TargeTing of The pro-oxidanT-anTioxidanT balance in vitro and in vivo by 4-Thiazolidinone-based chemoTherapeuTics wiTh anTicancer poTenTial

oxidative stress is one of the main mechanisms by which anticancer chemotherapeutics damage normal tissues and organs. at the same time, it is an important biochemical mechanism of the neoplastic action of such medicines. the aim of the present study was to determine the pro-oxidant-antioxidant balance in vitro and in vivo under the influence of novel 4-thiazolidinone-based chemotherapeutics with anticancer potential. an advantage of using these compounds in vivo is their low general toxicity, compared to doxorubicin (Kobylinska L. et al., 2014, 2015, 2016). The 4-thiazolidinone derivatives (Les-3288, Les-3833, Les-3882) with previously established anti-neoplastic activity in vitro (Kobylinska L. et al., 2016) and antitumor effect in vivo (Kobylinska L. et al., 2018) were synthesized, dissolved in dimethyl sulfoxide, and administered intraperitoneally to Wistar rats daily for 20 days. Doses of the injected drugs equaled 10% of the LD50, namely doxorubicin 5.5 mg/kg, Les-3882 and Les-3833 10.7 mg/kg, and Les-3288 24.3 mg/kg. the radical scavenging of 1,1-diphenyl-2-picrylhydrazil (DPPH) activity was measured. Concentrations of thio-barbituric acid-active products were assessed in blood serum, liver, heart and kidney tissues of treated rats. additionally, the activities of superoxide dismutase, catalase and glutathione peroxidase were measured in blood serum and these tissues. We found that administration for 20 days of Les-3288, Les-3833 and Les-3882 compounds disturbed the pro-oxidant-antioxidant balance in the treated rats. Increased amounts of products of reactions of lipid peroxidation and exhaustion of the enzymatic antioxidant system in liver, heart and kidney tissues were detected. In general, Les-3288, Les-3833 and Les-3882 compounds exhibited less pro-oxidant action, compared with the effect of doxorubicin. According to the results of influencing the pro-oxidant-antioxidant balance in the selected tissues, the studied compounds can be ranked in the following order: doxorubicin >> Les-3833 > Les-3288 >> Les-3882. the results of measuring direct scavenging ability of these compounds observed in 24 h suggests their lower toxic effect compared with the effect of the doxorubicin. The obtained results are in correspondence with the results of our recent experiments demonstrating their antineoplastic effect in vitro (Kobylinska L. et al., 2016) and anticancer action in vivo (Kobylinska L. et al., 2018), as well as their lower general toxicity in vivo compared with doxorubicin (Kobylinska L. et al., 2014, 2015, 2016).

oxidative stress is one of the main mechanisms by which anticancer chemotherapeutics damage normal tissues and organs.at the same time, it is an important biochemical mechanism of the neoplastic action of such medicines.the aim of the present study was to determine the pro-oxidant-antioxidant balance in vitro and in vivo under the influence of novel 4-thiazolidinone-based chemotherapeutics with anticancer potential.an advantage of using these compounds in vivo is their low general toxicity, compared to doxorubicin (Kobylinska L. et al., 2014(Kobylinska L. et al., , 2015(Kobylinska L. et al., , 2016)).The 4-thiazolidinone derivatives (Les-3288, Les-3833, Les-3882) with previously established anti-neoplastic activity in vitro (Kobylinska L. et al., 2016) and antitumor effect in vivo (Kobylinska L. et al., 2018) were synthesized, dissolved in dimethyl sulfoxide, and administered intraperitoneally to Wistar rats daily for 20 days.Doses of the injected drugs equaled 10% of the LD 50 , namely doxorubicin -5.5 mg/kg, Les-3882 and Les-3833 -10.7 mg/kg, and Les-3288 -24.3 mg/kg.the radical scavenging of 1,1-diphenyl-2-picrylhydrazil (DPPH) activity was measured.Concentrations of thio-barbituric acid-active products were assessed in blood serum, liver, heart and kidney tissues of treated rats.additionally, the activities of superoxide dismutase, catalase and glutathione peroxidase were measured in blood serum and these tissues.We found that administration for 20 days of Les-3288, Les-3833 and Les-3882 compounds disturbed the pro-oxidant-antioxidant balance in the treated rats.Increased amounts of products of reactions of lipid peroxidation and exhaustion of the enzymatic antioxidant system in liver, heart and kidney tissues were detected.In general, Les-3288, Les-3833 and Les-3882 compounds exhibited less pro-oxidant action, compared with the effect of doxorubicin.According to the results of influencing the pro-oxidant-antioxidant balance in the selected tissues, the studied compounds can be ranked in the following order: doxorubicin >> Les-3833 > Les-3288 >> Les-3882.the results of measuring direct scavenging ability of these compounds observed in 24 h suggests their lower toxic effect compared with the effect of the doxorubicin.The obtained results are in correspondence with the results of our recent experiments demonstrating their antineoplastic effect in vitro (Kobylinska L. et al., 2016) and anticancer action in vivo (Kobylinska L. et al., 2018), as well as their lower general toxicity in vivo compared with doxorubicin (Kobylinska L. et al., 2014(Kobylinska L. et al., , 2015(Kobylinska L. et al., , 2016)).

