Cytotoxic action of maleimide derivative 1-(4-Cl-benzyl)-3-chloro-4-(CF(3)-phenylamino)-1H-pyrrole-2,5-dione toward mammalian tumor cells and its capability to interact with DNA

Development of chemical compounds capable to supress tumor progression is a perspective strategy of cancer treatment. heterocyclic compounds possess a broad spectrum of biological activities, including anticancer one. according to the previous results of in silico modeling maleimide derivative 1-(4-cl-benzil)-3cl-4-(cF3-phenylamino)-1Н-pyrrole-2,5-dione (MI-1) has a potential effect as an inhibitor of tyrosine protein kinases. The present study was aimed at in vitro evaluation of MI-1 cytotoxic effects toward tumor cells of various lines. The viability of tumor cells after incubation with MI-1 was measured by means of 3,4,5-dymetyltiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) test. The MI-1 compound was shown to be toxic for a majority of studied tumor cell lines with Ic50 value ranging from 0.8 to 62.2 μg/ml depending on the tissue origin of cells. The most prominent effect of MI-1 towards human cervix carcinoma (KB3-1 and KBC-1) cells with six times higher toxicity towards the multidrug resistant sub-line kBc-1 cells comparing with the action of Doxorubicin was demonstrated. MI-1 inhibited the viability of human pancreatic, hepatocarcinoma, and colon carcinoma cells only in high doses, while human and rat glioblastoma cells were not sensitive to MI-1. thus, the MI-1 anticancer activity dropped in the following rank of tumor cells: cervix > breast > pancreatic carcinoma > liver carcinoma > colon carcinoma > glioblastoma. experiments on replacement of methyl green dye from DNa-methyl green complex showed that MI-1 intercalated into DNa molecule structure. the increase of the amount of the additional band of super-spiral DNa in the presence of MI-1 was revealed by means of DNA retardation at electrophoresis in the agarose gel and this effect was more pronounced than the effect of doxorubicin. The data presented indicate a new DNA-targeting mechanism of maleimide derivative 1-(4-cl-benzil)-3-cl-4-(cF3-phenylamino)-1Н-pyrrole-2,5-dione anticancer action. k e y w o r d s: 1-(4-cl-benzil)-3-cl-4-(cF3-phenylamino)-1Н-pyrrole-2,5-dione, anticancer activity, МТТ.


