INHIBITOR OF PROTEIN KINASES 1-(4-CHLOROBENZYL)-3-CHLORO-4-(3-TRIFLUOROMETHYLPHENYLAMINO)-1 H -PYRROLE-2,5-DIONE INDUCES DNA DAMAGE AND APOPTOSIS IN HUMAN COLON CARCINOMA CELLS

Н -pyrrole-2,5-dione induces DNA 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-trifluoromethylpheny l-amino)-1 Н -pyrrole-2,5-dione (MI-1) might be a promising agent for suppression of growth of colon tumor cells.

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

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. Besides, they can be easily 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].
Several commercial and experimental anticancer drugs act through the apoptotic signaling pathways [3,29,30], targeting cell proliferation, causing DNA damage (cleavage of DNA by endonucleases, strand DNA breaks, changes in chromatin condensation) [13], and microtubule surplus Ca 2+ [29]. It was reported that the induction of apoptosis is one of the most successful non-surgical approach in cancer treatment [14,30,31]. Agents with anti-proliferative action might affect the extrinsic (via death receptors) and intrinsic (mitochondria mediated) pathways of apoptosis [29]. The phosphatidylinositol-3 kinase (PI3K)-protein kinase B (Akt)-mammalian target of the rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathways might be involved in the cancer cells growth inhibition [8,16]. The anticancer agents trigger the TNF receptor superfamily receptors, Fas and/or Fas ligand, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), activate caspase-8 or caspase-10, affect the transcription nuclear factor-B (NF-κB), and mitogen-activated protein kinases (MAPKs) [1,29]. Mitochondria and mitochondrial proteins are targets for numerous anticancer agents that act via the mechanisms of apoptosis [29]. The B-cell-lymphoma protein 2 (BCL-2) family members are key regulators of the intrinsic apoptotic pathway [27]. The agents that possess the anti-proliferative action can also affect the initiator caspases with next activation of the effector caspases that are considered to be the main regulators of apoptosis [1].
Diphenylamine assay of DNA fragmentation. For quantitative determination of DNA fragmentation, diphenylamine assay was applied. Diphenylamine interacts with deoxyribose that results in an increase of blue colored product. Human colon carcinoma cells of HCT116 line were treated for 24 h with MI-1 or Dox at 0.9 µg/mL. Cells were lysed in 0.5 mL Tris-EDTA buffer, pH 7.4, supplemented with 0.2 % Triton X-100 and centrifuged for 10 min at 12,000 g at 4 °C. The fragmented DNA (labelled as A) was separated from intact chromatin. The supernatant with fragmented DNA (labelled as B) was transferred into a new tube. 0.5 mL of 25 % Trichloroacetic acid (Sfera Sim, Lviv, Ukraine) was added to tubes A and B, mixed and incubated for 1 h at 56 °C. The DNA sample was centrifuged for 10 min at 14,000 g at 4 °C. 1 mL of freshly prepared diphenylamine reagent (150 mg diphenylamine (Sigma Aldrich, St. Louis, Missouri, USA), 10 mL glacial acetic acid, 150 mL concentrated H 2 SO 4 and 50 mL of acetaldehyde solution) was added to pellets A and B and incubated overnight at 37 ºC. Afterwards, 200 µL of the colored solution was transferred into a 96-well plate. The percentage of DNA fragmentation was calculated as {OD tube B / (OD tube A + OD tube B) × 100 %} [2,11]. The optical density (OD) was measured at 630 nm using Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, Vermont, USA).
Statistical analysis. The results were analyzed and illustrated with GraphPad Prism (version 6; GraphPad Software, La Jolla, California, USA), and presented as a mean ± standard deviation. ANOVA test (by Dunnett's test) was used for analysis of the statistical data. A P-value <0.05 was admitted as statistically significant.

RESULTS
Recently, we have demonstrated that MI-1 possessed high cytotoxicity towards human colon carcinoma HCT116 cells with the IC 50 of 0.9 µg/mL. The IC 50 value of Dox for these cells equaled 0.8 µg/mL [10]. The 0.9 µg/mL concentration of MI-1 and Dox was chosen as a working dose at Western-blot analysis of their pro-apoptotic effects in HCT116 cells.
It was found that MI-1 induced an increase in the content of the cleaved (active) form of caspase 3 in treated HCT116 cells. This effect was comparable with that under the action of Dox (Fig. 2).
MI-1 did not affect the level of the phosphorylated form of MAPK (Mitogen Activated Protein Kinases) in the treated HCT116 cells, while doxorubicin significantly reduced the level of the phosphorylated form of MAPK (Fig. 2).
In order to estimate the amount of DNA fragmentation in HCT116 cells under the treatment with MI-1, a colorimetric diphenylamine assay was applied. An increased content of the fragmented DNA was revealed in these cells at such action (Fig. 3). At 0.9 µg/mL dose, MI-1 induced DNA fragmentation at the level of 14.2 %, while Dox at the same dose caused 19.1 % of DNA fragmentation in HCT116 cells (Fig. 3). DNA comet analysis of the targeted cells at their horizontal electrophoresis under alkaline conditions is applied for the detection of single-strand breaks in the DNA molecule and under neutral conditions -for the detection of double-strand breaks in the DNA molecule. This method is widely used for investigation of DNA damage under the action of physical (gamma radiation) or chemical (antitumor drugs) agents [20,22]. We used an alkaline comet assay in order to identify the potential of MI-1 and Dox to induce single-strand DNA breaks in the treated HCT116 cells. At the dose of 0.9 µg/mL, MI-1 caused DNA damage in HCT116 cells with the OTM (Olive Tail Moment) of 13.2, while Dox induced OTM equal 10.9 (Fig. 4). A insignificant DNA damage (OTM = 1.8) was detected in the untreated HCT116 cells (Fig. 4).

