In Vitro Anti-Prostate Cancer Activity of Two Ebselen Analogues

Scientific research has been underway for decades in order to develop an effective anticancer drug, and it has become crucial to find a novel and effective chemotherapeutics in the case of prostate cancer treatment. Ebselen derivatives have been shown to possess a variety of biological activities, including cytostatic and cytotoxic action against tumor cells. In this study, the cytotoxic effect and anticancer mechanism of action of two organoselenium compounds— (N-allyl-1,2-benzisoselenazol-3(2H)-one (N-allyl-BS) and N-(3-methylbutyl)-1,2-benzisoselenazol-3(2H)-one) (N-(3-mb)-BS)—were investigated on two phenotypically different prostate cancer cell lines DU 145 and PC-3. The influence of analyzed compounds on the viability parameter was also assessed on normal prostate cell line PNT1A. The results showed that both organoselenium compounds (OSCs) efficiently inhibited cancer cell proliferation, whereas normal PNT1A cells were less sensitive to the analazyed ebselen analouges. Both OSCs induced G2/M cell cycle arrest and prompted cell death through apoptosis. The detection of cleaved Poly (ADP-ribose) Polymerase (PARP) confirmed this. In addition, N-allyl-BS and N-(3-m)-b-BS increased the level of reactive oxygen species (ROS) formation, however only N-allyl-BS induced DNA damage. Based on our data, we assume that OSCs’ anticancer action can be associated with oxidative stress induction and inactivation of the Akt- dependent signalling pathway. In conclusion, our data demonstrate that ebselen derivatives showed strong cytotoxic efficiency towards prostate cancer cells and may be elucidated as a novel, potent anticancer agent.


Introduction
The search for novel and more effective drugs for prostate cancer treatment has been going on for decades, yet the progress in curing tumors is marginal. Recently, prostate cancer has been listed as the second leading cause of male death in the US and western countries, but its incidence is also high in other regions of the world, according to GLOBOCAN statistics [1]. These figures highlight the urge of novel and potent chemotherapeutics against prostate malignancies.
In particular, over the last decades, much effort has been made to develop new anticancer drugs, which exhibit less toxicity towards noncancerous human cells. The potential therapeutic use of

Cell Line and Cell Culture
The DU145 cancer cell line purchased from the American Type Culture Collection (ATCC, Manassas, VA) was maintained in MEME medium that was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. PC-3 cells were also from ATCC. Noncancerous, prostate epithelial PNT1A cells were from the European Collection of Authenticated Cell Cultures (ECACC, France, Paris). The PC-3 and PNT1A cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All of the cell lines were maintained at 37 • C under 5% CO 2 in a humidified incubator. In addition, the PC-3 cells were cultured and treated under hypoxic (4% O 2 ) condition at 37 • C.

SRB Cell Viability Assay
Cell viability was determined by using the SRB assay. The cells were seeded into 96-well plates (6 ×10 3 cells/well) in 200 µL of culture medium for 24 h. The cells were then treated with various concentrations (2.5, 5, 10, 20, 30, or 40 µM) of N-allyl-BS or N-(3-m)-b-BS for additional 24 h. After incubation, the cells were fixed in 20% trichloroacetic acid for 1 h. The plates were then washed with distilled water, air-dried, and stained with 0.4% solution of SRB (Sigma-Aldrich) in 1% acetic acid, for 15 min. The cells were washed four times with 1% acetic acid and then dried. SRB was then solubilized in 10 mM Trisma-base solution and sample absorbance was measured at 570 nm while using an automated microplate reader. The data were obtained from at least three independent experiments with six samples for each treatment.

