Background: Calycosin, an active compound in plants, can promote the apoptosis of various cancer cells; however, the mechanism by which it regulates reactive oxygen species (ROS) in gastric cancer (GC) cells remains unclear. Purpose: In this study, we investigated the effects of calycosin on apoptosis, the cell cycle, and migration in GC cells under ROS regulation. Results: The results of the Cell Counting Kit-8 assay suggested that calycosin had significant cytotoxic effects on 12 gastric cancer cells, but no significant cytotoxic effects on normal cells. Hoechst 33342/propidium iodide (PI) double staining and flow cytometry showed that calycosin had clear pro-apoptotic effects on AGS cells. Western blotting revealed that the expression of cytochrome C and pro-apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), cleaved (cle)-caspase-3, and cle-poly (ADP-ribose) polymerase gradually increased, and the expression of anti-apoptotic protein Bcl-2 gradually decreased. Calycosin also decreased the expression of extracellular signal-regulated kinase, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation levels of p38, c-Jun N-terminal kinase, and inhibitor of NF-κB. In addition, calycosin markedly increased ROS accumulation, and pretreatment with active oxygen scavenger n-acetyl-l-cysteine (NAC) clearly inhibited apoptosis. Calycosin downregulated the cell cycle proteins cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclin D1, and cyclin E; upregulated p21 and p27; and arrested cells in the G0/G1 phase. Similarly, calycosin also downregulated Snail family transcriptional repressor 1, E-cadherin, and β-catenin and inhibited cell migration. However, pretreatment with NAC inhibited the calycosin-induced effects of cycle arrest and migration. Conclusion: In summary, calycosin induces apoptosis via ROS-mediated MAPK/STAT3/NF-κB pathways, thereby exerting its anti-carcinogenic functions in GC cells.

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up‐regulated. Transgenic mice were generated that express a dominant‐negative RhoA transgene in ameloblasts using amelogenin gene‐regulatory sequences. Transgenic and wild‐type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F‐actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho‐associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E‐cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E‐cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.