Development of chemical compounds capable to supress tumor progression is a perspective strategy of cancer treatment. heterocyclic compounds possess a broad spectrum of biological activities, including anticancer one. according to the previous results of in silico modeling maleimide derivative 1-(4-cl-benzil)-3cl-4-(cF 3 -phenylamino)-1Н-pyrrole-2,5-dione (MI-1) has a potential effect as an inhibitor of tyrosine protein kinases. The present study was aimed at in vitro evaluation of MI-1 cytotoxic effects toward tumor cells of various lines. The viability of tumor cells after incubation with MI-1 was measured by means of 3,4,5-dymetyltiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) test. The MI-1 compound was shown to be toxic for a majority of studied tumor cell lines with Ic 50 value ranging from 0.8 to 62.2 μg/ml depending on the tissue origin of cells. The most prominent effect of MI-1 towards human cervix carcinoma (KB3-1 and KBC-1) cells with six times higher toxicity towards the multidrug resistant sub-line kBc-1 cells comparing with the action of Doxorubicin was demonstrated. MI-1 inhibited the viability of human pancreatic, hepatocarcinoma, and colon carcinoma cells only in high doses, while human and rat glioblastoma cells were not sensitive to
thus, the MI-1 anticancer activity dropped in the following rank of tumor cells: cervix > breast > pancreatic carcinoma > liver carcinoma > colon carcinoma > glioblastoma. experiments on replacement of methyl green dye from DNa-methyl green complex showed that MI-1 intercalated into DNa molecule structure. the increase of the amount of the additional band of super-spiral DNa in the presence of MI-1 was revealed by means of DNA retardation at electrophoresis in the agarose gel and this effect was more pronounced than the effect of doxorubicin. The data presented indicate a new DNA-targeting mechanism of maleimide derivative 1-(4-cl-benzil)-3-cl-4-(cF 3 -phenylamino)-1Н-pyrrole-2,5-dione anticancer action. k e y w o r d s: 1-(4-cl-benzil)-3-cl-4-(cF 3 -phenylamino)-1Н-pyrrole-2,5-dione, anticancer activity, МТТ. C ancer is a worldwide health problem, and it is the second leading reason of human mortality after the cardiovascular diseases. A development of chemical compounds capable of specific blocking key regulatory proteins of tumor progression is a perspective strategy of cancer treatment [1,2].
Protein kinases are enzymes that regulate different cellular processes including cell proliferation, differentiation and migration, cell cycle regulation, and metabolism [2,12]. A deregulation of these processes via mutations or over-expression of kinases leads to various diseases [13,14]. Kinases play an important role in the carcinogenesis and metastases of different types of cancer [2,15]. Thus, a development of protein kinases inhibitors is of great interest for the cancer treatment. A number of protein kinases inhibitors, such as imatinib [16], benzotriazines, quinazolines, pyrazolopyrimidines, imidazo[1,5-a]pyrazines, pyridopyrimidinones and other heterocycles, ATP-phospho-peptide conjugates have been developed and investigated for the treatment of cancer [12,[17][18][19].
It is obvious that the multifunctional anticancer agents affecting different bio-molecules and biological processes could be more efficient treatment remedies comparing to such agents with only one bio-target in the tumor cells. The aim of our study was to carry out a search for novel biological targets at the cytotoxic action of maleimide derivative MI-1, namely evaluating a possibility that DNA molecule could also be such target. Besides, we addressed a potential toxic effect of MI-1 towards mammalian tumor cell lines, specifically the drug-resistant tumor cells not studied earlier.
cell culture and anti-proliferative Mtt assay. Human pancreatic ductal adenocarcinoma Capan-1 cells, human epidermoid cervix carcinoma KB3-1 cells and its colchicine-resistant sub-line KBC-1, that is characterized by over-expression of plasma membrane P-glycoprotein, were donated by a Collection of the Institute for Cancer Research at Vienna Medical University (Vienna, Austria). Human breast adenocarcinoma MCF-7 and MDA231, human colon carcinoma HCT116 cells, human hepatocarcinoma HepG2 cells were obtained from the Collection at the Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine). Human glioblastoma cells of U251, U373 and T98G lines, rat glioma C6 cells were obtained from a Collection at the Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine (Kyiv, Ukraine). Cells were grown in RPMI-1640 (Biowest, Nuaille, France) or Dulbecco's-modified Eagle's medium (DMEM, Biowest, Nuaille, France) culture medium supplemented with 10% fetal bovine serum (Biowest, Nuaille, France) at the standard conditions (37 °C in an atmosphere of 5% CO 2 ).
The MI-1 cytotoxicity towards tumor cells in vitro was measured using colorimetric MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich, USA) assay for estimating functional activity of the treated cells. Cells were plated at 5,000 cells/well (substrate-dependent cells) or 15,000 cells/well (suspension cells) in 100 µl of 96-well plates and allowed to grow overnight. The MI-1 or Doxorubicin at 1, 10, and 100 µg/ml was added in 100 µl of cultural medium, and cells were incubated for the next 72 h. After that, the MTT assay was performed according to the manufacturer's recommendations (Sigma-Aldrich, St. Louis, MO, USA). The absorbance of formazan was measured by an Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, VT, USA). The IC 50 level of MI-1 was calculated as the drug concentration that reduced cell viability by 50% [20].
DNa intercalation assay using methyl green replacement. Methyl green binds to DNA with an absorption maximum at 642 nm. Free methyl green does not show absorption at this wavelength, while the compounds that bind/intercalate with DNA replaced methyl green from the complex of methyl green-DNA and decreased the optical density at 642 nm wave length. 485 µl of salmon sperm DNA (50 μg/ml, Sigma-Aldrich, St. Louis, Missouri, USA) were incubated for 1 h at 37 °C with 15 µl of methyl green (Sigma-Aldrich, St. Louis, Missouri, USA) solution (1 mg/ml in water). 500 µl of MI-1 (1 and 10 μg/ml), or Dox (10 μM) were added to methyl green-DNA complex and incubated for 2 h at 37 °C in the dark. The Ethydium bromide (EtBr, 1 and 10 μg/ml) was used as a positive control. Absorption of methyl green was measured at 630 nm [22] using a fluorescence plate reader (Absorbance Reader BioTek ELx800, BioTek Instruments, Inc., Winooski, Vermont, USA).
Data analysis. The results were analyzed and illustrated using Origin software (version 7). All data are presented as the mean (M) ± standard deviation (SD) of three independent replications. A t test was used for statistical analysis. Statistical significance was identified at P ≤ 0.05.

results and discussion
cytotoxicity study. In this study, human tumor cells of different tissue origin, namely leukemia, cervix, breast, glioblastoma, and pancreas, were subjected to the action of the MI-1 compound applied in three doses -1, 10, and 100 μg/ml. The MTT assay was used for evaluation of the antineoplastic activity of the MI-1. It was found that the MI-1 inhibited growth of human breast adenocarcinoma MCF-7 cells with the IC 50 of 9.7 μg/ml (Fig. 2, Table), while the Doxorubicin demonstrated higher cytotoxicity towards these tumor cells (IC 50 = 1.9 μg/ml). However , human breast adenocarcinoma cells of the MDA231 line were relatively resistant to the action of MI-1 in dose up to 50 μg/ml (Fig. 2, Table).
Rapid development of the multidrug resistance is one of the main problems in cancer treatment. Among the main mechanisms of such resistance are the over-expression of the ABC transporters on plasma membrane, mutations in the BRca gene, DYNll1 gene and other mechanisms that promote drug inactivation and/or degradation [23,24]. In this study, we compared the anti-neoplastic activity of the MI-1 towards human epidermoid cervix carcinoma cells of KB3-1 line and its colchicine-resistant KBC-1 sub-line that is characterized by P-glycoprotein over-expression. The MI-1 proved to be six times more effective in growth inhibition of KBC-1 cells (IC 50 = 0.8 μg/ml), comparing to Doxorubicin (IC 50 = 4.8 μg/ml), while at targeting wild type KB3-1 line, the picture was opposite and these cells were more sensitive to the Doxorubicin (IC 50 = 0.3 μg/ml) than to the MI-1 (IC 50 = 7.5 μg/ml) (Fig. 2, Table). Thus, the MI-1 was shown to be capable of circumventing drug resistance mechanism dependent on Pglycoprotein over-expression.

