Mechanistic insights into the anti-tumor effects of neochlorogenic acid in hepatocellular carcinoma: in vitro and in vivo studies.

Background: Neochlorogenic acid (NCA), a naturally occurring polyphenolic compound, exhibits diverse biological activities. This study aimed to investigate the inhibitory effects of NCA on hepatocellular carcinoma (HCC) cells and elucidate its underlying molecular mechanisms.

Methods: The anti-proliferative activity of NCA on human HCC cell lines HepG2 and Huh-7 was assessed using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to analyze apoptosis and cell cycle distribution. Wound-healing assays were conducted to evaluate the effects of NCA on cell migration. Transcriptome sequencing was performed on NCA-treated and untreated Huh-7 cells to identify differentially expressed genes (DEGs) and signaling pathways. Western blot was used to validate the expression of key apoptosis-related proteins. In vivo experiments were carried out using a nude mouse xenograft model to assess the anti-tumor effects of NCA.

Results: NCA significantly inhibited the proliferation of HepG2 and Huh-7 cells, with half maximal inhibitory concentration (IC₅₀) values of 345.5 and 231.8 µM at 24 hours, and 244.0 and 199.2 µM at 48 hours, respectively. Flow cytometry revealed that NCA induced apoptosis and G1 phase cell cycle arrest. Wound-healing assays demonstrated that NCA effectively suppressed HCC cell migration. Transcriptome analysis revealed 2,297 DEGs in NCA-treated Huh-7 cells (Padj<0.01), with 1,162 upregulated and 1,135 downregulated. Pathway enrichment analysis indicated significant enrichments in pathways related to "Alcoholism", "MicroRNAs in cancer", "Hepatocellular carcinoma", and "TGF-beta signaling pathway". Western blot confirmed the upregulation of pro-apoptotic proteins [BCL2-associated X protein (BAX); cysteinyl aspartate specific proteinase 3 (CASP3), BH3 interacting domain death agonist (BID), and cytochrome C (CYCS)] and downregulation of the anti-apoptotic protein B-cell lymphoma 2 (BCL2). In vivo, NCA treatment significantly inhibited tumor growth.

Conclusions: This study provides compelling evidence that NCA inhibits HCC cell growth and migration both in vitro and in vivo through the induction of apoptosis and cell cycle arrest. Transcriptomic analysis reveals that NCA induces widespread changes in transcriptional networks and metabolic pathways within HCC cells, highlighting its potential as a promising therapeutic strategy for HCC.