Evaluation of the Anticancer Activities of Novel Transition Metal Complexes with Berenil and Nitroimidazole

Novel transition metal complexes (Au, Pd, Pt) with berenil and 2-(1-methyl-5-nitroimidazol-2-yl)ethanol were obtained through two-step synthesis. The cytotoxicity assay against MCF-7 and MDA-MB-231 breast cancer cells revealed that novel platinum and palladium complexes cause a reduction on the viability of MCF-7 and MDA-MB-231 breast cancer cells to a greater extent than cisplatin. The complexes showed lower cytotoxicity on normal MCF-10A human breast epithelial cells than on tumor cells. Furthermore, we observed that these complexes selectively concentrate in tumor cell mitochondria due to the characteristic for these cells increased membrane potential that may explain their increased proapoptotic activity. The activity of the synthesized compounds against topoisomerase type IIα and their increased impact on DNA defragmentation also were documented. The novel complexes also induced autophagosome changes and inhibited tumor growth in xenograft models (established using breast cancer cells).


Introduction
Despite the development of thousands of anticancer structures, cancer is still a major cause of death worldwide [1]. The main problem in the treatment of cancer is drug resistance from long-term treatment and the adverse side effects of the drugs along with non-specific toxicity [1,2]. In addition, the reported new anticancer compounds usually suffer from low stability under physiological conditions, interfering with delivery to the tumor tissues and often resulting in toxic side effects [3]. Therefore, there is a constant search for new compounds with potential anticancer effects that at the same time will be lack from these disadvantages.
Metal complexes offer unique characteristics and exceptional versatility, with the ability to alter their pharmacology through facile modifications of geometry and coordination number. This has prompted the search for metal-based complexes with distinctly different structural motifs and non-covalent modes of binding with a primary aim of circumventing current clinical limitations [4].

Biological Activity of Novel Series of Transition Metal Complexes with Nitroimidazole and Berenil (AuMet-1, PdMet-1, PtMet-1)
In the first stage, the effect of a series of transition metal (Au, Pd, Pt) compounds with nitroimidazole and berenil moiety on the viability of breast cancer cells MCF-7, MDA-MB-231 and non-tumorigenic epithelial breast cell line MCF-10A was analyzed using an MTT assay ( Figure 2). The IC50 values for the reference drug (cisplatin) were 93 µM in MCF-7 cells, 87 µM in MDA-MB-231 cells and >100 µM in MCF-10A cells after 24 h of incubation. It was shown that the AuMet-1 compound was less cytotoxic than cisplatin in all cell lines (IC50 > 100 µM). In the case of the PdMet-1 and PtMet-1 compounds in MCF-7 cells, the IC50 values were 40 µM for PdMet-1 and 73 µM for PtMet-1, while in MDA-MB-231 cells 49 µM for PdMet-1 and 68 µM for PtMet-1 after 24 h of incubation. In MCF-10A cells, the obtained IC50 values were 64 µM for PdMet-1 and 89 µM for PtMet-1. The compound that exhibited the highest cytotoxic properties was PdMet-1.

Novel Series of Transition Metal Compounds with Nitroimidazole and Berenil Moiety (AuMet-1, PdMet-1, PtMet-1) Induce Apoptosis by Decreasing Mitochondrial Membrane Potential
Dual annexin V and iodium propidium staining with flow cytometry were used to assess the apoptosis status of breast cancer MCF-7 and MDA-MB-231 cells after 24 h of treatment with a novel series of tested compounds (AuMet-1, PdMet-1, PtMet-1) and cisplatin ( Figure 3). Annexin V served to identify cells in the early stages of apoptosis, because it detects the externalized phosphatidylserin (PS) on the cell membrane (characteristic change during apoptosis), whereas iodium propidium (PI) identified late apoptotic and dead cells. PI stains cells with a disrupted cell membrane. Live cells don't bind to annexin V and PI (annexin-V − /PI − ), early apoptotic cells bind to annexin V but not PI (annexin-V + /PI − ), late apoptotic cells bind to both molecules (annexin-V + /PI + ), and death cells only bind to PI (annexin-V − /PI + ).

