Background. Realgar (REA), a Chinese herbal decoction, has been used to treat various tumors and has produced positive outcomes; however, there is a lack of convincing evidence for the treatment of esophageal cancer. The present study aimed to investigate the effects of REA on esophageal cancer (EC) and explore its mechanism. Methods. EC cells Eca109 and KYSE150 were selected for this study, and different groups of treated cells were set up. We studied the inhibition rate and half inhibition concentration (IC50) by CCK-8 method, the clone formation assay was used to detect the clone formation ability, the scratch assay is used to determine the cell migration ability, the Transwell assay was used to detect the cell invasion ability, the protein expressions of E-cadherin, Slug, N-cadherin, ASK1, p38 MAPK, p-p38 MAPK, and GPX4 were determined using Western blot, the mRNA expressions of ASK1 and p38 MAPK were assessed using qRT-PCR, transmission electron microscopy was used to observe the cellular ultrastructure, Prussian blue staining was used to observe the intracellular iron particle distribution, and biochemical analysis of cellular MDA, SOD, GSH, and GPXS activities, flow cytometric analysis of cellular ROS levels, immunofluorescence staining to detect cellular GPX4 expression, and JC-1 method to detect mitochondrial membrane potential were used. Results. REA inhibited the proliferation of Eca109 and KYSE150 cells in a time- and concentration-dependent manner, and REA significantly inhibited the migration and invasion of Eca109 and KYSE150 cells and activated the cellular ferroptosis and ROS-ASK1-p38 MAPK signaling pathways ( P < 0.05 ). Inhibition of activation of the ROS-ASK1-p38 MAPK signaling pathway promoted the inhibition of proliferation, migration, and invasion of Eca109 and KYSE150 cells and the induction of ferroptosis by REA. Conclusion. REA induced ferroptosis and inhibited the migration of EC cells by activating the ROS-ASK1-p38 MAPK signaling pathway.

Background: Realgar, a Chinese herbal decoction, has been used to treat various types of tumors with positive outcomes; however, there is a lack of convincing evidence on its use for the treatment of esophageal cancer (EC). In this study, the role of the p62-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the regulation of EC cell proliferation, migration, and ferroptosis in response to realgar was assessed Methods: Different concentrations of realgar (0, 10, 20, 40, 60, 80, and 100 µmol/L) were applied to the EC cell lines Eca109 and KYSE150. The inhibition rate and half-inhibitory concentration (IC50) were determined using the Cell Counting Kit-8 (CCK-8) method. Subsequently, the cells were treated with realgar (1/2IC50, IC50, 2IC50). Cell migration was measured using the scratch assay, and cell invasion was measured using the transwell assay. The mRNA expression of p62, Keap1, and Nrf2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expression of p62, Keap1, Nrf2, matrix metalloproteinase (MMP)-2, MMP-9, E-cadherin, Slug, N-cadherin, and vimentin was measured by Western blot. The control, 2IC50, shRNA-NC, shRNA-p62, 2IC50 + shRNA-NC, 2IC50 + shRNA-p62, shRNA-Keap1, 2IC50 + shRNA-Keap1, and 2IC50 + shRNA-p62 + shRNA-Keap1 groups were defined. The CCK-8 method was used to measure the cell inhibition rate, and the clone formation assay was used to measure the clone formation ability. Moreover, the scratch assay was used to detect the cell migration ability, and the transwell assay was used to detect the cell invasion ability. Transmission electron microscopy was used to observe the mitochondrial morphology, Prussian blue staining was used to observe the intracellular iron particle distribution, and flow cytometry was used to detect changes in intracellular reactive oxygen species. In addition, qRT-PCR was performed to detect p62, Keap1, Nrf2, and glutathione peroxidase 4 (GPX4) mRNA expression, and Western blot was performed to detect p62, Keap1, Nrf2, E-cadherin, Slug, N-cadherin, and GPX4 protein expression. Results: Realgar inhibited Eca109 and KYSE150 cell proliferation in a time- and concentrationdependent manner. It also significantly inhibited the migration and invasion of Eca109 and KYSE150 cells and affected the mRNA and protein expression of p62, Keap1, and Nrf2. In response to realgar, low p62 expression inhibited the proliferation, migration, and invasion of Eca109 and KYSE150 cells, as well as ferroptosis induction. Conclusion: The findings demonstrate that inhibiting the p62-Keap1-Nrf2 signaling pathway promotes the inhibitory effects of realgar on EC cells.

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