The role of oxidative stress in 63 T-induced cytotoxicity against human lung cancer and normal lung fibroblast cell lines

Summary It has been shown previously that molecules built on benzanilide and thiobenzanilide scaffolds possess differential biological properties including selective anticancer activity. In our previous study, we examined the cytotoxic activity and mechanism of action of the thiobenzanilide derivative N,N′-(1,2-phenylene)bis3,4,5–trifluorobenzothioamide (63 T) as a potential chemotherapeutic compound in an experimental model employing A549 lung adenocarcinoma cells and CCD39Lu non-tumorigenic lung fibroblasts. Since the results suggested oxidative stress as a co-existing mechanism of the cytotoxic effect exerted by 63 T on tested cells, studies involving the analysis of reactive oxygen species (ROS) generation and markers of oxidative stress in cells incubated with 63 T were carried out. It may be concluded that the selective activity of 63 T against cancer cells shown in our experiments is caused, at least in part, by the response of the tested cells to 63 T mediated oxidative stress in both tested cell lines. Electronic supplementary material The online version of this article (10.1007/s10637-018-0704-8) contains supplementary material, which is available to authorized users.


Introduction
Oxidative stress is a fundamental concept in redox biology and medicine. It is defined as Ban imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage [ 1]. Over the past several years, it has been shown that oxidative stress could be related to the pathomechanism of neurodegenerative [2], cardiovascular [3], and metabolic disorders [4] as well malignant diseases [5]. Numerous studies shed light on the important role of the overproduction of reactive oxygen species (ROS) in the development of different types of cancer, as this increases DNA mutations and damage, increasing the tendency for alterations in the genome and cancer cell growth and preventing cell death [6,7]. Moreover, it is well known that a moderate increase of ROS production may stimulate cell proliferation while an excessively high increase corresponds to several abnormalities and decreases viability [8].
Although an antioxidant treatment is considered as chemoprevention which may potentially protect from oxidative injuries, it should be emphasized that, depending on several circumstances, the use of antioxidants may have an adverse effect by promoting cancer cell survival [9,10]. In general, ROS levels are higher in cancer cells compared to non-transformed cells [10], thus targeting cancer cells by boosting ROS generation may enhance the cytotoxic effects of anticancer agents. This phenomenon has important implications for pro-oxidant therapies, including radiotherapy, photodynamic therapy and chemotherapeutic agents which are based on oxidative stress induction [8,11]. However, cancer cells adapt to continuous oxidative stress through several mechanisms such as the upregulation of antioxidative enzymes, intracellular antioxidants and reprogramming metabolic pathways [12,13]. Indeed, several studies have shown that a high level of intracellular antioxidants and antioxidative enzymes could be connected with the resistance of cancer cells to ROS-inducing agents [14][15][16]. The main ROS-detoxifying enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPx) play a role in the breakdown of the superoxide radical (O 2 -. ), hydrogen peroxide (H 2 O 2 ) as well other hydroperoxides [17]. Other proteins like peroxiredoxins, thioredoxins (Trx), glutaredoxins (Grx), as well low-molecular-weight antioxidants like glutathione (GSH), tocopherol and ascorbate, are involved in ROS scavenging and, thus, prevent oxidative modification of biologically important molecules [17]. Beyond ROS, an antioxidant defense is also important for maintaining the levels of reactive nitrogen species (RNS), a family of highly reactive, nitrogen-bearing molecules including the nitric oxide radical (NO or NO˙), nitrogen dioxide radical (NO 2˙) , nitrite (NO 2 − ) and peroxynitrite (ONOO − ) [18]. Considering that ROS production is lower in normal cells and the basal level of antioxidative enzymes is different, a therapeutic approach that targets the antioxidant system might significantly increase selectivity and enhance ROS-mediated cancer cell death [12].
To date, molecules built on benzanilide and thiobenzanilide scaffolds have been found to exert promising spasmolytic [19], antibacterial [20], antimycotic [21], antifungal [22] and estrogenic [23], as well anticancer [24,25], activity. In previous studies, the cytotoxic activity of the thiobenzanilide derivative N,N′-(1,2phenylene)bis3,4,5-trifluorobenzothioamide (63 T) has been broadly reviewed for a variety of cancer cell lines including breast and lung cancer [23]. Particularly noteworthy is that 63 T exhibited significant anticancer activity against the lung cancer cell line A549 with a half maximal inhibitory concentration (IC 50 ) of >1 μM, 0.41 μM and 0.36 μM after 24 h, 48 h and 72 h, respectively [25]. Moreover, experiments using the normal human fibroblast (CCD39Lu) and cancer cell lines confirmed the selective cytotoxic effect of 63 T. The calculated selectivity index after 48 h was 2.7 (IC 50 CCD39Lu/IC 50 A549), which is superior to results obtained in similar experiments for clinically approved drugs [25]. Our previous data showed that 63 T selectively induces cancer cell death in the caspase independent pathway, and the autophagic dose-dependent processes may be involved in the mechanism of cell death [25]. However, various aspects related to molecular targets and the antitumor effects of 63 T are still unknown [25]. In line with our preliminary studies, which indicated the generation of oxidative stress as a possible antitumor mechanism of 63 T, further experiments were designed to explore the anticancer effect of the aforementioned benzanilide derivative as an oxidative stress-inducing agent. In the current work, ROS generation, lipid peroxidation, detection of H 2 O 2 and NO, the activity of antioxidative enzymes (mitochondrial superoxide dismutase [MnSOD], CAT, glutathione-S-transferase [GST], GPx) as well the status of glutathione in cancer and normal cells have been assessed. Additionally, we investigated the effects of antioxidative enzyme inhibitors on 63 T cytotoxic activity and DNA damage as a potential effect of oxidative stress. A general overview of the performed experiments and the aims of this study are presented in Fig. 1.