K e y w o r d s: 4-thiazolidinone derivatives, doxorubicin, products of lipid peroxidation, enzymes of antioxi-
dant defense, pro-oxidant-antioxidant balance.
© 2019 Kobylinska L. et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
T he reactive oxygen species (ROS) are primary inducers of apoptosis that are switched on by chemotherapeutic drugs in tumor cells [1][2][3].However, neither the augmentation nor reduction of the amount of ROS can guarantee a positive therapeutic effect of an applied antitumor drug.ROS possess dual functions based on antagonism in the action of their different types and the activity of different systems of enzymatic and non-enzymatic antioxidant defenses.At a low level, ROS can activate signaling mechanisms that promote cell proliferation, whereas an excess of ROS irreversibly affects doi: https://doi.org/10.15407/ubj91.02.007 the molecular components of a cell and leads to its death [4].A balance between the production and neutralization of ROS provides a pro-oxidant-antioxidant balance or redox homeostasis in the cell [5,6].An increase in the ROS level destroys tumor cells, and, in general, these cells are considered more vulnerable to a disturbance of the oxidative-reductive processes and mitochondrial functions than normal cells.Consequently, an imbalance in the ROS and redox homeostasis in cancer cells plays a key role in anticancer chemotherapy [6].A direct influen ce of ROS on processes associated with apoptosis positively correlates with their overall toxic effect on cellular structures, in particular, an impairment of the mitochondrial respiratory chain [2,7].
ROS participate in the oxidative stress caused by anticancer drugs [4].They can cause the severe side effects induced by these drugs, as well as the side effects of non-steroidal anti-inflammatory drugs, anti-retroviral drugs, analgesics, and other medicines.[3].The oxidative stress-related cardiotoxicity mechanisms of doxorubicin (Dox) action are well known, and they include formation of free radicals, lipid peroxidation, mitochondrial dysfunction, impairment of calcium signaling, DNA damage, and activation of apoptotic signaling [2].Free radicals induced by oxidative stress can also lead to energy imbalance, whereas an inhibition of Dox-induced ROS production significantly inhibits the abovementioned toxic effects of this anticancer drug [3].
The anticancer drugs used in chemotherapy cause an excessive activation of lipid peroxidation (LPO) in biomembranes and shift the redox balance [8].Cell damage induced by peroxides can be prevented by the antioxidant system (AOS) that regulates the LPO response in membranes and controls the content of ROS and free radicals, by using both enzymatic and non-enzymatic mechanisms [8].The AOS not only deactivates the ROS and peroxides, but also keeps an optimal concentration of free radicals and a stationary level of LPO [9][10][11].The mobilization of the antioxidant activity (AOA) is an important mechanism in the adaptation process, although its effectiveness is not always sufficient to regulate activated LPO and counteract peroxide-induced tissue damage [6,9].
