Inhibition of Melanoma metastasis by Eristostatin

Eristostatin抑制黑色素瘤转移

• 批准号: 6316920
• 负责人: MARY ANN MCLANE
• 金额: $ 15万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-19 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6316920
• 关键词: alanine; animal poison; antineoplastics; athymic mouse; collagen; fibrinogen receptors; fibronectins; gene mutation; integrins; laminin; melanoma; metastasis; neoplasm /cancer chemotherapy; nonhuman therapy evaluation; protein structure function; site directed mutagenesis; vascular cell adhesion molecule; vitronectin

Metastasis (i.e. tumor spread) is the major obstacle to cancer cure. The metastatic process consists of many steps. If this cascade of events is interrupted at any step, metastasis will not occur. Malignant melanoma cells interacted with their environment using the same repertoire of molecules (e.g. receptors) found on normal cells surfaces. Since cell-to- cell interactions occur at many pints of the cascade, blockade of receptor(s) on melanoma cells, through which they interact with other cells, represents a rational target for interruption of tumor spread. Eristostatin, am member of the disintegrin family of viper venom proteins, has been found to inhibit melanoma metastasis in vivo using immunodeficient mice. To date, the basis for eristostatin's anti-metastatic effect remains unknown. The long term objective of this research is to identify molecular mechanisms by which melanoma metastasis may be inhibited. The specific goal proposed here is to investigate hoe eristostatin inhibits experimental melanoma metastasis by pursuing three specific aims: i) Create a panel of eristostatin mutations designed to alter single amino acid residues along its entire length. ii) Characterize the interactions of eristostatin and its mutants with four melanoma cell lines possessing distinct combinations of well-defined surface receptors. iii) Identify the structural sequence within eristostatin which is most critical for its anti-metastatic ability. Recombinant eristostatin mutations will be created by alanine scanning mutagenesis, and made as bacterial fusion proteins with glutathione-S transferase. These purified mutants will be used in a series of functional assays with four metastatic, human melanoma cell lines. We will identify the specific molecule with which eristostatin interacts on each type pf melanoma cell through crosslinking and immunological techniques. To determine which residue(s) of eristostatin are responsible for its cellular interactions, each mutant will be compared to wild type eristostatin's activity in cellular assays. Those mutants which show the greatest difference in cellular interactions will be used in experimental metastasis assays. After I.V. injection of immonodeficient mice with melanoma cells and mutated eristostatin, mice will be observed for 4 weeks, and then necropsied. Metastatic potential will be assessed by counting lung metastases. Cryosections of how lungs will be evaluated using immunohistochemical stains. Taken together, these studies will provide insights into how one naturally occurring protein possesses the "right fit" to bind melanoma cells and block their metastatic ability. This information will, in turn, lead to a rational design of therapeutic agents which would target these cells and triumph over tumor spread the major obstacle to cancer cure.

转移（即肿瘤扩散）是癌症治愈的主要障碍。 转移过程由许多步骤组成。 如果这一系列事件在任何步骤被中断，转移就不会发生。 恶性黑色素瘤细胞使用与正常细胞表面相同的分子（例如受体）与其环境相互作用。 由于细胞与细胞之间的相互作用发生在级联的许多阶段，因此阻断黑色素瘤细胞上的受体（它们通过该受体与其他细胞相互作用）代表了阻断肿瘤扩散的合理目标。 Eristostatin 是毒蛇毒蛋白解整合素家族的成员，已被发现可以在免疫缺陷小鼠体内抑制黑色素瘤转移。 迄今为止，埃立他汀抗转移作用的基础仍不清楚。 这项研究的长期目标是确定抑制黑色素瘤转移的分子机制。 这里提出的具体目标是通过追求三个具体目标来研究埃立他汀抑制实验性黑色素瘤转移：i) 创建一组旨在改变其整个长度上的单个氨基酸残基的埃立他汀突变。 ii) 表征埃立他汀及其突变体与四种黑色素瘤细胞系的相互作用，这些细胞系具有明确的表面受体的不同组合。 iii) 确定埃立他汀内的结构序列，这对于其抗转移能力至关重要。 重组毛立抑素突变体将通过丙氨酸扫描诱变产生，并与谷胱甘肽-S转移酶一起制成细菌融合蛋白。 这些纯化的突变体将用于对四种转移性人类黑色素瘤细胞系进行一系列功能测定。 我们将通过交联和免疫学技术来鉴定与埃立他汀在每种类型的黑色素瘤细胞上相互作用的特定分子。 为了确定毛立抑素的哪些残基负责其细胞相互作用，将在细胞测定中将每个突变体与野生型毛立抑素的活性进行比较。 那些在细胞相互作用中表现出最大差异的突变体将用于实验性转移测定。 静脉注射后 给免疫缺陷小鼠注射黑色素瘤细胞和突变的埃立他汀，观察小鼠4周，然后进行尸检。 将通过计数肺转移来评估转移潜力。 如何使用免疫组织化学染色评估肺部的冷冻切片。 总而言之，这些研究将深入了解一种天然存在的蛋白质如何“正确契合”地结合黑色素瘤细胞并阻止其转移能力。 这些信息反过来将导致治疗剂的合理设计，这些治疗剂将针对这些细胞并克服肿瘤扩散这一癌症治愈的主要障碍。