Chemicals
All chemicals used in experiments were obtained from Sigma-Aldrich (St. Louis, Mo, USA) unless otherwise stated.

Cell culture
All experiments were performed using two cell lines: A549 (human lung adenocarcinoma) and CCD39Lu (normal human lung fibroblast). Both cell lines were o b t a i n e d f r o m t h e E u r o p e a n C o l l e c t i o n o f Authenticated Cells Cultures (ECACC, Salisbury, UK) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine at 37°C Gibco Invitrogen Corp. (Grand Island, NY, USA), in a humidified atmosphere containing 5% CO 2 .
The stock solution of the tested compound 63 T (1 mM) was prepared by dissolving it in DMSO and then storing it in the dark at -20°C.

Detection of ROS
Two different methods were used to investigate the influence of 63 T on cellular radical generation: fluorescence microscopy and flow cytometry. For this purpose, the non-fluorescent 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed. DCFH-DA after de-esterification to 2′,7′dichlorodihydrofluorescein (DCFH), is further oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) by ROS as well RNS. For microscopic analysis, 25 × 10 4 cells/well were seeded into each of the wells of a 12-well plate. After 6 h and 24 h incubation with 63 T at concentrations of 0.25 μM, 0.5 μM and 1 μM, the cells were washed with phosphate buffered saline (PBS) and DCFH-DA (10 μM in PBS) was added to each well. The plates were incubated for 30 min at 37°C. Then, the plates were washed twice with PBS and observed under a fluorescence microscope (Nikon Eclipse TS100 microscope with attached fluorescence unit model C-SHG and digital camera DS-SMc).
For flow cytometry analysis, cells were seeded in a 25 cm 2 flask and incubated overnight under cell culture conditions. Then 63 T was added at concentrations of 0.25 μM, 0.5 μM and 1 μM and incubated for 24 h. DMSO was used as the negative control and the concentration did not exceed 0.1%. For the positive control, 1 μM of cumene hydroperoxide (CHPO) was used. Cells were harvested by trypsinization, then suspended in a DCFH-DA solution in PBS (0.1 μM) and incubated for 30 min at 37°C in dark conditions. Cells were centrifuged at 1300 rpm for 5 min and washed twice with PBS. ROS generation was determined by FACS Calibur (Becton & Dickinson, USA). The results are presented as the mean ± SD from three independent experiments.

Lipid peroxidation measurement
Lipid peroxidation was measured using the Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Extensive oxidative species generation can lead to the formation of malondialdehyde (MDA) as a result of the peroxidation of polyunsaturated fatty acids (PUFAs) which contain at least three double bonds. MDA can react with thiobarbituric acid to form a colorimetric, as well a fluorescent, product which is a well-known biomarker of the peroxidation of polyunsaturated lipids [26]. The A549 and CCD39Lu cells were seeded at a density of 5 × 10 5 cells per well. The cells were treated with 63 T at concentrations of 0.25 μM, 0.5 μM and 1 μM for 24 h. Then, cells were lysed on ice using MDA lysis buffer containing 3 μL of butylated hydroxytoluene (BHT) and samples were centrifuged at 13,000×g for 10 min. Next, a thiobarbituric acid (TBA) solution was added and incubated at 95°C for 60 min. The samples were chilled to room temperature in an ice bath for 10 min and transferred into a black bottom 96-well plate. The fluorescence of MDA-TBA adducts was measured using microplate reader Tecan Infinity 200 (Männedorf, Switzerland) at 532 nm and 553 nm for excitation and emission, respectively. The MDA solution was used as a standard at concentrations of 0.4 nM, 0.8 nM, 1.2 nM, 1.6 nM and 2.0 nM.