The liver, heart and kidney tissues of rats are good objects for studying the in vivo toxicity of anticancer agents [12].In liver tissue, the main reactions occur for neutralizing xenobiotics, in particular the anticancer drugs, while in kidney tissue the products of drug metabolism can be found.Assessing the toxic effect on heart tissue caused by chemotherapeutic agents is informative, since most of these agents possess cardiotoxicity.Thus, although liver, heart and kidney tissues participate in drug metabolism, they differ in the type of metabolic processes according to their individual functions.
Presently, chemotherapy is the main approach in the treatment of cancer patients.Therefore, there is a perpetual need to develop various anticancer agents that possess high treatment efficacy and minimal side effects [13].Synthetic heterocyclic 4-thiazolidinone derivatives have demonstrated high antineoplastic potential in vitro within the framework of the Developmental Therapeutic Program for new antitumor compounds at the National Cancer Institute (Bethesda, Maryland, USA) [14][15][16][17].As a result of such studies, the pyrazoline-thiazolidinone-isatin derivatives Les-3288, Les-3833 and Les-3882 were selected as the most promising candidates for further preclinical studies [18].
The aim of this study was to determine the effects of the 4-thiazolidinone-based potential anticancer chemotherapeutics -Les-3288, Les-3833, Les-3882 -on the indicators of the pro-oxidantantioxidant balance in the liver, heart and kidneys of labo ratory rats.We hypothesized that these compounds differentially affect the balance of the products of LPO and the activity of enzymes of the antioxidant defenses in the targeted organs.The direct scavenging ability of these compounds towards a reduced glutathione observed in 24 h was also measured.
animals.All experiments using experimental rats were conducted under the control of the Bio-Ethics Commission at Danylo Halytsky Lviv National Medical University (Protocol N4 dated by April 18, 2016) in Lviv, Ukraine.Mature white Wistar laboratory rats (200-220 g) were kept on standard fodder in the animal facility with adequate lighting and temperature conditions [12].
All compounds under study were administered to rats intraperitoneally every day in the morning before the first meal.Rats had 24-hour access to wa-ter.This procedure for drug administration is common in a pre-clinical study of medicines [12].The experiment lasted for 20 days.Dox was injected at initial doses of 5.5 mg/kg for Les-3833, 10.7 mg/kg for Les-3882 and 24.3 mg/kg for Les-3288.The dose was gradually increased by 1.5 times during 4 days in order to achieve a cumulative effect.The starting dose equaled 10% of the maximum injected dose calculated in experiments that determined the LC 50 [12].The rats were euthanized on the 20 th day by decapitation under thiopental anesthesia.Serum was obtained from blood samples.After taking organs from each animal the tissue samples were homogenized in phosphate buffer pH 6.0 1:4, centrifuged at 5000 rpm and supernatant was used to determine of biochemical parameters.
assay for scavenging activity.Radical scavenging activity of 1,1-diphenyl-2-picrylhydrazil (DPPH) was measured, as described by Asatiani et al. [20].A solution of 0.1 mM DPPH was prepared and 1 ml of this solution was added to 3 ml of solutions of the investigated drugs in concentrations 1 µg/ml.DMSO was used as a solvent.The reaction mixture was vortex-mixed at room temperature, and the absorbance was determined at 20 min after mixing and at 24 h by measurement at 520 nm with a multifunctional reader Infinite M1000PRO (Tecan, Switzerland).A decrease in absorbance of the resulting solution was then measured spectrophotometrically at 520 nm.All experiments were repeated three times with three parallels in each variant.Inhibition (%) of DPPH free radicals was calculated as follows: [(А 0 -А 1 )]/А 0 ]×100%, where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.
Measurement of tBa-active products.For measuring the amount of malonic dialdehyde, 0.5 ml of a 1% solution of thiobarbituric acid (TBA) in 50 mM NaOH and 0.5 ml of a 2.8% solution of trichloroacetic acid were added to aliquots of tissue homogenates.The mixture was kept for 20 min in a boiling water bath, cooled to room temperature, and then the optical density was read at 532 nm [21].The concentrations of the TBA-active products were presented as μmol/mg of protein.
