FXR activation suppresses NF-κB signaling, proliferation and migration in cervical cancer cells

Abstract

Background: The Farnesoid X receptor (FXR) is a nuclear receptor known for its role in inflammation regulation and tumor suppression in various cancers. However, its functional significance and underlying mechanisms in cervical cancer (CC) remain unclear. The persistent activation of the nuclear factor kappa B (NF-κB) signaling pathway due to inflammation is a key driver of cancer progression. This study investigates the effects of FXR activation in CC and its interaction with the NF-κB pathway.

Methods: CC cells were treated with GW4064, an FXR agonist (3 µM), and xenograft tumor models were assigned to receive 30 mg/kg GW4064. NF-κB-mediated transcriptional activity was assessed using a dual-luciferase reporter assay. Gene expression in CC cells and mouse tissues was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR), while key proteins in the NF-κB and STAT3 signaling pathways were examined using Western blotting. Cell proliferation, migration, and invasion were evaluated through methylthiazolyldiphenyl-tetrazolium bromide (MTT), wound healing, and real-time cellular analysis (RTCA), respectively. Apoptosis was measured using a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I.

Results: FXR deletion in 6- to 8-week-old C57B/6 female mice led to abnormal upregulation of inflammatory genes in the cervix and aberrant NF-κB activation. Treatment with GW4064 suppressed NF-κB-regulated gene expression in Hela and Siha CC cells and inhibited NF-κB activity at the transcriptional level. Mechanistically, FXR activation suppressed tumor necrosis factor alpha (TNFα)-induced phosphorylation of NF-κB inhibitor alpha (IκBα) by directly binding to the promoter of inhibitor of nuclear factor kappa B kinase regulatory subunit gamma (IKBKG), thereby inhibiting its transcription. Additionally, FXR activation reduced CC cell proliferation and migration. In vivo, xenograft experiments in Hela cell-bearing Bagg's albino (BALB/c) nude female mice confirmed that FXR activation significantly suppressed tumor growth.

Conclusions: These findings highlight FXR activation as a potential therapeutic strategy for CC by targeting the NF-κB pathway as shown in both in vitro and in vivo.

Keywords: Farnesoid X receptor (FXR); GW4064; cervical cancer (CC); nuclear factor kappa B signaling (NF-κB signaling); proliferation.

Figure 1

FXR deficiency promotes cervical inflammation response. (A) Relative mRNA levels of pro-inflammatory genes in the cervix from the WT and FXR-KO mice, n=6. (B) Western blot showing p-IκBα, T-IκBα, p-STAT3, and T-STAT3 protein levels in the cervix from the WT and FXR-KO mice. β-actin served as a loading control. CXCL-2, chemokine (C-X-C motif) ligand 2; FXR-KO, farnesoid X receptor-knockout; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IL-10, interleukin 10; MCP-1, chemokine (C-C motif) ligand 2; MMP2, matrix metallopeptidase 2; MMP7, matrix metallopeptidase 7; MMP10, matrix metallopeptidase 10; mRNA, messenger RNA; p-IκBα, phosphorylated NF-κB inhibitor alpha; p-STAT3, phosphorylated STAT3; T-IκBα, NF-κB inhibitor alpha; T-STAT3, total STAT3; TNFα, tumor necrosis factor alpha; WT, wild-type.

Figure 2

FXR activation impairs the proliferation and migration of human CC cells. (A,B) FXR activation by GW4064 inhibited the proliferation of the Hela (A) and Siha (B) cells. The proliferation of the cells was analyzed by MTT assay. (C) Left panel: following the activation of FXR, the Hela cells exhibited a lower scratch closure rate than the control cells. Magnification: ×40. Right panel: the scratch closure rate was scanned using Image J software. (D) FXR activation by GW4064 inhibited the proliferation of the Hela cells. The proliferation of the cells was analyzed by RTCA. (E,F) FXR activation by GW4064 inhibited the migration of the Hela (E) and Siha (F) cells. The migration of cells was analyzed by RTCA. Cell index = (impedance at time point n − impedance in the absence of cells)/nominal impedance value. The value of cell index is positively correlated with the ability of cell proliferation, migration and invasion. *, P<0.05. CC, cervical cancer; CON, control; FXR, farnesoid X receptor; MTT, methylthiazolyldiphenyl-tetrazolium bromide; OD, optical density; RTCA, real-time cellular analysis.

Figure 3

Activation of FXR promotes apoptosis in CC cells. (A,B) GW4064 treatment for 24 (A) and 48 (B) hours promoted the apoptosis of Hela cells in a dose-dependent manner, and apoptosis was analyzed using flow cytometry. (C) GW4064 treatment for 48 hours promoted the apoptosis of Siha cells in a dose-dependent manner. (D) Relative mRNA levels of the pro-apoptotic genes in the GW4064-treated Hela cells and control cells. “-”: GW4064 untreated group. *, P<0.05. CC, cervical cancer; CON, control; FITC, fluorescein isothiocyanate; FXR, farnesoid X receptor; mRNA, messenger RNA; PI, propidium iodide.

