Screening of spiro-substituted thiopyrano[2,3-d]thiazoles for their cytotoxic action on tumor cells

N. I. Zelisko, N. S. Finiuk, V. M. Shvets © 2017 N. I. Zelisko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 547.818:547.489.4:542.91:615.359


Introduction
Chemotherapy is one of the most effective ways of treating cancer patients.Conventional anticancer drug discovery and development have been focused on the cytotoxic agents.The drug discovery paradigms selected agents that had significant cytostatic or cytotoxic activity towards tumor cell lines in vitro and caused tumor regression in vivo [1].However, their application in cancer treatment is accompanied by frequent non-addressed actions lea ding to numerous negative side effects in the organism [2].Due to these effects, they de mon strate toxicity toward different normal cells of tissues and organs, including the bone marrow cells, mucous layer of the intestine, reproduction glands, and hair follicles.
4-Thiazolidinones and related heterocycles have been demonstrated to be a perspective source of the innovative anticancer agents [3,4].In the previous studies, we identified a large group of substances with high anticancer potential and low toxicity [5,6].According to the obtained data [6], most of the studied substances influenced cancer cell lines.5-Ene-4thiazolidinones were identified to possess the highest antitumor activity towards leukemia cells and induction of apoptosis in the mammalian leukemia cells [7].However, the mechanisms of the anticancer activity of these compounds have not been studied yet.
Previously it has been reported that PPARγ agonists, namely 4-thazolidinone derivatives, have a therapeutic potential in treatment of the inflammation and some cancers.This scientific assumption was successfully proved [8].Among 4-thiazolidinones, there were also identified the effective inhibitors of the anti-apoptotic proteins Bcl-XL and BH3 interactions [9], the inhibitors of the interaction between TNFα and the receptor TNFRc-1 [10], and the inhibitors of translation initiation [11].A significant influence on the above mentioned pathogenic mechanisms of tumor growth determines perspectives of 4-thiazolidinones as potential anticancer drugs.
The main aim of this study was to measure the toxic effect in vitro of novel spiro-substituted thiopyrano [2,3-d]thiazoles towards tumor cells of different tissue origin.

Materials and Methods
All chemicals used in the study were of the analytical grade and commercially available.All reagents and solvents were used without further purification and drying.5-triones (1a-e) for screening cytotoxi ci ty were synthesized via hetero-Diels-Alder reaction (Scheme 1) starting with trans-aconitic acid, corresponding aromatic amine and appropriate 5-arylidene-4-thioxo-2-thiazoli di no ne in the presence of the catalytic amount of hydroquinone (2-3 mg) for preventing polymerization processes, as it was described in our previ-ous work [22].The 5-arylidene-4-thioxo-2-thiazolidinones were synthesized via Knoevenagel condensation of 4-thioxo-2-thiazolidinone with the aromatic aldehydes [23].

Screening of cytotoxic activity. Materials
10 mM stock solution of synthesized compounds was prepared in the dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA), and additional ly dissolved in culture medium prior to its addition to the cell culture.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was 99.5 % pure.Cell culture medium DMEM was obtained from Sigma-Aldrich (USA), and RPMI -from APP (Austria).Fetal bovine serum (FBS) was obtained from APP (Austria), and Doxorubicin -from Pharmache mie B.V. (the Netherlands).Mel-28 line, human non-small-cell lung cancer cells of SW-1573 line, transformed mouse fibroblasts of L929 line, human embryonic kidney cells of HEK293 line were obtained from Cell culture collection at R.E.Kavetsky Institute of Experimental Pathology, Oncology and Radiology (Kyiv, Ukraine).Cells were grown in the RPMI or DME culture medium supplemented with 10 % fetal bovine serum.Cells were cultivated in the CO 2 -thermostate at 37 °C in atmosphere of 95 % air and 5 % CO 2 at 100 % humidity.

Cytotoxicity assay
Cytotoxic activity of the synthesized compounds and doxorubicin used as a reference drug control towards cancer cell lines cultured in vitro was measured by the MTT test Tumor cells were seeded for 24 h in 96-well microtiter plates at a concentration of 2,000 of substrate-dependent cells/well or 10,000 of suspension cells/well in 100 μL.After that, cells were treated for 72 h with various additions of the synthesized compounds (0; 1; 10; 50; 100 μM).The MTT reagent, which is converted by mitochondrial dehydrogenases to dark blue, water insoluble MTT formazan, was used to measure the vitality of cells according to the manufacturer's protocol (Sigma-Aldrich, USA).The IC 50 of tested compounds was calculated as a lethal concentration of drug decreasing cell vitality by 50 % in comparison with drug-free culture medium.

