Trans-vaccenic acid reduces HT-29 colorectal cancer cell viability and is associated with changes in apoptosis-, lipid metabolism-, and survival-related gene and candidate miRNA expression.

BACKGROUND

Colorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide, necessitating the identification of novel bioactive compounds with therapeutic potential. Trans-vaccenic acid (TVA), a primary ruminant-derived trans fatty acid, possesses diverse biological activities; however, its cellular effects in CRC and its association with miRNA expression and downstream targets remain to be fully characterized.

METHODS

In this study, HT-29 human CRC cells were exposed to increasing concentrations of TVA (0-800 µM). Cell viability was evaluated using the MTT assay, and apoptotic cell populations were quantified using Annexin V-FITC/PI flow cytometry alongside Hoechst 33,342 nuclear staining. The expression levels of apoptosis-, lipid metabolism-, and cell survival-related genes (BAX, CASP3, CASP8, CASP9, MCL1, BCL2, SCD1, and GPR34), as well as candidate miRNAs predicted via miRWalk 2.0, miRDB, and TargetScan, were analyzed by RT-qPCR and ELISA.

RESULTS

TVA reduced HT-29 cell viability in a dose-dependent manner, with an IC₅₀ value of 251.13 µM at 24 h. Hoechst staining revealed apoptotic nuclear changes and apoptotic body formation, while Annexin V-FITC/PI flow cytometry confirmed a predominant increase in late apoptotic cells (p < 0.001). At the molecular level, TVA treatment significantly increased the expression of pro-apoptotic markers (BAX, CASP3, CASP8, and CASP9) and reduced anti-apoptotic (MCL1, BCL2), lipid homeostasis/tumor survival (SCD1), and oncogenic survival signaling (GPR34) markers (p < 0.05). Concurrently, TVA administration was associated with increased expression of candidate miRNAs (let-7e-5p, let-7b-5p, miR-519d-3p, miR-582-5p, and miR-381-3p; p < 0.05), accompanied by changes in the expression of their predicted target transcripts.

CONCLUSION

TVA exerts cytotoxic and pro-apoptotic effects in HT-29 cells, accompanied by favorable shifts in lipid metabolism/survival gene expression and candidate miRNA profiles. While these correlative findings support the bioactive potential of TVA in CRC, further studies incorporating multiple cell lines and functional miRNA validation (e.g., mimic/inhibitor transfections) are required to establish direct mechanistic causality.