EffEct of a novEl thiazolE dErivativE and its complEx with a polymEric carriEr on stability of dna in human brEast cancEr cElls

thiazole derivatives are perspective antitumor compounds characterized by a broad range of bioactivity, while polymeric carriers are widely used to enhance the efficiency of biological action of drugs, improve their biocompatibility and water solubility. Previously, we identified that the thiazole-based derivative BF1 (N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide) possessed differential toxicity towards targeted tumor cell lines. the aim of the present work was to investigate the action in vitro of BF1 and its complex with the polymeric carrier (PC) poly(PEGMA-co-DMM) (BF1-РС complex) towards human breast adenocarcinoma cells of the MDa-MB-231 and McF-7 lines. DNa comet analysis, diphenylamine DNa fragmentation assay, gel retardation assay of plasmid DNa, DNa intercalation assay using methyl green dye and fluorescent microscopy were used to study the effects of BF1 on DNA stability in breast cancer cells. The ІС50 of cytotoxic action towards MDA-MB-231 cells was 26.5 ± 2.9 μМ for BF1, while the ІС50 for the BF1PC complex was 6.9 ± 0.4 μМ, and the PC demonstrated low toxicity (ІС50 ˃ 50 μМ). The BF1-PC complex possessed higher toxicity towards MCF-7 cells than free BF1, with ІС50 of 9.6 ± 0.8 μМ and 15.8 ± 0.9 μМ, respectively. BF1 and BF1-pc induced an increase in the number of damaged cells of the MDa-MB-231 line with blebbing of plasma membrane, condensed chromatin and/or fragmented nucleus and micronuclei formation. Both BF1 and the BF1-pc complex induced single-strand breaks in DNa and its fragmentation in treated MDa-MB-231 cells. the studied compounds were not bound to plasmid DNa and did not intercalate into DNa molecules.

quantum dots, biodegradable polymeric materials, and hybrid inorganic/organic nanomaterials are of great clinical and commercial interest [16,17]. The polymeric carriers are water-soluble, biocompatible, stable during long periods of storage, and can be easily modified [14,18]. They are currently being tested in several preclinical and clinical trials [14,19].
In the present study, we further investigated the action of thiazole derivative BF1 in complex with the PC poly(PEGMA-co-DMM) compared to free BF1 and free poly(PEGMA-co-DMM) towards proliferation and DNA damage in breast cancer cells.
The PC poly(PEGMA-co-DMM) ( Fig. 1) was synthesized at the Department of Organic Chemistry of Lviv Polytechnic National University (Ukraine). The PC was synthesized in dioxane by the method of radical(co)polymerization of PEG-containing macromere after the initiation of azobis(isobutyronitrile) in the presence of isopropyl benzene as the polymer chain length regulator [23].
The PC and BF1 were dissolved in DMSO, and the solutions were subsequently transferred to water, forming the BF1-PC complexes. Initially, 45 mg of PC were dissolved in 0.15 ml of DMSO and 1.5 g of BF1 were dissolved in 0.1 ml of DMSO. The PC and BF1 solutions were mixed, added to 4.25 ml of 0.9% NaCl water solution (physiological solution), and subjected to ultrasound (US) dispersion for 10 s. The free PC solution was prepared by dilution of 45 mg of PC in 0.45 ml of DMSO. Then, it was added to 4.0 ml of physiological solution and subjected to US dispersion for 10 s [24].
cell culture. Human breast adenocarcinoma cells of the MDA-MB-231 and MCF-7 lines were kindly provided by a Collection at the Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine (Kyiv, Ukraine) that had received those cells from the American Type Culture Collection (ATCC). Cells were cultivated in Dul- becco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum (both were purchased from Biowest, Nuaille, France) in a CO 2 thermostat system at 37°C with atmosphere of 95% air and 5% CO 2 [21].