Novel Series of Transition Metal Compounds with Nitroimidazole and Berenil Moiety (AuMet-1, PdMet-1, PtMet-1) Induce Apoptosis by Decreasing Mitochondrial Membrane Potential
Dual annexin V and iodium propidium staining with flow cytometry were used to assess the apoptosis status of breast cancer MCF-7 and MDA-MB-231 cells after 24 h of treatment with a novel series of tested compounds (AuMet-1, PdMet-1, PtMet-1) and cisplatin ( Figure 3). Annexin V served to identify cells in the early stages of apoptosis, because it detects the externalized phosphatidylserin (PS) on the cell membrane (characteristic change during apoptosis), whereas iodium propidium (PI) identified late apoptotic and dead cells. PI stains cells with a disrupted cell membrane. Live cells don't bind to annexin V and PI (annexin-V − /PI − ), early apoptotic cells bind to annexin V but not PI (annexin-V + /PI − ), late apoptotic cells bind to both molecules (annexin-V + /PI + ), and death cells only bind to PI (annexin-V − /PI + ).  Following 24 h of incubation with the tested compounds (concentration of 50 µM), we observed that all of them significantly induced apoptosis in MCF-7 and MDA-MB-231 cells compared with the control. In the control cells, there were 8.9 ± 2.4% apoptotic cells * and 1.3 ± 0.7% necrotic cells in the population of MCF-7 cells and 6.4 ± 1.6% apoptotic cells * and 1 ± 0.3% necrotic cells in the MDA-MB-231 cells. Following 24 h of incubation with the tested compounds (concentration of 50 µM), we observed that all of them significantly induced apoptosis in MCF-7 and MDA-MB-231 cells compared with the control. In the control cells, there were 8.9 ± 2.4% apoptotic cells* and 1.3 ± 0.7% necrotic cells in the population of MCF-7 cells and 6.4 ± 1.6% apoptotic cells* and 1 ± 0.3% necrotic cells in the MDA-MB-231 cells.
As one of the earliest changes in the apoptotic process, a decrease in the mitochondrial membrane potential (MMP) is observed. Apoptosis, which proceeds through the mitochondrial pathway, shows an increase in the permeability of the internal and external mitochondrial membrane, which is associated with changes in the transmembrane mitochondrial potential (∆Ψm) [12]. The change of ∆Ψm was determined using lipophilic fluorochrome JC-1 and flow cytometry analysis. JC-1 occurs in two forms, monomers and aggregates, which emit different fluorescence color. Fluorochrome emits green fluorescence in healthy cells (monomers) and red fluorescence in cells with disturbed mitochondrial potential (aggregates).
As shown in Figure 4, a 24-h incubation with the tested compounds (concentration of 50 µM) caused an increase in the proportion of breast cancer cells MCF-7 and MDA-MB-231 with depolarized mitochondria. In the control group (untreated cells), the MMP decrease was 9.0 ± 0.8% in MCF-7 and 7.3 ± 1.7% in MDA-MD-231 cells, respectively. The highest MMP decrease was observed in PdMet-1 and it was 67.1 ± 3.5% in MCF-7 cells and 63.8 ± 2.7% in MDA-MB-231 cells. The MMP decrease induced by the remaining compounds was higher than the reference compound-cisplatin (MMP decrease: 13.2 ± 2.8% in MCF-7 and 10.9 ± 2.5% in MDA-MB-231). It was proved that the tested compounds had a greater effect on programmed cell death measured by annexin V binding and an MMP decrease than cisplatin. The mitochondrial membrane potential results are consistent with those obtained in the annexin V/iodium propidium test and show that programmed cell death induced by the novel series of transition metal (AuMet-1, PdMet-1, PtMet-1) compounds may go through the mitochondrial pathway.

