Proanthocyanidins,Novel bioactive components for prevention of melanoma invasion

描述(申请人提供)：黑色素瘤是皮肤病死亡的主要原因，很大程度上是由于它的转移倾向。由于黑色素瘤的转移是皮肤病导致死亡的主要原因，降低其侵袭性或转移能力的方法可能有助于制定有效的治疗和/或治疗策略。 预防。一些药物已经显示出治疗黑色素瘤的潜力，但由于长期的安全性、耐药性和毒性问题，它们作为化学预防药物的使用是值得怀疑的。具有低毒性的饮食生物活性成分作为化学保护剂是有意义的，包括原花青素，它们对小鼠没有明显毒性。原花青素在葡萄籽中高度丰富，在小鼠模型中具有抗皮肤致癌作用，但其对转移的影响尚未被探索。我们发现用葡萄籽原花青素(GSPs)处理一些转移的人黑色素瘤细胞(A375，Hs294t)，抑制了它们的迁移，这一结果通过Boyden小室实验确定，这表明GSPs可能在抑制转移方面有效。由于b-catenin突变是与黑色素瘤发生和转移相关的最常见的改变之一，并且突变的b-catenin与侵袭性肿瘤生长有关，我们研究了b-catenin状态对黑色素瘤细胞侵袭的影响，发现GSPs介导的抑制黑色素瘤细胞侵袭与b-catenin失活有关，并且激活b-catenin的黑色素瘤细胞(MEL1241)表现出比失活b-catenin的黑色素瘤细胞(MEL1011)更大的侵袭力。基于我们的初步数据，我们提出了一个创新的假设，即GSPs抑制黑色素瘤细胞的迁移或侵袭至少部分是通过灭活b-catenin介导的，这是其化学预防黑色素瘤转移的关键。我们的长期目标是充分检验这一假设。这一探索性R21应用的目的是利用体外细胞培养和体内裸鼠模型来验证和扩展我们的初步结果，以便生成设计研究所需的数据，这些研究将在小鼠模型中严格测试中心假设，并为观察到的数据建立分子基础。我们提出了两个互补的特异性目标：(1)确定GSPs是否抑制具有不同突变的人黑色素瘤细胞系的细胞侵袭，以及GSPs抑制细胞侵袭是否需要灭活b-catenin及其信号转导。以正常人皮肤黑素细胞为对照，(2)检测膳食GSPs对裸鼠体内人黑色素瘤细胞侵袭潜能的影响，并探讨GSPs抑制裸鼠黑色素瘤细胞转移的作用是否与该模型中b-catenin失活有关。相关性：这些研究将产生关于GSP在体内对黑色素瘤侵袭/转移的影响的知识，以及对其潜在的长期用于抑制转移性黑色素瘤的未来研究所必需的作用机制。

DESCRIPTION (provided by applicant): Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. Since metastasis of melanoma is the leading cause of death due to skin diseases, an approach that decreases its invasiveness or metastatic ability may facilitate the development of an effective strategy for its treatment and/or prevention. Some drugs has demonstrated potential in treatment of melanoma but their use as chemopreventive agents is questionable due to long-term safety, resistance and toxicity concerns. Dietary bioactive components that have low toxicity are of interest as chemopreventives, including the proanthocyanidins, which do not exhibit gross toxicity in mice. Proanthocyanidins, which are highly enriched in grape seeds, have anti-skin carcinogenic effects in mouse models, but their effects on metastasis have not been explored. We have found that treatment of some metastatic human melanoma cells (A375, Hs294t) with grape seed proanthocyanidins (GSPs) inhibited their migration, as determined using a Boyden chamber assay, suggesting the possibility that GSPs may be effective in inhibiting metastasis. As mutations in b-catenin are one of the most common alterations associated with melanoma development and metastasis and mutated b- catenin is associated with aggressive tumor growth, we examined the effects of the b-catenin status and found that the GSPs-mediated inhibition of melanoma cell invasion was associated with inactivation of b-catenin and that melanoma cells that have activated b-catenin (Mel 1241) showed significantly greater invasion than those melanoma cells that have inactivated b-catenin (Mel 1011). Based on our preliminary data, we have formulated the innovative hypothesis that the inhibition of migration or invasiveness of melanoma cells by GSPs is mediated, at least in part, through inactivation of b-catenin and that is critical for their chemopreventive effects on melanoma metastasis. Our long-term goal is to fully test this hypothesis. The objectives of this exploratory R21 application are o verify and extend our preliminary results using in vitro cell culture and in vivo athymic nude mouse model in order to generate the data necessary for the design of studies that will rigorously test the central hypothesis in mouse model and establish the molecular basis for the observed data. We propose two complementary Specific Aims: (1) To determine whether GSPs inhibit cell invasion of human melanoma cell lines with different mutations, and whether inhibition of cell invasion by GSPs requires inactivation of b-catenin and its signaling. Normal human epidermal melanocytes will serve as a control, and (2) To determine the effect of dietary GSPs on the invasive potential of human melanoma cells in vivo in athymic nude mice, and to ascertain whether the inhibitory effect of GSPs on cell metastasis is associated with inactivation of b-catenin in this model. RELEVANCE: These studies will generate knowledge concerning the effects of GSPs on melanoma invasiveness/metastasis in vivo and the mechanism of action that is necessary for future studies of their potential long-term use for suppression of metastatic melanoma.