In vitro and in silico evaluation of yamogenin and acid stimulation axis via Otopetrin-1 in the acid receptors–colorectal cancer linkage identified by virtual screening

Objective: The aim of this thesis is to investigate whether yamogenin, a steroidal saponin identified through virtual screening, regulates the proliferation and migration capacities of colorectal cancer cells via Otopetrin proteins. Another objective is to elucidate the potential effect of environmental acidity on yamogenin's interaction with the Otopetrin-1 protein. Method: The effects of yamogenin, both alone and in combination with lactic acid stimulation, were examined in vitro on Caco-2 cells. Proliferation, migration, and OTOP1 protein levels were evaluated using MTT, wound-healing, and ELISA assays, respectively. Additionally, the interaction between yamogenin and the Otopetrin protein family was analyzed using molecular doking methods. Results: The antiproliferative effect of yamogenin on Caco-2 cells was observed even at low concentrations, and acidic conditions further enhanced this effect. Low-dose lactic acid stimulation increased proliferation, whereas high-dose lactic acid exhibited cytotoxic effects. Migration analysis revealed that both yamogenin and high-dose lactic acid inhibited Caco-2 cell migration. The expression of the OTOP1 protein was found to be inversely correlated with proliferation levels. Molecular doking results supported these findings, showing that yamogenin binds to all Otopetrin proteins with high affinity, with the highest binding affinity observed in the central pocket of Otopetrin-2. Conclusion: The findings demonstrate that yamogenin exhibits antiproliferative and antimetastatic effects on Caco-2 cells under acidic microenvironmental conditions. The acidic environment enhanced the cytotoxic effects of yamogenin and suppressed cell migration. The expression of the Otopetrin-1 protein in colorectal cancer cells was demonstrated for the first time in this study. Furthermore, it was confirmed that yamogenin interacts strongly with the Otopetrin-1 protein following lactic acid stimulation. These results suggest that Otopetrin-1 may serve as a potential target protein in colorectal cancer therapy.