AuMet-1, PdMet-1 and PtMet-1 Induce Autophagy
Autophagy is one of the most important processes to maintain internal cell homeostasis. In healthy cells, it occurs at a low level because its role is to prevent the accumulation of damaged cell organelles, but its level increases during starvation or stress [13]. Autophagy Probe, Red staining with flow cytometry was used to assess the state of autophagy of breast cancer cells MCF-7 and MDA-MB-231 after 24 h of exposure to the tested compounds in a concentration of 50 µM ( Figure 5). Autophagic Probe, Red identifies autophages and autolysosomes by introducing molecules into the lipid membranes of these structures and then bright fluorescence. and it was 67.1 ± 3.5% in MCF-7 cells and 63.8 ± 2.7% in MDA-MB-231 cells. The MMP decrease induced by the remaining compounds was higher than the reference compound-cisplatin (MMP decrease: 13.2 ± 2.8% in MCF-7 and 10.9 ± 2.5% in MDA-MB-231). It was proved that the tested compounds had a greater effect on programmed cell death measured by annexin V binding and an MMP decrease than cisplatin. The mitochondrial membrane potential results are consistent with those obtained in the annexin V/iodium propidium test and show that programmed cell death induced by the novel series of transition metal (AuMet-1, PdMet-1, PtMet-1) compounds may go through the mitochondrial pathway.   Autophagy is one of the most important processes to maintain internal cell homeostasis. In healthy cells, it occurs at a low level because its role is to prevent the accumulation of damaged cell organelles, but its level increases during starvation or stress [13]. Autophagy Probe, Red staining with flow cytometry was used to assess the state of autophagy of breast cancer cells MCF-7 and MDA-MB-231 after 24 h of exposure to the tested compounds in a concentration of 50 µM ( Figure 5). Autophagic Probe, Red identifies autophages and autolysosomes by introducing molecules into the lipid membranes of these structures and then bright fluorescence.
After 24 h of incubation, we observed that all the tested compounds induced autophagy in MCF-7 and MDA-MB-231 cells with a different intensity compared to the control (untreated cells). There was 94.2 ± 2.8% of non-autophagic cells and 5.3 ± 3.0% of autophagic cells in the control population of MCF-7 cells and 94.6 ± 1.9% of non-autophagic cells and 5.3 ± 1.9% of autophagic cells in the MDA-MB-231 cells. The highest activation of autophagy on both cell lines after 24-h incubation was exhibited by PdMet-1, where we observed 30.9 ± 3.5% of autophagic cells in MCF-7 and 30.4 ± 3.1% in MDA-MB-231 cells. An insignificant intensity of autophagy was observed in cisplatin (9.4 ± 2.8% of autophagic cells in MCF-7 and 9.9 ± 2.4% in MDA-MB-231). In the case of the AuMet-1 compound, the activation of autophagy was higher than in cisplatin: 12.7 ± 2.0% of autophagic cells in MCF-7 and 13.6 ± 2.4% in MDA-MB-231; but more than two-fold lower than in PdMet-1 and PtMet-1: 30.9 ± 3.5% and 28.1 ± 3.3% in MCF-7, 30.4 ± 3.1% and 29.0 ± 2.3% in MDA-MB-231, respectively. Autophagy allows to maintain homeostasis in the cell, thus avoiding its death. If cellular stress is not overcome, which leads to a homeostasis disorder, the same signaling ultimately leads to the activation of programmed cell death [14]. Thanks to this knowledge we can conclude that compounds PdMet-1 and PtMet-1 cause large stress in cells, which leads to the disruption of their homeostasis and directing the cells to apoptosis, which is proved in the annexin-V/PI assay. After 24 h of incubation, we observed that all the tested compounds induced autophagy in MCF-7 and MDA-MB-231 cells with a different intensity compared to the control (untreated cells). There was 94.2 ± 2.8% of non-autophagic cells and 5.3 ± 3.0% of autophagic cells in the control population of MCF-7 cells and 94.6 ± 1.9% of non-autophagic cells and 5.3 ± 1.9% of autophagic cells in the MDA-MB-231 cells. The highest activation of autophagy on both cell lines after 24-h incubation was exhibited by PdMet-1, where we observed 30.9 ± 3.5% of autophagic cells in MCF-7 and 30.4 ± 3.1% in MDA-MB-231 cells. An insignificant intensity of autophagy was observed in cisplatin (9.4 ± 2.8% of autophagic cells in MCF-7 and 9.9 ± 2.4% in MDA-MB-231). In the case of the AuMet-1 compound, the activation of autophagy was higher than in cisplatin: 12.7 ± 2.0% of autophagic cells in MCF-7 and 13.6 ± 2.4% in MDA-MB-231; but more than two-fold lower than in PdMet-1 and PtMet-1: 30.9 ± 3.5% and 28.1 ± 3.3% in MCF-7, 30.4 ± 3.1% and 29.0 ± 2.3% in MDA-MB-231, respectively.
Autophagy allows to maintain homeostasis in the cell, thus avoiding its death. If cellular stress is not overcome, which leads to a homeostasis disorder, the same signaling ultimately leads to the activation of programmed cell death [14]. Thanks to this knowledge we can conclude that compounds PdMet-1 and PtMet-1 cause large stress in cells, which leads to the disruption of their homeostasis and directing the cells to apoptosis, which is proved in the annexin-V/PI assay.

