Gastrointestinal Digestion Enhances the In vitro Antitumor Activity of Red Grape Anthocyanins Against Colorectal Cancer Cells

Anthocyanins from red grapes possess notable anticancer properties, yet their biological activity after gastrointestinal digestion remains poorly characterized. In this study, we investigated the in vitro antitumor effects of a red grape anthocyanin extract digested using the INFOGEST protocol on colorectal cancer (CRC) cell lines. The study assessed cell viability, apoptosis induction, cell cycle progression, and confined 3D migration, and integrated whole-transcriptome microarray profiling to elucidate the underlying molecular mechanisms.Cell viability assays showed that simulated digestion markedly increased the extract’s potency, reducing the IC50 values by 2.25-fold in DLD1 cells and 1.59-fold in Caco2 cells. Transcriptomic profiling by microarray analysis demonstrated substantial gene expression changes upon treatment in DLD1 cells, with 873 genes showing a fold regulation greater than |±1.5|. Gene Set Enrichment Analysis (GSEA) identified enrichment of pro-apoptotic gene sets in treated cells, including p53 signaling, prolonged unfolded protein response activation and suppression of RNA metabolism. These findings were supported by the Annexin V/PI assay demonstrating apoptosis induction, and RT-qPCR validation of key apoptosis-regulating genes. The extract also displayed significant antiproliferative activity with cells arrested in the S phase, as evidenced by AlamarBlue and cell cycle analyses. Downregulation of key cell cycle promoters and upregulation of proliferation-inhibitory genes were also observed. Furthermore, the extract significantly impaired 3D migration in a microfluidic model, reducing migration speed, directionality, and number of migratory cells. GSEA revealed suppression of RHO GTPases and MIRO-mediated signaling, suggesting disruption of cytoskeletal and mitochondrial dynamics, strongly impacting cell migration. Together, these findings indicate that digested red grape anthocyanins exert a multifaceted anticancer effect in vitro by promoting apoptosis, inhibiting proliferation, and impairing cell migration, highlighting their potential as functional bioactive compounds for CRC management.