Isoliquiritigenin induces HMOX1 and GPX4-mediated ferroptosis in gallbladder cancer cells

Abstract

Background: Gallbladder cancer (GBC) is the most common malignant tumor of biliary tract. Isoliquiritigenin (ISL) is a natural compound with chalcone structure extracted from the roots of licorice and other plants. Relevant studies have shown that ISL has a strong anti-tumor ability in various types of tumors. However, the research of ISL against GBC has not been reported, which needs to be further investigated.

Methods: The effects of ISL against GBC cells in vitro and in vivo were characterized by cytotoxicity test, RNA-sequencing, quantitative real-time polymerase chain reaction, reactive oxygen species (ROS) detection, lipid peroxidation detection, ferrous ion detection, glutathione disulphide/glutathione (GSSG/GSH) detection, lentivirus transfection, nude mice tumorigenesis experiment and immunohistochemistry.

Results: ISL significantly inhibited the proliferation of GBC cells in vitro . The results of transcriptome sequencing and bioinformatics analysis showed that ferroptosis was the main pathway of ISL inhibiting the proliferation of GBC, and HMOX1 and GPX4 were the key molecules of ISL-induced ferroptosis. Knockdown of HMOX1 or overexpression of GPX4 can reduce the sensitivity of GBC cells to ISL-induced ferroptosis and significantly restore the viability of GBC cells. Moreover, ISL significantly reversed the iron content, ROS level, lipid peroxidation level and GSSG/GSH ratio of GBC cells. Finally, ISL significantly inhibited the growth of GBC in vivo and regulated the ferroptosis of GBC by mediating HMOX1 and GPX4 .

Conclusion: ISL induced ferroptosis in GBC mainly by activating p62-Keap1-Nrf2-HMOX1 signaling pathway and down-regulating GPX4 in vitro and in vivo . This evidence may provide a new direction for the treatment of GBC.

Figure 1

ISL inhibits GBC cell proliferation. (A) NOZ and SGC996 cells were treated with ISL (0 μmol/L, 20 μmol/L, 40 μmol/L, 60 μmol/L, 80 μmol/L, and 100 μmol/L) for 24 h, 48 h, and 72 h. CCK-8 assays were performed to assess cell viability. The IC50 for each time point is shown in the upper right corner. (B) ISL suppressed NOZ and SGC996 cell colony formation. Cells were exposed to ISL (0 μmol/L, 20 μmol/L, 40 μmol/L, 60 μmol/L, and 80 μmol/L) and allowed to form colonies for 15 days (Original magnification × 1). (C) NOZ and SGC996 cells were treated with control or ISL (60 μmol/L and 70 μmol/L, respectively) for 48 h, and the morphology was analyzed with a fluorescence microscope (Original magnification × 400). Representative images from three independent experiments are shown. (D) IC50 analysis of HiBECs and L-2F7 cells. All data are presented as the means ± SDs, and each experiment was repeated thrice. Significant differences compared to the control are indicated by ^*P <0.05, ^†P <0.01, and ^‡P <0.001. CTL: Control; GBC: Gallbladder cancer; HiBECs: Human intrahepatic biliary epithelial cells; IC50: Half maximal inhibitory concentration; ISL: Isoliquiritigenin; SDs: Standard deviations.

Figure 2

RNA-seq analysis of GBC cells treated with ISL. (A) RNA was isolated from ISL-treated NOZ and SGC996 cells. The differentially expressed genes and samples were clustered by two-way hierarchical clustering and displayed in a heatmap. Red and blue represent high and low expression, respectively. (B) KEGG pathway enrichment scatter plot. Based on the sequence information of the enrichment factor, the top enriched KEGG pathways are displayed. (C) Heatmap of GSEA shows different expression levels of ferroptotic pathway genes between the control and ISL groups. Control/ISL 01 to 06 refer to the numbering order of the 3 biological repeats of two cell lines (01 to 03 for NOZ cells, 04 to 06 for SGC-996 cells ) in the two groups. GBC: Gallbladder cancer; GSEA: Gene set enrichment analysis; ISL: Isoliquiritigenin; KEGG: Kyoto Encyclopaedia of Genes and Genomes; RNA-seq: RNA sequencing.