Western Blot Analysis
The cells were treated with 40 µM N-allyl-BS or N-(3-mb)-BS for different periods of time and then lysed with a solution of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and protease and phosphatase inhibitor cocktails (Roche Diagnostics). The cell lysates were cleared by centrifugation at 16,000× g for 20 min. The proteins were resolved on 10-12% SDS-PAGE and then transferred onto polyvinylidene fluoride membrane, or separated using Trans-Blot ® Turbo TM mini-size transfer stacks (Bio-Rad Laboratories, Hercules, California, using Trans-Blot ® Turbo TM transfer system M (Bio-Rad Laboratories). The membranes were incubated with a TBST solution containing Tris-buffered saline, 0.05% Tween 20, and 5-10% (w/v) nonfat dry milk, and then exposed to the appropriate primary antibody overnight at 4 • C. After four washes in TBST, the membranes were treated with the appropriate secondary antibody for 1 h at 22 • C. Next, the protein bands were visualized while using ImageQuant LAS 500 (GE Healthcare). The changes in protein levels were quantified by densitometry using the LASImage software. β-Actin was used as a reference control.

Cell Death Assay
The cells were seeded into six-well plates (3 × 10 5 /well) for 24 h. Next, the cells were incubated with 40 µM N-allyl-BS or N-(3-mb)-BS for 24 h. The medium and trypsynized cells were collected and centrifuged at 300× g for 10 min. The early and late apoptotic, and necrotic cells were detected using Muse ® cell analyzer, and Muse annexin-V and dead cell assay kit (Muse, Darmstadt, Germany), according to the manufacturer's instructions.

Cell Cycle Assay
For the experiment, the cells (3 × 10 5 cells/well) were seeded in six-well plates for 24 h and then treated with 40 µM N-allyl-BS or N-(3-mb)-BS for 8 h. Subsequently, both floating and attached cells were collected, trypsynized, washed with PBS, and then fixed with 70% ethanol overnight at −20 • C. The cells were stained using the Muse cell cycle kit (Muse), according to the manufacturer's instructions, and then analyzed by Merck Muse ® Cell Analyzer.

Cell Viability Assay
Prior to the experiment, the cells cultured in both normoxic and hypoxic conditions were seeded (3 × 10 5 cells/well) in six-well plates for 24 h. After 24 h of culture in normal growth medium, the cells were treated with 40 µM N-allyl-BS or N-(3-mb)-BS for another 24 h. The medium and trypsinized cells were collected and centrifuged at 500× g for 5 min. Next, the cells were washed with 1 mL of PBS and then resuspended in 50 µL of fresh medium. In order to determine the viability, 15 µL of the cells were added to freshly prepared ViaCount (merck Millipore) solution (135 µL), according to manufacturer's instructions, in the final volume of 150 µL for each sample. The cells were analyzed while using Guava Easycyte HT Flow Cytometer.

Detection of Intracellular ROS
The generation of intracellular ROS was determined by flow cytometry with H2DCFDA. Prior to the experiment, cells (3 × 10 5 cells/well) were seeded in a 6-well plate, allowed to attach overnight, and then exposed to 40 µM N-allyl-BS or N-(3-mb)-BS for 2 h. Subsequently, the cells were stained with 10 µM H2DCFDA for 30 min. at 37 • C in a complete medium. Floating and attached cells were collected and centrifuged, washed twice with PBS, and then placed on ice. Immediately afterwards, cell fluorescence was analyzed using a BD LSR II flow cytometer. The fluorescence of unstained cells was also measured and subtracted as the background.

