Abstract
BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy with the efficacy of current treatments limited. Nitidine chloride (NC), a natural alkaloid, exhibits antitumor potential, but its mechanism in CRC remains unclear. OBJECTIVE: To investigate the antitumor effects and mechanisms of NC in CRC for clinical therapeutic strategies. METHODS: A Cell Counting Kit-8 was used to assess the viability of HCT116 cells. Xenograft mice received NC (2, 4, 8 mg/kg, intraperitoneal, once every other day for 2 weeks). Transmission electron microscopy and hematoxylin-eosin staining analyzed tumor ultrastructure and pathology. RNA sequencing, real-time quantitative polymerase chain reaction, and molecular dynamics simulation (MDS) examined gene expression and NC-budding uninhibited by benzimidazoles 1 (NC-BUB1) interaction. Single-cell RNA sequencing, spatial transcriptomics, immunohistochemistry, and chromatin immunoprecipitation sequencing explored BUB1 expression and RAD21 regulation. Immune correlation analysis was conducted using TISIDB, TIMER2.0, and deconvolution algorithms (CIBERSORT, CIBERSORT-ABS, EPIC, ESTIMATE, MCPcounter, quanTIseq, TIMER, xCell) to evaluate associations between BUB1 and immune factors or infiltration. RESULTS: NC inhibited HCT116 cells with time- and concentration-dependent half-maximal inhibitory concentration reduction. NC-treated tumors displayed apoptosis and reduced angiogenesis. Transcriptomics identified BUB1 as a core downregulated gene. Multi-omics confirmed BUB1 overexpression in CRC (1293 vs. 2299 samples; standardized mean difference = 1.77, area under the curve = 0.94) in proliferative/malignant regions. Immune analyses revealed that high BUB1 expression correlated negatively with antigen presentation molecules, chemokines/receptors, and immune cell infiltration (B cells, M2 macrophages, cancer-associated fibroblasts, endothelial cells), suggesting immune microenvironment suppression. BUB1 knockout inhibited CRC cells; NC reduced BUB1 expression. MDS showed a stable NC-BUB1 complex (- 8.8 kcal/mol). The RAD21-BUB1 axis exerted key regulatory roles. CONCLUSION: NC exerts anti-CRC effects by downregulating BUB1, disrupting the RAD21-BUB1 axis, and alleviating BUB1-mediated immune suppression.