Inhibitor of protein kinases 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione induces DNA damage and apoptosis in human colon carcinoma cells

N S Finiuk,S P Vashchuk,H M Kuznietsova,V K Rybalchenko,R S Stoika,O Yu Klyuchivska

doi:10.30970/sbi.1404.636

Abstract

Background. The heterocyclic scaffolds are in the list of key structural blocks used at synthesis of novel biologically active compounds. Materials and Methods. The present study addressed the evaluation of the mechanisms of the DNA damaging and pro-apoptotic actions in vitro of the maleimide derivative 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione (MI-1) targeting human colon carcinoma cells of HCT116 line. The Western-blot analysis was used to study changes in apoptosis-associated proteins, DNA comet assay under alkaline conditions was applied for evaluation of the DNA-damaging events, and Barton’s assay with diphenylamine was applied for measuring the level of DNA fragmentation in human colon carcinoma cells treated with MI-1 compound. Results. The results of the Western-blot analysis demonstrated that MI-1 induced the apoptosis in HCT116 cells via mitochondria-dependent pathway. It activated caspase 3 via its cleavage in the treated human colon carcinoma cells. Besides, MI-1 increased the content of mitochondria-specific proteins: endonuclease G (EndoG) and the pro-apoptotic cytosolic protein protease-activating factor 1 (Apaf1). At the same time, MI-1 reduced the level of the anti-apoptotic Bcl-2 protein in HCT116 cells. The DNA comet analysis under alkaline conditions of the targeted human colon carcinoma cells of HCT116 line demonstrated that MI-1 induced DNA single-strand breaks in line with the olive tail moment of 13.2. The results of the colorimetric diphenylamine assay in HCT116 cells have shown that cell treatment with MI-1 increased the content of fragmented DNA to 14.2 %. Conclusions. The anti-proliferative action of MI-1 in human colon carcinoma cells of HCT116 line is associated with apoptosis induction via mitochondria-dependent pathway, as well as the DNA damage through single-strand breaks and DNA fragmentation. These data suggest that the 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione (MI-1) might be a promising agent for suppression of growth of colon tumor cells. Keywords: 1Н-pyrrole-2,5-diones, apoptosis, Western-blot assay, comet assay, single-strand breaks, Barton’s assay, DNA fragmentation

Highlights

The heterocyclic structures with specific physicochemical properties are key components of the commercially available pharmaceuticals, including the anti-cancer agents approved by the FDA [19, 24]
The anti-proliferative action of MI-1 in human colon carcinoma cells of HCT116 line is associated with apoptosis induction via mitochondria-dependent pathway, as well as the DNA damage through single-strand breaks and DNA fragmentation
These data suggest that the 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenyl amino)-1Н-pyrrole-2,5-dione (MI-1) might be a promising agent for suppression of growth of colon tumor cells

Summary

Introduction

The heterocyclic structures with specific physicochemical properties are key components of the commercially available pharmaceuticals, including the anti-cancer agents approved by the FDA [19, 24]. The heterocyclic scaffolds are characterized by the lipophilicity, polarity, and bio-isosteric replacements They can be modified for construction of the targeted pharmaceutical agents [15]. Different heterocyclic scaffolds such as azole, imidazole, pyrazole, indole, pyrrolidine, pyridine, pyrrole, pyrimidines, quinoline, oxadiazole, and others were addressed as key structural blocks for development of the biologically active compounds [19]. Agents with anti-proliferative action might affect the extrinsic (via death receptors) and intrinsic (mitochondria mediated) pathways of apoptosis [29]. The agents that possess the anti-proliferative action can affect the initiator caspases with activation of the effector caspases that are considered to be the main regulators of apoptosis [1]. The heterocyclic scaffolds are in the list of key structural blocks used at synthesis of novel biologically active compounds

Methods

Results

Discussion

Conclusion