Comet Assay
The comet assay was performed to evaluate DNA damage. Briefly, the aliquots of treated and control cells (10,000 cells per sample) were transferred to 1.5-mL centrifugation tubes, and then centrifuged for 10 min. at 800× g at 4 • C. The supernatant was removed and the cells were resuspended in 0.7% low-melting agarose of which 0.035 mL was placed on pre-coated, high-throughput comet assay slides (Trevigen). These slides have a clean area separated by silicon barriers to allow simultaneous layering of ten different samples on each slide. The clean areas are manufactured with a dried agarose coating, in order to enhance adhesivity. The microgels on slides were allowed to solidify at 4 • C. Subsequently, the slides were immersed overnight at 4 • C in the dark in ice-cold, freshly prepared lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO, with pH adjusted to 10) to lyse the embedded cells and to allow DNA to unfold. After incubation in the lysis solution, the slides were placed in an alkaline buffer (1 mM Na2EDTA and 300 mM NaOH buffer, pH 7.4 [13] for 30 min. to allow DNA to unwind. Electrophoresis was then performed for 20 min. at 1 V/cm in the same buffer. After neutralization in a Tris buffer (pH 7.5) and dehydration in 75% methanol, DNA on each slide was stained with 0.015 mL of ethidium bromide (20 µg/mL) and then viewed under fluorescent light using an Olympus BX51 fluorescence microscope connected to a computer.

Analysis of the Comet Assay Data
For each sample, 15 randomly acquired images were recorded and processed using custom-made software. The Department of Life and Environmental Sciences developed the software in collaboration with the Engineering Department of the Polytechnic University of Marche [13]. It is based on the Labview programming platform (National Instruments), which enables the automatic identification of comets, thus greatly reducing operator-dependent variability. A key feature of the software is the ability to distinguish the comets from the background, and determine the commonly used DNA damage indices, including tail length, tail intensity, and tail moment. Comet-specific DNA damage indices and images of 150 nucleoids from each slide were fed to a Microsoft Access database. The data and images were then easily traceable for subsequent evaluation and statistical analysis. Three slides were analyzed for each treatment condition and a total of 450 comets were saved.

Statistical Analysis
The data were analyzed using GraphPad Prism 6. T-test, One-way ANOVA, followed by Dunnett's or Bonferroni's multiple comparison test were used to determine statistical significance of the differences in measured variables between the control and treated groups. Differences were considered to be significant at P < 0.001, P < 0.01, and P < 0.05.

N-Allyl-BS and N-(3-mb)-BS Inhibit the Viability of DU145 and PC-3 Prostate Cancer Cell
A series of N-alkyl-1,2-benzisoselenazol-3(2H)-ones were synthesized and their potential anticancer activity tested. Two compounds with the strongest anti-proliferative activity against prostate cancer cells were selected and further examined ( Figure 1) [7]. The anticancer capacity of the N-allyl-BS and N-(3-mb)-BS was studied while using two phenotypically different cancer cell lines: PTEN-positive (DU145) with normal Akt kinase protein levels and PTEN-negative (PC-3) with high Akt kinase activity. The cells were exposed for 24 h to increasing concentrations (2.5-40 µM) of the analyzed compounds and the cytotoxic effect of N-allyl-BS and N-(3-mb)-BS was evaluated using the SRB assay. The inhibition of cell proliferation was significant at all tested concentrations in both cancer cell lines. The highest anti-proliferative activity was observed after incubation in the presence of 40 µM, with the viability of DU145 and PC-3 cells reduced by 60 and 80%, respectively (Figure 2A-D). Further, the two-way Anova analysis indicates that the cytotoxicity of N-allyl-BS and N-(3-mb)-BS in the normal prostate cell line (PNT1A) was significantly lower when compared with the cancer cell lines, especially at 40 µM (p < 0.05) (not shown).
Pharmaceuticals 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/pharmaceuticals mL of ethidium bromide (20 μg/mL) and then viewed under fluorescent light using an Olympus BX51 fluorescence microscope connected to a computer.

Analysis of the Comet Assay Data
For each sample, 15 randomly acquired images were recorded and processed using custom-made software. The Department of Life and Environmental Sciences developed the software in collaboration with the Engineering Department of the Polytechnic University of Marche [13]. It is based on the Labview programming platform (National Instruments), which enables the automatic identification of comets, thus greatly reducing operator-dependent variability. A key feature of the software is the ability to distinguish the comets from the background, and determine the commonly used DNA damage indices, including tail length, tail intensity, and tail moment. Comet-specific DNA damage indices and images of 150 nucleoids from each slide were fed to a Microsoft Access database. The data and images were then easily traceable for subsequent evaluation and statistical analysis. Three slides were analyzed for each treatment condition and a total of 450 comets were saved.

