DIRECT INTERACTION OF ERISTOSTATIN WITH MELANOMA CELL SURFACE MOLECULES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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，包括涉及运动和粘连的步骤。如果这一连串的事件在任何一步被中断，就不会发生转移。Eristostatin是蛇毒蛋白去整合素家族的一员，可以抑制黑色素瘤细胞在小鼠或人黑色素瘤细胞注射小鼠肺部的定植。我们已经证明，eristostatin的存在可以引起黑色素瘤细胞粘附性和运动性的变化，但不会影响细胞的生长或存活。到目前为止，eristostatin的抗转移作用的基础尚不清楚，但这些功能研究已经为解开这种天然蛋白质的抗癌机制开辟了道路。这项研究的长期目标是确定黑色素瘤转移被抑制的机制。这里提出的具体目标是研究eristostatin影响黑色素瘤细胞功能的分子机制。我们将通过追求三个具体目标来做到这一点：(I)表征eristostatin与黑色素瘤细胞分子的直接相互作用(S)。我们将使用原子力显微镜、共聚焦显微镜和化学交联来确定eristostatin的结合伙伴，eristostatin存在于6种不同程度转移潜力的黑色素瘤细胞系中。(Ii)比较Eristostatin和黑色素瘤细胞在二维和三维环境中的相互作用差异。组织培养中的细胞反应(2-D)与体内发生的反应(3-D)可能有很大的不同。我们将使用含有纤维连接蛋白和/或胶原的基质，在三维环境中研究eristostatin-黑色素瘤细胞之间的相互作用，包括黏附、增殖和信号转导。(Iii)确定与eristostatin与黑色素瘤细胞相互作用有关的细胞内信号事件。Eristostatin抑制黑色素瘤细胞活性的最明显的例子是当放置在纤维连接蛋白上时具有运动性。我们将对黑色素瘤细胞暴露于eristostatin时发生的细胞内信号进行免疫化学评估，重点放在与运动相关的已知途径上。综上所述，这些研究将提供关于一种自然产生的蛋白质--eristostatin--如何拥有与黑色素瘤细胞结合并阻止其转移能力的“合适”的洞察力。这些信息反过来将导致针对这些细胞的治疗剂的合理设计。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Metastasis (i.e., tumor spread) is the major obstacle to cancer cure. The metastatic process consists of many steps, including those involving motility and adhesion. If the cascade of events is interrupted at any step, metastasis will not occur. Eristostatin, a member of the disintegrin family of viper venom proteins, can inhibit melanoma cell colonization in the lungs of mice injected with either murine or human melanoma cells. We have shown that the presence of eristostatin can cause changes in adhesion and motility in melanoma cells, but does not affect cell growth or survival. To date, the basis for eristostatin's anti-metastatic effect remains unknown, but these functional studies have opened the way to unlocking this natural protein's anti-cancer mechanism. The long term objective of this research is to identify mechanisms by which melanoma metastasis may be inhibited. The specific goals proposed here are to investigate the molecular mechanism used by eristostatin to affect melanoma cell function. We will do this by pursuing three specific aims: (i) Characterize the direct interaction of eristostatin with melanoma cell molecule(s). We will use atomic force microscopy, confocal microscopy and chemical crosslinking to identify the binding partner for eristostatin which is present on 6 different melanoma cell lines with varying degrees of metastatic potential. (ii) Compare the interaction differences between eristostatin and melanoma cells in a 2-dimensional versus 3-dimensional environment. Cellular responses can be very different in tissue culture (2-D) compared with what happens in the body (3-D). We will investigate eristostatin-melanoma cell interactions involving adhesion, proliferation and signaling in a 3-dimensional environment using matrices containing fibronectin and/or collagen. (iii) Identify intracellular signaling events associated with eristostatin's interaction with melanoma cells. The clearest example of melanoma cell activity inhibition by eristostatin is with motility when placed on fibronectin. We will perform immunochemical assessment of the intracellular signaling occurring when melanoma cells are exposed to eristostatin, focusing on known pathways associated with motility. Taken together, these studies will provide insights into how one naturally occurring protein, eristostatin, possesses the "right fit" to bind melanoma cells and block their metastatic ability. The information will, in turn, lead to a rational design of therapeutic agents which will target these cells.