The activity of superoxide dismutase (SOD, EC 1.15.1.1)was measured in the reduction reaction of nitrotetrazolium blue to nitroformazan.The assay medium contained 0.1 ml of blood serum or a homogenate sample (1:10 dilution), 0.9 ml of distilled water, 0.5 ml of absolute ethyl alcohol, 0.25 ml of chloroform and 0.3 g of KH 2 PO 4 .The mixture was shaken vigorously, centrifuged (5,000 rpm, 30 min), and then 0.1 ml of NADPH and 0.05 ml of the incubation solution (37 mg EDTA, 330 mg p-iodonitrotetrazolium violet, 55 mg phenazine methosulfate) were added to the supernatant and incubated for 10 min at 20°C.The absorbance was read immediately at 540 nm.SOD activity was presented as μmol/min per ml or mg of protein [22].
The activity of catalase (CAT, EC 1.11.1.6)was determined by measuring a decrease in the hydrogen peroxide (H 2 O 2 ) concentration at 410 nm.The assay medium consisted of 1 ml Tris-HCl buffer solution (0.05 mmol, pH 7.8), 0.1 ml of blood serum or homogenate sample and 2 ml of 0.03% H 2 O 2 .The reaction was stopped after 10 min of incubation by adding 1 ml of 4% ammonium molybdate.The measurement was conducted at 410 nm, and the activity of CAT of blood serum was presented as micromoles of H 2 O 2 /min per ml or per mg of protein [23].
The activity of glutathione peroxidase (GPO, EC 1.11.1.9)was measured at 262 nm, as the quantity of reduced glutathione.It was presented as micromoles of reduced glutathione per 1 min in 1 ml for blood serum and in 1 mg of protein for tissues [24].
Data analysis and statistics.The results were analyzed using GraphPad Prism 7 (GraphPad Software, Inc, San Diego, CA, USA).Statistical analysis was performed using a t-test or two-way analysis of variance (ANOVA) for comparison of groups.All data are presented as the mean ± SD.P values less than 0.05 were considered statistically significant and marked with asterisks: * P < 0.05; *** P < 0.01.

results and discussion
The content of TBA-active products is considered to be an integrated indicator of the activity of the reactions of LPO induced under the action of various toxic agents [8].Here we demonstrated a similar pattern of content of LPO products in three organs -liver , heart and kidney of rats treated with each of the three 4-thiazolidinone-based derivatives, with Dox used as a positive control.In liver, Dox increased by 58% the content of TBA-active products (Fig. 1).The same tendency was detected in the liver of rats treated with Les-3833, but the increase in content of TBA-active products was lower at 44% (Fig. 1).In heart tissue of rats treated for 20 days with Les-3833, the content of these products increased by 94% (Fig. 1).A 20-day administration of Les-3833 increased by 77% the content of the LPO products in kidney tissue of the treated rats.The effects of Les-3288 and Les-3882 on the content of TBA-active products in liver, heart and kidney tissues were significantly less than those of Dox.For example, the content of LPO products after 20 days of Les-3882 administration was increased by 52% in heart tissue and after of Les-3288 administration by 40% in kidney tissue (Fig. 1).
Next, we measured the activities of the enzymes of the antioxidant defense system -SOD, CAT and GPO -in liver, heart and kidney homogenates of the experimental rats treated with different 4-thiazolidinone-based derivatives, with Dox used as a positive control.Dox administration decreased SOD activities in homogenates of all organs under investigation: by 42% -in liver, 41% -in heart, and 35% -in kidney (Fig. 2).These effects could be explained by the action of free radicals induced by Dox, because the cardiotoxic effect of Dox is based on the increased generation of the superoxide anion Fig. 1. concentrations of tBa-active products in blood serum, liver, heart and kidney of rats treated with doxorubicin and Les-3288, Les-3833 and Les-3882.*P ≤ 0.05; ***P ≤ 0.01 (difference compared to control).tBa, thiobarbituric acid radical in mitochondria.In contrast, after 20 days of administration, Les-3288 decreased SOD activi ty by 23% in heart tissue and 27% in kidney tissue (Fig. 2).An injection of Les-3833 decreased SOD activi ty by 26% in rat liver, by 48% -in rat heart, and by 42%in rat kidney (Fig. 2).A 20-day admini stration of Les-3882 decreased SOD activity by 75% only in kidney tissue, while in heart tissue, this compound slightly increased the activity of SOD (Fig. 2).
