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

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