Mecanismos de ação anticâncer colorretal in vitro e toxicidade em modelo de peixe-zebra do derivado acridínico AMTAC-19

Abstract

Colorectal Cancer (CRC) is a major global public health concern. In light of the need to develop more effective agents and/or those with lower toxicity, the acridine derivative (E)-1'-((4-bromobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrol]-4'-carbonitrile (AMTAC-19) was previously synthesized and tested for its in vitro antitumor potential. AMTAC-19 exhibited significant cytotoxicity against the HCT-116 human colorectal cancer cell line (half-maximal inhibitory concentration – IC50 = 10.35 ± 1.66 μM). Accordingly, this study aimed to characterize the anti-CRC mechanisms of action of AMTAC-19 through in silico and in vitro assays, as well as its embryotoxicity in a zebrafish model. The results of molecular docking demonstrated favorable interactions between AMTAC-19 and the crystallographic structures of Extracellular Signal-Regulated Kinase 1 (ERK1), c-Jun N-terminal Kinase 1 (JNK1), p38α Mitogen-Activated Protein Kinase (p38α MAPK), Protein Kinase B (PKB/AKT), Nuclear Factor kappa B (NF-κB; p50/p65), and B-cell Lymphoma-2 (BCL-2). In vitro assays using specific antibody labeling revealed that, after 48 hours of treatment with AMTAC-19, there was a significant increase in the percentage of cells labeled with anti-p-ERK1/2 and anti-p-JNK1/2 antibodies, as well as a significant reduction in cells labeled with anti-p-BCL-2 antibodies. Pre-treatment with ERK1/2 or JNK inhibitors significantly prevented AMTAC-19-induced cytotoxicity in HCT-116 cells. In redox state analysis, AMTAC-19 stimulated the production of Reactive Oxygen Species (ROS) after 30 minutes and 1 hour of treatment, and pre-treatment with N-acetyl-L-cysteine (NAC) significantly prevented its cytotoxicity. Using the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride (JC-1) dye, it was observed that AMTAC-19 induces changes in the mitochondrial membrane potential (Δψm) of HCT-116 cells. Laser confocal microscopy analysis of apoptotic morphological characteristics using Hoechst 34580 dye showed that AMTAC-19 significantly increased the number of fluorescent cells after 48 hours of treatment, with chromatin condensation observed, supporting the pro-apoptotic effect induced by this molecule. Additionally, the clonogenic assay used to evaluate changes in cell survival and proliferation induced by AMTAC-19 demonstrated that the test compound, at a concentration of 1.56 μM (equivalent to 1/8 of the IC50), significantly reduced the colony-forming ability of HCT-116 cells. Finally, non-clinical toxicity evaluation using a zebrafish model showed that treatment of embryos and larvae with AMTAC-19 (100 μM – approximately 10 times the IC50 for HCT-116 cells) for 96 hours did not cause animal mortality, suggesting that the compound's median lethal concentration (LC50) is greater than 100 μM. In summary, AMTAC-19 exhibits in vitro anti-CRC effects through apoptosis induction, oxidative stress, and modulation of ERK1/2, JNK, and BCL-2 proteins.