The Wnt/β-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/β-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/β-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/β-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (β-catenin) was not decreased, suggesting that 1 inhibited the Wnt/β-catenin signaling pathway without affecting β-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-β-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/β-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/β-catenin signaling pathway.

Graphical abstract

中文翻译：

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Linderapyrone 类似物 LPD-01 通过靶向导入作为癌症治疗剂7

Wnt/ β-连环蛋白信号通路在多种癌细胞中发挥重要作用，包括细胞增殖和发育。我们此前成功合成了一种抑制Wnt/ β -catenin信号通路的小分子化合物，命名为LPD-01( 1 )，1可抑制人结直肠癌(HT-29)细胞的生长。在这项研究中，我们发现1对 HT-29 细胞生长的抑制作用强于另一种人类结直肠癌 (SW480) 细胞。因此，我们试图鉴定HT-29细胞中1的靶蛋白。首先，我们研究了对Wnt/ β -catenin信号通路相关蛋白表达水平的影响。结果，1抑制了Wnt/ β -catenin信号通路靶蛋白（c-Myc和Survivin）及其基因的表达，而转录辅激活因子（ β -catenin）的量没有减少，表明1抑制 Wnt/ β -catenin 信号通路而不影响 β-catenin。接下来，我们使用 FG 磁珠研究了1的靶蛋白。化学下拉分析与质谱分析相结合表明1直接与 importin7 结合。正如预期的那样，1抑制了 TGF- β刺激的 HT-29 细胞中 importin7 货物（例如 Smad2 和 Smad3）的核转位。此外，siRNA敲低importin7降低了Wnt/ β -catenin信号通路靶基因的表达。这些结果表明 importin7 是1抑制 Wnt/ β -catenin 信号通路的靶蛋白之一。

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