Detection of DNA damage
Double stranded breaks (DSBs) can be associated with the phosphorylation of histone H2A variant H2AX. Because phosphorylation of H2AX at Ser 139 (γH2AX) is abundant, fast, and correlates well with each DSB, it is one of the most sensitive markers used to determine DNA damage [27].
DNA damage after 63 T treatment was detected using an EpiQuik™ In Situ DNA Damage Assay Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer's protocol. A549 and CCD39Lu cells were seeded on 96-well plates at a density of 2 × 10 4 cells per well and incubated overnight. Then, cells were exposed to 63 T at concentrations of 0.25 μM, 0.5 μM and 1 μM for 24 h. Cells were fixed using paraformaldehyde, permeabilized and blocked. The phosphorylation of H2AX at serine 139 was detected by the primary anti-phospho H2AX Ser139 antibody and HRP conjugated secondary antibody. The absorbance was measured at 490 nm using a plate reader (Biotek Instruments, Elx-800). The results are presented as a percent of control (mean ± SD from two independent experiments).
Evaluation of H 2 O 2 production H 2 O 2 was detected using a Hydrogen Peroxide Cell-based Assay Kit according to the manufacturer's protocol. A549 and CCD39Lu cells were seeded at a density of 2 × 10 4 cells per well on 96-well plates and incubated overnight. Next, the tested compound 63 T was added at concentrations of 0.25 μM, 0.5 μM and 1 μM for 12 h and 24 h under cell culture conditions. Subsequently, plates were centrifuged at 400 x g for 5 min, and 80 μl of the medium was transferred to another plate and the assay buffer and reaction mixture were added to the samples and standards. Plates were incubated with gentle shaking on an orbital shaker for 15-30 min at room temperature. The resorufin fluorescence produced was measured using a Tecan Infinite 200Pro (excitation: 530 nm; emission: 590 nm). The protein concentration was measured using the BCA Protein Assay.

Measurement of CAT activity
CAT activity was measured using a commercially available kit purchased from Cayman Chemicals (Ann Arbor, MI, USA). The method was based on the reaction of CAT with methanol in the presence of an optimal concentration of H 2 O 2 . The formaldehyde produced in this reaction was measured spectrophotometrically at 540 nm with purpald (4-amino-3hydrazino-5-mercapto-1,2,4-triazole) as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to a purple color. A549 and CCD39Lu cells were seeded in 25 cm 2 flasks and incubated under cell culture conditions. Then 63 T was used at concentrations of 0.25 μM, 0.5 μM and 1 μM and incubated for 12 h, 24 h, and 48 h. Cells were collected using a rubber policeman and centrifuged (1300 rpm, 10 min at 4°C). Cells were sonicated in 1 ml of cold buffer and centrifuged at 10,000 rpm for 15 min at 4°C and supernatants were transferred into the new tubes. As a standard, formaldehyde was used. The samples and standards were mixed with methanol and an assay buffer, and then H 2 O 2 was added and incubated on a shaker for 20 min at room temperature. To terminate the reaction, potassium hydroxide was added, and then CAT purpald (chromogen) was added to each well and plates were incubated for 10 min on a shaker. Next, potassium periodate was added and incubated for 5 min on a shaker. The absorbance at 540 nm was measured using a BioTek plate reader.

Assessment of MnSOD activity
MnSOD activity was analyzed using a Manganese Superoxide Dismutase Assay HCS232 (Millipore) according to the manufacturer's protocol. A549 and CCD39Lu cells were seeded at a density of 2 × 10 4 cells on 96-well plates and incubated under cell culture conditions. On the next day, the 63 T was added at concentrations of 0.25 μM, 0.5 μM and 1 μM and incubated for 24 h and 48 h. As a positive control, etoposide at a concentration of 100 μM was used. After incubation, cells were fixed at room temperature for 30 min, washed and the primary antibody was added for 1 h at room temperature. Then, cells were washed three times and incubated for 1 h with the secondary antibody/Hoechst dye. Cells were washed four times and analyzed under a fluorescence microscope (Nikon Eclipse TS100 microscope with attached fluorescence unit model C-SHG and digital camera DS-SMc).

