RSL3 induces ferroptosis by activating the NF-κB signalling pathway to enhance the chemosensitivity of triple-negative breast cancer cells to paclitaxel

Abstract

Chemotherapy resistance in triple-negative breast cancer (TNBC) leads to poor therapeutic effects and a poor prognosis. Given that paclitaxel-based chemotherapy is the main treatment method for TNBC, enhancing its chemosensitivity has been a research focus. Induced ferroptosis of tumour cells has been proven to increase chemosensitivity, but its ability to sensitize TNBC cells to paclitaxel (PTX) is unknown. In our experiments, measurements of viability and proliferation validated the synergistic effect of PTX combined with RSL3 on TNBC cells. The accumulation of intracellular Fe²⁺ and lipid reactive oxygen species, as well as the expression of malondialdehyde, illustrated that RSL3 enhanced the chemosensitivity of TNBC to PTX by inducing ferroptosis. Through transcriptome sequencing, a series of differentially expressed genes were identified, in which the expression of cytokines, such as CXCLs, was significantly increased in the treatment group, and the effect of combination therapy on TNBC was enriched mainly in the NFκB signalling pathway. In subsequent validation experiments, the use of the NF-κB inhibitor BAY11-7082 reversed the inhibitory effects of PTX and RSL3 on TNBC cell activity. In a xenograft immunodeficient mouse model, the inhibitory effects of PTX and RSL3 on TNBC in vivo were further verified. Our research validated the synergistic effects of PTX and RSL3 both in vivo and in vitro, with RSL3 inducing ferroptosis by activating the NF-κB signalling pathway, thereby increasing the chemosensitivity of TNBC to PTX. This study provides new insights for improving the therapeutic efficacy of treatment strategies.

Keywords: Chemosensitivity; Ferroptosis; Triple-negative breast cancer.

Fig. 1

RSL3 Enhances the Chemosensitivity of TNBC to PTX in vitro. (A, B) TNBC MDA-MB-231 and MDA-MB-468 cells were treated with various concentrations of PTX (0, 5, 50, 500 nM) combined with different concentrations of RSL3 (0, 0.1, 0.5, 1.5 μM) for 24 h, and cell viability was assayed using the CCK-8 assay. (C) Colony formation experiments were performed on MDA-MB-231 cells treated with PTX (5 nM), RSL3 (0.5 μM) or their combination for 24 h and on MDA-MB-468 cells treated with PTX (5 nM), RSL3 (0.1 μM) or their combination for 24 h. A histogram showing colony formation is presented. (D–G) Migration and invasion of treated cells were assayed by Transwell assays. (D, F) MDA-MB-231 cells were treated with PTX (5 nM), RSL3 (0.5 μM) or their combination for 24 h. Scale bar: 100 μm. (E, G) MDA-MB-468 cells were treated with PTX (5 nM), RSL3 (0.1 μM), or their combination for 24 h. Scale bar: 200 μm. The numbers of migrated and invaded cells were plotted in histograms. *P < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

RSL3 enhances the chemosensitivity of TNBC cells to PTX by inducing ferroptosis. (A, B) MDA-MB-231 cells were treated with PTX (5 nM) and RSL3 (0.5 μM) combined with Ac-DEVD-CHO (20 μM), necrostatin-1 (10 μM), 3-MA (10 μM), ferrostatin-1 (Fer-1) (10 μM), liproxstatin-1 (LIP) (200 nM), or deferoxamine (DFO) (5 μM) for 24 h, and cell viability was assayed using the CCK-8 assay. MDA-MB-468 cells were treated with PTX (5 nM) and RSL3 (0.1 μM) combined with the same inhibitors for 24 h, and cell viability was also assayed using the CCK-8 assay. (C) The level of Fe2⁺ was measured by FerroOrange. Scale bar: 50 μm. (D) Cellular lipid ROS levels were analysed by flow cytometry. (e) Intracellular MDA levels were measured using the MDA detection kit. **P < 0.01; *P < 0.05; ****p < 0.0001; ***p < 0.001. NS: Not significant.

Fig. 3

Transcriptomics reveals the molecular mechanism by which RSL3 enhances the chemosensitivity of TNBC cells to PTX. (A, B) Heatmaps and volcano plots depict the expression of differentially expressed genes among the different treatment groups, with red representing highly expressed genes and blue representing genes with low expression. (C) A Venn diagram shows overlapping and unique genes among the different treatment groups. (D) A bubble chart presents the results of the KEGG enrichment analysis of 75 differentially expressed genes in the Venn diagram, with the size of the circle indicating the number of enriched genes in each project and each colour representing a different p value (www.kegg.jp/kegg/kegg1.html). (E) The infiltration of immune cells in different groups. (F) GSEA pathway enrichment analysis of cancer-related genes, with red circles representing significantly upregulated pathways (www.kegg.jp/kegg/kegg1.html).

Fig. 4

Combination treatment with PTX and RSL3 activates NF-κB and promotes TNBC cell death. (A) The viability of MDA-MB-231 and MDA-MB-468 cells was assayed using the CCK-8 assay after combination treatment with PTX or RSL3 and an NF-κB inhibitor for 24 h. (B) After the treatments, the expression levels of cytokines were detected by RT-qPCR. (C) After the treatments, the expression levels of cytokines were detected by Western blotting and ELISA. (D) The protein expression levels of P65, P-P65 and GPX4 were measured by Western blotting, and a grayscale map of protein expression is displayed. **P < 0.01; *P < 0.05; ****p < 0.0001; ***p < 0.001. NS: Not significant.

Fig. 5

RSL3 enhanced the chemosensitivity of TNBC to PTX in vivo. (A) Experimental design. Day -10: MDA-MB-231 cells (6 × 10⁶ cells/100 μl) were inoculated subcutaneously into the right mammary fat pads of severely immunocompromised mice. Day 0: paclitaxel (5 mg/kg) and RSL3 (40 mg/kg) were administered via intraperitoneal injection at a volume of 100 μl, with administration every 3 days. (B) Anatomical images of excised tumours on Day 18. (C) Volumes of excised tumours on Day 18. (D) The tumour weights of the mice in each group were calculated every 3 days. (E) The body weights of the mice in each group were calculated every 3 days. (F) Haematoxylin and eosin staining of mouse organs and tumours; scale bar: 100 μm. (G) Ki67 and GPX4 expression was detected by immunohistochemical staining; scale bar: 50 μm. *P < 0.05.