Trametes versicolor laccase induces apoptosis and DNA damage in colorectal cancer via reactive oxygen species-mediated signaling disruption.

OBJECTIVE

Copper-containing laccases oxidize various phenolic and non-phenolic compounds. Among them, fungal laccases have been extensively studied in industry, but their biomedical potential remains largely unexplored. We aimed to investigate the anticancer activity of the laccase enzyme, which we purified from Trametes versicolor without loss of activity using the three-phase partitioning (TPP) technique-an easy, inexpensive method that allows high activity recovery-on colorectal cancer (CRC) cells for the first time and to elucidate its mechanism of action.

METHODS

The effects of the pure laccase enzyme were investigated on two CRC cell lines (HCT-116 and Caco-2) and normal endothelial cells (HUVEC). Cytotoxicity was assessed using the WST-1 assay. Apoptosis and cell cycle progression were analyzed by flow cytometry. Genotoxicity was determined by using micronucleus and comet assays. Intracellular DCFH-DA fluorescence intensity was measured, and the expression of apoptosis- and DNA repair-related genes was analyzed by qRT-PCR. The MAPK (p38, JNK) and NF-κB signaling pathways were examined by immunofluorescence.

RESULTS

Laccase treatment exhibited a dose-dependent cytotoxic effect on CRC cells, with minimal impact on HUVECs. It induced apoptosis, oxidative stress-associated responses, DNA damage, and G0/G1 cell cycle arrest. Gene expression analysis revealed upregulation of Bax and Caspase-3 and downregulation of Bcl-2, RAD51, and NF-κB. Immunofluorescence confirmed activation of p38/JNK and suppression of NF-κB signaling.

CONCLUSION

TPP-purified fungal laccase exhibits selective cytotoxicity and pro-apoptotic effects in CRC cells via redox-mediated DNA damage and modulation of signaling pathways, supporting its potential as a novel anticancer agent.