AuMet-1, PdMet-1, and PtMet-1 Increase Topoisomerase IIα Activity
Topoisomerase IIα is an enzyme responsible for the correct process of replication, transcription and recombination. It takes part, among others, in chromosome condensation or separation of chromatids. Scientific research has proved that the overexpression of this enzyme generates double-strand breaks of DNA (DSB) induced by topoisomerase IIα, directing cells to the apoptosis pathway [15]. The activity of topoisomerase IIα was measured using anti-topoisomerase IIα antibody conjugated with phycoeritin and flow cytometer analysis of breast cancer cells MCF-7 and MDA-MB-231 after 24 h of treatment with AuMet-1, PdMet-1, and PtMet-1 ( Figure 6). All of them in a concentration of 50 µM. The antibody was binding with topoisomerase IIα in cancer cells and brightly fluoresced. It was observed that all the tested compounds induced topoisomerase IIα activity in MCF-7 and MDA-MB-231 cells with a different intensity compared to the control (untreated cells). In the control group, there were 94.2 ± 2.0% in MCF-7 and 95.3 ± 2.8% in MDA-MB-231 cells without antibody and 5.7 ± 1.2% in MCF-7 and 4.5 ± 2.5% in MDA-MB-231 cells with anti-topoisomerase IIα antibody. The highest activity of topoisomerase IIα on both cell lines after 24-h incubation was exhibited by PdMet-1, where we observed 77.2 ± 2.9% of cells without antibody and 22.7 ± 2.2% with antibody in MCF-7 and 76.5 ± 2.0% of cells without antibody and 23.3% ± 2.1 with antibody in MDA-MB-231. The compound exhibiting the lowest activation of topoisomerase IIα was AuMet-1; after 24-h incubation, there was 85.6 ± 2.2% of cells without antibody and 14.0 ± 2.1% of cells with anti-topoisomerase IIα antibody in MCF-7 and 84.3 ± 2.1% and 15.5 ± 2.1% in MDA-MB-231. The PtMet-1 compound induced topoisomerase IIα activity insignificantly more than the AuMet-1 compound and was less effective than PdMet-1: PtMet-1: 81.0 ± 2.8% in MCF-7 and 81.5 ± 2.5% in MDA-MB-231 cells without antibody and 18.8 ± 2.9% in MCF-7 and 18.3 ± 2.5% in MDA-MB-231 cells with antibody. These results are consistent with those obtained in the annexin V/iodium propidium and MMP tests and show that programmed cell death induced by PdMet-1 may also be related to increased topoisomerase IIα activity.
2.6. AuMet-1, PdMet-1 and PtMet-1 Promote DNA Fragmentation DNA damage can be the result of external factors such as ionizing and ultraviolet radiation, cytotoxic or internal factors, which are: reactive oxygen species and DNA polymerase errors during replication [16]. The above factors may lead to single-and double-strand DNA breaks, which are identified by the TUNEL assay. This test uses the incorporation of FITC-labelled nucleotides by using an exogenous terminal deoxynucleotide transferase (TdT). The FITC-labelled nucleotides incorporated into DNA emit green fluorescence. As shown in Figure 7 topoisomerase IIα antibody in MCF-7 and 84.3 ± 2.1% and 15.5 ± 2.1% in MDA-MB-231. The PtMet-1 compound induced topoisomerase IIα activity insignificantly more than the AuMet-1 compound and was less effective than PdMet-1: PtMet-1: 81.0 ± 2.8% in MCF-7 and 81.5 ± 2.5% in MDA-MB-231 cells without antibody and 18.8 ± 2.9% in MCF-7 and 18.3 ± 2.5% in MDA-MB-231 cells with antibody. These results are consistent with those obtained in the annexin V/iodium propidium and MMP tests and show that programmed cell death induced by PdMet-1 may also be related to increased topoisomerase IIα activity.

AuMet-1, PdMet-1 and PtMet-1 Promote DNA Fragmentation
DNA damage can be the result of external factors such as ionizing and ultraviolet radiation, cytotoxic or internal factors, which are: reactive oxygen species and DNA polymerase errors during replication [16]. The above factors may lead to single-and double-strand DNA breaks, which are identified by the TUNEL assay. This test uses the incorporation of FITC-labelled nucleotides by using an exogenous terminal deoxynucleotide transferase (TdT). The FITC-labelled nucleotides incorporated into DNA emit green fluorescence. As shown in Figure 7   In the untreated cells (control), the TUNEL positive cells was 4.4 ± 2.1% in MCF-7 and 3.8 ± 1.9% in MDA-MB-231, respectively. In case of PdMet-1 we detected the highest percentage of TUNEL positive cells − it was 42.5 ± 2.5% in MCF-7 and 39.2 ± 3.5% in MDA-MB-231, respectively. AuMet-1 and PtMet-1 showed a lower percentage of TUNEL positive cells compared with PdMet-1 in both cell lines: 24.5 ± 2.7% (MCF-7) and 22.1 ± 2.5% (MDA-MB-231), 30.6 ± 3.6% (MCF-7) and 30.2 ± 2.5% (MDA-MB-231), respectively. We found that all the tested compounds caused DNA strand breaks to a greater extent than the reference compound (cisplatin).

