Thymoquinone triggers DNA hypomethylation in human colorectal adenocarcinoma (CACO-2) cells.

Abstract

DNA hypermethylation is a frequent feature of colorectal cancer, where it has been linked to the silencing of tumour suppressor genes and cancer progression. Thymoquinone (TQ) is a bioactive compound found in black cumin (Nigella sativa), and displays promising anti-cancer effects; however, its epigenetic effect in colorectal cancer is uncertain. This study investigated the impact of TQ on global DNA methylation in the human colorectal adenocarcinoma (Caco- 2) cell line. Caco-2 cells were cultured and treated with TQ for 24 hours. The MTT assay was conducted to assess cell viability and obtain an IC50. Global DNA methylation was quantified using an ELISA kit. Changes in mRNA expression of DNMT1, DNMT3a, DNMT3b, UHRF1, MBD2, TET1, TET2, TET3, and miR-29b were determined with qPCR. Changes in DNMT1,DNMT3a, DNMT3b, and MBD2 protein expression were assessed by Western blotting or ELISA. TQ dose-dependently decreased cell viability and yielded an IC50 of 504 μM. TQ brought about global DNA hypomethylation (p < 0.005) in Caco-2 cells. TQ reduced mRNA expression of DNMT1 (p < 0.05), DNMT3a (p < 0.05), and DNMT3b (p < 0.005), as well as UHRF1 (p < 0.05) and MBD2 (p < 0.05). TQ increased TET1 (p < 0.05), TET2 (p < 0.05), and TET3 mRNA expression (p < 0.005). MiR-29b expression was also increased (p < 0.05). TQalso reduced protein expression of DNMT1 (p < 0.0001), DNMT3a (p < 0.0001), DNMT3b (p< 0.05) and MBD2 (p < 0.005). Together, these results suggest that TQ induced global DNA hypomethylation in Caco-2 cells by down-regulating DNMT1, DNMT3a, DNMT3b, UHRF1,and MBD2, and up-regulating TET1-3 and miR-29b expression. This highlights the potential of TQ as a promising anti-cancer agent.