Fucoxanthin Induces Ferroptosis in Cancer Cells via Downregulation of the Nrf2/HO-1/GPX4 Pathway

Abstract

This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.

Keywords: Nrf2/HO−1/GPX4 pathway; anti−cancer; ferroptosis; fucoxanthin.

Figure 1

Effects of different fucoxanthin concentrations (0, 1, 2, 3, 4, 5, 6, 7, or 8 nM) for 6, 12, 18, or 24 h on SCC−25 tongue cancer and 293T embryonic kidney cell viability. (A) Chemical structure of fucoxanthin. (B) SCC−25 cell viability after fucoxanthin treatment relative to control group (no drug treatment). (C) 293T cell viability after fucoxanthin treatment relative to control group. Data are means ± standard deviations (SDs; n = 5). * p < 0.05, ** p < 0.01, compared with control.

Figure 2

Effects of different fucoxanthin concentrations (0, 2, 4, or 6 nM) for 24 h on ROS production in SCC−25 cells. (A) Control group, (B) 2 nM fucoxanthin, (C) 4 nM fucoxanthin, (D) 6 nM fucoxanthin, and (E) quantitative analysis of fluorescence intensity in SCC−25 cells. (F) Fold change of fluorescence intensity (relative to control) in SCC−25 cells. * p < 0.05, ** p < 0.01, compared with control.

Figure 3

Effects of different fucoxanthin concentration (0, 2, 4, or 6 nM) for 24 h on antioxidant enzyme activities, Fe level, and MDA level in SCC−25 cells. (A) MDA level, (B) GSH activity, (C) SOD activity, and (D) Fe level. * p < 0.05, ** p < 0.01, compared with control.

Figure 4

Fucoxanthin−induced apoptosis in SCC−25 cells. Data are means ± SDs (n = 3). (A) Flow cytometry was utilized to evaluate apoptosis after 24 h of treatment with 0, 2, 4, and 6 nM of fucoxanthin. (B–D) Graphs showing the effects of fucoxanthin on the overall, early, and late apoptosis rates in SCC−25 cells. * p < 0.05, ** p < 0.01 compared with the control group.

Figure 5

Effects of fucoxanthin for 24 h on mitochondrial membrane potential in SCC−25 cells. Data are means ± SDs (n = 3). (A) Control group, (B) 2 nM fucoxanthin, (C) 4 nM fucoxanthin, (D) 6 nM fucoxanthin, (E) percentages of JC−1 multimers and monomers, and (F) JC−1 red/green fluorescence ratio. * p < 0.05, ** p < 0.01, compared with control.

Figure 6

Effects of different fucoxanthin concentrations (0, 2, 4, or 6 nM) for 24 h on ferroptosis−related gene expression in SCC−25 cells. Data are means ± SDs (n = 3). (A–G) Quantitative measurements of the effects of fucoxanthin on Nrf2, SLC7A11, HO−1, Keap1, GPX4, TFR1, and p53 mRNA expression in SCC−25 cells, respectively. * p < 0.05, ** p < 0.01 compared with the control group.

Figure 7

Effects of different fucoxanthin concentrations (0, 2, 4, or 6 nM) for 24 h on the Keap1/Nrf2/HO−1 signaling pathway in SCC−25 cells. Data are means ± SDs (n = 3). (A) Qualitative depiction of the effects of fucoxanthin on ferroptosis−related proteins in SCC−25 cells. (B–H) Quantitative measurements of the effects of fucoxanthin on Keap1, Nrf2, p53, SLC7A11, HO−1, GPX4, and TFR1 protein expression in SCC−25 cells, respectively. * p < 0.05, ** p < 0.01, compared with control.

Figure 8

Molecular docking of fucoxanthin with target proteins. Optimal conformations of fucoxanthin binding to p53 (A), SLC7A11 (B), GPX4 (C), Keap1–Nrf2 (D), HO−1 (E), and TFR1 (F), with key residues around each binding site. Hydrogen bonds are presented as yellow dashed lines with distances in angstroms (Å). Two−dimensional schematic diagrams depict interactions of fucoxanthin with active sites of target proteins. Dotted lines represent hydrogen bonds (dark green), π–σ T−shaped forces (dark purple), and hydrophobic interactions (purple) with surrounding amino acid residues. Acidic residues in light green denote van der Waals forces.