Thiosemicarbazide Derivatives Targeting Human TopoIIα and IDO-1 as Small-Molecule Drug Candidates for Breast Cancer Treatment

In 2020, breast cancer became the most frequently diagnosed type of cancer, with nearly 2.3 million new cases diagnosed. However, with early diagnosis and proper treatment, breast cancer has a good prognosis. Here, we investigated the effect of thiosemicarbazide derivatives, previously identified as dual inhibitors targeting topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1), on two distinct types of breast cancer cells (MCF-7 and MDA-MB-231). The investigated compounds (1–3) selectively suppressed the growth of breast cancer cells and promoted apoptosis via caspase-8- and caspase-9-related pathways. Moreover, these compounds caused S-phase cell cycle arrest and dose-dependently inhibited the activity of ATP-binding cassette transporters (MDR1, MRP1/2 and BCRP) in MCF-7 and MDA-MB-231 cells. Additionally, following incubation with compound 1, an increased number of autophagic cells within both types of the investigated breast cancer cells was observed. During preliminary testing of ADME-Tox properties, the possible hemolytic activities of compounds 1–3 and their effects on specific cytochrome P450 enzymes were evaluated.


Introduction
Currently, cancer is recognized as a leading cause of death worldwide, being responsible for 10 million deaths in 2020 [1]. Additionally, in the same year, breast cancer (BC) became the most frequently diagnosed type of cancer, with nearly 2.3 million new cases. However, with early diagnosis and proper treatment, BC has a good prognosis. One of the most widely recognized classifications of BC is based on the immunohistochemical expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptors (HER2). Among the four classified subtypes of BC, luminal A and luminal B subtypes are ER and/or PR positive and the HER2 subtype is characterized by high HER2 expression and a lack of ER/PR, while triple-negative BC (TNBC) is characterized by the absence of ER/PR and HER2 overexpression [2]. Depending on the type and grade of BC, its treatment includes surgery, radiation and systemic therapy (chemotherapy, hormone therapy, immunotherapy and targeted biological therapy). Pharmacological treatment can be applied before or after the surgical intervention, i.e., as neoadjuvant or adjuvant therapy, respectively. The main objective of chemotherapy in BC is to destroy cancer cells and to reduce their ability to spread from the primary tumor to other parts of the body. Traditional cytotoxic agents still remain an important part of therapy in BC; however, their efficacy is seriously confined by their low selectivity and dose-limiting toxic effects against human normal cells. Since one of the objectives of the WHO, in relation to cancer policy, is to primary tumor to other parts of the body. Traditional cytotoxic agents still remain an important part of therapy in BC; however, their efficacy is seriously confined by their low selectivity and dose-limiting toxic effects against human normal cells. Since one of the objectives of the WHO, in relation to cancer policy, is to reduce BC mortality by 2.5% annually, there is a vital need to improve the diagnosis and treatment of patients suffering from BC [3].
In our recently published work, we identified the firstin-class dual inhibitors targeting both human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1) ( Figure 1) [4]. These compounds exerted strong cytotoxic and antiproliferative activity, up to 90times higher than that of etoposide, in various human cancer cell lines. Among the two breast cancer cell types (MCF-7 and MDA-MB-231) investigated during our preliminary studies, MCF-7 cells turned out be more sensitive to the above-mentioned agents. Compound 1, when examined against MCF-7 cells in aBrdU assay, showed a 46times stronger antiproliferative activity than etoposide and was much more selective against cancer cells than normal (non-cancerous) cells, as indicated by its selectivity index (SI = 46.8). As shown by the example of etoposide, topoisomerase IIα inhibitors can be important elements ofBC treatment. Due to the relatively low toxicity and beneficial clinical response, orally administered etoposide is regarded as a safe option for patients with heavily pre-treated metastatic breast cancer, especially for those not responding to other medical therapies [5,6]. Bearing in mind that compounds 1-3 ( Figure 1) possess additional IDO 1 inhibitory activity, they may constitute an interesting option in cancer treatment. It is known from the literature that co-administration of IDO 1 inhibitors with radiotherapy or systemic therapy improves the results of the treatment [7]. IDO 1 inhibitors havealso been comprehensively examined in clinical trials as potentially useful drugs to overcome tumor-induced immunosuppression [8]. Both topoIIα and IDO 1 were reported to be highly upregulated in breast cancer patients [9,10]. The measurement of topoIIα in invasive BC has an important clinical value, since it gives information on the quantity of cycling tumor cells [11]. Additionally, significant correlation was observed between HER-2/neu oncoprotein overexpression and the increased topoIIα level in breast tumors [9]. The increased expression of topoIIα was observed in higher stage tumors [12]. Likewise, IDO overexpression is frequent among Both topoIIα and IDO 1 were reported to be highly upregulated in breast cancer patients [9,10]. The measurement of topoIIα in invasive BC has an important clinical value, since it gives information on the quantity of cycling tumor cells [11]. Additionally, significant correlation was observed between HER-2/neu oncoprotein overexpression and the increased topoIIα level in breast tumors [9]. The increased expression of topoIIα was observed in higher stage tumors [12]. Likewise, IDO overexpression is frequent among high-grade TNBC specimens [13]. High levels of IDO 1 within BC cells are also correlated with microvessel density and a worse clinical prognosis [14]. Therefore, there is a strong rationale for the use of topoIIα/IDO 1 inhibitors as possible anti-BC agents.
In the present study, we aimed to investigate the effects of thiosemicarbazide derivatives 1-3 on the mechanisms affecting breast cancer cells functions, including their different effects on ER/PR positive cells (MCF-7) and TNBC cells (MDA-MB-231). Moreover, an in vitro screening of some ADME-Tox properties of compounds 1-3 was carried out in order to better characterize them as possible drug candidates.