Statistical Analysis
The data were analyzed using GraphPad Prism 6. T-test, One-way ANOVA, followed by Dunnett's or Bonferroni's multiple comparison test were used to determine statistical significance of the differences in measured variables between the control and treated groups. Differences were considered to be significant at P ˂ 0.001, P < 0.01, and P < 0.05.     Viability was determined using SRB assay. Data are presented as mean ± SE (n = 3); significance of variation was calculated using one-way ANOVA followed by Dunnett's multiple comparison test. (*P < 0.01) (**P < 0.001).

N-allyl-BS and N-(3-mb)-BS Induce G2/M Cell Cycle Arrest in Prostate Cancer Cells
Next, the inhibitory effect of N-allyl-BS on the cell cycle was determined while using flow cytometry. The experiment revealed that the 8h incubation of tumor cells with 40 μM N-allyl-BS and N- (3-  Viability was determined using SRB assay. Data are presented as mean ± SE (n = 3); significance of variation was calculated using one-way ANOVA followed by Dunnett's multiple comparison test. (* P < 0.01) (** P < 0.001).

N-allyl-BS and N-(3-mb)-BS Induce G2/M Cell Cycle Arrest in Prostate Cancer Cells
Next, the inhibitory effect of N-allyl-BS on the cell cycle was determined while using flow cytometry. The experiment revealed that the 8h incubation of tumor cells with 40 µM N-allyl-BS and N-(3-mb)-BS significantly increased the percentage of cells in the G2/M phase. This phenomenon was observed in both cancer cell lines tested ( Figure 3A-D). Therefore, N-allyl-BS and N-(3-mb)-BS induced G2/M cell cycle arrest. Moreover, the cytostatic effect on PC-3 cells was stronger than that exerted on DU145 cells ( Figure 3C,D).  Significance of variations compared with control was calculated using one-way ANOVA followed by Bonferroni's multiple comparison test. (*P < 0.01).

N-allyl-BS and N-(3-mb)-BS Induce Apoptosis and Necrosis of Cancer Cells
We  Significance of variations compared with control was calculated using one-way ANOVA followed by Bonferroni's multiple comparison test. (*P < 0.01).

N-allyl-BS and N-(3-mb)-BS Induce Apoptosis and Necrosis of Cancer Cells
We next investigated whether N-allyl-BS and N-(3-mb)-BS could induce cell death in prostate cancer cells since the DU145 and PC-3 cells differ with respect to the Akt kinase activity. Both cancer cell lines were treated for 24 h with 40 µM N-allyl-BS or N-(3-mb)-BS and then analyzed using flow cytometry. Indeed, N-allyl-BS induced apoptosis and necrosis in DU145 and PC-3 cells ( Figure 4A-D). The pro-apoptotic effect of N-allyl-BS was more pronounced in PC-3 cells than in DU145 cells ( Figure 4A,C The level of caspase-dependent cleaved PARP, an apoptosis marker and enzyme responsible for DNA repair, was evaluated in cells exposed to N-allyl-BS or N-(3-mb)-BS for different periods of time to confirm that N-allyl-BS and N-(3-mb)-BS indeed caused cell death via apoptosis. The highest level of cleaved PARP was observed after 4 h of treatment with 40 µM N-allyl-BS or N-(3-mb)-BS in PC-3 cells, and after 8 h of treatment in DU145 cells, as shown in (Figure 5A,B and Figure 6A,B). Interestingly, the level of PARP cleavage significantly dropped after 24 h treatment.

