Study of antineoplaStic action of novel iSomeric derivativeS of 4-thiazolidinone

Pyrazoleand aryl-substituted derivatives of 4-thiazolidinone belong to a perspective group of compounds with potential antitumor action. earlier, we have demonstrated high toxicity in vitro of several 4-thiazolidinones derivatives towards tumor cell lines. to further enhance the antitumor activity of novel 4-thiazolidinones, their chemical scaffold was optimized, and new pyrazole-thiazolidinones were synthesized. that allowed us to combine in one molecule the potential pharmacophore centres of previously tested compounds. as a result, "hybrid" 4-thiazolidinones exhibit higher toxicity in vitro toward tumor cells of various origin. the molecular mechanisms of antineoplastic activity of these compounds and intensity of induction of apoptosis strongly depended on the position of the substituent in the thiazolidinone cycle. in particular, les-3661 compound, containing pyrazoline fragment in the 4th position of thiazolidinone core, exhibits 14 times higher cytotoxic activity towards tumor cells (lc50 = 3 μM) in comparison to its 2-substituted isomer les-3713 (lc50 = 42 μM). it is demonstrated that in terms of underlying molecular mechanisms for cytotoxic effect the les-3661 compound induced caspase-8 and caspase-9 dependent mixed-type of apoptosis, while les-3713 induced apoptosis mediated only by the caspase-8.


D
espite the influx of data in the latter deca des and experience in medical science concerning oncotherapy, the search for novel chemical agents remains a high priority task, potentiated by high rate at which tumor cells attain drug resistance.Furthermore, the application of most of the existing antitumor pharmaceuticals is accom panied by the negative side effects due high unspe cific toxicity.
The chemistry of 4thiazolidinones and their related compounds, which has been considered phar macologically significant since the beginning of the 20 th century, has recently experienced an accelerated growth.These substances demonstrate a characteris tically wide spectrum of biological effects, including antimycotic, antibacterial, hypoglycemic, and anti neoplastic [1].Moreover, the high capacity of thiazo lidinone core used to accept chemical modifications opens a possibility for development of multitude of novel derivatives potentiating the biological activity of the compound.
The search for new potent antitumor pharma ceuticals possessing high selectivity and low toxicity to normal cells is currently strongly prioritized [2].Necrostatin7 (Nec7) was identified as heterocyclic derivative of 4thiazolidinone that suppresses TNF βinduced necroptosis in Jurkat cells (human Tlym phocyte leukemia cell line) deficient in FADD gene [3].Therefore, the combination of several functional groups in a compound's molecule allows for broader spectrum of its antitumor application.
Novel derivatives of 4thiazolidinone are syn thesized at the Department of Pharmaceutical, Or ganic and Bioorganic Chemistry of the Danylo Halytsky Lviv National Medical University.The antineoplastic potential of these compounds has been demonstrated [4], particularly by the in vitro studies at the National Cancer Institute (Bethesda, USA).Although the antitumor potential of these compounds is the primary focus of research, it is complemented with various additional effects, e.g.antibacterial, antimycotic, immunemodulating, an tidiabetic activity [5][6][7].
The present study is intended to characterize novel compounds noted as Les3661 and Les3713, which are 4thiazolidinone isomeric derivatives.
The algorithm of synthesis of these compounds has been established in our previous studies of biologi cal activity in vitro of certain other 4thiazolidinone derivatives [8].In order to improve that activity, we have combined parts of Les3120 molecule contain ing 3,5diarylpyrazoline fragment with Les3372, which is 4aryliminothiazolidinone derivative with chloro3(4nitrophenyl)allylidene group.Such combination was done in accordance with hybridpharmacophore approach and was validated by the proven high antitumor potential of pyrazolinethi azolidinone conjugates as well as by the fact -as it has been demonstrated in our studies -that the structure of arylidene group at 5 th position of thio zolidinone core plays an essential role in its antineo plastic activity [9,10].
We have demonstrated that the cytotoxic effect of the new Les3661 compound is much more po tent than that of Les3120 and Les3372, which had been characterized earlier.These findings allowed us to assume that Les3661 possesses high biologi cal activity and might be a potential antineoplastic medicine (Fig. 1).
The aim of the present study is to investigate the effect of novel isomeric 4thiazolidinone deriva tives on viability of tumor cells of various lines in vitro, and to establish potential mechanisms under lying the realization of cytotoxic activity of these compounds.

materials and methods
cell lines and cell culture.The following cell lines were used in the study: Jurkat (human acute T cell leukemia), HL60 (human acute lymphoblastic leukemia), MCF7 and MDAMB231 (human breast adenocarcinoma), HeLa (human cervical carcinoma).
