Involvement of EGFR-AKT signaling in hemin-induced neurotoxicity

Abstract

Intracerebral hemorrhage (ICH), as bleeding from ruptured vessels within the brain, is the second leading neuropathological problem following ischemic stroke. In the present study, the involvement of epithelial growth factor receptor (EGFR)-tyrosine kinase (TK) signaling underlying ICH-related neurodegeneration was investigated using afatinib, a clinically available EGFR-tyrosine kinase inhibitor (EGFR-TKI). We employed hemin (a breakdown product of hemoglobin) to mimic the pathophysiology of ICH in primary cultured cortical neurons. Using a lactate dehydrogenase (LDH) assay, incubation of hemin concentration- and time-dependently induced neuronal death. Simultaneous incubation of afatinib (10 nM) significantly inhibited hemin (30 μM)-induced neuronal death. Immunofluorescent data demonstrated that co-treatment of afatinib for 1 h attenuated hemin (30 μM)-induced elevation in phosphorylated-EGFR (p-EGFR) immunoreactivity and neurite impairment. Western blot assay demonstrated that co-incubation of afatinib for 16 h diminished hemin-induced elevation in p-EGFR and p-AKT, tumor necrosis factor-α and cyclooxygenase 2 (two proinflammatory biomarkers) as well as heme oxygenase-1 (HO-1, an enzyme catalyzing heme/hemin), glutathione hydroperoxidase 4 and receptor-interacting protein 3 (two biomarkers of ferroptosis and necroptosis). In addition, co-treatment of afatinib for 24 h inhibited hemin-induced NO production in the culture medium. In conclusion, our study shows that afatinib via blocking EGFR-AKT signaling inhibits hemin-induced EGFR-AKT activation, neuroinflammation, HO-1 expression and programed cell death, suggesting that EGFR-AKT signaling is involved in hemin-induced neurotoxicity and may be a druggable target for ICH.

Keywords: EGFR-AKT signaling; afatinib; ferroptosis; hemin; primary cultured cortical neurons.

FIGURE 1

Effects of afatinib on hemin-induced cytotoxicity. (A) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 16 h and 24 h. (B) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) simultaneously for 16 h. Cell death was measured by LDH assay. Values are the mean ± S.E.M. (n = 3/each group). *p < 0.05 statistically significant in the hemin groups compared with the control groups; ^#P < 0.05 in hemin plus afatinib compared with hemin alone by one-way ANOVA followed by the LSD test as post-hoc method.

FIGURE 2

Effects of afatinib on hemin-induced EGFR-AKT activation. (A,B) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 1 h (C,D) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 1h. Western blot assay was employed to measure phosphorylated EGFR (p-EGFR) (A,C), and phosphorylated AKT (p-AKT) (B,D). Each lane contained 30 μg protein for all experiments. Graphs show statistical results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *p < 0.05 statistically significant in the hemin groups compared with the control groups; ^#P < 0.05 in hemin plus afatinib compared with hemin alone by one-way ANOVA followed by the LSD test as post-hoc method.

FIGURE 3

Effects of afatinib on hemin-induced impairment of neurite outgrowth. (A) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 1 h. Representative immunofluorescent data show concentration-dependent damages of hemin on neurite outgrowth. (B) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 1 h. The neurites were immunostained with phosphorylated EGFR (p-EGFR) and MAP-2. Calibration: 10 μm. The results were duplicated.

FIGURE 4

Effects of afatinib on hemin-induced neuroinflammation. (A, B) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 16 h. (C,D) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 16 h. Western blot assay was employed to measure TNF-α (A,C) and COX2 (B,D). Each lane contained 30 μg protein for all experiments. Graphs show statistical results from relative optical density of bands on the blots. (E) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 24 h. The levels of NO in culture medium were measured using Griess reaction. Values are the mean ± S.E.M. (n = 3/each group). *p < 0.05 statistically significant in the hemin groups compared with the control groups; ^#P < 0.05 in hemin plus afatinib compared with hemin alone by one-way ANOVA followed by the LSD test as post-hoc method.

FIGURE 5

Effects of afatinib on hemin-induced HO-1 expression. (A) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 16 h. (B) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 16 h. Western blot assay was employed to measure HO-1. Each lane contained 30 μg protein for all experiments. Graphs show statistical results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *p < 0.05 statistically significant in the hemin groups compared with the control groups; ^#P < 0.05 in hemin plus afatinib compared with hemin alone by one-way ANOVA followed by the LSD test as post-hoc method.

FIGURE 6

Effects of afatinib on hemin-induced programmed cell death. (A,B) Primary cultured cortical neurons were treated with hemin (10–60 μM) for 16 h. (C,D) Primary cultured cortical neurons were treated with hemin (30 μM) plus afatinib (10 nM) for 16 h. Western blot assay was employed to measure GPX4 (A,C) and RIP3 (B,D). Each lane contained 30 μg protein for all experiments. Graphs show statistical results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *p < 0.05 statistically significant in the hemin groups compared with the control groups; ^#P < 0.05 in hemin plus afatinib compared with hemin alone by one-way ANOVA followed by the LSD test as post-hoc method.