Statistical analysis
All data are presented as the mean ± standart deviation (SD).Results were analyzed and illustrated with GraphPad Prism (version 6; GraphPad Software, San Diego, CA).Statis tical analyses were performed using two-way ANOVA with Bonferroni post-tests (tumor growth).P-value of <0.05 was considered as statistically significant.

Measuring of cytotoxic activity
In vitro screening of cytotoxic activity of the synthesized compounds towards various tumor cell lines (human acute T-cell leukemia cells of Jurkat line, human breast adenocarcinoma cells of MCF-7 line, human ovarian carcinoma cells of Skov3 line, human melanoma cells of SK-Mel-28 lines, and human non-small-cell lung cancer cells of SW-1573 line) was performed using the MTT assay.The tested compounds were added to cultured cells in different final concentrations (0-100 μM), and then the cells were treated for 72 h (Fig. 1).The IC 50 was calculated and used while comparing the values of the cytotoxic effect.
The tested compounds were found to possess different cytotoxic action towards human tumor cells of various tissue origin.The tumor cells of epithelial origin (MCF-7, Skov3, SK-Mel-28 and SW-1573 lines) were relatively resistant to the action of compounds 1a-e, 2 used in concentrations up to 100 μM (Fig. 1).
The anticancer cytotoxic effect in vitro of the compounds 1a-e, 2 was also studied using human acute leukemia T-cells of Jurkat line.The compound 2 possessed cytotoxic activity towards Jurkat cells with the IC 50 = 33.5 μM (Fig. 2).
Thus, the cytotoxic activity of synthesized compounds 1a-e, 2 seems to depend on the targeted tumor cell line, although the leukemia cells appeared to be the most sensitive to the action of studied derivatives.
Most of chemotherapeutic drugs demonstrate toxicity toward normal cells of tissues and organs, namely the bone marrow cells, cells of mucous layer of the intestine, reproduction glands, and hair follicles [25].We have measured the cytotoxic effect of the compounds under study towards non-tumor cells, such as transformed mouse fibroblasts of L929 line and pseudo-normal human embryonic kidney cells of HEK293 line (Fig. 3).There was no significant cytotoxic effect of used compounds towards HEK293 and L929 cells.Thus, one could suggest the lack of general  toxicity of novel synthetic compounds towards normal cells of tissues and organs.
We found that the cytotoxic effect of compound 2 towards human acute T-cell leukemia cells of Jurkat line was dose-and time-dependent (3, 6, 24, 48 and 72 h).Compound 2 showed such effect as soon as in 6 h after starting cell treatment (IC 50 = 66 μM), whereas its toxicity towards Jurkat cells was more prominent after 24 h treatment (IC 50 = 40 μM) (Fig. 4).

Fig. 2 .
Fig. 2. Results of testing cytotoxicity of compounds towards human acute T-cell leukemia cells of Jurkat line.After a total time of experiment (72 h) cytotoxic effect was measured by the MTT assay.

Fig. 1 .
Fig. 1. Results of testing cytotoxicity of compounds towards different tumor cell lines.After a total time of experiment (72 h), cytotoxic effect was measured by the MTT assay.
thiazole derivatives were synthesized and tested for their cytotoxic activity towards human tumor cells in vitro.Generally, they possessed low toxicity towards tumor cells.Compound 2 was found to be the most cytotoxic agent specifically towards the human acute T-cell leukemia cells of Jurkat line (IC 50 = 33.5 μM).A low toxicity of this compound towards the pseudo-normal human embryonic kidney cells of HEK293 line should be noted.Acknowledgement The publication contains the results of studies conducted by President's of Ukraine grant for competitive projects F-63 of the State Fund for Fundamental Research.The investigation was also supported by Cedars Sinai Medical Center's International Research and Innovation in Medicine Program, 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 (CRRC).

Fig. 4 .
Fig. 4. Exposure time-dependency of compound 2 cytotoxicity.Pulsing experiments were performed using human acute T-cell leukemia cells of Jurkat line treated for 3, 6, 24, 48 and 72 h with compound 2. Cytotoxic action was determined using MTT assay.

Fig. 3 .
Fig. 3. Results of testing cytotoxicity of compounds towards non-tumor mammalian cells.After a total time of experiment (72 h) cytotoxic effect was measured by the MTT assay.