The Interaction of Structural Analogues of Phenothiazines in p53-Dependent Cellular Signaling Pathways

The cytostatic effect of structural analogues of phenothiazines, in which benzene rings were replaced by azine systems (pyridines - <b>BM1</b>, <b>BM2</b>, and quinoline - <b>MJ1</b>, <b>MJ2</b>), on the HCT116 colon cancer cell line with wild-type p53 protein status and with mutated p53 protein, was studied. The participation of the tested substances in p53-dependent cell signaling pathways was assessed. Based on the performed colorimetric MTT assays, IC₅₀ values for the tested chemical compounds were calculated. The best IC₅₀ values for all lines were found for the <b>MJ2</b> compound. For the HCT116 line with wild-type p53 protein, IC₅₀ = 36.37 μM, and IC₅₀ = 57.82 μM for HCT116 with mutated p53 protein were estimated. 72 h microscopic observations of the survival of HCT116 line cells after exposure to the tested analogues were performed, which revealed apoptotic changes in the cells. Real-time polymerase chain reaction was carried out for marker genes from p53-dependent pathways. The studies were conducted under the reference gene RPL41 for the tested genes, which included apoptosis-inducing factor mitochondria-associated 2 (AIFM2), blocker of programmed apoptotic cell death (BCL2), and primary negative regulatory factor of the p53 protein (MDM2), respectively. Two HCT116 lines were used to compare the effect related to cellular p53 protein status on the relative increase in tested gene expression. The modulation of p53 protein-dependent signaling pathways by the tested phenothiazine analogues was confirmed.