Administration of Dox led to a decrease in CAT activity in homogenates of all investigated organs of rats (Fig. 3).Whereas the effect of 20 days of administration of Les-3288 was characterized by an increase of CAT activity by 27% in liver, by 52% -in kidney, and by 43% -in heart (Fig. 3).Les-3833 caused an increase of CAT activity by 20% in liver and by 28% -in heart (Fig. 3).After 20 days of administration, the Les-3882 compound acted differentially on CAT activity by decreasing it by 28% in heart and kidney, but by increasing it by 15% in liver (Fig. 3).
The activity of the antioxidant enzyme GPO was decreased under the action of all agents under study (Fig. 4).Dox reduced the GPO activity by 34% in liver, by 36% -in heart, and by 68% -in kidney (Fig. 4).After 20 days of administration, Les-3288 reduced the GPO activity by 39% in liver, by 41%in heart, and by 65.5% -in kidney (Fig. 4).After 20 days of administration, Les-3833 decreased the activity of GPO by 28% in liver, by 41% -in heart, and by 56% -in kidney (Fig. 4).The Les-3882 compound had an inhibitory effect toward the activity of GPO in all studied organs of the rats (Fig. 4).
We calculated the OSI index that shows the difference in the oxidative balance compared to a normal state that has an equilibrium between the prooxidant and antioxidant components of the oxidative balance [25].Comparing the values of the OSI index calculated as LPO/SOD+CAT+GPx ratio simplified the interpretation of the obtained results.Low values (<1) of the OSI index suggest an oxidation status that is closer to oxidative balance and better antioxidant defense.The OSI index increases proportionally with the oxidative imbalance.Such an increase can be caused by either an elevation of the pro-oxidant species or a decrease of the antioxidant protection.The higher values (>>1) of the OSI index suggest a greater deviation of the pro-oxidant status from the norm.The results of calculations of the OSI index are presented in Table .As one can see in Table, Dox possesses prooxidant properties and the highest OSI was observed in heart tissue.The compound Les-3833 has a pro-oxidant effect in heart and kidney.While the compounds Les-3288 and Les-3882 demonstrated a smaller pro-oxidant action in blood and tissues, even Les-3288 had a small antioxidant effect in blood.
In a final set of experiments, we investigated possible free radical scavenging activity of the 4-thiazolidinone-based derivatives and reduced glutathione (GSH).The results presented in Fig. 5 demonstrate that the scavenging activity of the investigated substances depend significantly on both their species and duration of action.After 20 min of incubation, the highest anti-radical scavenging activity was demonstrated by Dox and the Les-3833 compound.However, after 24 h of incubation, all investigated 4-thiazolidinone derivatives increased their antioxidant activities, while the radical scavenging activity of Dox was decreased significantly.
The results of these in vitro experiments suggest that the investigated 4-thiazolidinone-based
In a previous study, we showed that Dox and the heterocyclic 4-thiazolidinone-based derivative Les-3833 increased the level of the malonic dialdehyde in blood serum of laboratory rats, while the related derivatives Les-3288 and Les-3882 did not affect the amount of TBA-active products [26].We also showed that 4-thiazolidinone derivatives reduced the activity of SOD and GPO in blood serum of rats, and that Les-3833 and Dox induced much more potent inhibition of the activity of these en- The results of the present study demonstrated that within 20 days the 4-thiazolidinone-based derivatives (Les-3833, Les-3288, and Les-3882), which possess antineoplastic activity in vitro and antitumor activity in vivo, also cause changes in the balance of the LPO products and the enzymatic system of the antioxidant defense in rats.The detected increase in the amount of products of LPO reactions and the depletion of the activity of enzymes of the antioxidant defense (SOD, CAT and GPO) in liver, heart and kidney tissues of rats treated with the 4-thiazolidinone derivatives are less pronounced than such L. Kobylinska, o.Klyuchivska, R. Lesyk, R. Stoika changes induced by the known anticancer drug, Dox.One can suggest that the general toxicity of the Les-3288, Les-3833 and Les-3882 compounds would be lower compared to that of Dox.This possibility was confirmed in our former studies that demonstrated much lower levels of the biochemical indicators of cardiotoxicity, hepatotoxicity, and nephrotoxicity in blood serum of rats treated with these compounds compared to rats treated with Dox [27][28][29].Accor ding to the results of measuring the amount of products of the LPO reaction and the levels of decreased activi ty of enzymes of the antioxidant defense in three organs (liver, heart and kidney) of treated rats, the studied agents can be ranked as: Dox >> Les-3833 > Les-3288 >> Les-3882.These data are in good correspondence with our previous data that were obtained when the cytotoxicity and antitumor effect of these agents were measured in vitro and in vivo, correspondingly [18,26,30].