Measurement of GSH content
The GSH level in the A549 and CCD39Lu cells incubated with 63 T was measured using ThioGlo-1 (Calbiochem, San Diego, CA). ThioGlo-1 is a maleimide reagent, which produces highly fluorescent adducts upon reaction with thiol groups, chiefly with intracellular GSH [28]. Briefly, A549 and CCD39Lu cells were seeded in 96-well plates at a density of 2 × 10 4 cells per well and incubated overnight. Cells were treated with 63 T alone or with 63 T and buthionine sulphoximine (BSO) -a gamma-glutamylcysteine synthetase (γ-GCS), GSH synthesis inhibitor (1.125 mM) for 24 h and 48 h under cell culture conditions. Then, cells were lysed and incubated for 30 min at 4°C. 50 μL of lysates were transferred to white 96-well plates, and 50 μL of the freshly diluted ThioGlo-1 (Calbiochem, San Diego, CA) reagent in PBS (5 μM) was added to the standards and samples. Plates were mixed using a plate shaker and incubated in the dark for 5 min and measured (λ EX 384 nm, λ EM 513 nm) using a microplate reader, Tecan Infinity 200 (Männedorf, Switzerland). The total GSH was calculated using a standard curve with GSH standard solution of concentrations (0.3-1 μM). The data are expressed as the amount of GSH per total protein (mean ± SD) from two independent experiments.

Evaluation of GST and GPx activity
After exposure, the cells were scraped, washed twice with icecold PBS and centrifuged at 200 x g for 5 min at 4°C. Then the cells were resuspended in PBS with 0.05% Triton X-100 (v/v) and sonicated three times for 5 s intervals in ice-cold ultrapure water. After that, the suspension was centrifuged at 10,000 x g at 4°C, and the supernatant aliquots were stored at −80°C. GST activity measurement was based on the spectrophotometric determination of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate formed in a GSH coupled reaction at 340 nm. The activity was expressed as nmol CDNB conjugated with GSH per min per mg protein and compared with activity measured in the control cells. GPx activity was determined by measuring the disappearance of β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) at 340 nm, as the substrate cumene peroxide was used. Activity was calculated and expressed as nmol of NADPH oxidized per min per mg protein [29]. Treatment of inhibitors -Impact on antioxidative enzymes A549 and CCD39Lu cells were seeded at a density of 2 × 10 4 cells per well in 96-well plates and incubated overnight under cell culture conditions. Cells were treated with 63 T at concentrations of 0.25 μM, 0.5 μM and 1 μM and with antioxidative enzymes: 2-methoxyestradiol (2-ME)a MnSOD inhibitorat a concentration of 1 μM, diethyldithiocarbamate (DDC)a CuZn-superoxide dismutase (CuZnSOD) inhibitor at a concentration of 10 μM, 3-Amino-1,2,4-triazole (3AT)a CAT inhibitorat a concentration of 5 mM, and BSOa γ-GCS (GSH synthesis) inhibitorat a concentration of 1.5 μM. Inhibitors and their concentrations were selected after literature search [30] and after preliminary studies. Cells were incubated for 24 h and 48 h. DMSO was used as the negative control. The cell viability was measured using the MTT assay.

Protein measurement
Protein concentration was measured according to the Lowry method using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Melville, NY, USA). Bovine serum albumin (BSA) was used as the standard.

Statistical analysis
Statistical analyses were carried out using one-way ANOVA with Dunnett's multiple comparison tests. The results are presented as the mean ± SD from two or three independent experiments. The values were calculated using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

Detection of ROS
A DCFH-DA assay was employed to investigate the influence of 63 T on cellular radical generation. ROS generation was evaluated using fluorescence microscopy and flow cytometry. The flow cytometry showed a significant dose-dependent increase of the ROS level in A549 cells. Compared with the control, 0.5 μM and 1 μM of 63 T increased the ROS generation by 113% and 309%, respectively. Although the increase of ROS in CCD39Lu cells was also observed for doses of 0.25 μM, 0.5 μM and 1 μM by 61%, 90%, 50% respectively, compared with the control, the ROS generation was lower compared to A549 cells. Increased ROS production was also observed by fluorescence microscopy and, interestingly, Bhot spots^were observed in these pictures (Fig. 3). These results suggest that the cytotoxic effect of 63 T is dependent on the generation of ROS. Furthemore, our results demonstrated that 63 T may target preferentially cancer cells by ROS-mediated mechanism.
Lipid peroxidation measurement ROS may easily react with lipids and, thus, lipid peroxidation is one of the most widely used markers of free radical formation. The peroxidation of membrane lipids may dramatically change the physical properties of lipid bilayers such as lipidlipid interactions, ion gradients, membrane fluidity, and permeability [31]. In our study, MDA, the well-known biomarker of lipid oxidation, was measured after the cells were incubated with 63 T. At a concentration of 0.5 μM, the MDA level in A549 cells was significantly increased and was similar to the level measured in cells incubated with CHPO (the positive control). In contrast, 63 T at a concentration of 1 μM induced lipid peroxidation comparable to that observed for the control cells (Fig. 4a). Interestingly, no significant difference in MDA level was observed for the CCD39Lu cell line. These findings suggest that cell lipid peroxidation may be selectively induced in cancer cells which are more sensitive to oxidative stress induced by 63 T.