N-Allyl-BS and N-(3-mb)-BS Respectively Enhance ROS Generation in Cancer Cells
The intracellular level of ROS was determined to investigate whether oxidative stress was involved in the cytotoxic activity of N-allyl-BS and N- (3-

N-Allyl-BS and N-(3-mb)-BS Respectively Enhance ROS Generation in Cancer Cells
The intracellular level of ROS was determined to investigate whether oxidative stress was involved in the cytotoxic activity of N-allyl-BS and N-

N-allyl-BS Induces DNA Damage
The comet assay was performed on DU145 and PC-3 cell lines to elucidate whether the increased cell death and oxidative damage via elevated ROS generation was linked with DNA damage. The cells that were treated for 24h with 40 μM N-allyl-BS concentration efficiently induced DNA damage in PC-3 cells, as highlighted by a significant increase of the upper third (+33%) and fourth quartile (+88%) of tail intensity, a parameter indicating the percentage of DNA fluorescence in the tail of comet images proportional to the amount of damaged DNA, on the contrary in the same experimental conditions N-allyl-BS had no significant effects on the DU145 cells ( Figure 8A

N-allyl-BS Induces DNA Damage
The comet assay was performed on DU145 and PC-3 cell lines to elucidate whether the increased cell death and oxidative damage via elevated ROS generation was linked with DNA damage. The cells that were treated for 24 h with 40 µM N-allyl-BS concentration efficiently induced DNA damage in PC-3 cells, as highlighted by a significant increase of the upper third (+33%) and fourth quartile (+88%) of tail intensity, a parameter indicating the percentage of DNA fluorescence in the tail of comet images proportional to the amount of damaged DNA, on the contrary in the same experimental conditions N-allyl-BS had no significant effects on the DU145 cells ( Figure 8A

The Cytotoxic Activity of N-Allyl-BS and N-(3-mb)-BS are Similar under Hypoxic and Normoxic Conditions
Cells growing within a tumor are exposed to a much lower concentration of oxygen than cells growing in cell cultures (REF). Cytotoxicity experiments were performed in an atmosphere of 4 and 21% oxygen concentration to test whether different oxygen concentrations affected cell sensitivity to N-allyl-BS. The cells were treated for 24h with 40 μM N-allyl-BS and N-(3-mb)-BS under hypoxic and normoxic conditions. A similar fraction of apoptotic and necrotic cells was observed under the two conditions ( Figure 9A-D). Hence, a low oxygen concentration did not affect the cytotoxic activity of N-allyl-BS.

The Cytotoxic Activity of N-Allyl-BS and N-(3-mb)-BS are Similar under Hypoxic and Normoxic Conditions
Cells growing within a tumor are exposed to a much lower concentration of oxygen than cells growing in cell cultures (REF). Cytotoxicity experiments were performed in an atmosphere of 4 and 21% oxygen concentration to test whether different oxygen concentrations affected cell sensitivity to N-allyl-BS. The cells were treated for 24 h with 40 µM N-allyl-BS and N-(3-mb)-BS under hypoxic and normoxic conditions. A similar fraction of apoptotic and necrotic cells was observed under the two conditions ( Figure 9A-D). Hence, a low oxygen concentration did not affect the cytotoxic activity of N-allyl-BS.  presented as mean ± SE (n = 3); significant difference was calculated using one-way ANOVA followed by Dunnett's multiple comparison test (*P < 0.01).

N-Allyl-BS and N-(3-mb)-BS Reduce Akt Phosphorylation
Decreased Akt phosphorylation induces cell death in prostate cancer cells [14]. The cells were treated with 40 μM N-allyl-BS ( Figure 10) or N-(3-mb)-BS ( Figure 11) for different time periods and Akt phosphorylation was analyzed by western blotting to examine whether N-allyl-BS and N-(3-mb)-BS decreased the level of phosphorylated Akt (p-Akt). Indeed, Akt phosphorylation was slightly reduced after 1-h treatment and highly reduced after 4-h treatment, in the case of N-allyl-BS(10a,b), however N-(3-mb)-BS already slightly reduced the level of Akt phosphorylation after 30 min. of treatment. (Figure 11a,b). The changes in Akt phosphorylation were similar in the DU145 and PC-3 cells.

