Acrylamide induces human chondrocyte cell death by initiating autophagy‑dependent ferroptosis

Abstract

Acrylamide (ACR) is formed during heat treatment of foodstuffs and ACR may serve as a probable malignant neoplastic disease agent in all organs and tissues of the human body. However, it is unknown if ACR is associated with ankylosing spondylitis (AS) pathogenesis. Cell viability and proliferation were determined using CCK-8 assay and EdU staining. Flow cytometry was used to determine cell death and cell cycle arrest. Intracellular lipid reactive oxygen species, Fe²⁺ and mitochondrial membrane potential (MMP) were analyzed using a C11-BODIPY581/591 fluorescent probe, FerroOrange staining and a JC-1 MMP Assay kit, respectively. The present study showed that ACR decreased chondrocyte cell viability in a dose-dependent manner and that ACR significantly promoted chondrocyte senescence. ACR also elevated the expression of cell cycle arrest-associated proteins, including p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein, in human chondrocytes. Similarly, DNA damage was also enhanced following ACR treatment in chondrocytes. In addition, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the autophagy inhibitor 3-methyladenine abolished ACR-induced cell death in chondrocytes. ACR was shown to activate autophagic flux and induce mitochondrial dysfunction by increasing the MMP. Western blot analysis of ferroptosis-related proteins demonstrated that ACR decreased the expression of glutathione peroxidase 4, solute carrier family 7 member 11, transferrin receptor protein 1 and ferritin heavy chain 1 in chondrocytes whereas Fer-1 abolished these effects. ACR treatment significantly elevated the phosphorylation levels of AMP-activated protein kinase (AMPK) and serine/threonine-protein kinase ULK1 in human chondrocytes. Notably, the effect of ACR was diminished by knockdown of AMPK, as evidenced by reduced lipid reactive oxygen species accumulation and Fe²⁺ levels. Hence, ACR inhibited cell proliferation and contributed to cell death by inducing autophagy-dependent ferroptosis while promoting autophagy by activating AMPK-ULK1-mTOR signaling in human chondrocytes. It was hypothesized that the presence of ACR in foodstuffs may increase the risk of AS and that decreasing ACR in food products is of importance.

Keywords: AMPK/ULK1/mTOR signaling; acrylamide; ankylosing spondylitis; autophagy; ferroptosis.

Figure 1

Cell Counting Kit-8 assay determination of IC₅₀ of acrylamide in human chondrocytes. ^*P<0.05, ^**P<0.01; ^***P<0.001 vs. 0 µg/ml ACR. IC₅₀, half maximal inhibitory concentration.

Figure 2

ACR suppresses chondrocyte proliferation. (A) EdU staining showed that ACR decreased the number of EdU-positive cells in human chondrocytes (magnification, x20; scale bar, 30 µm). (B) Flow cytometry analysis showed that ACR induced cell cycle arrest in human chondrocytes. ^***P<0.001 vs. Con. ACR, acrylamide; Con, control.

Figure 3

ACR induces human chondrocyte senescence. (A) SA-β-gal staining showed that 0.35 µg/ml ACR significantly increased the number of SA-β-gal-positive cells (magnification, x20; scale bar, 20 µm). (B) Immunofluorescence staining showed that the relative fluorescence of γ-H2A histone family member X was increased in human chondrocytes treated with ACR (magnification, x40; scale bar, 10 µm). (C) Flow cytometry showed that ACR increased the number of dead cells. (D) Western blot assay showed that expression levels of p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein were significantly increased in human chondrocytes treated with ACR. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Con. ACR, acrylamide; Con, control; SA-β-gal, senescence-associated β-galactosidase; γ-H2AX, γ-H2A histone family member X.

Figure 4

Fer-1 and 3-MA reverse ACR-induced cell death in human chondrocytes. (A) CCK-8 assay showed that 0.35 µg/ml ACR decreased human chondrocyte cell viability at 24, 48 and 72 h. (B) Fer-1 and 3-MA significantly reversed ACR-induced cell death. ^**P<0.01 and ^***P<0.001 vs. Con. ^##P<0.01 vs. ACR and ^###P<0.001. Fer-1, ferrostatin-1; 3-MA, 3-methyladenine; ACR, acrylamide; Con, control; Nec-1, necrostatin-1; Z-VAD-MFK, benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone.

Figure 5

ACR induces autophagy in human chondrocytes. (A) Transmission electron microscopy demonstrated that ACR induced rupture of the mitochondrial membrane in human chondrocytes (magnification, x400; scale bar, 2 µm). (B) ACR increased the number of red puncta in human chondrocytes whereas preincubation with 3-MA reversed these effects (magnification, x40; scale bar, 10 µm). (C) Western blot analysis showed that ACR increased LC3II/LC3I ratio whereas it decreased sequestosome 1 expression in human chondrocytes. ^**P<0.01 and ^***P<0.001 vs. Con. ^##P<0.01 and ^###P<0.001 vs. ACR. ACR, acrylamide; Con, control; p62, sequestosome 1; 3-MA, 3-methyladenine; LC, light chain; M, mitochondria.

Figure 6

ACR promotes ferroptosis in human chondrocytes. (A) Cell mitochondrial membrane potential was detected in human chondrocytes treated with ACR (magnification, x20; scale bar, 20 µm). (B) FerroOrange staining showed that ACR increased intracellular Fe²⁺ levels in human chondrocytes (magnification, x20; scale bar, 20 µm). (C) C11 BODIPY fluorescent staining showed that ACR increased lipid-ROS accumulation (magnification, x20; scale bar, 20 µm). (D) Fer-1 attenuates ACR-induced upregulation of ROS and malondialdehyde content in human chondrocytes. (E) Western blot analysis demonstrated that ACR decreased expression of GPX4, SLC7A11, TfR1 and FTH1 in chondrocytes. ^*P<0.05 vs. Con, ^**P<0.01 and ^***P<0.001; ^#P<0.05, ^##P<0.01 vs. ACR. ACR, acrylamide; Con, control; ROS, reactive oxygen species; MDA, malondialdehyde; GPX4, glutathione peroxidase 4; TfR1, transferrin receptor protein 1; SLC7A11, solute carrier family 7 member 11; FTH1, ferritin heavy chain 1; Fer-1, ferrostatin-1.

Figure 7

ACR activates AMPK/ULK1 signaling in human chondrocytes. (A) Western blot analysis showed that ACR increased phosphorylation of AMPK and ULK1 while suppressing the activation of mTOR in human chondrocytes at 24 and 48 h. (B) Western blot assay showed that transfection with siRNA-AMPK significantly suppressed expression of AMPK. (C) Immunofluorescence staining showed that siRNA AMPK significantly suppressed fluorescence density of AMPK in human chondrocytes even in the presence of ACR (magnification, x20; scale bar, 20 µm). (D) Transmission electron microscopy showed that ACR-induced mitochondrial membrane rupture was notably reversed by silencing AMPK in human chondrocytes (magnification, x400; scale bar, 2 µm). Upregulation of (E) lipid ROS and (F) Fe²⁺ was blocked by transfection with siRNA-AMPK (magnification, x20; scale bar, 20 µm). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Con. AMPK, AMP-activated protein kinase; ACR, acrylamide; Con, control; NC, negative control; p-, phosphorylated; si, small interfering.