Preparation of the Investigated Compounds
Thiosemicarbazide derivatives 1-3 were prepared via reaction of the carboxylic acid hydrazides and isothiocyanates in one-step reaction performed in boiling ethanol. Their structures were designed using in silico methods and are presented in Figure 1. The design, synthesis and spectral characterization of the compounds have been described in our previously published paper [4].

Viability of Human Normal Breast Epithelial Cells (MCF-10A) Exposed to Compounds 1-3 for 24 h
The cytotoxic effect of compounds 1-3 against MCF-7 and MDA-MB-231 cells was evaluated previously and described in [4]. However, the selectivity of newly designed anticancer agents towards cancer cells compared to normal cells is crucial to develop new treatment options. In order to examine the selectivity of the tested compounds towards MCF-7 and MDA-MB-231 breast cancer cells, the viability of human normal breast epithelial cells (MCF-10A) incubated for 24 h with increasing concentrations of compounds 1-3 was determined using MTT assays. The median inhibitory concentrations (IC 50 ) of compounds 1-3 were 25.31 ± 2.27, 42.74 ± 3.16 and 40.45 ± 4.59 µg/mL, respectively (Table 1). In turn, the values of the selectivity index (SI) ranged from 2.57 to 5.30 for MCF-7 and from 2.75 to 5.60 for MDA-MB-231. Human normal MCF-10A cells were less sensitive to thiosemicarbazide derivatives 2 and 3 when compared to compound 1.

Effect of Compounds 1-3 on the Apoptosis in MCF-7 and MDA-MB-231 Cells
The investigated thiosemicarbazide derivatives (1-3) induced apoptosis in MCF-7 and MDA-MB-231 cells in a dose-dependent manner ( Figure 2). The pro-apoptotic activities of 1-3 were much higher than that of etoposide. It is worth mentioning that the ability of the tested compounds to induce apoptosis in breast cancer cells was correlated with their cytotoxic properties observed in MTT assays. Compound 3, which induced apoptosis in nearly 68% of MCF-7 cells, also most potently impaired their viability, with an IC 50 of 7.67 µg/mL. Similarly, MDA-MB-231 cells were most sensitive to derivative 2 (IC 50 = 7.64 µg/mL), which caused the greatest increase in the number of apoptotic cells to 70%. At the same time, MCF-7 and MDA-MB-231 cells incubated with etoposide (20 µg/mL) contained about 8.5% apoptotic cells (measured as a sum of early and late apoptotic cells). 7.64 µ g/mL), which caused the greatest increase in the number of apoptotic cells to 70%. At the same time, MCF-7 and MDA-MB-231 cells incubated with etoposide (20 µ g/mL) contained about 8.5% apoptotic cells (measured as a sum of early and late apoptotic cells).

