Inhibitory effect of daphnetin on gastric cancer cells and its mechanism

Abstract

Objective To explore the inhibitory effect of daphnetin (Dap) on gastric cancer cells and its mechanism. Methods Gastric cancer AGS cells were treated with Dap at concentrations of 0, 30, 60, 90, 120, and 150 μmol/L, and the effect on cell viability was evaluated after 48 h using the MTT assay. AGS cells were divided into groups A to E, and each group was treated with Dap at concentrations of 0, 30, 60, 90, 120, and 150 μmol/L, respectively, for 48 h. Each group was observed for changes in cell count and morphology using optical microscopy and crystal violet staining. DCF staining was used to evaluate the effect of Dap on intracellular reactive oxygen species (ROS) levels in AGS cells in groups A to D, while JC-1 staining was used to evaluate the effect of Dap on the level of mitochondrial membrane potential (MMP) in AGS cells in groups A to D. Western blot was conducted to measure the expression levels of LC3 and P62 proteins in AGS cells in groups A to D, and changes in autophagic flux in AGS cells were measured by Ad-mCherry-GFP-LC3B staining. Results Dap significantly inhibited AGS cell viability in a concentration-dependent manner (F=321.50,t=12.44-34.77,P<0.05), with an IC50 of approximately 90 μmol/L at 48 h of treatment. After 48 h of Dap treatment, the number of AGS cells in groups A to E gradually decreased, and the number of twisted and wrinkled AGS cells gradually increased with the increase in Dap concentration. Intracellular ROS in AGS cells in groups A to D also gradually increased with the increase in Dap concentration. The ratio of JC-1 green/red fluorescence intensity in AGS cells in groups C and D was significantly higher than that in group A (F=10.92,t=3.09,4.96,P<0.05). Western blot revealed that groups B to D had a significant increase in the ratio of LC3B/LC3A (F=36.46,t=3.17-9.78,P<0.05) and in the expression of P62 (F=109.90,t=12.38-16.78,P<0.05) compared to group A. Ad-mCherry-GFP-LC3B staining revealed that Dap inhibited the fusion of autophagosomes with lysosomes, resulting in the blockage of autophagic flux. Conclusion Dap can inhibit the proliferation of gastric cancer cells, and its mechanism of action may be related to inducing significant production of ROS within gastric cancer cells and blocking the autophagic flux.

Keywords

• stomach neoplasms|daphnetin|autophagy|reactive oxygen species|membrane potential, mitochondrial