Discovery of vitexin as a novel VDR agonist that mitigates the transition from chronic intestinal inflammation to colorectal cancer

Abstract

Colitis-associated colorectal cancer (CAC) frequently develops in patients with inflammatory bowel disease (IBD) who have been exposed to a prolonged state of chronic inflammation. The investigation of pharmacological agents and their mechanisms to prevent precancerous lesions and inhibit their progression remains a significant focus and challenge in CAC research. Previous studies have demonstrated that vitexin effectively mitigates CAC, however, its precise mechanism of action warrants further exploration. This study reveals that the absence of the Vitamin D receptor (VDR) accelerates the progression from chronic colitis to colorectal cancer. Our findings indicate that vitexin can specifically target the VDR protein, facilitating its translocation into the cell nucleus to exert transcriptional activity. Additionally, through a co-culture model of macrophages and cancer cells, we observed that vitexin promotes the polarization of macrophages towards the M1 phenotype, a process that is dependent on VDR. Furthermore, ChIP-seq analysis revealed that vitexin regulates the transcriptional activation of phenazine biosynthesis-like domain protein (PBLD) via VDR. ChIP assays and dual luciferase reporter assays were employed to identify the functional PBLD regulatory region, confirming that the VDR/PBLD pathway is critical for vitexin-mediated regulation of macrophage polarization. Finally, in a mouse model with myeloid VDR gene knockout, we found that the protective effects of vitexin were abolished in mid-stage CAC. In summary, our study establishes that vitexin targets VDR and modulates macrophage polarization through the VDR/PBLD pathway, thereby alleviating the transition from chronic colitis to colorectal cancer.

Keywords: Colitis-associated colorectal cancer; Macrophage; Vitamin D receptor; Vitexin.

Fig. 1

VDR plays an important role in the transformation of chronic intestinal inflammation-induced cancer. A) AOM/DSS induced mid-cycle CAC modeling protocol. B) Macroscopic view of colon obtained by mid-stage CAC, Scale bar, 1 cm. C) H&E staining of the colon, Scale bar, 200 μm. D) Immunohistochemistry of VDR protein and statistics in colon (Scale bar, 100 μm, **P < 0.01). E) Heatmap of the colon. F) Immunofluorescence double staining of iNOS and CD206 protein in colon tissue, Scale bar, 100 μm. G) Schematic diagram of the experimental design of VDR^ΔMΦ mice. H) Body weight in the mice, n = 6. I) DAI scores, n = 6. J) macroscopic image of the colon. K) H&E staining of the colon. The data are presented as the mean ± SEM (n = 3). ^##P < 0.01 vs. WT group

Fig. 2

Vitexin targets VDR and binds amino acid motifs in its VDR-LBD region. A) Vitexin promotes the resistance of VDR to different temperature gradients as detected by CETSA in THP-1 cells. B) Vitexin enhances the resistance of VDR to proteolytic enzymes as investigated by DARTS. C) Chemical structure of Biotin-vitexin (Bio-vitexin). D) Co-localization of biotin-vitexin (green) and VDR (red) by immunofluorescence, Scale bar, 10 μm. E) Protein blotting analysis of biotin-vitexin binding to the VDR-LBD structural domain. Recombinant proteins VDR and VDR-LBD structural domains were incubated with either biotin- vitexin-loaded magnetic beads or biotin-loaded magnetic beads. The top panel shows the structure of VDR and the bottom panel shows the results of the pulled-down proteins. F) SPR analysis showing the interaction between vitexin and recombinant VDR-LBD protein (left). Different concentrations of vitexin were added and K_D values were calculated (right). G) ITC analysis of VDR-LBD binding to vitexin, representative images are shown. Representative titration temperature plots are shown on the left, and data integration with the fitted curve (independent model) of vitexin versus VDR-LBD is shown on the right. H) The presentative conformation of VDR protein binding with vitexin upon molecular dynamics simulation. I) free energy landscape. J) The left panel shows the carbon atoms of the side chains of the 11 key residues, with vitexin indicated as green and yellow bars, respectively. The right panel shows a box plot of the per-residue catabolic energy for the 11 residues. K) Pull-down analysis of biotin-vitexin binding to mutant VDRs containing T287A. HEK 393T cells were transfected with wild-type VDR or mutants. Lysates were used to assay for binding to biotin-vitexin. The data are presented as the means ± SEM (n = 3). **, P < 0.01; ns, no significance

