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已经获得了相当大的兴趣。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，以深刻地抑制肺癌细胞的侵袭和转移，但不是实体瘤的生长。抗侵袭和抗转移作用由FAAH底物模拟，包括内源性大麻素2-花生四烯酸甘油（2-AG）。在初步实验中，JZL 184，一种主要的2-AG降解酶单酰基甘油脂肪酶（MAGL）的抑制剂，被发现可以抑制几种肺癌细胞系的侵袭，并抑制肿瘤细胞相关的人内皮细胞（HUVEC）的血管生成能力，同时不改变肿瘤细胞的活力。在无胸腺裸鼠中，JZL 184被证明能够抑制转移，并且与FAAH抑制剂相反，实体瘤生长与肿瘤血管生成的抑制相关。JZL 184可诱导基质金属蛋白酶组织抑制因子-1（TIMP-1）的表达，下调基质金属蛋白酶-2（MMP-2）和胰岛素样生长因子-1（IGF-1）的表达。本提案解决了MAGL抑制在肺癌细胞中的抗肿瘤发生机制。将使用肺癌细胞系以及来自肺癌患者活检的原发性癌细胞进行改良的Boyden室测定，以表征JZL 184对肿瘤侵袭的影响。将通过分析来自癌细胞的条件培养基对内皮细胞的迁移、活力、管形成和三维芽形成的影响来测试JZL 184的抗血管生成影响。JZL 184和2-AG的抗侵袭和抗血管生成作用的研究重点将分别放在TIMP-1，MMP-2和IGF-1的作用上，分别使用siRNA和add-back方法。此外，大麻素受体和瞬时受体电位香草素1（TRPV 1）在抗肿瘤反应中的作用将被表征。此外，还将使用缺氧肺癌细胞的条件培养基模拟启动血管生成途径的低氧癌症生长，评估血管生成特征。为了进一步了解由MAGL抑制触发的转移和血管生成相关的细胞内途径，将进行TaqMan-Array分析。为了鉴定HUVEC中或HUVEC上的血管生成相关靶点，将分析条件培养基对HUVEC中迁移参数的影响，如CD 63、VE-钙粘蛋白、桩蛋白和粘着斑激酶。动物实验将最终揭示JZL 184与2-AG联合给药的可能协同作用以及大麻素受体和TRPV 1对JZL 184和2-AG的抗转移和肿瘤消退作用的贡献。来自异种移植实验的样品将用于分析JZL 184和2-AG的体内蛋白调节。