Combined treatment of thymoquinone and embelin epigenetically attenuates epithelial-to-mesenchymal transition inducing tumor regression by efficiently suppressing stemness factors through EGFR-RAS-MAPK axis colonospheres

This study aims to investigate the effects of thymoquinone (TQ) and embelin (EB), both individually and in combination on cancer stem cells (CSC)-enriched colonospheres derived from HT29 and HCT15 cell lines, thereby offering valuable insights for future therapeutic strategies. To address this three-dimensional (3D) colonospheres were cultured and used as experimental model to assess the effect of individual and combinatorial treatment of drugs. Computational analysis of edible phytochemicals, bioinformatic analyses for prediction of stemness markers and prominent signaling axis suggested that, TQ & EB would be most effective against colon cancer. Hence, cells were treated with these two drugs. Subsequently, RNA isolation, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, Molecular docking (MD) and simulation were performed, and data garnered in this way revealed the impact of TQ in colonospheres derived from both HT29 and HCT15 cell lines. Co-treatment exacerbates better regression of colonospheres and colonoid by inhibiting the EGFR-RAS-MAPK pathway, leading to a reduction in cell proliferation and a decrease in stemness through the downregulation of KI67, CD44, CD133, SOX2, OCT4, NANOG, and KLF4. Compelling facts are; TQ & EB treatment affects the epigenetic DNA methylation pattern of the genome by downregulating DNMT1, DNMT3A, DNMT3B, EZH2, UHRF1 and PARP1, induces E-cadherin gene expression and Vimentin gene repression, and thus augments mesenchymal-to-epithelial transition (MET). Co-treatment inhibits the EGFR-RAS-MAPK signaling pathway at multiple effector level and stimulates the expression of certain collagen-synthesizing genes in colonospheres. MD simulations have confirmed that TQ and EB can stably interact with the DNMTs and HDACs, which could influence their enzymatic activity near their target genes. Therefore, the combined use of TQ and EB offers a promising new therapeutic approach as epidrugs to inhibit DNMT1, DNMT3A, DNMT3B, HDAC1, and HDAC2, rendering them safe as therapeutic medicines for the prevention of colon cancer.