Mtt assay. The effects of free BF1, free PC, BF1-PC complex, and doxorubicin (Dox, Actavis, Bucharest, Romania) on proliferation of the MDA-MB-231 and MCF-7 cell lines were examined using the MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were seeded overnight into 96-well plates in 100 μl at concentrations of 5,000 cells/well. Aliquots of 100 µl of experimental compounds (0-50 µM) were added to the cell cultures. Cells were incubated for the next 72 h. MTT was added to the cells. The reaction products were measured by an Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, VT, USA). The IC 50 of tested compounds was calculated as the drug concentration that caused 50% reduction in cell viability [25].
DNa intercalation assay using methyl green method. 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 H 2 O). 500 µl of the compounds BF1, or BF1-PC, or PC, or Dox (all at 10 µM) were added to the methyl green-DNA complex and incubated for the next 2 h at 37°C in the dark. EtBr (10 µM) was used as a positive control. Absorption of methyl green was measured at 630 nm using a fluorescence plate reader (Absorbance Reader BioTek ELx800, BioTek Instruments, Inc., Winooski, Vermont, USA) [25,26].

Diphenylamine assay of DNa fragmentation.
The MDA-MB-231 cells were treated for 24 h with tested compounds at concentrations that corresponded to their IC 50 value: BF1 (27 µM), BF1-PC (7 µM), PC (7 µM), Dox (0.6 µM); and DMSO (270 μg/ml corresponding to the solvent concentration of BF1 at 27 µM). Cells were lysed in 0.5 ml of Tris-EDTA buffer, pH 7.4, supplied with 0.2% Triton X-100 and centrifuged for 10 min at 12,000 g at 4°C. Supernatant that contained fragmented DNA (labelled as "B") was transferred into a new tube. The tube with a sediment which contained intact chromatin was labelled as "A". The 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 samples were 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) diluted in 10 ml of glacial acetic acid, 150 ml of concentrated H 2 SO 4 , and 50 ml of acetaldehyde solution) was added to pellets "A" and "B" and incubated overnight at 37°C. The optical density (OD) of the colored solution was measured at 630 nm using Absorbance Reader BioTek ELx800 (BioTek Instruments, Inc., Winooski, Vermont, USA). The percentage of DNA fragmentation was calculated as {OD tube B/(OD tube A + OD tube B)×100%} [25,27].
ethical committee. This research did not involve human or animal investigations.
Statistical analysis. The results were analyzed and illustrated with GraphPad Prism 6 (GraphPad Software, La Jolla, California, USA), and presented as the mean ± standard deviation of two repeats each with two parallels (n = 4). The ANOVA test (by Dunnett's test) was used for analysis of statistical data. The p-value of < 0.05 was considered to be statistically significant. Similar dose-dependent inhibition of cell viability of both MDA-MB-231 and MCF-7 cells was found under the effects of BF1, BF1-PC, PC, and Dox. BF1 achieved its IC 50 for MDA-MB-231 cells at the dose of 26.5 µМ ± 2.9 μM, while the IC 50 of BF1-PC was 6.9 ± 0.4 μM (Fig. 2). The IC 50 of BF1 was 15.8 ± 0.9 μM for MCF-7 cells, while the IC 50 of BF1-PC was 9.6 ± 0.8 μM. The PC did not reach an IC 50 value at concentrations up to 50 µM (Fig. 2). It was observed that 83.2% of MDA-MB-231 cells and 77.2% of MCF-7 cells were alive after treatment with PC at 50 µM. The IC 50 of Dox was 0.63 ± 0.05 μM for MDA-MB-231 cells, and 0.62 ± 0.04 μM for MCF-7 cells (Fig. 2).
Thus, the BF1-PC complex possessed higher activity in growth inhibition of MDA-MB-231 and MCF-7 cells. The cytotoxic action of BF1-PC was approximately 4 times higher (p < 0.001) compared to the action of free BF1 towards MDA-MB-231 cells, and approximately 1.6 times higher (p < 0.001) compared to the action of free BF1 towards MCF-7 cells.