Figure 3

Ferroptotic genes were validated by RT-qPCR and Western blotting in ISL-treated cells. (A) Total RNA was extracted from NOZ and SGC996 cells treated with 60 μmol/L and 70 μmol/L ISL, respectively, for 48 h and used for RT-qPCR analysis of gene expression. The mRNA expression levels were normalized to the level of β-actin. The RNA-seq data are also shown in parallel. Each reported value represents the mean ± standard error of mean from three independent experiments. (B) GBC cells were treated with the indicated concentrations of ISL for 48 h. Cells were then harvested for Western blotting of ferroptosis markers. The image is representative of three independent experiments. (C) GBC cells were pretreated with various inhibitors, including Z-VAD-FMK (VAD, 50 μmol/L), necrostatin-1 (Nec, 50 μmol/L), ferrostatin-1 (Ferro, 1 μmol/L), liproxstatin-1 (Lipro, 1 μmol/L), and deferoxamine (DFO, 50 μmol/L), for 1 h followed by treatment with ISL (90 μmol/L in NOZ cells and 130 μmol/L in SGC996 cells) for 48 h. Cell viability was assessed by the CCK-8 assay. ^*P <0.05, ^†P <0.01 and ^‡P <0.001 compared to ISL alone. NS indicates no significant difference. CTL: Control; GBC: Gallbladder cancer; ISL: Isoliquiritigenin; RNA-seq: RNA sequencing RT-qPCR: Quantitative real-time polymerase chain reaction.

Figure 4

ISL induces ferroptosis via HMOX1 upregulation or GPX4 downregulation. (A) GBC cells were treated with ISL or combination of ISL and zinc protoporphyrin 9 (ZnPP, 4 μmol/L or 10 μmol/L) for 48 h. Cell viability was assessed by the CCK-8 assay. ^‡P <0.001.(B) Western blotting analysis of HMOX1 and GPX4 expression levels in NOZ and SGC996 cells after infection with shRNA1-HMOX1 and OE-GPX4 lentiviruses. (C,D) HMOX1-deficient (shRNA1-HMOX1) or GPX4-overexpressing (OE-GPX4) GBC cells were treated with ISL for 48 h followed by a cell viability assay. ^*P <0.05 and ^†P <0.01 compared to CTRL (sh-control + OE-control). NS indicates no significant difference. (E,F) HMOX1-deficient (shRNA1-HMOX1) or GPX4-overexpressing (OE-GPX4) GBC cells were treated with ISL for 48 h followed by Western blotting. CTL: Control; GBC: Gallbladder cancer; ISL: Isoliquiritigenin; shRNA: Short hairpin RNA; OE: Overexpressed.

Figure 5

HMOX1 knockdown or GPX4 overexpression alleviates ISL-induced iron overload, ROS accumulation, and lipid peroxidation. (A) HMOX1-deficient or GPX4-overexpressing GBC cells were treated with ISL for 48 h. After incubation with 1 μmol/L FerroOrange for 30 min, cells were washed with PBS and detected by fluorescence microscopy (scale bar = 100 μm). The representative figure shows one of three independent experiments. The fluorescence intensity was quantified using ImageJ software. (B) The ROS level in GBC cells after ISL treatment was detected using the DCFH-DA dye. The sample preparation was the same as in (A). (C) The lipid peroxide content in GBC cells after ISL treatment was measured using the Liperfluo probe. The sample preparation was the same as in (A). (D) GSH and GSSG levels were determined using a GSSG/GSH Quantification kit, and the GSH/GSSG ratio was calculated. The sample preparation was the same as in (A). All the data are shown as the mean ± standard error of mean of three independent experiments. ^*P <0.01 and ^†P <0.001. CTRL: Control; DCFH-DA: 2,7-dichlorofluorescein diacetate; GBC: Gallbladder cancer; GSSG/GSH: Glutathione disulphide/glutathione; ISL: Isoliquiritigenin; ROS: Reactive oxygen species.

Figure 6

HMOX1 knockdown or GPX4 overexpression attenuates tumor inhibition in mice treated with ISL. (A) CTRL (sh-control + OE-control), HMOX1-deficient, and GPX4-overexpressing NOZ cells (1 × 10⁶ cells in 100 μL) were injected subcutaneously into mice followed by ISL treatment (50 mg/kg, every other 2 days via gavage) for 28 days. Following 28 days of observation, the tumors were removed and photographed (n = 5). (B) Tumor volume was measured. Each experimental group contained five mice (n = 5, ^*P <0.01 and ^†P <0.001). (C) Paraffin sections of tumor samples were used for IHC staining with antibodies against HMOX1 and GPX4 (200× and 400× magnification; scale bar = 100 μm). ^*P <0.01 and ^†P <0.001. CTRL: Ccontrol; NS indicates no significant difference. IHC: Immunohistochemistry; ISL: Isoliquiritigenin; OE: Overexpressed; sh: Short hairpin.

Figure 7

Proposed model of the HMOX1- and GPX4-dependent ferroptosis signaling pathway in response to ISL. GSH: Glutathione; GSSG: Glutathione disulfide; ISL: Isoliquiritigenin; ROS: Reactive oxygen species.