DISCUSSION
In the previous study, we showed that MI-1 possessed a toxic action towards tumor cells of different origin (cervix, colon, breast, liver, pancreatic carcinomas). The most prominent effect was demonstrated at treatment of human cervix carcinoma (KB3-1 and KBC-1) and human colon carcinoma (HCT116) cells [10]. In this study, we have demonstrated that MI-1 induced the activation of caspase 3 via its cleavage in the treated HCT116 cells. As known, the effector caspases, in particular caspase 3, cleave various intracellular molecular targets that regulate the metabolism of DNA, histones and other proteins with biologically significant functions [25].
The development of antitumor agents that affect the mitochondrial signaling pathway of apoptosis is a promising strategy in the development of targeted antitumor chemotherapy [12,27]. The Western-blot analysis showed that MI-1 induces the mitochondria-dependent pathway of apoptosis in HCT116 cells. MI-1 reduced the level of the anti-apoptotic protein Bcl-2, increased the level of Apaf1 protein and EndoG in human colon carcinoma cell of HCT116 line. The overexpression of Bcl-2 and Bcl-xL proteins inhibits apoptosis by reducing the production of the reactive oxygen species via stabilization of the mitochondrial membrane potential (∆ψ) and blocking the release of proapoptotic proteins (e.g. cytochrome c) from mitochondria [5]. The Apaf1 is one of central elements in the mitochondrial pathway of apoptosis activated by a wide range of proapoptotic stimuli. The formation of the apoptosome of a circular structure that includes Apaf1, cytochrome c, ATP, and initiating pro-caspase-9 is critical for the activation of the caspase cascade in the targeted cells [32].
MI-1 did not affect the content of the phosphorylated form of MAPK in HCT116 cells. MAPK is a group of protein serine-threonine kinases that play an important role in the regulation of cell proliferation and differentiation, as well as the survival and drug resistance of tumor cells [21]. Рис. 4. Показник ДНК-пошкоджувального ефекту (за результатами ДНК-комет аналізу за лужних умов) досліджуваних чинників у пухлинних клітинах лінії HCT116: А -контроль, B -Dox (0,9 мкг/мл), C -МІ-1 (0,9 мкг/мл), D -кількісні дані щодо пошкодження ДНК у клітинах на 24-ту годину дії цих чинників. *** -P <0,001 (суттєві зміни порівняно з контролем -необробленими клітинами). Лінійка відповідає 20 мкм DNA fragmentation is catalyzed by the endonucleases and it is one of the indicators of the catabolic changes in the apoptotic pathway of cell death [29]. MI-1 induces DNA fragmentation and single-strand DNA breaks in the treated HCT116 cells. It is known that antitumor agents induce apoptosis that is accompanied by DNA fragmentation, in particular under the action of EndoG, while the inhibition of the endonuclease activity protects tumor cells from death [4]. As reported, the reactive oxygen species produced in the targeted cells under the action of the antitumor drugs cause the oxidative damage of DNA molecule and, consequently, increase the efficiency of the cytotoxic effects of these drugs [26]. The capability of MI-1 to activate caspase 3 together with the above noted elevation of the EndoG nuclease might be responsible for the degradation of the chromosomal DNA (DNA single-strand breaks and DNA fragmentation) in colon carcinoma cells. We propose a hypothetical scheme of signaling pathways induced by MI-1 during its inhibition of human colon carcinoma cells of HCT116 line (Fig. 5). Concluding, we have shown that the anti-proliferative action of protein kinase inhibitor 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione is associated with apoptosis induction via mitochondria-dependent pathway. Particularly, MI-1 activated caspase 3 and increased the level of mitochondria-specific endonuclease EndoG and Apaf1 protein. At the same time, it reduced the level of Bcl-2 protein in human colon carcinoma HCT116 cells. Besides, MI-1 induced DNA single-strand breaks and DNA fragmentation in these cells. Thus, 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione (MI-1) might be a promising agent for killing tumor colon cells.

ACKNOWLEDGEMENT
This research was supported by the Ministry of Education and Science of Ukraine grant (registration number 0119U100331).

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Animal Rights: This article does not contain any studies with animal subjects performed by the any of the authors.