(1) Background: Decitabine and azacitidine are cytosine analogues representing the class of drugs interfering with DNA methylation. Due to their molecular homology and similar clinical application, both drugs are often regarded as interchangeable. Despite their unique mechanism of action the studies designed for observation and comparison of the prolonged activity of these drugs are rare. (2) Methods: The short-time (20-72 h) and long-term (up to 20 days) anti-cancer activity of decitabine and azacitidine has been studied in colorectal cancer cells. We observe the impact on cell culture's viability, clonogenicity, proliferation, and expression of CDKN1A, CCND1, MDM2, MYC, CDKN2A, GLB1 genes, and activity of SA-β-galactosidase. (3) Results: Decitabine has much stronger anti-clonogenic activity than azacitidine. We show that azacitidine, despite significant immediate toxicity, has negligible long-term effects. Contrary, decitabine, which does not exert initial toxicity, profoundly worsened the condition of the cells over time. On the 13th day after treatment, the viability of cells was decreased and proliferation inhibited. These functional changes were accompanied by up-regulation of expression CDKN1A, CCND1, and CDKN2A genes and increased activation of SA-β-galactosidase, indicating cellular senescence. (4) Conclusions: Our head-to-head comparison revealed profound differences in the activities of decitabine and azacitidine important in their anti-cancer potential and clinical application. The effects of decitabine need relatively long time to develop. This property is crucial for proper design of studies and therapy concerning decitabine and undermines opinion about the similar therapeutic mechanism and interchangeability of these drugs.
Keywords: azacitidine; cellular senescence; chemotherapy; colon cancer; decitabine; epigenetic drug; vidaza.