Kobayashi Y, Ohtsuki M, Murakami T, Kobayashi T, Sutheesophon K, Kitayama H, Kano Y, Kusano E, Nakagawa H, Furukawa Y
Division of Stem Cell Regulation, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi, Japan.
Oncogene. 2006 Jan 26;25(4):512-24. doi: 10.1038/sj.onc.1209072.
Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.
组蛋白脱乙酰酶（HDAC）抑制剂有望对难治性癌症有效，因为其作用机制与传统抗肿瘤药物不同。在本研究中，我们检测了HDAC抑制剂FK228对恶性黑色素瘤的作用及其分子机制。与其他常用药物相比，FK228对黑色素瘤具有高效性。通过比较黑色素瘤细胞和正常黑素细胞的基因表达谱，我们确定了一组在黑色素瘤细胞中被FK228特异性上调的基因，其中包括Rap1，一种Ras家族的小GTP结合蛋白。在FK228处理的黑色素瘤细胞中，Rap1 mRNA和蛋白的表达呈剂量和时间依赖性增加。在FK228处理的细胞和Rap1过表达的细胞中，c-Raf、MEK1/2和ERK1/2磷酸化的减少伴随着Rap1表达的增加。用小干扰RNA（siRNA）抑制Rap1上调可消除FK228处理的黑色素瘤细胞中凋亡的诱导和ERK1/2磷酸化的抑制。这些结果表明，FK228的细胞毒性作用是通过Rap1的上调介导的。此外，我们发现Rap1在具有B-Raf V599E突变的黑色素瘤细胞系中过表达，并与B-Raf形成复合物。siRNA介导的Rap1过表达的消除增加了这些细胞的活力，表明Rap1也是黑色素瘤中Ras-MAP激酶信号的内源性调节因子。