DNa binding and intercalation. The interaction of anticancer drugs with DNA is one of the mechanisms of their action. Electrophoresis was used to examine the ability of BF1, BF1-PC complex, free PC, and Dox (used as a reference drug) to bind with DNA. Dox (1 µM) inhibited the electrophoretic mobility of plasmid DNA pEGFPc-1 (Fig. 3, lane 2). The plasmid DNA pEGFPc-1 was not retarded by BF1, BF1-PC complex, or free PC (Fig. 3, lanes 3-5). Because compounds that intercalate into DNA replace methyl green dye from the DNA-methyl green complex [25,26], the methyl green assay was conducted to examine the ability of BF1, BF1-PC complex, free PC, and Dox to intercalate into the DNA molecule. BF1 (10 μM) replaced 2.1 ± 0.4% and BF1-PC (10 μM) replaced 3.2 ± 0.5% of the methyl green from the DNA-methyl green complex (Fig. 4). Free PC slightly replaced the methyl green (0.9 ± 0.1%). Dox (10 μM) used as a positive control replaced 64.2 ± 0.9% of the methyl green. The referen ce compound, EtBr (10 μM), demonstrated 83.6 ± 1.0% of methyl green replacement. Thus, BF1, BF1-PC complex and free PC did not bind and intercalate with DNA.
In the present study, we demonstrated that the BF1-PC complex BF1 with the PC poly(PEGMA-co-DMM)) possessed higher growth inhibition activity towards human breast adenocarcinoma cells of the MDA-MB-231 and MCF-7 lines. The cytotoxic action of the BF1-PC complex was approximately four times higher compared to the action of free BF1 towards MDA-MB-231 cells and approximately 1.6 times higher towards MCF-7 cells. It should be emphasized that the cytotoxic effect of the BF1-PC complex toward MDA-MB-231 cells (which are more malignant) was more pronounced compared to the effect of this complex toward MCF-7 cells. The PC did not demonstrate toxicity towards MDA-MB-231 or MCF-7 cells. In addition, the complex of BF1 with the PC poly(PEGMA-co-DMM) has shown higher activity in growth inhibition of human hepatocarcinoma HepG2 cells, human promyelocytic leukemia HL-60 cells and rat glioma C6 cells, compared to the activity of free BF1 [22].
Different mechanisms have been proposed for the antitumor action of the thiazole-based derivatives. It was reported that they acted as apoptosis inducers [6,21,25,29], affected inosine monophosphate dehydrogenase (IMPDH) [29], induced reactive oxygen species (ROS) production [30], inhibited lipid peroxidation and caused DNA damage [5]. In the present study, we investigated the effect of thiazole derivative BF1 and its complexes with poly(PEGMA-co-DMM) on the stability of DNA in human breast adenocarcinoma MDA-MB-231 cells. It was found that these cells were more sensitive to the growth-inhibiting action of the BF1-PC complex compared with MCF-7 cells. The difference in response by two lines of human breast cancer cells -MDA-MB-231 and MCF-7 -to the cytotoxic action  factor receptor-2 (HER2) [4]. The MDA-MB-231 cell line belongs to highly metastatic breast cancer cells [33] and it is a promising object for studying antineoplastic agents and systems for improvement of their antitumor action.
Several anticancer drugs that are currently used in chemotherapy were shown to interact with DNA either via covalent or non-covalent mechanisms. DNA interacting agents can change DNA conformation, interrupt DNA-protein interaction, or in-duce DNA strand breaks [34]. Unfortunately, some of them demonstrated high toxicity for both tumor and normal cells [10,34]. Based on the results of the electrophoretic retardation of plasmid DNA and methyl green replacement assay, we concluded that BF1 and the BF1-PC complex could not bind DNA or intercalate into its molecule.
