Fig. 5

Transcriptomic analysis of HCT116 cells treated with HJ-4, emphasizing the activation of p53 signaling and apoptosis pathways. (A–B) Heat map (A) and volcano map (B) of differentially expressed genes in HCT116 cells treated with HJ-4 (16 µM). A total of 257 differentially expressed genes were screened compared with the control group, among which pro-apoptotic genes (e.g., PUMA, also known as BBC3) were significantly up-regulated (p < 0.05, |log2-fold change| > 1.2). (C) Ridge plot reveals the enrichment of p53 signaling, apoptosis and cell cycle regulation pathways. (D) GSEA plots. Positive enrichment of p53 signaling (NES = 1.55, p = 0.0017) and apoptosis pathways (NES = 1.61, p = 0.0017) indicates activation of tumor suppressive mechanisms by HJ-4. Negative enrichment of E2F targets (NES = -2.00, p = 0.0025) and Wnt/β-catenin signaling (NES = -1.70, p = 0.0067) reveals inhibition of oncogenic pathways. (E) KEGG pathway analysis of differentially expressed genes. Significant pathways include p53 signaling, DNA replication, and cell cycle regulation, underscoring HJ-4’s dual role in promoting apoptosis and suppressing proliferation. (F–G) GO enrichment analysis of up-regulated (F) and down-regulated (G) genes. Up-regulated genes were associated with apoptosis and stress response, while the down-regulated genes were involved in mitotic spindle organization and chromatin regulation. (H) Comprehensive GO analysis of all differentially expressed genes in the ontology, including biological process (BP), molecular function (MF), and cell component (CC). The bar chart highlights the regulation of pathways involved in apoptosis and nuclear tissue.