZL184的抗肿瘤机制

• 批准号: 323010209
• 负责人: Professor Dr. Burkhard Hinz
• 金额: --
• 依托单位: Institut für Toxikologie und Pharmakologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/323010209?language=en
• 关键词: Antitumorigenic; mechanisms; monoacylglycerol; lipase; inhibitor

The endocannabinoid system has gained considerable interest as pharmacological target in cancer treatment. Recently, we reported inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) to profoundly suppress lung cancer cell invasion and metastasis, but not growth of solid tumors. Anti-invasive and antimetastatic effects were mimicked by FAAH substrates, including the endocannabinoid 2-arachidonoylglycerol (2-AG). In preliminary experiments, JZL184, an inhibitor of the major 2-AG-degrading enzyme monoacylglycerol lipase (MAGL), was found to inhibit invasion in several lung cancer cell lines and to suppress tumor cell-related angiogenic capacities of human endothelial cells (HUVEC), while not altering the viability of tumor cells. In athymic nude mice, JZL184 was demonstrated to confer suppression of metastasis and, in contrast to FAAH inhibitors, solid tumor growth associated with inhibition of tumor angiogenesis. Several invasion- and angiogenesis-related regulations have been found as response to JZL184 such as induction of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and downregulation of matrix metalloproteinase-2 (MMP-2) and insulin-like growth factor-1 (IGF-1). The present proposal addresses the antitumorigenic mechanisms of MAGL inhibition in lung cancer cells. Modified Boyden chamber assays will be performed using lung cancer cell lines as well as primary cancer cells from lung cancer patients´ biopsies to characterize the impact of JZL184 on tumor invasion. The anti-angiogenic impact of JZL184 will be tested by analyzing the impact of conditioned media from cancer cells on migration, viability, tube formation and 3-dimensional sprout formation of endothelial cells. The particular focus in investigating anti-invasive and anti-angiogenic effects of JZL184 and 2-AG will be set on the role of TIMP-1, MMP-2 and IGF-1 by use of siRNA and add-back approaches, respectively. Moreover, the role of cannabinoid receptors and transient receptor potential vanilloid 1 (TRPV1) in antitumorigenic responses will be characterized. Angiogenic features will additionally be assessed using conditioned media of oxygen-deprived lung cancer cells to mimic hypoxical cancer growth that initiates angiogenic pathways. To provide further insight into metastasis- and angiogenesis-related intracellular pathways triggered by MAGL inhibition, TaqMan-Array analyses will be carried out. To identify angiogenesis-linked targets in or on HUVEC, the impact of conditioned media on migration parameters in HUVEC, such as CD63, VE-cadherin, paxilin and focal adhesion kinase, will be analyzed. Animal experiments will finally reveal a probable synergism of combinational administration of JZL184 with 2-AG and the contribution of cannabinoid receptors and TRPV1 to the antimetastatic and tumor-regressive effects of JZL184 and 2-AG. Samples from xenograft experiments will be used to analyze in vivo protein regulations by JZL184 and 2-AG.

内源性大麻素系统作为癌症治疗的药理学靶点引起了相当大的兴趣。最近，我们报道了内源性大麻素降解酶脂肪酸酰胺水解酶（FAAH）的抑制剂可以深度抑制肺癌细胞的侵袭和转移，但不能抑制实体瘤的生长。 FAAH 底物（包括内源性大麻素 2-花生四烯酰甘油 (2-AG)）模拟了抗侵袭和抗转移作用。在初步实验中，JZL184是主要2-AG降解酶单酰基甘油脂肪酶（MAGL）的抑制剂，被发现可以抑制多种肺癌细胞系的侵袭并抑制人内皮细胞（HUVEC）的肿瘤细胞相关血管生成能力，同时不改变肿瘤细胞的活力。在无胸腺裸鼠中，JZL184 被证明能够抑制转移，与 FAAH 抑制剂相比，实体瘤生长与抑制肿瘤血管生成相关。已发现一些与侵袭和血管生成相关的调节作为对 JZL184 的反应，例如诱导基质金属蛋白酶-1 组织抑制剂 (TIMP-1) 以及下调基质金属蛋白酶-2 (MMP-2) 和胰岛素样生长因子-1 (IGF-1)。本提案解决了肺癌细胞中 MAGL 抑制的抗肿瘤机制。将使用肺癌细胞系以及来自肺癌患者活组织检查的原发性癌细胞进行改良的博伊登室测定，以表征 JZL184 对肿瘤侵袭的影响。 JZL184 的抗血管生成作用将通过分析癌细胞的条件培养基对内皮细胞的迁移、活力、管形成和 3 维芽形成的影响来测试。研究 JZL184 和 2-AG 的抗侵袭和抗血管生成作用的特别重点将分别通过使用 siRNA 和回加方法来研究 TIMP-1、MMP-2 和 IGF-1 的作用。此外，还将描述大麻素受体和瞬时受体电位香草酸 1 (TRPV1) 在抗肿瘤反应中的作用。此外，还将使用缺氧肺癌细胞的条件培养基来评估血管生成特征，以模拟启动血管生成途径的缺氧癌症生长。为了进一步了解 MAGL 抑制引发的转移和血管生成相关的细胞内途径，将进行 TaqMan 阵列分析。为了识别 HUVEC 内或之上的血管生成相关靶点，将分析条件培养基对 HUVEC 迁移参数（例如 CD63、VE-钙粘蛋白、桩蛋白和粘着斑激酶）的影响。动物实验最终将揭示 JZL184 与 2-AG 联合给药可能产生的协同作用，以及大麻素受体和 TRPV1 对 JZL184 和 2-AG 的抗转移和肿瘤消退作用的贡献。异种移植实验的样品将用于分析 JZL184 和 2-AG 的体内蛋白质调节。