Figure 4

Activation of FXR antagonizes the NF-κB signaling pathway in CC cells. (A) Hela and Siha cells were grown in 6-well plates for 16 hours and treated with 3 μM of GW4064 or DMSO for 24 hours, and then stimulated with TNFα (10 ng/mL) for another 1 hour. The cells were harvested, and the transcript levels of the NF-κB target genes were examined by qRT-PCR. (B) Hela cells were grown in 6-well plates for 16 hours, and transfected with p65 expression vector or control plasmid. After 24 hours of incubation, the cells were treated with 3 μM of GW4064 or DMSO for another 24 hours. The cells were harvested, and the transcript levels of the following cytokines were examined by qRT-PCR. (C) Relative luciferase activities of Hela cells that were co-transfected with the NF-κB reporter plasmid (pNF-κB-Luc), control phRL-TK plasmid, p65 expression plasmid, and FXR expression plasmids. The cells were treated with GW4064 (3 μM) or DMSO for 24 hours. (D,E) Representative immunoblots showing p-IκBα, T-IκBα, P-STAT3, and T-STAT3 protein levels in the Hela (D) and Siha (E) cells treated with DMSO, TNFα (10 ng/mL), or IL-6 (10 ng/mL), GW4064 (3 μM), TNFα + GW4064. β-actin served as a loading control. *, P<0.05. CC, cervical cancer; CON, control; DMSO, dimethyl sulfoxide; FXR, farnesoid X receptor; ICAM-1, intercellular adhesion molecule 1; IL-1α, interleukin 1 alpha; IL-1β, interleukin 1 beta; IL-6, interleukin 6; INOS, inducible nitric oxide synthase; IP-10, interferon-inducible protein-10; MCP-1, chemokine (C-C motif) ligand 2; MMP2, matrix metallopeptidase 2; mRNA, messenger RNA; NF-κB, nuclear factor kappa B; p-IκBα, phosphorylated NF-κB inhibitor alpha; p-STAT3, phosphorylated STAT3; qRT-PCR, quantitative real-time polymerase chain reaction; RLU, relative light unit; T-IκBα, NF-κB inhibitor alpha; T-STAT3, total STAT3; TGFβ2, transforming growth factor beta 2; TNFα, tumor necrosis factor alpha.

Figure 5

FXR antagonizes the NF-κB signaling pathway by inhibiting IKBKG transcription. (A) Relative mRNA levels of IKBKG in GW4064 or 6-eCDCA treated Hela and Siha cells. (B) The online tool NUBIS can predicted the binding site of FXR on the IKBKG promoter. (C) Relative luciferase activities of the Hela cells that were co-transfected with the IKBKG reporter plasmid (WT), control phRL-TK plasmid, and FXR expression plasmids. (D) Relative luciferase activities of the Hela cells that were co-transfected with the IKBKG reporter plasmid (WT) or mutant IKBKG reporter plasmid (MUT), control phRL-TK plasmid, and FXR expression plasmids. (E) Hela and Siha cells were pre-transfected with Flag-tagged FXR overexpression plasmids, followed by ChIP with anti-Flag antibodies to confirm the interaction between FXR and the IKBKG promoter. *, P<0.05; ns, no significance. ChIP, chromatin immunoprecipitation; CON, control; FXR, farnesoid X receptor; FXRE, farnesoid X-receptor-responsive element; IgG, immunoglobulin G; IKBKG, inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; MUT, mutant; mRNA, messenger RNA; NF-κB, nuclear factor kappa B; pCMV, control vector; RLU, relative light unit; TSS, transcription start site; WT, wild-type.

Figure 6

Activation of FXR inhibits CC progression in vivo. Nude mice were subcutaneously inoculated with Hela cells and then intraperitoneally injected with GW4064 (30 mg/kg) or the vehicle (n=5). (A) Photographs of tumors in the GW4064-treated and control groups. (B) Tumor growth curve. (C) Statistics of tumor weight. (D) Body weight change curve of mice after GW4064 injection. (E) Relative mRNA levels of pro-inflammation genes in GW4064-treated tumors and control tumors. (F) The levels of IL-6, IL-1β and TNFα in GW4064-treated and control tumors were detected using ELISA assays. n=5. *, P<0.05. CC, cervical cancer; CON, control; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; FXR, farnesoid X receptor; IL-1α, interleukin 1 alpha; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IL-8, interleukin 8; IP-10, interferon-inducible protein-10; MCP-1, chemokine (C-C motif) ligand 2; mRNA, messenger RNA; TNFα, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor.