Prassas Ioannis, Paliouras Miltiadis, Datti Alessandro, Diamandis Eleftherios P
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Clin Cancer Res. 2008 Sep 15;14(18):5778-84. doi: 10.1158/1078-0432.CCR-08-0706.
Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value.
A cell-based high-throughput screening (HTS) of three small molecule libraries ( approximately 4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs.
The initial screening resulted in 66 "putative hits" that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 "validated hits." These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC(50)s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression.
We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides.
人组织激肽释放酶（KLK）由15种同源分泌型丝氨酸蛋白酶组成一个亚群。KLK主要因其作为癌症生物标志物（如前列腺特异性抗原）的临床用途而为人所知，最近它们被直接牵涉到与癌症相关的过程中，包括侵袭、血管生成和肿瘤生长调节。因此，鉴定能够调节KLK表达的化合物可能具有相当大的治疗价值。
对三个小分子文库（约4500种化合物）进行了基于细胞的高通量筛选（HTS）；用灵敏的酶联免疫吸附测定法（ELISA）评估乳腺癌细胞系MDA-MB-468中KLK的表达。
初步筛选产生了66个“假定命中物”，与对照相比，它们使KLK5的表达降低了至少50%。二次筛选和微量剂量反应测定产生了21个“验证命中物”。这21种化合物仅聚集在三个不同的功能家族中，并在体外进一步分析以确定它们的有效性（半数抑制浓度[IC(50)]）。未显示剂量反应性或干扰细胞活力的命中物被排除。发现强心苷家族的多个成员是KLK表达的新型抑制剂，在低浓度（10 - 50纳摩尔/升）下起作用。此外，同一家族的成员以与KLK抑制相关的剂量依赖性方式诱导c-MYC和c-FOS表达显著降低，提示存在调节KLK表达的转录机制。
我们得出结论，强心苷可显著抑制KLK的转录，且这些作用可能与原癌基因（c-myc/fos）的表达有关。这些发现可能部分解释了强心苷最近所显现出的抗肿瘤作用。
