3.1 ISX9 activates the Wnt/β-catenin signaling pathway
In order to identify novel small molecules that modulate the
Wnt/β-catenin signaling pathway, a cell-based SuperTOPFlash reporter
system was used to screen a known-compound library. Our screening
identified ISX9, a small molecule with the isoxazole scaffold, as a
potential activator of Wnt/β-catenin signaling (Figure 1A and B). To
determine the agonistic effect on the Wnt/β-catenin pathway, the
luciferase reporter SuperTOPFlash was transfected into HEK293T cells.
Treatment with 2.5-40 µM ISX9 strongly increased the transcriptional
activity of the SuperTOPFlash reporter in a dose-dependent manner. ISX9
at 40 µM could induce the reporter activity ~2700-fold
above the basic levels (Figure 1C). However, ISX9 had little effect on
the activity of negative control reporter SuperFOPFlash, which contains
mutated TCF-binding elements. As anticipated, lithium chloride (LiCl), a
well-known activator of Wnt signaling, remarkably increased the activity
of the SuperTOPFlash reporter (Figure 1D).
To further confirm the effect of ISX9 on Wnt signaling, we performed the
SuperTOPFlash reporter assay in human keratinocyte HaCaT cells.
Similarly, ISX9 dose-dependently enhanced the activity of the
SuperTOPFlash reporter in HaCat, while it did not affect the
SuperFOPFlash reporter activity (Figure 1E). Together, these results
demonstrated that ISX9 is capable of activating the Wnt/β-catenin
pathway.