Inhibition of p62-Keap1-Nrf2 Pathway Activation by Realgar Promotes
the Inhibition of Esophageal Cancer Cell Proliferation, Migration, and
Ferroptosis



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



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.



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.



The findings demonstrate that inhibiting the p62-Keap1-Nrf2 signaling pathway promotes
the inhibitory effects of realgar on EC cells.
