Abstract
Purpose :
One key precursor to Limbal Stem Cell Deficiency is insufficient Wnt-β-catenin signaling pathway; however, therapeutic tools are underdeveloped as manipulation of hydrophobic WNT proteins proves challenging. This study explored the effect of our small molecule WNT-mimic(MFH-ND) on Wnt-β-catenin signaling pathway using an experimental model of 293STF cells treated with MFH-ND and Wnt3a.
Methods :
HEK 293 (293STF) cells were treated with vehicle (DMSO), 0-4000 ng/mL recombinant human Wnt3a protein with carrier, 0-800 µM of MFH-ND for 18 hours. Cell viability and firefly luciferase activity was measured using TOP-Flash assays following manufacturer’s protocol. Microplate Reader was used to measure cell viability and firefly luciferase activity. The Wnt pathway activity was expressed as the ratio of fluorescence intensity to firefly luciferase. Figure 1 is displayed as Mean ± Standard Error of Mean (SEM). Figure 2 graphs were interpolated from sigmoidal standard curves(Model: Sigmoidal, 4PL, X is concentration) using GraphPad. Hill Coefficient(Model: Specific binding with Hill slope) using Graphpad.
Results :
Per. Figure 1
At MFH-ND concentrations starting from 200 uM to 800 uM, across all the Wnt concentrations, there is a drastic increase in WNT Signaling Activation.
Hill Coefficient was calculated to be 1.113.
Conclusions :
MFH-ND alteration of the Wnt-β-catenin pathway seemed to be an enhancement of the Wnt3a ability to activate the Wnt pathway. Evidence of cooperative binding(n=1.113 and Figure2) as well as boosted Wnt pathway activation(Figure1) point to evidence of signalosome assembly. Wnt3a alone was sufficient to activate the signalosome complex but had a much lower activation when compared to the high concentrations MFH-ND at the same Wnt3a concentration, suggesting MHF-ND was able to recruit a higher activation through a second mechanism, possibly cooperative binding. Figure 1 shows how Wnt pathway activation can be finely altered, which is important when trying to reach a certain therapeutically ideal target. Prospective animal trials will provide more in-vivo understanding of MFH--ND with in-vivo Wnt-β-catenin signaling to see if MFH-ND could be used as potential therapeutic target to Limbal Stem Cell Deficiency, caused by reduced Wnt-β-catenin pathway activation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.