In our search for bioactive mushrooms native to British Columbia, we determined that the ethanol extracts from fruiting bodies of the terrestrial polypore Albatrellus flettii had potent anti-cell viability activity. Using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance, we successfully isolated three known compounds (grifolin, neogrifolin and confluentin). These compounds represent the major anti-cell viability components from the ethanol extracts of A. flettii. We also identified a novel biological activity for these compounds, specifically in down-regulating KRAS expression in two human colon cancer cell lines. Relatively little is known about the anti-cell viability activity and mechanism of action of confluentin. For the first time, we show the ability of confluentin to induce apoptosis and arrest the cell cycle at the G2/M phase in SW480 human colon cancer cells. The oncogenic insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) has been previously shown to regulate KRAS mRNA expression in colon cancer cells, possibly through its ability to bind to the KRAS transcript. Using a fluorescence polarization assay, we show that confluentin dose-dependently inhibits the physical interaction between KRAS RNA and full-length IMP1. The inhibition also occurs with truncated IMP1 containing the KH1 to KH4 domain (KH1to4 IMP1), but not with the di-domain KH3 and KH4 (KH3&4 IMP1). In addition, unlike the control antibiotic neomycin, grifolin, neogrifolin and confluentin do not bind to KRAS RNA. These results suggest that confluentin inhibits IMP1-KRAS RNA interaction by binding to the KH1&2 di-domains of IMP1. Since the molecular interaction between IMP1 and its target RNAs is a pre-requisite for the oncogenic function of IMP1, confluentin should be further explored as a potential inhibitor of IMP1 in vivo.

中文翻译：

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灰质多孔孢子藻的Grifolin，neogrifolin和confluentin抑制人结肠癌细胞中KRAS的表达。

在寻找不列颠哥伦比亚省本土的生物活性蘑菇时，我们确定了从陆地多孢子藻（Albaterllus flettii）子实体中提取的乙醇具有有效的抗细胞活力。使用生物测定指导的分馏，质谱和核磁共振，我们成功分离了三种已知化合物（grifolin，neogrifolin和confluentin）。这些化合物代表了A. flettii乙醇提取物中主要的抗细胞活力成分。我们还确定了这些化合物的新生物活性，特别是在下调两个人结肠癌细胞系中的KRAS表达中。关于confluentin的抗细胞活力活性和作用机理的了解相对较少。首次，我们展示了confluentin诱导SW480人结肠癌细胞凋亡并阻止G2 / M期细胞周期的能力。先前已显示，致癌性胰岛素样生长因子2 mRNA结合蛋白1（IMP1）可能通过其结合KRAS转录物的能力来调节结肠癌细胞中KRAS mRNA的表达。使用荧光偏振测定，我们表明，confluentin剂量依赖性抑制KRAS RNA和全长IMP1之间的物理相互作用。截短的IMP1包含KH1至KH4结构域（KH1to4 IMP1），但双结构域KH3和KH4（KH3＆4 IMP1）不存在抑制作用。此外，与对照抗生素新霉素不同，格雷夫林，新格列佛灵和融合蛋白不与KRAS RNA结合。这些结果表明，融合蛋白通过与IMP1的KH1＆2双结构域结合而抑制IMP1-KRAS RNA相互作用。由于IMP1及其靶RNA之间的分子相互作用是IMP1致癌功能的先决条件，因此应进一步探索confluentin作为体内IMP1的潜在抑制剂。