N-Allyl-BS and N-(3-mb)-BS Reduce Akt Phosphorylation
Decreased Akt phosphorylation induces cell death in prostate cancer cells [14]. The cells were treated with 40 µM N-allyl-BS ( Figure 10) or N-(3-mb)-BS ( Figure 11) for different time periods and Akt phosphorylation was analyzed by western blotting to examine whether N-allyl-BS and N-(3-mb)-BS decreased the level of phosphorylated Akt (p-Akt). Indeed, Akt phosphorylation was slightly reduced after 1-h treatment and highly reduced after 4-h treatment, in the case of N-allyl-BS (Figure 10a,b), however N-(3-mb)-BS already slightly reduced the level of Akt phosphorylation after 30 min. of treatment (Figure 11a,b). The changes in Akt phosphorylation were similar in the DU145 and PC-3 cells.   Results are presented as mean ± SE (n = 3). The statistical significance of differences between respective samples was determined by one-way ANOVA followed by Bonferroni's multiple comparison test, where (a) indicates significant difference between control and treated cells P < 0.01    Results are presented as mean ± SE (n = 3). The statistical significance of differences between respective samples was determined by one-way ANOVA followed by Bonferroni's multiple comparison test, where (a) indicates significant difference between control and treated cells P < 0.01

Discussion
In the current study, we investigated the cytotoxic and antitumor potential of N-allyl-BS and N-(3-mb)-BS against two phenotypically different cancer cell lines, the PTEN-positive DU145 cells and PTEN-negative PC-3 cells, and against a healthy epithelial cell line PNT1A. We have previously shown that N-allyl-BS, which is one of recently synthesized OSCs, exerts a strong antiproliferative effect on prostate and breast cancer cells [7,8]. In the present study, we confirmed these observations by studying the cytotoxic effect of N-allyl-BS in prostate cancer cells. Here, we demonstrated that both tested tumor cell lines were sensitive to the analyzed compounds. In addition, the DU 145 and PC-3 cells were more sensitive to N-allyl-BS and N-(3-m)-b-BS than the non-tumor cell line PNT1A. As mentioned above, the PC-3 cells are PTEN-negative and it has been demonstrated that the growth of cancer cells depends mainly on the activation of the Akt pathway [15]. Hence, we hypothesized that some of the N-allyl-BS and N-(3-mb)-BS anticancer activities (apoptosis induction and/or cell-cycle arrest) could be associated with the inhibition of Akt kinase. In fact, the presented data confirmed that the levels of the active form of Akt (p-Akt) were significantly reduced in the DU145 and PC-3 cells treated with both compounds. This suggested that the higher sensitivity of PC-3 than DU145 cells to N-allyl-BS was associated with Akt inactivation. Furthermore, PC-3 cells accumulated significant DNA damage upon N-allyl-BS exposure, as determined by the comet assay, while the changes in DU145 cells did not reach statistical significance. Interestingly, N-(3-mb)-BS did not induce DNA damage in both cancer cell lines, which indicates the different molecular mechanism of cell death. As above, this suggested that N-allyl-BS induced genotoxic stress in PC-3 cells. As ROS can potentiate DNA damage, the effect of the studied compounds on ROS production has been evaluated. The data clearly shows that both OSCSs induce ROS formation, in both cancer cell lines. Therefore, the differences in the extent of DNA damage could not be explained by changes in ROS production. Interestingly, the hyperactivity of Akt might also affect DNA repair. It has been demonstrated that PTEN-deficient HCT116 cells that exhibit high Akt activity fail to repair DNA as efficiently as the wild type after irradiation [16]. In PTEN-deficient cells, the DNA damage response complex MRE11 is highly unstable, and this might explain impaired DNA repair in these cells [16]. Consequently, it can be concluded that N-allyl-BS induced genotoxic stress; however, that was only apparent in PC-3 cells, in which DNA damage repair could be impaired. However, this was not confirmed with PC-3 cells