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

## TL;DR

This study examines how phenothiazine analogues affect p53-dependent pathways in colon cancer cells with wild-type and mutated p53.

## Contribution

The study identifies a phenothiazine analogue (MJ2) that shows strong cytostatic effects and modulates p53-dependent gene expression.

## Key findings

- MJ2 had the lowest IC50 values in both wild-type and mutated p53 HCT116 cells.
- Apoptotic changes were observed in HCT116 cells after exposure to the analogues.
- Phenothiazine analogues modulated the expression of p53-dependent genes like AIFM2, BCL2, and MDM2.

## Abstract

The cytostatic effect of structural analogues of phenothiazines,
in which benzene rings were replaced by azine systems (pyridines – BM1, BM2, and quinoline – MJ1, MJ2), 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 MJ2 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.