Ott_a_237474 5057..5067

*These authors contributed equally to this work Background: Diosgenin, a natural steroidal saponin isolated from Trigonella foenumgraecum, has been reported to exert anti-cancer effects. Inhibitors of enhancer of zeste homology 2 (EZH2) have been widely used in treatment of cancers. However, the effects of combined treatment with diosgenin and an EZH2 inhibitor on gastric cancer (GC) cells, and the mechanism for those effects are not fully understood. Methods: AGS and SGC-7901 gastric cancer cells were treated with diosgenin (0 to 8 μM), followed by treatment with either diosgenin or an EZH2 inhibitor, GSK126 alone. Afterwards, an EZH2 overexpression plasmid and Rho inhibitor, GSK429286Awas involved in cells. Cell proliferation, cell cycle distribution,andcell apoptosis,migration, and invasionwereexaminedbyCCK-8assays,flowcytometry, and transwell assays. Western blotting was performed to detect the relative levels of protein expression. Results: Treatment with diosgenin alone caused a dose-dependent decrease in the cell viability, and combined treatment with an EZH2 inhibitor plus GSK126 caused a further significant decrease. A further analysis revealed that treatment with either diosgenin or GSK126 alone induced significant increases in G0/G1 cell cycle arrest and apoptosis, and combined treatment with both agents induced further increases in those parameters. In addition, combined treatment with diosgenin and GSK126 synergistically induced even stronger effects on impaired cell proliferation, G0/G1 phase arrest, and cell apoptosis when compared to treatment with either diosgenin or GSK126 treatment alone. At the molecular level, we demonstrated that inhibition of Rho/ROCK signaling by combined treatment with diosgenin andGSK126could downregulate the expressionof epithelial–mesenchymal transition (EMT)-relatedmolecules.We also found that EZH2 overexpression reversed the anti-tumor effect of diosgenin by inducing cell survival, blocking G1-phase arrest, and promoted EMT. While, these biological properties were further reversed by GSK429286A. Conclusion: Collectively, combined treatment with diosgenin and GSK126 produced even more significant effects on GC cell inhibition by targeting EZH2 via Rho/ROCK signalingmediated EMT, which might be a therapeutic strategy for improving the poor therapeutic outcomes obtained with GSK126 monotherapy.