that were treated with N-(3-mb)-BS, since no significant DNA damage was observed. Most of the data obtained on cell culture, including our own, are performed in 21% oxygen, while the tissue oxygen level is between 3 to 8% and there are some evidences that the high oxygen concentration routinely used in cell culture might lead to altered cellular pathways in vitro [17,18]. Hence, another goal of the current study was to compare the cytotoxicity of N-allyl-BS and N-(3-mb)-BS towards prostate cells that were cultured under 21% and 4% oxygen. We hypothesized that the cytotoxic activity of both organoselenium compounds would be lower under 21% oxygen (because of adaptation to greater free radical formation under such conditions) than under 4% oxygen [19,20]. Nevertheless, this assumption was not confirmed, as the cytotoxicity of N-allyl-BS and N-(3-mb)-BS was the same under 21% oxygen and 4% oxygen. Previously, we demonstrated that, in vitro, N-allyl-BS exhibits higher antioxidant activity than ebselen in vitro [7]. Conversely, in the current study, we observed that N-allyl-BS induced cell death via apoptosis and enhanced ROS formation. However, the cellular sources and mechanisms underpinning the enhanced ROS generation are unknown. Santofimia-Castano and co-workers [21] reported that ebselen induced apoptosis in pancreatic AR42J cancer cells by increasing mitochondrial ROS production. These data are in agreement with previous studies that showed that ebselen and selenadiazole, which are well-defined antioxidants, induce ROS production in cancer cells [22,23]. The use of apoptosis-and necrosis-inducing agents is being increasingly as a therapeutic approach in cancer treatment, because the induction of these processes would result in a pronounced reduction of tumor mass. In the current study, we showed that N-allyl-BS and N-(3-mb)-BS induce apoptotic and necrotic cell death, which was confirmed by flow cytometry and western blot analysis of cleaved PARP levels. PARP is an abundant enzyme that is present in all somatic cells. It detects and signals DNA damage to the cellular repair mechanisms. It is well established that PARP cleavage, as catalyzed by caspase 3, occurs during programmed cell death that is induced by a variety of apoptotic stimuli [24]. In the current study, western blot analysis of an N-allyl-BS and N-(3-mb)-BS-induced PARP cleavage in prostate cancer cells revealed the extensive degradation of PARP after 4-h and 8-h exposure of both cancer cell lines to this compound. These results are in agreement with data showing that ebselen promoted apoptosis in cancer cells by an intrinsic pathway [22]. However, the level of cleaved PARP dramatically decreased after 24-h exposure of PC-3 and DU145 cells to N-allyl-BS and N-(3-mb)-BS. This phenomenon might be a consequence of necrosis induction. It has been proven that depletion of intracellular ATP levels switches the energy-requiring apoptotic cell death to necrosis [25]. It has been also reported that, during necrosis, further PARP cleavage occurs, with small PARP fragments (Mr 50,000, 40,000, and 35,000) being detected [26]. Although, necrosis, as an uncontrolled modality of cell death, is generally associated with damage to peripheral tissues and increased systemic inflammation; recent observations highlight a positive role of necrosis induction during cancer therapy. In fact, HMGB1 (biochemical marker of necrosis) might be capable of initiating antitumor immunity. Moreover, in vivo studies suggest that immunization with HMGB1 can enhance antitumor immunity against poorly immunogenic apoptotic tumors [27].

Conclusions
In conclusion, the presented data demonstrate that N-allyl-BS and N-(3-mb)-BS are active cytotoxic compounds that can inhibit the growth of prostate cancer cells. The cytotoxicity mechanism is different in DU145 and PC-3 cells. Both of the compounds induce oxidative stress, however DNA damage was only observed in PC-3 after treatment with N-allyl-BS. Interestingly, partial oxygen pressure had no effect on the cytotoxic activity of the studied compounds.