The cells were cultured in the RPMI1640 and DMEM media (Sigma, USA) supplemented with de complemented fetal bovine serum (Sigma, USA) and 50 μg/ml of Gentamicin (Sigma, USA) in CO 2 incu bator at 37 °C and 5% CO 2 content.The cells were passaged every other day at the density of 0.5×10 6 to 1×10 6 per 1 ml of culture medium.
analysis of cytotoxic activity.The cells were subcultured into 24well plates (Greiner Bio One, Germany) in the RPMI/DMEM medium with 10% fetal bovine serum at 0.5×10 6 cells per ml for suspen sion cultures or 0.1×10 6 cells per ml for adherent cul tures.The subject compounds were added to cultural medium at various concentrations.After 24hour in cubation, the cells were counted in hemocytometer chamber.The number of dead cells was counted af ter their staining with 0.1% solution of trypan blue.This dye colors dead cells, whereas living ones re main colorless [11].
cytomorphological characterization of ultrastructure of cellular chromatin (staining with DaPi (4',6-diamidino-2-phenylindole) fluorescent dye).Cells were subcultered into 6well plates (0.1×10 6 cells per well) on glass in DMEM medium with 10% fetal bovine serum.After 24hour growth period, the cells were incubated with the investigated com pounds for 24 hours, then fixed with 100 μl of for malin and 10 μl Triton X100 per sample, and stained with 1% (in final volume) DAPI (Sigma, USA).The samples were washed with phosphate buffered saline (PBS) and imaged with a digital camera mounted on Zeiss AxioLam A1 microscope (Carl Zeiss, Ger many) [12].
Flow cytometry study of cell cycling.The cells subjected to the compoundcontaining medium were sampled (2×10 6 cells per sample), sedimented by centrifugation for 5 min at 151 g, and washed twice with PBS.One millilitre of cold PBS (0 °C) was then added to the cells, and the sample was fixed with 4 ml of absolute ethanol at 20 °C.The cells were stored in this solution at 20 °C for no longer than a fortnight [13].Prior to the flow cytometry proce dure, the samples were centrifuged, supernatant liq uid was decanted, and the sediments were suspended in 1 ml of PBS.The samples were then incubated with 100 μl of RNAse (200 μg/ml concentration) for 30 min at 37 °C, followed by incubation with 100 μl of the propidium iodide (1 mg/ml) for 510 min at room temperature [14].The samples were transferred to conical polypropylene test tubes and analyzed by FACSCalibur flow cytometer (BectonDickinson, USA).
Western Blot analysis of cellular proteins.The cells of the centrifugation sediment were washed with PBS and then lysed in buffered solution (20 mM TrisHCl, 1% TritonX100, 150 mM NaCl, 50 mM NaF, 0.1% SDS, protease inhibitors mix (Complete™, Roche); pH 7.6) in proportion of 20 μl buffer per 1×10 6 cells.After lysis, the supernatant liquid was separated, mixed with 1/3 part of quad ruple Laemmli buffer, and heated in boiling water for 5 min.The samples prepared in this way were afterwards stored at 20 °C and analyzed electropho retically at convenience.
Protein concentration was determined by Peter son's modification of Lowry method [16].Proteins from PAGE were transferred onto the nitrocellulose membrane in Mini TransBlot Cell (BioRad, Sweden) at 90 v for 90 min in transfer buffer (0.192 M glycine, 0.1% SDS, 20% methanol, 0.025 M Tris, pH 8.3).Afterwards, the membrane was blocked at room temperature for 1 h with 5% so lution of nonfat dry milk in PBS containing 0.05% Tween 20.
Protein identification on the nitrocellulose membrane was achieved via incubation with speci fic monoclonal rat or rabbit antibodies raised against pro and antiapoptotic proteins, followed by incu bation with peroxidaselinked antibodies specific for mouse (or rabbit) immunoglobulin (Amersham Pharmacia Biotech, USA).Antibodies were diluted in the blocking solution at 1 : 5000.The incubation was performed with shaking for 1 h at 4 °C.The membrane was washed three times (5 min each) in the PBS supplemented with 0.05% Tween 20.
Specific antibody protein binding was detec ted via chemiluminescence caused by 1 min mem brane incubation in the detection buffer containing 1.25 mM luminol (5amino2,3dihydro1,4phthala zinedione (Sigma)), 2.72 mM pcoumaric acid (4hy droxycinnamic acid (Sigma)), and 0.01% hydrogen peroxide in 0.1 M TrisHCl (pH 8.5).Photographic film (Fujifilm, Japan) was exposed on the membrane for 110 min and then developed to visualize immu noreactive bands.Protein amount distribution across samples was equalized through βactin level in the samples [15].statistical analysis.The experiments were con ducted in three parallel samples each.Mean value m was obtained through triple repetition of a typi експериментальні роботи cal assay.Mean deviation m was obtained through standard deviation σ.