Fig. 3

Vitexin promotes nuclear translocation of VDR protein in macrophages and regulates nuclear transcription. Both RAW264.7 and THP-1 cells were treated with different concentrations of vitexin and Calcitriol. A) Gene levels of VDR in RAW264.7 cells (top) and THP-1 cells (bottom) were detected by RT-PCR. B) Immunofluorescence was used to examine the levels of VDR in the nucleus and cytoplasm, RAW264.7 cells (left) and THP-1 cells (right). Scale bar, 10 μm. C-D) Western blot was used to examine the levels of VDR in the nucleus and cytoplasm. E) Detection of transcription factor VDR against CYP24A1 promoter activity using dual luciferase in HEK293T cells. F) ChIP-PCR amplification showing VDR activity against the promoter of CYP24A1. The data are presented as the means ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ns, no significance

Fig. 4

In the tumor microenvironment, vitexin promotes the nuclear translocation of VDR. A) After 48 h incubation with vitexin, CT26 was co-cultured with BMDM for 24 h, which was at the transwell insert, and CT26 was located at the bottom of the cell plate. The experiments related to this section are all co-culture assays. B) Left: RNA-seq analysis of THP-1 cells (THP-1 and HCT116) co-cultured with or without treatment with vitexin of differential genes (n = 3). Right: volcano plot of differential gene enrichment. C) Gene levels of VDR in BMDM cells (top) and THP-1 cells (bottom) were detected by RT-PCR. D) Western blot was used to examine the levels of VDR in the nucleus and cytoplasm. E) Immunofluorescence was used to examine the levels of VDR in the nucleus and cytoplasm. Scale bar, 10 μm. The data are presented as the means ± SEM (n = 3). ^#, P < 0.05; ^##, P < 0.01; *, P < 0.05; **, P < 0.01; ns, no significance

Fig. 5

Vitexin regulates polarization in macrophages in co-culture, dependent on VDR. A) Schematic diagram of the in vitro Transwell-based coculture system. B) Heatmap of gene expression levels detected in macrophage arrays of co-cultured THP-1 cells for 24 h with or without vitexin treatment (n = 3 each). C) Data are presented as representative FACS plots. D) Flow cytometry analysis of iNOS or CD206 levels in BMDM cells from WT mice. E) Western blot detection of VDR expression in BMDM from WT, VDR^flox, and VDR^ΔMΦ. F) Flow cytometry analysis of iNOS or CD206 levels in BMDM cells. G) Data are presented as representative FACS plots and in summary plots. H) The levels of Nos2 and Arg-1 mRNA. The data are presented as the mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ns, no significance

Fig. 6

Vitexin regulates macrophage polarization in the tumor microenvironment via the VDR/PBLD pathway. A) Schematic diagram of the in vitro Transwell-based coculture system. B) Binding maps of VDR to the PBLD gene were analyzed from ChIP-Seq data in the control and vitexin groups and visualized by IGV software. C) The abundance of gene fragments in the input and immunoprecipitates was evaluated using designated primers through real-time PCR, and the position of the primers was detected by ChIP. D) Schematic representation of the PBLD promoter containing three VDREs, with the promoter’s mutation strategy shown in the figure. E) Transcriptional regulatory activity of VDR on full-length PBLD promoter and doubly VDRE-mutated PBLD promoter in HEK293T cells with or without Vitexin treatment measured by dual-luciferase reporter gene assay. F) Western blot assay for VDR expression. G) Flow cytometry analysis of CD86 and CD206 levels. Data are presented as representative FACS plots. H) Statistical results of macrophage typing. I) mRNA levels of INOS and ARG-1. Data are expressed as SEM ± mean (n = 3). ^#, P < 0.05; ^##, P < 0.01; *, P < 0.05; **, P < 0.01; ns, no significance

Fig. 7

Vitexin mitigates the transition from chronic intestinal inflammation to cancer by modulating macrophage polarization and is dependent on the VDR/PBLD pathway. A) Flow chart of drug administration. B) Graph of body weight changes (The data are presented as the means ± SEM (n = 6). In the VDR^flox group, ^##P < 0.01 compared with the NC group and **P < 0.01 compared with the CAC group.). C) DAI score. D) Tumor number. E) Colon length (left), colon thickness (right). F) Macroscopic view of colon, Scale bar, 1 cm. G) H&E staining of the colon, Scale bar: 1000 μm and 200 μm, n = 3). H) Multicolour immunohistochemistry of colon tissue (VDR-green, F4/80 red, CD86 pink, CD163 rose. Scale bar, 500 μm, n = 3). I-J) The expression of PBLD and PCNA proteins and statistical plots (n = 3). The data are presented as the means ± SEM (n = 6). ^#P < 0.05, ^##P < 0.01;*P < 0.05, **P < 0.01; ns, no significance