Evaluation of DNA damage
Oxidative stress-induced DNA damage is well known as a major cause leading to cell death. We found that incubation with 63 T led to DNA damage in A549 cells at concentrations of 0.5 μM and 1 μM (Fig. 4b). By contrast, in CCD39Lu cells, DNA damage did not occur (Fig. 4b). These results demonstrate that 63 T may induce DNA damage at the mild and high doses within 24 h of treatment.

H 2 O 2 measurement
H 2 O 2 is one of the major redox metabolites involved in physiological and pathological processes such as redox sensing, signaling and redox regulation. Our previous study has shown that treatment with 63 T increased the caspase protein level. Therefore, we tested whether H 2 O 2, as a main substrate for CAT, plays an important role in 63 T-mediated oxidative stress induction. It was found that incubation with 63 T at a concentration of 1 μM for 12 h increased the level of H 2 O 2 (by 33% of control). The level of H 2 O 2 measured after 24 h was significantly increased in cells incubated with 63 T at a concentration of 0.5 μM (by 63% of control) and 1 μM (by 140% of control). In CCD39Lu fibroblasts, a significantly higher level of H 2 O 2 was present after 12 h and 24 h only in cells incubated with 63 T at a concentration of 1 μM (by 54% and 146% of control, respectively) ( Fig. 5a and b).

Nitric oxide production
For both tested cell lines, incubation with 63 T at a concentration of 0.5 μM and 1 μM led to an increase in the level of NO ( Fig. 5c and d) (j, l), respectively. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 of 0.5 μM. Interestingly a clear correlation between the concentration of NO and the 63 T dose was observed in A549 cells for all concentrations. The same trend was observed in CCD39Lu cells only for mild and moderate doses (Fig. 1S).

Evaluation of MnSOD, CAT, glutathione-S-transferase, glutathione peroxidase activity
Next, we analyzed the activity of GSH-dependent enzymes (GPx, GST), CAT and MnSOD in order to obtain a comprehensive view of the role of antioxidant machinery in cancer and non-cancerous cell lines.
MnSOD activity was analyzed using a MnSOD Assay. A sample of images is presented in Fig. 6. Data are also presented as histograms (analyzed using Nikon BR software). The blue (nuclei) to red (MnSOD) ratios were calculated and presented in charts. Using this method, a dose-dependent increase in MnSOD activity in A549 cells incubated with 63 T was noticed, while, in adjacent CCD39Lu cells, a significant increase of MnSOD activity was found in cells incubated with 63 T at the higher concentration (1 μM) (Fig. 6). The CAT activity was significantly increased only in A549 cells incubated with 63 T at a concentration of 1 μM after 24 h, while incubation for 48 h resulted in decreased activity. A significant loss of activity was also observed for CCD39Lu cells incubated with 63 T at a concentration of 1 μM (Fig. 7). The same trend was found for GST and GPx which significantly increased in A549 and CCD39Lu cell lines after incubation with 1 μM of 63 T for 24 h. A significant depletion of enzymatic activity was observed after the next 24 h (Fig. 7). Increased activity of GPx and CAT supports our findings obtained for H 2 O 2 detection. Indeed, GPx and CAT are known as the two main enzymes involved in H 2 O 2 detoxification. Therefore, these correlations between experiments suggest that 63 T at a concentration of 1 μM, particularly in cancer cells, may increase activity of CAT and GPx in response to overproduction of H 2 O 2 .

Assessment of reduced glutathione (GSH) content
24 h incubation of A549 cells with 63 T resulted in a significant increase in the GSH level. However, after the next 24 h, a significantly lower level of this important peptide was measured. Interestingly, in CCD39Lu cells the concentration of GSH in cells incubated with 63 T significantly declined after 24 h, while after the next 24 h its level was restored and was similar or even higher in comparison to the concentration measured in the control cells. To confirm the important role of GST in 63 T-mediated oxidative stress induction, BSO was used to inhibit GSH biosynthesis in both cell lines; BSO significantly reduced the concentration of GSH (Figs. 8 and 9).