Discussion
Despite the fact that currently cisplatin has a leading, well-established position among drugs used in the treatment of cancer, its use is limited by the toxic side effects. Therefore much research aims at the synthesis of new complexes directed at different targets. The presence of a less labile ligand in the structure of new generation platinum drugs may contribute to the lower toxicity of these compounds, especially neuro-and nephron-toxicity, while maintaining the same, and in some cases greater, antitumor activity [17,18]. The compounds synthesized by our team seem to confirm this thesis.
In our research, we conducted studies on the anti-tumor activity of three transition metal (Au, Pd, Pt) complexes with nitroimidazole ligand. The cytotoxicity assay against breast cancer cells MCF-

Discussion
Despite the fact that currently cisplatin has a leading, well-established position among drugs used in the treatment of cancer, its use is limited by the toxic side effects. Therefore much research aims at the synthesis of new complexes directed at different targets. The presence of a less labile ligand in the structure of new generation platinum drugs may contribute to the lower toxicity of these compounds, especially neuro-and nephron-toxicity, while maintaining the same, and in some cases greater, antitumor activity [17,18]. The compounds synthesized by our team seem to confirm this thesis.
In our research, we conducted studies on the anti-tumor activity of three transition metal (Au, Pd, Pt) complexes with nitroimidazole ligand. The cytotoxicity assay against breast cancer cells MCF-7 and MDA-MB-231 revealed that the novel compounds were more effective than the hitherto used agent, cisplatin. At the same time, what is worth emphasizing is that the tested compounds showed lower cytotoxicity on normal human breast epithelial cells MCF-10A than on tumor cells. Moreover, the fact that they have high cytotoxic activity at lower levels than cisplatin is important. This may suggest the high activity of the nitroimidazole ligand present in the complex. Our research confirms the observation of earlier research teams that suggested that nitroimidazoles may have antiproliferation activity in an appropriate chemical system [19]. In addition, we observed that this activity varies depending on the current central atom present in the structure. The cytotoxic activity of these compounds with a different central atom increased in the following order: PdMet-1 > PtMet-1 > AuMet-1.
The anticancer action of PdMet-1 and PtMet-1 was also investigated in a zebrafish embryo xenograft model. The xenograft experiment using MCF-7 and MDA-MB-231 human breast cancer cells grafted into the zebrafish yolk showed the significant antitumor effect of these compounds compared with the control group. According to the research carried out by Rawera et al. [20], the higher anti-tumor activity of compounds with these central atoms may be due to their greater intracellular penetration capacity. These authors noted that platinum(II) complexes with imidazole ligands have the ability to decrease the side effects and regulate the hydrophilicity/hydrophobicity ratio, which makes them easy to penetrate into biofilms, thus leading to a better curative effect.
The search for new drugs based on cisplatin is mainly associated with the mechanisms of cellular resistance to this medicine. The resistance of cancer cells to platinum compounds is a process that probably consists of many factors. First of all, the mechanisms of intracellular resistance, which include: increased activity of DNA repair processes and increased tolerance to DNA deformations, should be considered [21]. The key enzymes involved in DNA repair are topoisomerases. They perform their function by introducing transient protein-bridged DNA breaks on one (topoisomerase I) or both DNA strands (topoisomerase II). They have been recognized as a valuable and particularly intriguing target for chemotherapeutics because of their critical roles in cell progression, apoptosis, transcription, and other cellular regulation. Topoisomerases targeting agents could not only stabilize the cleavable complex formed between the enzyme and DNA, but also control the replication and transcription of DNA in malignant tumor cells [22].
Our study using topoisomerase IIα antibody showed that the tested compounds induced excessive expression of this enzyme. The imidazole ring may be responsible for this activity. Its unique structural feature with desirable electron rich characteristic is beneficial, because it enables to readily bind with a variety of enzymes and receptors in biological systems through diverse weak interactions, thereby exhibiting broad bioactivities [22]. Our thesis seems to be confirmed by the research of Shinohar at al., who reported that imidazole polyamides could competitively inhibit nuclear factor-Y (NF-Y) binding to the ICB2 site in the topoisomerase IIα promoter region and cause the upregulation of this enzyme [23]. Topoisomerase IIα increased activity could potentially lead to DNA fragmentation resulting in cell death by apoptosis [24]. The induction of apoptosis as result of DNA damage in precancerous lesions can remove potentially harmful cells, thereby blocking tumor growth [25]. We observed inhibited tumor growth as well as increased induction of apoptosis and DNA defragmentation under the influence of the tested compounds in our research. While examining the effect of the tested compounds on the process of apoptosis, we noticed significant accumulation of apoptotic breast cancer cells with externalized phosphatidylserine after incubation with the tested compounds. To explore the cellular mechanism underlying the synthetized compounds inducing apoptosis, we examined the alterations of the mitochondrial transmembrane potential (MMP) by using flow cytometry analysis. We found that all of the new derivatives increased the number of cells with decreased levels of MMP compared with the control cells in both lines. In cancer, the apoptotic pathway is typically inhibited through a wide variety of means including overexpression of antiapoptotic proteins and underexpression of proapoptotic proteins. Many of these changes cause intrinsic resistance to the most common anticancer therapy, chemotherapy. Therefore compounds that exhibit anticancer activity through activating the apoptotic pathway are the promising new anticancer therapies [26].
Researchers have already confirmed that novel compounds with potential anticancer activity based on cisplatin exert antitumor effects by inducing autophagy [27]. Moreover some effective anticancer agents, such as rapamycin and nilotinib, also repress cancer cell growth through the induction of autophagy [28,29]. Autophagy exhibits either a protumorigenic or antitumorigenic function, depending on the cell type, developmental stage of cancer, and stimulator [14]. Previously, Chen K. et al. demonstrated that stimulation of autophagy could be a potential strategy for the treatment of breast cancer [30]. In this study, we showed that autophagy induced by the tested compounds had an anticancer effect against breast cancer. We found that the induction of autophagy was involved in the cytotoxicity of a novel series of transition metal compounds. Considering the cross link between autophagy and apoptosis, we assume that the autophagy pathway could be involved in cytotoxicity and ultimately tumor reduction by the tested compounds.

