Hydroxytyrosol induced ferroptosis through Nrf2 signaling pathway in colorectal cancer cells

Abstract

In recent years, the incidence of colorectal cancer is still on the rise. The killing of tumor cells through chemotherapy and/or radiation therapy is the mainstay of clinical anticolorectal cancer therapy, but is limited by drug and radiation resistance of tumor cells. Ferroptosis, a novel mode of programmed cell death, plays an important role in antitumor therapy. Ferroptosis inducers have been extensively studied as a strategy to target drug-resistant cancers. The aim of this study is to investigate the mechanism by which hydroxytyrosol (HT) induces ferroptosis in colorectal cancer cells via the Nrf2 signaling pathway. The goal of this study is to use network pharmacology and molecular docking approaches to screen and confirm hydroxytyrosol targets for the treatment of colorectal cancer. The response of colorectal cancer cells to hydroxytyrosol was assessed by cell viability, colony formation assay and scratch assay. Additionally, molecular techniques, including Western blotting and fluorescent probe technology, were employed. The network pharmacological screen identified 14 core targets. Among these genes, nuclear factor-erythroid 2 related factor 2 (Nrf2) was identified as the top target. Molecular docking revealed enhanced binding activity for HT with targets related to oxidative stress, including Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), thioredoxin reductase 1 (TrxR1), prostaglandin-endoperoxide synthase 2 (PTGS2) and aldo-keto reductase 1C3 (AKR1C3). HT-induced ferroptosis elevates iron levels, lipid peroxidation (LPO) and reactive oxygen species (ROS), while decreasing glutathione (GSH) and mitochondrial membrane potential. Moreover, HT reduced the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) proteins while increasing the expression of Tfr1 protein. Changes in the expression levels of these proteins led to an increase in soluble iron pools, which in turn promoted lipid peroxidation. Notably, the ferritin deposition inhibitor ferroprostatin-1 (Fer-1) significantly reversed this process. Additionally, the levels of protein expression of Nrf2 and NQO1 were reversed by two activators of Nrf2, bardoxolone (CDDO) and sulforaphane (SFN). In summary, we provide evidence that HT may induce ferroptosis in colorectal cancer cells. Mechanistically, HT induces ferroptosis via the Nrf2 signaling pathway.

Keywords: Colorectal cancer; Ferroptosis; Hydroxytyrosol; Nrf2 signaling pathway.

Fig. 1

Network pharmacology and molecular docking predict HT-induced pathway in colorectal cancer cells. (A) Potential targets of HT; (B) Key targets of HT colorectal cancer; (C) Venn diagram plot screening on key targets of HT colorectal cancer ferroptosis; (D) PPI network topology analysis; (E) Degree value ranking of the 14 core targets; (F) HT with Nrf2 (NFE2L2), NQO1, TXNRD1, PTGS2, AKR1C3 molecular docking results; (G) molecular docking binding energy ranking.

Fig. 2

HT inhibited the proliferation, cloning, and migration of cells, NAC pretreatment reduced the cytotoxicity of HT, and HT treatment disrupted the cytoskeleton. (A,B) Cell viability was measured using the CCK8 method after treatment with HT (0µM, 50µM, 100µM, 150 µM, and 200µM for HCT116 cells; 0µM, 40µM, 80µM, 160 µM, and 320µM for SW480 cells) for 24 and 48 h; (C,D) NAC (3mM) pretreatment significantly reduced the cytotoxicity induced by HT; (E,F) HT inhibited the clonogenicity of HCT116 and SW480 cells; (G, H) the inhibitory effect of HT on migratory ability was examined in HCT116 and SW480 cells; (I,J) HT treatment caused gradual blurring and shrinkage and deformation of the cytoskeleton in HCT116 and SW480 cells. Statistical analyses of HT-treated and control groups: *p ≤ 0.05, **p ≤ 0. 01.

Fig. 3

HT-induced ferroptosis in HCT116 and SW480 cells. (A,B) HT treatment caused morphological changes in cellular mitochondria, including smaller size, reduced cristae, and even membrane rupture (transmission electron microscopy). (C,D) HT promoted intracellular iron production in colorectal cancer cells. (E,F) HT inhibited intracellular GSH production in colorectal cancer cells. (G,H) HT promoted intracellular LPO production. (I, J) HT treatment promoted intracellular ROS production in colorectal cancer cells. (K,L) HT treatment caused a decrease in mitochondrial membrane potential in colorectal cancer cells. Statistical analyses of HT-treated and control groups: *p ≤ 0.05, **p ≤ 0.01.

Fig. 4

HT inhibited Nrf2 signalling pathway-induced ferroptosis in colorectal cancer cells. (A) After 48 h of HT treatment, the expression level of Tfr1 protein was elevated while the expression level of SLC7A11 and GPX4 protein were reduced; at the same time, the expression levels of the key proteins of the Nrf2 signalling pathway, Nrf2 and NQO1, were reduced; (B) HT treatment notably increased Tfr1 protein levels while reducing SLC7A11 and GPX4 protein expression in SW480 cells. Similarly, Nrf2 and NQO1 protein expression were suppressed in these cells. Statistical analyses were performed for the HT-treated and control groups: * p ≤ 0.05, ** p ≤ 0.01.

Fig. 5

Fer-1, a ferroptosis inhibitor, inhibits HT-induced ferroptosis in colorectal cancer cells. (A) Fer-1 effectively attenuated HT cytotoxicity in HCT116 cells (HCT116 cells were exposed to 1µM Fer-1 for 2 h before treatment with specified concentrations of HT, followed by cell viability assessment using the CCK8 method). (B) Fer-1 reduced the toxic effects of HT in SW480 cells. Cells were also pretreated with (1µM) Fer-1 and then treated with HT (45, 90,and 180 µM) and cell viability was determined using the CCK8 assay. (C) In HCT116 cells, HT treatment raised Trf1 protein levels while reducing SLC7A11 and GPX4 protein levels; the addition of 1 µM Fer-1 with HT reversed these protein expression levels. (D) The expression levels of Trf1, SLC7A11, and GPX4 proteins were similarly reversed by 1µM Fer-1 in SW480 cells. Statistical analyses were performed for the HT-treated and control groups: ^nsp > 0.05.

Fig. 6

HT triggers ferroptosis in colon cancer cells by suppressing Nrf2 pathway. (A) (20µM) SFN pre-treated HCT116 cells for 2 h, followed by HT treatment for 48 h. Compared to HT alone, SFN changed the expression levels of the key proteins Nrf2 and NQO1 in the Nrf2 signaling pathway. (B) In SW480 cells, co-treatment with 20µM SFN and HT similarly reversed the protein expression levels of Nrf2 and NQO1. (C) Cells were pre-exposed to 1µM CDDO for 2 h before being treated with HT. This reversed the protein expression levels of Nrf2 and NQO1 in HCT116 cells compared to when only HT was used. (D) Co-treatment of SW480 cells with (1µM) CDDO in combination with HT produced the same results as HCT116 cells. Statistical analyses of HT-treated and control groups *p ≤ 0.05, **p ≤ 0.01, ^nsp > 0.05.