The influence of MnSOD, CuZnSOD, 2-ME and glutathione synthetase inhibition on 63 T induced toxicity
In this study, we used a panel of antioxidative enzymes inhibitors: 3-AT (a CAT inhibitor), DDC (a CuZn-SOD inhibitor), 2-ME (an inhibitor of Mn-SOD) and BSO (a γ-GCS inhibitor), to investigate whether combining 63 T with a blockage of each of the mentioned enzymes may synergistically potentiate cytotoxicity. We found that 3-AT significantly enhanced the toxicity of 63 T in A549 cells incubated for 48 h at a concentration of 1 μM (Fig. 10). BSO significantly increased the toxicity of 63 T in CCD39Lu lung fibroblasts (Fig. 9) after 48 h of incubation for the dose of 1 μM. This finding showed that the GSH synthesis inhibitor can effectively enhance 63 T activity in CCD39Lu cells and confirmed that the GSH content in non-cancerous cells is important in the anticancer effect of 63 T, as has been suggested by previous studies.
Other tested inhibitors (MnSOD and CuZnSOD inhibitors) did not change the cytotoxic effect of 63 T for both cell lines (Figs. 2S and 3S).

Discussion
ROS play a crucial role in the death and survival of both normal and cancer cells. ROS production in living cells is controlled by several systems employing antioxidant enzymes such as SOD, CAT or GPx and other small-molecule antioxidants including GSH [32]. The level and cellular turnover of GSH is strictly controlled by several enzymatic systems [33][34][35][36]. It is commonly accepted that in cancer cells the enzymatic systems controlling their redox status are even more important than in normal cells, since cancer cells exist in conditions of continuous oxidative stress [37,38]. The increased ROS generation in cancer cells is linked to a turbulent cellular metabolic activity stimulated by oncogenic signals [39]. Additionally, the oxidative stress in cancer cells may be a consequence of mitochondrial malfunction which is often present in cancer cells [40,41].
The downregulation or inhibition of cellular antioxidative systems with parallel production of ROS may disturb the redox balance in cancer cells and cause apoptotic and/or necrotic death [41][42][43], which has been previously reported by our group [29,44]. On the other hand, the overexpression of antioxidative enzymes is observed in some malignant cells as a result of ROS generating anticancer therapy [45]. Our previous experiments performed with A549 and CCD39Lu cells suggested a ROS-driven mechanism of cell death induced by 63 T. Therefore, in this presented study, the impact of this promising benzanilide derivative on the redox balance in A549 lung cancer and CCD39Lu non-tumorigenic lung fibroblasts was investigated. The proposed mechanism and the consequences of oxidative stress in normal fibroblasts and cancer cells line are presented in Fig. 11.
The production of ROS was investigated in a DCHF assay where, in both cell types, an increased fluorescence of DCF was registered using flow cytometry and fluorescence microscopy. In both cell lines, the dose-response analysis suggested different mechanisms of ROS generation and antioxidant defense activity in the two tested types of cells. Since DCF assays conducted using only plate readers or flow cytometry are sometimes criticized due to a lack of morphological analysis [46], additional experiments employing fluorescence microscopy were performed. These experiments discovered Bhotspots^in cells and suggest the existence of subcellular structures where ROS generation was particularly intensive (Fig. 3). DCFH-DA is very useful a redox indicator probe that responds to changes in intracellular iron signaling or peroxynitrite formation [47]. Nevertheless, it should be taken into consideration that the intracellular DCF level depends on several factors such as the different esterase activity, a basal redox state as well an expression of MDR (multidrug resistance)-associated ABC transporters [48]. Moreover, the DCF fluorescence intensity may change due to (i) relocation of mitochondrial cytochrome c to cytosol from mitochondrial membrane permeation, (ii) relocation of lysosomal iron to cytosol from lysosomal membrane permeation, (iii) the presence of oxygen, H 2 O 2 and redox active metals, and (iv) a reaction with oxygen that led to the production of superoxide and, in effect, increased the H 2 O 2 concentration [46,48,49]. Therefore, due to non-specific properties of DA-DCFH, it was used in our study to measure the overall intracellular redox For both analyses, data are presented as mean ± SD from two separated experiments. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 status, and further studies employing more specific redox probes are necessary to fully explain the Bhotspots^phenomenon observed by us.
