Bile acids inhibit ferroptosis sensitivity through activating farnesoid X receptor in gastric cancer cells

Figure 1 Bile acids enhanced proliferation and inhibited erastin-induced ferroptosis sensitivity in gastric cancer cells. A and B: Cell viability assay for HGC-27 and MKN-45 cells treated with three Bas; C and D: Cell viability assay for HGC-27 and MKN-45 cells treated with different concentration of BAs together with erastin (5 μM); E-H: Cell viability assay for two gastric cancer cell lines stimulated with erastin followed by chenodeoxycholic acid (50 μM) or control for 24 and 48 h; I and J: Malondialdehyde production in HGC-27 and MKN-45 cells; K: BODIPY-589/591 C11 staining to identify lipid reactive oxygen species in the cell lines under different treatments. Scale bar: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. These experiments were repeated three times. BAs: Bile acids; CA: Cholic acid; DCA: Dehydrocholic acid; CDCA: Chenodeoxycholic acid; MDA: Malondialdehyde; NS: Not significant.

Figure 2 Bile acids significantly upregulated glutathione and glutathione peroxidase 4 in gastric cancer cells. A and B: Cell viability assay of two gastric cancer cell lines treated with RSL3 together with chenodeoxycholic acid (CDCA) or control; C and D: Malondialdehyde production in HGC-27 and MKN-45 cells treated with RSL3 (0.2 μM for HGC-27, 10 μM for MKN-45) followed by CDCA or control; E: BODIPY-589/591 C11 staining to identify lipid reactive oxygen species in the cell lines treated with RSL3 (0.2 μM for HGC-27, 10 μM for MKN-45) followed by CDCA or control; F and G: The glutathione (GSH) concentrations were measured in cells treated with CDCA; H and I: The GSH/oxidized GSH ratio was measured in cells treated with CDCA; J and K: Western blot analysis of GSH peroxidase 4 protein expression in HGC-27 and MKN-45 cells under different stimuli. Scale bar: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. These experiments were repeated three times. CDCA: Chenodeoxycholic acid; MDA: Malondialdehyde; GPX4: Glutathione peroxidase 4; NS: Not significant.

Figure 3 Bile acids inhibited ferroptosis sensitivity of gastric cancer cells by activating farnesoid X receptor. A and B: Cell viability of erastin-treated HGC-27 and MKN-45 cells with or without GW4064 treatment; C and D: HGC-27 and MKN-45 cells were transfected with shFXR or shNC plasmid. Successful construction was confirmed by western blot analysis; E and F: Cell viability assay of GC cells treated with different concentrations of erastin and CDCA (50 μM) transfected with shFXR or shNC for 24 h; G-I: Malondialdehyde (MDA) production and BODIPY-589/591 C11 staining of GC cells transfected with shFXR or shNC plasmid and treated with erastin together with or without CDCA for 24 h; J and K: GC cells were transfected with control or FXR-coding plasmid and confirmed through western blot analysis; L and M: Cell viability assay of GC cells treated with different concentrations of erastin and CDCA (50 μM) transfected with control or FXR-coding plasmid for 24 h.; N-P: MDA production and BODIPY-589/591 C11 staining of GC cells transfected with control or FXR-coding plasmid and treated with erastin together with or without CDCA for 24 h. Scale bar: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. These experiments were repeated three times. FXR: Farnesoid X receptor; NC: Negative control; CDCA: Chenodeoxycholic acid; MDA: Malondialdehyde; NS: Not significant.

Figure 4 Farnesoid X receptor significantly promoted the synthesis of glutathione and the level of glutathione peroxidase 4 in gastric cancer cells. A-D: Alterations of glutathione (GSH) concentrations and the GSH/oxidized GSH (GSSG) ratio in HGC-27 and MKN-45 cells transfected with the shNC or shFXR plasmid; E and F: Protein expression of GCLC, GSS, GCLM, and GSH peroxidase 4 (GPX4) in HGC-27 and MKN-45 cells transfected with the shNC or shFXR plasmid; G-J: Alterations of GSH concentrations and the GSH/GSSG ratio in HGC-27 and MKN-45 cells transfected with the control or farnesoid X receptor (FXR)-coding plasmid; K and L: Protein expression of GCLC, GSS, GCLM, and GPX4 in HGC-27 and MKN-45 cells transfected with the control or FXR-coding plasmid. ^aP < 0.05, ^bP < 0.01. These experiments were repeated three times. FXR: Farnesoid X receptor; GPX4: Glutathione peroxidase 4.

Figure 5 Farnesoid X receptor exerted anti-ferroptosis effects by inhibiting BTB and CNC homology 1. A and B: Protein expression of BTB and CNC homology 1 (BACH1) in gastric cancer (GC) cells transfected with the shNC or shFXR plasmid for 24 h; C and D: Western blot (WB) analysis of BACH1 protein expression in GC cells transfected with the shNC or shFXR plasmid for 24 h; E and F: HGC-27 and MKN-45 cells were transfected with the control or BACH1-coding plasmid and confirmed through WB analysis; G-I: Malondialdehyde production and BODIPY-589/591 C11 staining of GC cells after transfection with the farnesoid X receptor (FXR)-coding plasmid together with or without the BACH1-coding plasmid and erastin treatment (5 μM) for 24 h; J-M: Alterations of glutathione (GSH) concentrations and the GSH/oxidized GSH ratio in HGC-27 and MKN-45 cells after transfection with the FXR-coding plasmid together with or without the BACH1-coding plasmid; N and O: WB analysis of GCLC, GSS, GCLM, and GSH peroxidase 4 protein expression after transfection with the FXR-coding plasmid together with or without the BACH1-coding plasmid. Scale bar: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. These experiments were repeated three times. FXR: Farnesoid X receptor; BACH1: BTB and CNC homology 1; GSH: Glutathione; GSSG: Oxidized glutathione; GPX4: Glutathione peroxidase 4.

Figure 6 Farnesoid X receptor promoted proliferation of gastric cancer cells. A-F: Malignant proliferation assays, including cell viability (A and B), 5-ethynyl-2′-deoxyuridine (Edu) staining (C and D), andcolony formation assays (E and F), were performed in gastric cancer (GC) cells after transfection with the shNC or shFXR plasmid; G-L: Cell viability (G and H); Edu staining (I and J), andcolony formation assays (K and L) were performed in GC cells after transfection with the control or farnesoid X receptor-coding plasmid. Scale bar: 100 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. These experiments were repeated three times. FXR: Farnesoid X receptor; Edu: 5-ethynyl-2′-deoxyuridine; NC: Negative control; NS: Not significant.