Physical Measurements
The structure of a synthesized compound was confirmed by 1 H-NMR (200 MHz) and 13 C-NMR (50 MHz) spectra recorded on an AC 200F instrument (Bruker Hamburg, Germany) in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were expressed as a δ value (ppm). The multiplicity of resonance peaks was indicated as a singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Infrared spectra were recorded on a Spectrum 100 FT-IR spectrometer (Perkin-Elmer, Houston, TX, USA) as KBr pellets (4000-450 cm −1 ). Mass spectra were recorded using a Mariner mass spectrometer (company, Waltham, MA, USA). Melting points were determined on a 535 melting-point apparatus (Buchi, Essen, Germany), and are uncorrected. Elemental analysis of C, H, and N was performed on a Perkin-Elmer 240 analyzer and satisfactory results within ± 0.4% of the calculated values were obtained. KAuCl 4 (1.00 mmol) was dissolved in deionized water (30 mL). Then, 2-(1-methyl-5-nitroimidazol-2-yl)ethanol (2.00 mmol) was added to the reaction mixture and gradual dissolution of the substrate was observed. Stirring was continued for 24 h and the newly formed precipitate was then collected by filtration. This compound (Au(2-(1-methyl-5-nitroimidazol-2-yl)ethanol) 2 Cl 2 ) was filtered, then washed with 20 mL deionized water and dried under vacuum. Au(2-(1-methyl-5-nitroimidazol-2yl)ethanol) 2 Cl 2 (0.62 mmol) was suspended in an aqueous solution of silver nitrate (AgNO 3 , 5 mL, 1.25 mmol). The reaction mixture was stirred for 24 h at room temperature in the dark. The AgCl precipitate was filtered off. Berenil (0.62 mmol) and a solution of 10% NaCl (5 mL) were added to the filtrate and stirred for 24 h until a precipitate of the final gold complex formed. Afterwards, the product was filtered off and washed with a small amount of diluted HCl, deionized water, and ethyl ether, and dried under vacuum. Yield   (30 mL). KI (7.2 mmol) was added and the reaction mixture was stirred for 30 min. Then, 2-(1-methyl-5-nitroimidazol-2-yl)ethanol (2.00 mmol) was added to the reaction mixture and gradual dissolution of the substrate was observed. Stirring was continued for 24 h and the newly formed precipitate was then collected by filtration. This compound [Pt(2-(1-methyl-5-nitroimidazol-2-yl)ethanol) 2 I 2 ] was filtered, then washed with deionized water (20 mL) and dried under vacuum. Pt(2-(1-methyl-5-nitroimidazol-2-yl)ethanol) 2 I 2 (0.57 mmol) was suspended in an aqueous solution of silver nitrate (AgNO 3 , 5 mL, 1.14 mmol). The reaction mixture was stirred for 24 h at room temperature in the dark. The AgI precipitate was filtered off. Berenil (0.57 mmol) and a solution of 10% NaCl (5 mL) were added to the filtrate and stirred for 24 h until a precipitate of the final platinum complex formed. Afterwards, the product was filtered off and washed with a small amount of diluted HCl, deionized water, methanol, and ethyl ether, and dried under vacuum. Yield: 76.5% (389.1 mg); yellow powder; mp 184-186 • C; 1 H-NMR (DMSO-d 6    . All medias were complemented by 10% of fetal bovine serum (FBS) and 1% of antibiotics: penicillin/streptomycin (both Gibco). The cells were maintained in an incubator that provides optimal growth conditions for the cell culture: 5% CO 2 , 37 • C, and humidity in a range of 90−95%. The cells were cultured in 100 mm plates (Sarstedt, Newton, NC, USA). Subsequently after obtaining a subconfluent cell culture, the cells were detached with 0.05% trypsin and 0.02% EDTA phosphate buffered saline without calcium and magnesium (Gibco). Then, utilizing a hemocytometer, the number of cells was quantified and seeded at a density of 5 x 10 5 cells per well in six-well plates ("Nunc") in 2 mL of the growth medium (Dulbecco's Modified Eagle Medium or Mammary Epithelial Cell Growth Medium, respectively). In the present study, cells that obtained 80% of confluence were used.