A particularly important finding is that 63 T may selectively induce lipid peroxidation in cancer cells. We observed that the malondialdehyde (MDA) level in cancer cells is relatively higher than in normal fibroblasts. Therefore, 63 T may selectively deregulate redox homeostasis in cancer cells by increasing levels of lipid peroxidation. In general, lipid peroxides act as cytotoxic agents via different mechanisms such as affecting the function of lipid membranes, generating further ROS, modifying DNA andproteins [31].Our resultsdemonstratedthat 63 Tmay increase MDA production accompanied by the phosphorylation of γH2AX which is a marker of DNA double-strand breaks (DSBs) [50]. The oxidative lesions can lead to DNA DSB formation by different mechanisms: (i) when two DNA single strand breaks (SSBs) form close to each other on opposite strands, (ii) as a result of cleavage sites close to SSBs on the opposite strand by topoisomerases, (iii) when ROS-induced DNA damage affects DNA replication or transcription, and (iv) during the repair of two lesions in a cluster at the same time or when the modified base located close to an unrepaired SSB on the opposite strand is removed [50]. Although the appearance of DSBs is still considered to be less than that of other ROS-induced DNA lesions such as 8-hydroxydeoxyguanosine (8-OHdG), ROS such as H 2 O 2 may induce H2AX formation [51]. Work by Zhao et al. showed that there is a close association between DNA replication and H2AX phosphorylation in A549 cells [52]. This finding suggested that H2AX phosphorylation may be caused by stalled replication forks and perhaps also by induction of DNA doublestrand breaks at the primary DNA lesions induced by H 2 O 2 [52]. Also, authors reported that activation of Ataxia Telangiectasia Mutatedproteinkinase (ATM) and inductionof H2AXare among the early events of the DDR induced by exposure of cells to H 2 O 2 in human pulmonary carcinoma A549 cells [52]. On the other hand, Gao et al. found that activation of ATM by H 2 O 2 in human primary fibroblasts was observed to occur with no evidence of H2AX phosphorylation [53,54].
Since in our previous studies a significantly high expression of catalase has been measured in cells incubated with 63 T, we have measured hydrogen peroxide production in cells exposed to 63 T. The increased CAT activity may be considered as a possible cellular response to H 2 O 2 production reported by other authors [55,56]. In both cell lines incubated with 63 T at a concentration of 1 μM, a significantly increased level of H 2 O 2 was observed (Fig. 5). It was reported by some authors that the production of H 2 O 2 in cancer cells incubated with anticancer compounds, e.g. 2-methoxy-6-acetyl-7-methyl juglone, may by followed by increased NO production mainly via the activation of Jun-N-terminal kinase (JNK) and the expression of inducible nitric oxide synthase (iNOS) [48], thereby leading to NO generation in multiple cancer cells or by xanthine oxidoreductase [57,58]. Therefore, the next molecule to be measured in our experiments was NO. NO is a pleiotropic regulator, synthesized by a few nitric oxide synthetases (NOS -inducible iNOS, endothelial eNOS and neuronal nNOS) and plays a key role in numerous biological processes, including neurotransmission [59], vasodilatation [60], and macrophage-mediated immunity [61]. NOS activity has been detected in tumor cells of various histological origins and has been associated with tumor grade, proliferation rate and expression of important signaling components associated with cancer development such as the estrogen receptor. Consequently, NO has been described as a Janus-facedmolecule [62] which modulates several cancer-related events including angiogenesis, apoptosis, cell cycle, invasion, and metastasis in both cell death and cell survival directions [63]. In our experiment, the level of NO increased in both cell lines in a concentration-dependent manner. Interestingly, the basic concentration of NO was four times higher in CCD39Lu cells (Fig. 5). In general, the lung interstitium consists mainly of fibroblasts which are responsible for the production of extracellular matrix (ECM) proteins and also play an important role during injury repair [64]. A number of other studies show Statistical significance corresponding to increased activity is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, while statistical significance of a decrease in activity is shown with hashtags #p < 0.05, ###p< 0.001 the role of NO produced by fibroblasts during inflammation and wound-repair [65][66][67]. It was reported that exposure to a different proinflammatory stimulus (LPS, IFN, cytokine mix) may modify the response of lung epithelial cells to produce nitric oxide. Importantly, when lung fibroblasts were cocultured with cancer cells, it was observed that the NO production was associated with a higher ratio of fibroblasts [66]. Moreover, as mentioned by White et al., NO and NO donors increase cellular GSH in a rat lung fibroblast cell line (RFL6 cells) and, thus, NO could have a protective effect against oxidant injury by increasing cellular GSH synthesis [68].