Fig. 2. the anti-proliferative activity of 1-(4-clbenzil)-3-cl-4-(cF 3 -phenylamino)-1Н-pyrrole-2,5dion (MI-1) towards human breast adenocarcinoma (McF-7 and MDa 231) cells, human epidermoid cervix carcinoma cells of kB3-1 line and its colchicine-resistant kBc-1 sub-line. cell viability was examined using the Mtt assay after 72 h cell exposure to the MI-1 compound
Study of interaction of the MI-1 with DNa. The 1-(4-Cl-benzil)-3-Cl-4-(CF 3 -phenylamino)-1Нpyrrole-2,5-dion (MI-1) was designed in silico as an inhibitor of tyrosine kinases [3] that are important targets in the action of many anticancer drugs [4,5]. However, other biological targets of the MI-1's effect cannot be excluded. In order to address such possibility, we have suggested that the DNA molecule could be another target that is used at the cytotoxic action of the MI-1 compound. It is known that several FDA approved anticancer therapeutics, such as doxorubicin, cisplatin, bleomycin, chlorambucil, etoposide, mephalan can bind the DNA and in such a way affect its structure and functions [22,25,26]. Besides, the Reactive Oxygen Species (ROS) induced in treated cells by the above noted anticancer drugs can cause the oxidative damage of DNA molecule, and thus, enhance the efficacy of cytotoxic ac-  tion of these drugs [27]. The DNA-interacting agents can also affect the cell-cycle checkpoints leading to their block [26]. It was shown that the MI-1 used at 1 and 10 μM concentrations replaced the methyl green dye in the DNA-methyl green complex by 21.2 and 16.5%, respectively (Fig. 5). The Doxorubicin (10 μg/ml) also known as the DNA-intercalating agent [26], replaced by 72.1% the methyl green in its complex with the DNA, while the Ethydium bromide (1 and 10 μg/ml), a well-known DNA-intercalating agent, demonstrated 54.1 and 80.1% replacement of the methyl green, respectively (Fig. 5).

Fig. 3. the anti-proliferative activity of 1-(4-clbenzil)-3-cl-4-(cF 3 -phenylamino)-1Н-pyrrole-2,5dion (MI-1) towards human colon cancer cells of hct116 line, human pancreatic ductal adenocarcinoma capan-1 cells, and human hepatocarcinoma hepG2 cells. cell viability was examined using the Mtt assay after 72 h cell exposure to the MI-1 compound
As one can see on Fig. 6 (lanes 4-6), plasmid DNA was not retarded at electrophoresis by the MI-1 used in 1, 10 and 100 µg/ml doses, while the Doxorubicin used in 1 and 10 µg/ml doses as a positive control, retarded the electrophoretic mobility in the agarose gel of the supercoiled form of plasmid DNA (Fig. 6, lane 2). It should be noted that three bands of plasmid DNA were detected after its 1 h incubation with the MI-1 compound that suggests its potential to induce supercoiling of the DNA molecule that could cause its functional deterioration. Taken together, the results of the methyl green replacement assay and the results of the electrophoretic retardation study of plasmid DNA definitely indicate a possibility of a direct interaction of the MI-1 with the DNA molecule. However, additional experiments are necessary in order to confirm or exclude a specificity of such binding of the MI-1 to the DNA.
In this study, we detected that the maleimide derivative 1-(4-Cl-benzil)-3-Cl-4-(CF 3 -phenylamino)-1Н-pyrrole-2,5-dione (MI-1 compound) possessed a remarkable cytotoxic activity towards several human tumor cell lines with the inhibition indicator (IC 50 ) ranging from 0.8 to 62.2 μg/ml for different cell lines. The most prominent cytotoxic effect was demonstrated by the MI-1 towards human cervix carcinoma cells of KB3-1 and KBC-1 lines. It should be stressed that the MI-1 showed six times higher toxicity towards the multidrug resistant KBC-1 subline of these cells, comparing with the cytotoxic action of doxorubicin. The MI-1 also inhibited the viability of human pancreatic, hepatocarcinoma, colon carcinoma cells, however, did that at high doses than for the above mentioned human cervix carcinoma cells. Human glioblastoma cells were poorly sensitive to a cytotoxic action of the MI-1. Thus, the anti-neoplastic activity of the MI-1 dropped in the following rank of cells: cervix carcinoma > breast carcinoma > pancreatic carcinoma > liver carcinoma > colon carcinoma > glioblastoma.