Coumestrol facilitates apoptosis in colorectal cancer cells by interacting with ZIP8 protein via the ferroptosis pathway

Abstract

Objective: So far, there have been no reports of coumestrol inhibiting colorectal cancer (CRC) through the ferroptosis pathway. This study is to investigate the mechanism of the traditional Chinese medicine monomer coumestrol in the treatment of CRC. Methods: Data on CRC transcriptome sequencing was obtained from the GEO database and TCGA database. Bioinformatics analyses were conducted to screen for CRC prognostic-related key genes and their potential binding monomers in traditional Chinese medicine. The inhibitory effect of coumestrol on CRC cell lines (COLO 205 & HCT 116) was determined using the CCK-8 assay, and cell apoptosis was assessed by flow cytometry. The content of ferrous ions was measured using the Ferrous Ion Content Assay Kit. The expression of ferroptosis pathway-related genes SLC39A8, NCOA4, VDAC2, and NOX2 before and after small interference RNA (siRNA) was examined through real-time PCR and Western blotting. Results: SLC39A8 was found to be associated with CRC clinical progression staging, and its encoded protein ZIP8 may bind to coumestrol. KEGG enrichment analysis suggested that ZIP8 plays a role in iron transmembrane transport and may affect the expression of ferroptosis pathway-related genes NCOA4, VDAC2, and NOX2. Coumestrol was found to induce apoptosis in CRC cell lines by upregulating the expression of ferroptosis pathway-related genes SLC39A8, NCOA4, VDAC2, and NOX2. However, coumestrol was unable to upregulate the expression of ferroptosis pathway-related genes in CRC cell lines after SLC39A8 interference. Conclusion: Coumestrol facilitates apoptosis in CRC cells by interacting with ZIP8 protein via the ferroptosis pathway.

Keywords: SLC39A8; ZIP8; colorectal cancer; coumestrol; ferroptosis pathway.

Figure 1

SLC39A8 was associated with survival of CRC. Wayne's analysis revealed that four genes intersected across the three datasets: TCGA, GSE39582, and GSE17538 (A). In paired differential analysis, GDI1 demonstrated significant up-regulation, while GPX3 and SLC39A8 were down-regulated in tumor samples compared with normal samples, and no significant difference was observed in NOS2 (B-E).

Figure 2

The survival analysis of GDI1, GPX3, and SLC39A8 in the three datasets TCGA, GSE39582, and GSE17538 revealed that SLC39A8 displayed distinct survival outcomes between the high and low expression groups.

Figure 3

Results of correlation analysis between the expression of SLC39A8 and clinical classification indicators. In the GSE39582 dataset, a significant difference was noted in the expression of SLC39A8 among patients with different stages (A) and N-stages (B), but not M-stage (C). The GSE17538 dataset indicated significant variations (P<0.05) in the expression of SLC39A8 between patients at early and late stages (D). In the TCGA dataset, a significant disparity was observed in the expression of SLC39A8 across patients with varying stages (E), N-stages (F), and M-stage (G). Single factor independent prognostic analysis showed that SLC39A8 could serve as an independent prognostic factor for CRC (H-J).

Figure 4

Structural modeling revealed that coumestrol and ZIP8 could form a hydrogen bond of 2.5 Å.

Figure 5

Functional enrichment analysis indicated the involvement of ferroptosis pathway-related genes in CRC development. GO analysis indicated that ZIP8 primarily functions as a transporter (A). Paired differential analysis of ferroptosis pathway-related genes NCOA4 and VDAC2 using TCGA data revealed significant differences between high and low groups.

Figure 6

Cell inhibition and apoptosis analysis results. Drug concentration screening using CCK-8 showed better cell inhibition rates of 100 µM coumestrol for 96 hours (A-D). The CCK-8 results show that coumestrol had a weaker inhibitory effect on the normal cell line NCM460 (E). Immunofluorescence staining demonstrates that coumestrol can inhibit the proliferation of CRC cell lines (FG). Immunofluorescence staining suggests that coumestrol could promote apoptosis in CRC cell lines (H). Increased cell apoptosis was noted in both COLO 205 and HCT 116 cells in coumestrol group while compared with the DMSO control group (I-J), ordinate: Comp-Propidium lodide-A:: Propidium lodide-A.

Figure 7

Effects of coumestrol on the expression of ferroptosis pathway genes in CRC cells. Ferrous ions increased in both COLO 205 and HCT 116 cells after coumestrol treatment (A). The RT-PCR (B-C) and Western blot (D-E) analyses revealed that there was an increase in the expression of ferroptosis pathway-related genes, namely SLC39A8, NCOA4, VDAC2, and NOX2, at both the mRNA and protein levels following treatment with coumestrol.

Figure 8

Coumestrol affects ferroptosis pathway in CRC cell lines. SLC39A8 expression decreased at both mRNA (A) and protein levels (B) in both COLO 205 and HCT 116 cell lines after 96 hours of siRNA interference. Coumestrol could not upregulate ferroptosis pathway-related genes in CRC cell lines treated with SLC39A8 interference (C-D).