In the case of ROS overproduction and induction of oxidative stress, the compensative mechanism of antioxidative defense is expected [45]. The basic enzymatic cellular antioxidative system comprises several strictly collaborating enzymes: superoxide dismutase, CAT, and GPx. The mitochondria are Fig. 8 Changes in intracellular glutathione levels in A549 and CCD39Lu cells (panels a, c and b, d respectively) incubated simultaneously with BSO (glutathione synthetase inhibitor) and 63 T alone. Intracellular concentration of GSH in A549 and CCD36Lu was measured after 24 h (panels a and b) and 48 h (panels c and d). Statistical significance is indicated with asterisks: *p < 0.05 Fig. 9 Effect of GSH synthesis inhibition on A549 and CCD39Lu cells viability. The impact of BSO co-treatment on cell viability is in A549 cells is shown in panels (a) and (c), (aafter 24 h, cafter 48 h), while (b) and (d) show the parallel effect exerted by 63 T in CCD39Lu cells (bafter 24 h, dafter 48 h). Statistical significance is indicated with asterisks: *p < 0.05 the most sensitive cellular organelles where free radicals may induce cell death. In our experiments, a significant increase in MnSOD activity was observed in A549 cells, while in CCD39Lu cells its increase was less efficient. Although mitochondria are equipped with a relatively efficient antioxidant system, they are generally devoid of CAT. However, the expression of this enzyme, which is very important in the regulation of redox status, is very high in the cytoplasm. A significant induction of CAT was shown in our previous studies using immunoassay [25]; however, in the present study an assay measuring CAT enzymatic activity in cell lysates was used. Using this assay, a very high induction of CAT was noted only in A549 cells incubated for 24 h with 63 T at a concentration of 1 μM. Similarly, high activity of two key glutathione-dependent enzymes, GPx and GST, was measured in cells incubated for 24 h with 63 T. Analogously to CAT, the activity of these enzymes declined sharply after the next 24 h suggesting progression in the disintegration of cellular structures and the degradation of enzymes followed by cell death.
Since a significant increase of GSH-dependent redox buffers was observed as a result of 63 T toxicity, we further assayed the effect of this compound on the GSH level. In order to evaluate GSH recycling machinery, the GSH synthesis inhibitor BSO was used. As shown in Fig. 8, the reaction of CAT, catalase; GPx, glutathione peroxidase, GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione-S-transferase; H 2 O 2 , hydrogen peroxide; MnSOD, manganese superoxide dismutase, NO, nitric oxide; O2 .-, superoxide; OH . , hydroxyl radical; ONOO-, peroxynitrite; ROS, reactive oxygen species GSH synthesis in A549 cells was very fast and could be observed after 24 h. The synthesis of new GSH was efficiently inhibited by BSO. In CCD39Lu cells, a significant decrease in the level of GSH was followed by its resynthesis which was observed after 48 h of incubation. It was also observed that CCD39Lu cells were much more sensitive to BSO treatment and, in these cells, BSO significantly potentiated the cytotoxic activity of 63 T, while in A549 cells the synergistic action of 63 T and BSO was not observed. This result suggests significant differences in ROS defense systems in both cell lines.
Following this link, we assayed the impact of a few inhibitors of antioxidant enzymes: 2-ME for MnSOD, DDC for CuZnSOD and 3-AT for CAT on the cytotoxicity of 63 T. From these inhibitors, only 3-AT slightly potentiated the cytotoxic activity of 63 T, again indicating the role of H 2 O 2 in the cytotoxic activity of 63 T.
In conclusion, it may be summarized from this stage of experiments that 63 T is absorbed by cancer cells and nontumorigenic cells and is probably metabolized to short living reactive metabolites. The incubation of tested cells with 63 T resulted in changes in the expression of several antioxidative and drug-metabolizing enzymes as well as enzymes responsible for cell death or survival. It should be stressed that the concentration range used in our experiments was very narrow, however, the observed cytotoxic effects ranged from relatively mild effects at 0.25 μM through significant effects observed at 0.5 μM to severe toxicity observed in cells incubated with 63 T at a concentration of 1 μM, where the whole cellular machinery was quickly inactivated and destroyed leading to cell death. The selective activity against cancer cells showed in our experiments is caused at least partially by the different response for ROS in both tested cell lines as well as different metabolic pathways leading to oxidative stress. In further studies, metabolic pathways involved in the formation of reactive metabolites as well the pathways involved in their detoxification should be investigated and explained. Additionally, metabolites responsible for the cytotoxic activity of 63 T should be identified.