Omega-3 DHA Targets P53-Mutant CRC Stem-like Cells via Membrane Disruption, Modulation of Differential Marker Genes, and Suppression of Self-Renewal Capacity

P53-mutant colorectal cancer stem cells (CRCSCs) are characterized by multidrug-resistance, high self-renewal, and limited differentiation potential, which necessitates the development of novel compounds capable of targeting the molecular drivers of differentiation and stemness maintenance in CRCSCs, independent of p 53 status. Given this, docosahexaenoic acid (DHA) was selected as a promising therapeutic candidate and its ability to modulate differentiation marker genes and stemness maintenance in CRC stem-like cells was evaluated.

P53-mutant (E204X) Caco2 cells, serving as a well-established model of CRCSCs, were treated with 50-150 μM DHA for 48 h. We then evaluated multiple parameters including cell number, metabolic activity, proliferation rates, and LDH release. Additionally, the expression of stemness (CD133) and intestinal differentiation marker genes (Muc-2 and CK-20) as well as clonogenic capacity were assessed. Bioinformatic analysis was performed to predict p 53-binding sites within the promoters of CD133, Muc-2, and CK-20 for wild-type p 53 and its mutant form.

DHA significantly decreased cell numbers, metabolic activities, and proliferation rates after 48 h of treatment. Concurrently, DHA treatment disrupted cell membrane integrity, as evidenced by a marked increase in LDH release into the supernatant. The initially low expression levels of Muc-2 and CK-20 increased dramatically, whereas significant decreases were observed in CD133 gene expression. This was confirmed by significant reductions in self-renewal, plating efficiency, and survival fraction after 2 weeks. Bioinformatics analyses showed that while wild-type p 53 binds to the promoters of three differentiation marker genes, the mutant-p53 (E204X) lacks such binding.

DHA disrupts cell membrane integrity, modulates stemness and differentiation marker genes, and targets stemness maintenance through a p 53-independent pathway, highlighting its cytotoxic and therapeutic potential against p 53-mutant CRCSCs.