It was reported that bleomycin, a bisthiazolyl derivative, acted as an antitumor agent due to its DNA binding [10]. However, the platinum(II)and platinum(IV)-pyrophosphato-complexes (e.g. trans-1,2-cyclohexanediamine and pyrodach-2) that exhibit cytotoxicity towards ovarian cell lines do not bind to DNA molecules [35]. The NB-506, isolated from Streptoverticillium species culture supernatants, did not intercalate into DNA and possessed high toxicity towards a panel of tumor cells [36]. Grozav et al. [37] reported about the toxicity of 2-(2-benzyliden-hydrazinyl)-4-methylthiazole derivative (97a) towards MDA-MB-231 (IC 50 = 3.92 μg/ ml) and HeLa (IC 50 = 11.4 μg/ml) cell lines. The toxicity of this compound was comparable to that found for the commercial chemotherapeutic agents cisplatin and oxaliplatin. The IC 50 for cisplatin was 17.28 μg/ml for MDA-MB-231 cells and 26.12 μg/ ml -for HeLa cells, while the IC 50 for oxaliplatin was 14.09 μg/ml for MDA-MB-231 cells and 23.17 μg/ ml for HeLa cells. The results of gel electrophoresis of DNA suggest that the antiproliferative activity of the 97a derivative towards MDA-MB-231 and HeLa cells was independent of DNA intercalation [35].
Thus, several anticancer agents do not bind or intercalate into DNA molecules, however, such intercalation may be an alternative mechanism of the anticancer activity of some agents.
DNA fragmentation is another indicator of the catabolic changes related to the apoptotic pathway [38]. DNA comet analysis under the alkaline conditions is widely used for the investigation of DNA damage in cells under the action of different agents and drugs [28]. We have shown that the thiazolebased derivative BF1 and the BF1-PC complex induced significant DNA damage in the MDA-MB-231 cells, however, only minor DNA damage was detected in the MDA-MB-231 cells treated with free PC or the DMSO solvent. Cytomorphological investigation of the treated MDA-MB-231 cells showed that BF1 and the BF1-PC complex induced nuclear fragmentation, chromatin condensation, micronuclei formation and plasma membrane blebbing. The free PC and DMSO did not cause these significant morpho-logical changes. These findings correlate with previously reported data that 2-amino-5-benzylthiazole derivatives (2,8-dimethyl-7-(3-trifluoromethyl-benzyl)pyrazolo [4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)one; and 7-benzyl-8-methyl-2-propylpyrazolo [4,3-e] thiazolo[3,2-a]pyrimidin-4(2H)-one) induced DNA single-strand breaks and DNA fragmentation in human leukemia cells without significant DNA binding and DNA intercalation [25]. The furan-thiazole hybrid (compound 3d) exhibited its anticancer activity via DNA binding and DNA damage [12].
Taking into account the results of our study, one cannot exclude that in addition to the DNA damaging mechanisms of the cytotoxic action of the thiazole-based derivative BF1 and its complex with the PC towards human breast adenocarcinoma cells (MDA-MB-231 line), other mechanisms of action of these compounds should also be considered. We plan to undertake such corresponding investigations for our future work, particularly on tumor-bearing laboratory animals.
conclusion. Thiazole-based derivative BF1 demonstrates cytotoxic action towards human breast adenocarcinoma cells of the MDA-MB-231 and MCF-7 lines. The immobilization of BF1 on the PC poly(PEGMA-co-DMM) increased fourfold the cytotoxicity of the BF1 towards MDA-MB-231 cells and doubled its cytotoxicity towards MCF-7 cells, while free PC did not demonstrate significant cytotoxic action. The BF1 compound and BF1-PC complex did not intercalate into DNA or bind with its molecule. However, they induced morphological changes in the treated MDA-MB-231 cells, such as nuclear fragmentation, chromatin condensation, micronuclei formation and plasma membrane blebbing.

Conflict of interest.
Authors have completed the Unified Conflicts of Interest form at http://ukrbiochemjournal.org/wp-content/uploads/2018/12/ coi_disclosure.pdf and declare no conflict of interest.
Funding. This research was supported by the Ministry of Education and Science of Ukraine grants (registration number 0119U002201 and 0118U000260), and partially by Cedars-Sinai Medical Center's International Research and Innovation in Medicine Program and the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) and the participating Cedars-Sinai Medical Center -RECOOP Research Centers (CRRCs).