Proanthocyanidins,Novel bioactive components for prevention of melanoma invasion

描述（由申请人提供）：黑色素瘤是皮肤病死亡的主要原因，这在很大程度上是由于其转移倾向。由于黑色素瘤的转移是由于皮肤病导致死亡的主要原因，因此降低其侵袭性或转移能力的方法可以促进开发用于其治疗和/或治疗的有效策略。 预防一些药物已被证明有潜力治疗黑色素瘤，但由于长期安全性，耐药性和毒性问题，它们作为化学预防剂的使用是值得怀疑的。具有低毒性的膳食生物活性组分作为化学预防剂是令人感兴趣的，包括原花色素，其在小鼠中不表现出总毒性。葡萄籽中高度富集的原花青素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。我们已经发现，葡萄籽原花青素（GSPs）的一些转移性的人黑色素瘤细胞（A375，Hs 294 t）的治疗抑制其迁移，如使用Boyden室测定，这表明GSPs可能是有效的抑制转移的可能性。由于b-连环蛋白的突变是与黑素瘤发展和转移相关的最常见的改变之一，并且突变的B-连环蛋白与侵袭性肿瘤生长相关，我们检测了β-连环蛋白状态的影响，发现GSPs介导的黑色素瘤细胞侵袭的抑制与β-连环蛋白的失活有关，（Mel 1241）显示出比具有失活的b-连环蛋白的那些黑素瘤细胞（Mel 1011）显著更大的侵袭。基于我们的初步数据，我们已经制定了创新的假设，即抑制迁移或侵袭的黑色素瘤细胞的GSP介导的，至少部分地，通过失活的b-连环蛋白，这是至关重要的化学预防作用对黑色素瘤转移。我们的长期目标是充分验证这一假设。该探索性R21应用的目的是使用体外细胞培养和体内无胸腺裸鼠模型验证和扩展我们的初步结果，以生成研究设计所需的数据，这些研究将严格检验小鼠模型中的中心假设，并为观察到的数据建立分子基础。我们提出了两个互补的具体目的：（1）确定是否GSPs抑制细胞侵袭的人黑色素瘤细胞系与不同的突变，以及是否通过GSPs抑制细胞侵袭需要失活的β-catenin及其信号转导。正常人表皮黑素细胞将作为对照，以及（2）确定饮食GSPs对无胸腺裸鼠体内人黑素瘤细胞侵袭潜力的影响，并确定GSPs对细胞转移的抑制作用是否与该模型中的b-连环蛋白失活相关。相关性：这些研究将产生有关GSP对体内黑色素瘤侵袭/转移的影响以及其潜在长期用于抑制转移性黑色素瘤的未来研究所需的作用机制的知识。

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.