Curcumenol inhibits malignant progression and promotes ferroptosis via the SLC7A11/NF‑κB/TGF‑β pathway in triple‑negative breast cancer

Abstract

Triple‑negative breast cancer (TNBC) exhibits a high degree of malignancy and a propensity for metastasis, ultimately resulting in unfavorable patient outcomes. Curcuma phaeocaulis Valeton is a common herb used in traditional Chinese medicine to treat TNBC. Curcumenol (Cur) is a natural compound derived from C. phaeocaulis Valeton, the effects of which on breast cancer remain under‑reported. The present study elucidated that Cur could effectively inhibit the survival ability of TNBC cells and enhance their sensitivity to paclitaxel. Western blotting (WB) further revealed that Cur modulated apoptosis and epithelial‑mesenchymal transition (EMT) in TNBC. Findings from animal experiments further validated these observations. In the established TNBC mouse model, Cur was shown to exert an inhibitory effect on tumor growth, effectively attenuate EMT and substantially reduce the incidence of lung metastasis. Integrated analyses using RNA sequencing, WB and reverse transcription‑quantitative polymerase chain reaction demonstrated that Cur markedly downregulated the expression levels of solute carrier family 7 member 11 (SLC7A11), phosphorylated‑NF‑κB and TGF‑β. Molecular docking studies further validated that Cur can establish stable interactions with SLC7A11. In‑depth bioinformatics analysis revealed a positive association between high SLC7A11 expression and reduced disease‑free survival in patients with breast cancer. Additionally, in TNBC cells, Cur was revealed to reduce the mitochondrial membrane potential and promote the accumulation of lipid reactive oxygen species. Subsequent experimental investigations demonstrated that Cur can counteract the inhibitory influence of ferrostatin‑1 on ferroptosis. These findings strongly implied a potential underlying mechanism, suggesting that Cur may impede the malignant progression of TNBC via the modulation of ferroptosis. In conclusion, the findings of the present study underscore the marked efficacy of Cur in hampering the progression of TNBC by suppressing the SLC7A11/NF‑κB/TGF‑β signaling pathway.

Keywords: curcumenol; epithelial‑mesenchymal transition; ferroptosis; lung metastasis; triple‑negative breast cancer.

Figure 1

Research flowchart. CCK-8, Cell Counting Kit-8; EMT, epithelial-mesenchymal transition; RNA seq, RNA sequencing; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2

Effects of Cur on the viability, migration, invasion and colony formation of triple-negative breast cancer cells. (A) Chemical structure of Cur. (B) Viability of 4T1 and MDA-MB-231 cells treated with 6.25, 12.5, 25, 50, 100, 200 and 400 µM Cur for 24 and 48 h detected by CCK-8 assay. (C) Effects of 0, 25, 50 and 100 µM Cur, and 50 and 500 nM PTX on cell viability, as detected by CCK-8 assay. (D) Wound healing assays were performed to analyze cell migration after 4T1 and MDA-MB-231 cells were treated with 25, 50 and 100 µM Cur for 24 and 48 h. Scale bar, 100 µm. (E) Transwell assays were performed to evaluate cell invasion after 4T1 and MDA-MB-231 cells were treated with 25, 50 and 100 µM Cur for 24 h. Scale bar, 200 µm. (F) Colony formation assay of 4T1 and MDA-MB-231 cells grown for 7-10 days in the presence of the indicated concentrations of Cur or Ctrl. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Ctrl group or as indicated. CCK-8, Cell Counting Kit-8 ; Ctrl, control; Cur, curcumenol; PTX, paclitaxel.

Figure 3

Effects of Cur on the apoptosis and epithelial-mesenchymal transition of triple-negative breast cancer cells. (A) Western blot analysis was performed to detect the protein expression levels of C-caspase 9, C-caspase 3, BAX and BCL-2 in 4T1 and MDA-MB-231 cells following treatment with the indicated concentrations of Cur or Ctrl for 24 h. (B) Protein expression levels of E-cadherin, N-cadherin and Vimentin in 4T1 and MDA-MB-231 cells following treatment with the indicated concentrations of Cur or Ctrl for 24 h. (C) Immunofluorescence staining of E-cadherin (green) and Vimentin (red) in 4T1 and MDA-MB-231 cells treated with 100 µM Cur or Ctrl for 24 h. Scale bar, 20 µm. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Ctrl group. C-, cleaved; Ctrl, control; Cur, Curcumenol; ns, not significant.

