Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
PLoS One. 2011;6(6):e21539. doi: 10.1371/journal.pone.0021539. Epub 2011 Jun 27.
Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of grape seed proanthocyanidins (GSPs) on melanoma cancer cell migration and the molecular mechanisms underlying these effects using highly metastasis-specific human melanoma cell lines, A375 and Hs294t. Using in vitro cell invasion assays, we observed that treatment of A375 and Hs294t cells with GSPs resulted in a concentration-dependent inhibition of invasion or cell migration of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2 expression and prostaglandin (PG) E(2) production. Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of melanoma cells with COX-2 small interfering RNA, also inhibited melanoma cell migration. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, an inducer of COX-2, enhanced the phosphorylation of ERK1/2, a protein of mitogen-activated protein kinase family, and subsequently cell migration whereas both GSPs and celecoxib significantly inhibited 12-O-tetradecanoylphorbol-13-acetate-promoted cell migration as well as phosphorylation of ERK1/2. Treatment of cells with UO126, an inhibitor of MEK, also inhibited the migration of melanoma cells. Further, GSPs inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Additionally, inhibition of melanoma cell migration by GSPs was associated with reversal of epithelial-mesenchymal transition process, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin and cytokeratins) while loss of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in melanoma cells. Together, these results indicate that GSPs have the ability to inhibit melanoma cell invasion/migration by targeting the endogenous expression of COX-2 and reversing the process of epithelial-to-mesenchymal transition.
黑色素瘤是皮肤疾病导致死亡的主要原因，这在很大程度上是由于其转移的倾向。我们已经研究了葡萄籽原花青素（GSPs）对黑色素瘤癌细胞迁移的影响，以及这些影响的分子机制，使用了高度转移特异性的人黑色素瘤细胞系 A375 和 Hs294t。通过体外细胞侵袭实验，我们观察到 GSPs 处理 A375 和 Hs294t 细胞会导致这些细胞的侵袭或细胞迁移呈浓度依赖性抑制，这与环氧化酶（COX）-2 表达和前列腺素（PG）E（2）产生水平降低有关。用 COX-2 抑制剂塞来昔布或用 COX-2 小干扰 RNA 瞬时转染黑色素瘤细胞处理细胞，也抑制黑色素瘤细胞迁移。用 12-O-十四烷酰佛波醇-13-乙酸酯（COX-2 的诱导剂）处理细胞会增强丝裂原激活蛋白激酶家族蛋白 ERK1/2 的磷酸化，随后促进细胞迁移，而 GSPs 和塞来昔布均可显著抑制 12-O-十四烷酰佛波醇-13-乙酸酯促进的细胞迁移以及 ERK1/2 的磷酸化。用 MEK 的抑制剂 UO126 处理细胞也抑制了黑色素瘤细胞的迁移。此外，GSPs 抑制黑色素瘤细胞中 NF-κB/p65 的激活，NF-κB/p65 是 COX-2 的上游调节剂，用 NF-κB 的抑制剂咖啡酸苯乙酯处理细胞也抑制了细胞迁移。此外，GSPs 通过逆转上皮-间充质转化过程抑制黑色素瘤细胞迁移，导致上皮标志物（E-钙粘蛋白和细胞角蛋白）水平升高，同时黑色素瘤细胞中间充质标志物（波形蛋白、纤维连接蛋白和 N-钙粘蛋白）丢失。总之，这些结果表明 GSPs 通过靶向 COX-2 的内源性表达并逆转上皮-间充质转化过程，具有抑制黑色素瘤细胞侵袭/迁移的能力。
