Analysing the effects of epigenetic silencing inhibitors on senescence related genes in a colorectal cancer cell line

Abstract

Cancer can only occur when tumor suppressors fail to induce senescence (stable cell-cycle arrest). In some cancers the reactivation of p16 triggers proliferative arrest; thus such tumor suppressors are suitable candidates for pro-senescence cancer therapy. This investigation’s aims were to find a candidate drug, which can reactivate silenced tumor suppressors, to rescue senescence specifically in colorectal cancer, and to further understand what families of HDACs regulate tumor suppressors. These were all screened on their potential to reactivate hyper-methylated stably integrated GFP reporters in HeLaGFP cells. Twelve of these drugs significantly increased GFP expression. Two of the top hits (HC toxin and CAY10603), and one of the non-hits (bromosporine), were taken on to further experiments to test their effect on p16 and other related genes via qRT-PCR in HCT116 human colon cancer. All three of these drugs significantly increased levels of p16 and p21 mRNA. However, only HC toxin (1μM) was found to de-repress p21 expression at the protein level; bromosporine at 1μM had no effect on p21 expression at the protein level. Senescence associated beta-galactosidase stains reveals an increase in lysosomal activity when exposed to HC toxin (50nM), and CAY10603 (500nM), while bromosporine had no noticeable affect. Furthermore, presto blue assays suggest HCT116 cells are more susceptible to drug-toxicity than the non-cancerous fibroblast cell-line, IMR90. HC toxin shows a lot of potential as a de-repressor of p21. While there is a specific affect on growth, whether or not the activation of senescence and/or apoptosis is specific to HCT116 cancer cells has yet to be elucidated.