Cell Viability Assay
The vitality of the cultured cells was investigated using enzymatic reduction of the tetrazolinum dye MTT to formazan. Basically, seed MDA-MB-231, MCF-7, and MCF-10A cells lines at an initial concentration of 1 x 10 5 cells/well were incubated at 37 • C, 5% CO 2 for 24 h. Then, the cultured cells were treated with a medium containing various amounts of AuMet-1, PdMet-1, and PtMet-1 (5, 10, 25, 50, and 100 µM) and incubated at 37 • C, 5% CO 2 for 24 h. After the incubation period, MTT was added (final concentration 0.5 mg/mL) to each well and the plate was incubated at 37 • C, 5% CO 2 for 4 h. Next, the medium was removed and 200 µL of DMSO was added into each well to dissolve the formazan (0.5% DMSO). The absorbance of the formazan solution was measured at a wavelength of 570 nm (reference -630 nm) in an Evolution 201 reader (Thermo Scientific, Waltham, MA, USA).

Flow Cytometry Assessment of Annexin V Binding
To determine the mechanism of cell death induced by AuMet-1, PdMet-1, and PtMet-1, the exposure of phosphatidylserine was assessed by annexin V-FITC (phospholipid-binding protein) using the FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. A flow cytometer (BD FACSCanto II flow cytometer; BD Biosciences Systems, San Jose, CA, USA) was used to analyze the tested cells (10,000 events measured). Phosphatidylserine was bound with high affinity by Annexin V and thus identification of cells at all stages of apoptosis could be performed using this protein. Propidine iodide (PI) was used to identify late-apoptotic and dead cells because it stains cells with a disrupted cell membrane. The control was cells cultured in a medium without drugs. A positive control (cells incubated with 3% formaldehyde in buffer for 30 min on ice) was used to find the optimal parameter settings. An analysis of the results was performed using FACSDiva software (BD Biosciences Systems).

Analysis of Mitochondrial Membrane Potential
Disruption of the mitochondrial membrane potential (MMP) was measured using the lipophilic cationic fluorochrome (JC-1 MitoScreen kit; BD Biosciences Systems) as described previously [31]. In brief, the unfixed cells were washed, then resuspended in PBS containing 10 mg/mL of JC-1. Next, the cells were incubated for 15 min at room temperature and protected from light, washed and resuspended in PBS. Thus prepared samples were analyzed immediately by flow cytometry BD FACSCanto II, and then the percentage of cells with disturbed MMP was calculated using FACSDiva software (both from BD Biosciences Systems).

Measuring the Number of Autophagosomes and Autolysosomes by Autophagy Assay, Red
To determine the effects of AuMet-1, PdMet-1 and PtMet-1 compounds on the autophagy process of breast cancer cells MCF-7 and MDA-MB-231, an autophagy assay was performed according to the protocol from the manufacturer. The probe was a cell-permeant aliphatic molecule that fluoresces brightly when inserted in the lipid membranes of autophagosomes and autolysosomes (Autophagy Assay, Red kit; ImmunoChemistry Technologies, Bloomington, MN, USA). In short, the unfixed cells were washed and then resuspended in PBS with the added Autophagy Probe, Red solution. Afterwards, the cells were incubated for 30 min at 37 • C in the dark, washed, resuspended in 1× Cellular Assay Bufor and the provided Fixative was added at a volume/volume ratio of 1:5. The samples were measured immediately after preparation by flow cytometry using a BD FACSCanto II system (BD Biosciences Systems). The percentage of cells with autophagy was calculated using FACSDiva software (BD Biosciences Systems).

