Inhibition of Melanoma metastasis by Eristostatin

转移（即肿瘤扩散）是癌症治愈的主要障碍。 转移过程包括许多步骤。 如果这种级联反应在任何一步被中断，转移就不会发生。 恶性黑色素瘤细胞使用在正常细胞表面上发现的相同分子库（例如受体）与它们的环境相互作用。 由于细胞与细胞的相互作用发生在级联反应的许多阶段，因此阻断黑色素瘤细胞上的受体（它们通过受体与其他细胞相互作用）代表了中断肿瘤扩散的合理目标。蛇毒蛋白去整合素家族的成员之一，在免疫缺陷小鼠体内发现能抑制黑色素瘤的转移。 迄今为止，蛇毒抑制素的抗转移作用的基础仍然未知。 本研究的长期目标是确定抑制黑色素瘤转移的分子机制。 本文提出的具体目标是通过追求三个具体目标来研究蛇毒抑制素抑制实验性黑素瘤转移的作用：i）创建一组蛇毒抑制素突变，其被设计成改变沿其整个长度的单个氨基酸残基沿着。ii）表征蛇毒抑制素及其突变体与具有明确定义的表面受体的不同组合的四种黑素瘤细胞系的相互作用。iii）鉴定对于其抗转移能力最关键的蛇毒抑素内的结构序列。 通过丙氨酸扫描诱变将产生重组的蛇毒蛋白突变体，并将其制备为具有谷胱甘肽-S转移酶的细菌融合蛋白。 这些纯化的突变体将用于四种转移性人黑素瘤细胞系的一系列功能测定。 我们将通过交联和免疫学技术鉴定与每种类型的黑色素瘤细胞相互作用的特定分子。 为了确定蛇毒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种突变体与野生型蛇毒抑素的活性进行比较。 在细胞相互作用中显示最大差异的那些突变体将用于实验转移测定。 免疫缺陷小鼠静脉注射黑色素瘤细胞和突变的蛇毒抑制素后，观察小鼠4周，然后尸检。 将通过计数肺转移瘤来评估转移潜力。 使用免疫组织化学染色评价肺的冷冻切片。 总的来说，这些研究将为了解一种天然存在的蛋白质如何具有“正确的适合”来结合黑色素瘤细胞并阻断其转移能力提供见解。 这些信息将反过来导致治疗剂的合理设计，这些治疗剂将靶向这些细胞并战胜肿瘤扩散这一癌症治愈的主要障碍。

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.