results and discussion
The Les3661 and Les3713 compounds were synthesized as described [17].The primary stage of the present study was aimed at the investigation of the action of Les3661 and Les3713 towards tumor cells of various origin and at establishing of their effective acting doses (Fig. 2, a, B).The leukemic tumor cell lines Jurkat (human Tcell leukemia), HL60 (human promyelocytic leukemia), and also transformed secretory epithelium cell lines MCF7 and MDAMB231 (human breast adenocarcinoma) were used in the experiments.Median lethal con centration LC 50 for cells was determined for both 4thiazolidinone derivatives.LC 50 was 3 μM for Les 3661.LC 50 for Les3713 was 42 μM for HL60 and MDAMB231 cells, and 47 μM for Jurkat and MCF 7 cells.The cytotoxity experiments allowed us to es tablish for the first time the fact that substitutions in the 4 th position of the thiazolidinone leads to notable (14fold) increase in cytotoxicity of Les3661 com pared to 2 nd position substituted Les3713.Les3661  possessed higher cytotoxicity than its precursor substances.Namely, pyrazoline thiazolidinone Les 3661 is 3 times more active than Les3120 (LC 50 is 7.5 μM), and 6 times more active than Les3372 (LC 50 is 18 μM).We also demonstrated that Les3713 is 5.6 times less cytotoxic than Les 3120, and 2.3 times less cytotoxic than Les3372.Therefore, our idea on combination of diverse radicals within single compound for improving its cytotoxic effectiveness has been proven viable in the case of Les3661 com pound.
We investigated the state of chromatin in HeLa cells (cervical adenocarcinoma) by their staining with DAPI fluorescent dye in order to establish the pathways involved in cell death.The microphotog raphy (Fig. 3) demonstrates that these compounds induce chromatin hypercondensation that is charac teristic of apoptosis, which is the main pathway for transformed cell death under such conditions.Al though both compounds were used in concentrations corresponding to LC 50 for the cells of these lines, the presented results (Fig. 3) demonstrate that Les3661 causes more profound chromatin hypercondensation than Les3713.We suppose that cells treated with Les3661 for 24 h reach terminal stages of apoptosis, while Les3713 apparently activates apoptotic path way at later stages.
These results allow one to suggest that Les3661 and Les3713 caused transformed cell death via apoptosis, and the cytotoxic effect of Les3661 is much more profound than such effect of its isomer Les3713 and of other investigated compounds, such as Les3120 and Les3372.
We have also studied Les3661 effect on tumor cell cycle.Jurkat Tcells were treated with Les3661 for 24 h, and chromatin was examined using flow cy tometry.The data obtained (Fig. 4) demonstrate that Les3661 affects Jurkat cell cycle and increases the number of cells in G 0 /G 1 phase (19.71% more than in control), while doxorubicin increases the number of
Western blot analysis of proteins involved in initiation and execution of apoptosis was used to ex plore molecular mechanisms underlying the activi ty of the investigated heterocyclic compounds.The lysates were obtained from Jurkat line Tleukemia cells treated for 1, 3, 6, 12, and 24 h with Les3661 (3 μM) and Les3713 (42 μM), and with doxorubicin (0.5 μM, used as positive control).The experiments on initiation of apoptosis under the effect of 4thia zolidinone derivatives revealed that 12 h treatment of cells with Les3661 compound is enough to de crease the intracellular level of the procaspase8 (Fig. 5).That can be associated with activation of this proteolytic enzyme and the induction of recep tormediated apoptosis.The proteolysis product of the procaspase9 which activates mitochondrial apo  decreases content of Bid protein in the targeted cells.Therefore, only the mitochondrial apoptotic pathway is activated in cells under effect of Les3713.The effector phase proapoptotic proteins, such as caspase3, caspase7 and caspase6 are activated at 12 th h of cell treatment with Les3661 and Les 3713 (Fig. 6).This effect is accompanied by degrada tion of the reparation enzyme PARP1 (poly[ADP ribose]polymerase) and activation of the regulatory protein DFF45 (DNAfragmentation factor).
On Fig. 7 the pattern of possible mechanisms of action for various 4thiazolidinone derivatives is presented.As shown, Les3661 induces mixedtype apoptosis via caspase8 which initiates receptorme diated apoptosis, and via caspase9 participating in mitochondrial cell death pathway.This might lead to elimination of malignant cells with mutations in genes of programmed cell death regulation.Appar ently, extensive antitumor potential of Les3661 is determined by its ability to affect diverse apoptotic Les3661 and Les3713 induce apoptosis ini tiation and activate effector proteins earlier than doxorubicin does initiating apoptosis at 24 th h of cell treatment (Fig. 5 and 6).This fact provides grounds for further preclinical in vivo study of certain 4thia zolidinones, mainly Les3661 which is much more active than isomer.Thus, a combination of pyrazoline, 2chloro 3(4nitrophenyl)allylidene and thiazolidinone moieties in one molecular structure substantially po tentiates the antineoplastic efficiency of the precur sor compounds possessing such groups.Moreover, such a 'hybrid' molecule is more powerful and faster inducer of apoptosis in tumor cells.3,5Diaryl pyrazoline group which is present in 4 th position of Les3661's thiazolidinone fragment ensures its much enhanced antineoplastic efficiency in comparison to Les3713 isomer which has this group in 2 nd position of the thiazolidinone fragment.