Antibody Topoisomerase IIα Detection
To check whether the tested compounds induce cell death by damaging DNA directed to topoisomerases, the activity of topoisomerase IIα in breast cancer cells MCF-7 and MDA-MB-231 was measured using antibody Topoisomerase IIα, according to the manufacturer's instructions. The probe was anti-topoisomerase IIα antibody conjugated to phycoerythrin. In brief, the centrifuged cells were resuspended in 4% formaldehyde and incubated for 15 min at room temperature. The cells were washed by centrifugation with excess PBS. Afterwards, permeabilization was performed by adding ice-cold 90% methanol to the cells and incubating for 60 min in an ice bath. The cells were washed by centrifugation with excess PBS again. Then, they were resuspended in 100 µL of diluted primary antibody, prepared in PBS at a 1:100 dilution, and incubated for 60 min at room temperature in the dark, washed and resuspended in 300 µL PBS. The samples were measured immediately after preparation by flow cytometry using BD FACSCanto II. An analysis of the results was performed using FACSDiva software (both from BD Biosciences Systems).

DNA Fragmentation Assay
In order to identify and examine DNA fragmentation, the method called terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) was used. In our study we used a commercial assay kit (APO-Direct Kit; BD Pharmingen) in accordance with the manufacturer's instructions. In brief, the treated cells were fixed with 1% paraformaldehyde in PBS (4 • C, 30 min) and then washed in PBS. Afterwards, permeabilization was performed by adding ice-cold 70% ethanol to the cells. Fixed cells were washed twice using the kit wash buffer, then the cells were centrifuged and the supernatant was discarded. In the next step, the cells were resuspended in Labeling Solution DNA and incubated for 1 h at 37 • C with occasional shaking. At the end of the incubation time, rinse buffer was added, then cells were centrifuged, the supernatant was removed and cells were rinsed again.
After that, the cell pellet was suspended in the PI/RNase staining buffer and incubated for 30 min at room temperature in the dark. After this time, the cells were immediately analyzed in a flow cytometer BD FACSCanto II, and then the percentage of cells with distinctive DNA strand breaks was calculated using FACSDiva software (both from BD Biosciences Systems).

Zebrafish Handling, Establishment of Xenograft
Breast cancer MCF-7 and MDA-MB-231 cells were labeled before transplantation. The zebrafish embryos were manually dechorionated at 36 h post-fertilization (hpf) and after another 12 h were anesthetized by placement in 0.04 mg/mL ethyl 3-aminobenzoate methanesulfonate tricaine, which is a water soluble, fast-acting anesthetic agent. Zebrafish embryos were then transferred to a thin film of low-melting-point agarose to stabilize the fish in a lateral position. Breast cancer cells were loaded into a borosilicate glass needle pulled by a P-1000 Next Generation Micropipette Puller (Sutter Instrument Company, Novato, CA, USA). A suspension containing about 100-200 cells was injected into the inferior section of the yolk sac in a single injection by using an electronically regulated air-pressure microinjector (Narishige IM-300 Microinjector, Los Angeles, CA, USA). After injection, the zebrafish were washed once with fish water and transferred to a 6-well plate containing 2 mL of fresh fish water. MCF-7 and MDA-MB-231-xenografts (72 hpf) were incubated at 33 • C with PdMet-1 (100 µM) and PtMet-1 (100 µM) for 48 h. Although embryos are normally allowed to develop at 28.5 • C and human cells at 37 • C, a compromise at 33 • C works well.

Microscope Imaging
Living zebrafish embryos were anesthetized by tricaine and embedded in a lateral orientation. The animals were analyzed for cytoplasmic fluorescence intensity. Images of the cells were acquired using an EVOS M5000 Imaging System (Thermo Fisher Scientific Inc., Waltham, MA, USA) with the following filters: GFP (470/22 nm Excitation; 510/42 nm Emission) and Texas Red (585/29 nm Excitation; 624/40 nm Emission). Image analysis was performed using ImageJ v1.51 software (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis
All numerical data are presented as mean ± standard deviation (SD) from at least 3 independent experiments. Statistical analysis was conducted using the GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Statistical differences in multiple groups were determined by one-way ANOVA followed by Tukey's test. p < 0.05 was considered statistically significant.

Conclusions
In conclusion, the novel metal complexes may be excellent molecular frameworks that are amenable for optimization for two types of breast cancer cell death: apoptosis and/or autophagy. We discovered that topoisomerase IIα increased activity caused by the tested compounds could potentially lead to DNA fragmentation, resulting in cell death by apoptosis. The outcomes of our study suggest that the novel synthetic transition metal complexes with berenil and nitroimidazole may constitute a new group of anticancer agents for the treatment of breast cancer patients.