Figure 4

Effects of Cur on TNBC tumor growth in vivo. (A) Schematic diagram of the in vivo study on antitumor effects. (B) Representative images of tumors in different groups of 4T1-induced TNBC mouse models. (C) Tumor volumes of mice after treatment for 3 weeks. (D) Levels of Ki-67 and TUNEL in tumor tissues were determined using immunohistochemistry, which aimed to measure proliferation and apoptosis in the 4T1-induced TNBC mouse model. Scale bar, 50 µm. (E) Western blot analysis was performed to detect the protein expression levels of C-caspase 9, C-caspase 3, BAX, BCL-2, E-cadherin, N-cadherin and Vimentin. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Model group or as indicated. Cur, curcumenol; TNBC, triple-negative breast cancer; PTX, paclitaxel.

Figure 5

Cur alleviates triple-negative breast cancer lung metastasis in a mouse xenograft model. (A) Representative images of lung nodule images after Bouin's fixative solution and (B) lung nodule counts. (C) Hematoxlyin and eosin staining images of lung metastasis tissues from each group of mice. Scale bar, 100 µm. The arrows indicate lung metastases. (D) Protein expression levels of E-cadherin and Vimentin in lung metastatic lesions determined using immunohistochemistry. Scale bar, 50 µm. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Model group. Cur, curcumenol; PTX, paclitaxel.

Figure 6

Identification of Cur-regulated candidate target genes in TNBC tumor tissues by RNA sequencing. (A) Volcano plots and (B) heatmap of upregulated and downregulated genes in tumor tissues from the Cur group compared with the model group (n=5). (C) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. (D) GO annotation analysis of differentially expressed genes. (E) Effect of Cur on SLC7A11, APLNR, TGF-β, IL7, HCAR2 and NF-κBID in TNBC tumor tissues determined using reverse transcription-quantitative polymerase chain reaction. (F) Gene Set Enrichment Analysis. (G) Immunohistochemistry was performed to detect the expression of CD11b (dendritic cell marker) and CD69 (T-cell activation marker) in each group of tumor tissues (magnification, ×63). Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Model group. APLNR, angiotensin receptor like 1; Cur, curcumenol; GO, Gene Ontology; HCAR2, hydroxycarboxylic acid receptor 2; SLC7A11, solute carrier family 7 member 11; TNBC, triple-negative breast cancer.

Figure 7

Cur inhibits TNBC by modulating ferroptosis-related proteins. (A) Molecular docking of Cur with SLC7A11. (B) 3D structure prediction and comparison of human SLC7A11 (yellow) with mouse SLC7A11 (blue) using AlphaFold and PyMOL. (C) Survival analysis of the association between SLC7A11 expression and disease-free survival in breast cancer. (D) Effect of Cur on the ferroptosis-related proteins SLC7A11, GPX4 and ACSL4 in tumors. (E) Effect of Cur on NF-κB and TGF-β protein expression, and p-NF-κB levels. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Model group. ACSL4, acyl-CoA synthetase long-chain family member 4; Cur, curcumenol; GPX4, glutathione peroxidase 4; p-, phosphorylated; SLC7A11, solute carrier family 7 member 11; RMSD, root mean square deviation.

Figure 8

Cur can promote ROS accumulation and ferroptosis. (A) Fluorescence images of JC-1 aggregates (red) and JC-1 monomers (green) in 4T1 and MDA-MB-231 cells treated with 100 µM Cur or Ctrl for 24 h. Scale bar, 50 µm. (B) Labeling of 4T1 and MDA-MB-231 cells with the DCFH-DA fluorescent probe to assess the level of ROS release. Scale bar, 100 µm. (C) Flow cytometry of lipid ROS levels in MDA-MB-231 cells after 24 h of treatment with Fer-1 alone or in combination with Cur. (D) Western blot analysis was performed to detect the expression levels of the ferroptosis-related proteins SLC7A11, GPX4 and ACSL4 in MDA-MB-231 cells. Data are presented as the mean ± SD (n=3). ^*P<0.05, ^**P<0.01 vs. Ctrl group or as indicated. ACSL4, acyl-CoA synthetase long-chain family member 4; Ctrl, control; Cur, curcumenol; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11.

Figure 9

Potential mechanisms of Cur in the treatment of TNBC. Cur inhibits SLC7A11 expression, NF-κB and TGF-β signaling, and EMT, and promotes ROS accumulation and apoptosis in triple-negative breast cancer. Cur, curcumenol; E-cad, E-cadherin; EMT, epithelial-mesenchymal transition; p-, phosphorylated; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11.