A correlation between an increased level of the indicators of LPO in the biological membranes and mitochondria and a decreased activity of enzymes of the antioxidant defense induced by the anticancer drugs in liver, heart and kidney of the laboratory animals, on the one hand, and toxic effects (cardio-, hepato , and nephrotoxicities) of these drugs in treated animals, on the other hand, have been described in other studies [27][28][29].However, the novel 4-thiazolidinone derivatives used in our study induced a much lower amount of products of LPO and a much smaller decrease in the activity of enzymes of the antioxidant defense, compared to the effects of Dox -the "gold standard" of anticancer chemotherapy.Thus, the decreased general toxicity observed from using these derivatives in vivo allows their application at higher doses for treatment of tumor-bearing laboratory animals.The LPO-AOA balance in tissues and organs, specifically in liver, heart and kidney, can be used as prognostic criteria for evaluation of general toxic effects and treatment effects of applied anticancer drugs.
Here we also addressed an important issue that is rarely taken into consideration when evaluating the efficiency of the treatment action of anticancer drugs.This issue is the potential existence of inherent antioxidant properties of drugs, especially their radical scavenging activity.In this study, we described a unique capability of the 4-thiazolidinone derivatives for free radical scavenging.This capability depended on (increased with) the incubation time in vitro, while such action of Dox was decreased with the incubation time.Such a characteristic of the studied 4-thiazolidinone derivatives could explain why these compounds possess much less cardio-, hepato-, and nephrotoxicity compared with Dox, because the free radical scavenging potential of Dox was significantly lower after 24 h of incubation.Thus, in addition to antitumor activity, the Les-3833, Les-3288, and Les-3882 compounds possess free radical scavenging potential that could be very important for inactivation of deleterious free radicals in biological systems.Scavenging of the stable DPPH radical is a widely used method for evaluating AOA [20].DPPH is a stable free radical capable of accepting an electron or hydrogen radical to become a stable diamagnetic molecule.In this experimental model, glutathione (known antioxidant) was used as a positive control [31].Further studies are needed to determine in more detail the biochemical mechanisms regulating AOA action of the 4-thiazolidinone derivatives, as well as to establish the pharmacological efficacy of these promising anticancer agents.
Taking into account our results from evaluation of the OSI index, one can consider that Dox and the Les-3833 derivative have a well expressed pro-oxidant effect, while the effects of the Les-3288 and Les-3882 compounds are less pro-oxidant.Of course, the free radical scavenging activity of these compounds in a simple model system consisting of the anticancer compound and the DPPH radical scaven ger cannot fully reflect the pro-/anti-oxidant action of this compound in a complicated cellular system or living animal.
conclusion.The results of our in vivo studies suggest that 4-thiazolidinone derivatives (Les-3288, Les-3833 and Les-3882) possess different anticancer potential, since only Les-3833 is capable of significant activating free radical processes and decreasing the activity of the antioxidant enzymes (SOD, CAT, and GPO) in liver, heart and kidney tissues of laboratory rats.The pro-oxidant-antioxidant balance in these tissues is an informative criterion for elucidation of the mechanism of toxic lesions induced by the anticancer drugs in animals.The results of our in vitro study of free radical scavenging activity of various 4-thiazolidinone-based derivatives and Dox suggest a weak scavenging potential for free radicals that increases with incubation time.