Effect of Compounds 1-3 and Etoposide on Caspase-8 and Caspase-9 Activity
Caspases-8 and -9 are known as initiators of the apoptosis process. However, while activation of caspase-8 is a crucial step in the initiation of the death-receptor-mediated pathway of apoptosis (extrinsic pathway), caspase-9 plays a pivotal role in the intrinsic pathway mediated by mitochondria. Flow cytometric analyses showed that the reference drug (etoposide) statistically significantly increased caspase-8 activation in breast cancer cells only when used at a concentration of 20 µg/mL (Figure 3). However, the number of MCF-7 and MDA-MB-231 cells with activated caspase-8 was still as low as 6.73 and 7.2%, respectively. A significant and dose-dependent increase in caspase-8 activation was observed in breast cancer cells pretreated with compounds 1-3. Among the investigated thiosemicarbazide derivatives, compound 3 had the highest ability to induce caspase-8 activation in MCF-7 cells, while triple-negative MDA-MB-231 breast cancer cells were more sensitive to thiosemicarbazide derivative 2. Similar dependencies were observed when MCF-7 and MDA-MB-231 cells were tested for caspase-9 activity ( Figure 4). In order to investigate the ability of compounds 1-3 to induce apoptosis-related caspases-8 and -9, MCF-7 and MDA-MB-231 cells were pretreated with pan-caspase inhibitor (Z-VAD-FMK, 100 µM). Caspases-8 and -9 were strongly activated even after previous addition of Z-VAD-FMK (Figures S1 and S2). These observations collectively prove that apoptosis induced by compounds 1-3 in breast cancer cells is triggered via both intrinsic and extrinsic pathways. cells only when used at aconcentration of 20 µ g/mL( Figure 3). However, the number of MCF-7 and MDA-MB-231 cells with activated caspase-8 was still as low as 6.73 and 7.2%, respectively. A significant and dose-dependent increase in caspase-8 activation was observed in breast cancer cells pretreated with compounds 1-3. Among the investigated thiosemicarbazide derivatives, compound 3 had the highest ability to induce caspase-8 activation in MCF-7 cells, while triple-negative MDA-MB-231 breast cancer cells were more sensitive to thiosemicarbazide derivative 2. Similar dependencies were observed when MCF-7 and MDA-MB-231 cells were tested for caspase-9 activity ( Figure 4). In order to investigatethe ability of compounds 1-3 to induce apoptosis-related caspases-8 and -9, MCF-7 and MDA-MB-231 cells were pretreated with pan-caspase inhibitor (Z-VAD-FMK, 100 µ M). Caspases-8 and -9 werestrongly activated even after previous addition of Z-VAD-FMK ( Figure S1 and S2). These observations collectively prove that apoptosis induced by compounds 1-3 in breast cancer cells is triggered via both intrinsic and extrinsic pathways.

Thiosemicarbazide Derivative 1 Induces Autophagy in MCF-7 and MDA-MB-231
Besides apoptosis, cancer cells may be successfully eliminated from living organisms through the functionally distinct mechanism of programmed cell death, i.e., through the autophagy process. Cells undergoing autophagy display an increase in the number of autophagosomes and autolysosomes which, subsequently, can be labeled with a fluorescent autophagy probe. Such a method is widely used to monitor the occurrence and progress of autophagy. Among the investigated thiosemicarbazide derivatives, only compound 1 induced the autophagy process in MCF-7 and MDA-MB-231 breast cancer cells in a statistically significant manner. In the population of MCF-7 cells pretreated with 10 and 20 µ g/mLof compound 1, 2.5 ± 0.2% and 11.5 ± 1.2% of cells were observed to beundergoing autophagy, respectively ( Figure 5). In the case of MDA-MB-231 cells, these values ranged from 3.0 ± 0.2% to 5.5 ± 0.9%. Regarding both untreated and etopo-

Thiosemicarbazide Derivative 1 Induces Autophagy in MCF-7 and MDA-MB-231
Besides apoptosis, cancer cells may be successfully eliminated from living organisms through the functionally distinct mechanism of programmed cell death, i.e., through the autophagy process. Cells undergoing autophagy display an increase in the number of autophagosomes and autolysosomes which, subsequently, can be labeled with a fluorescent autophagy probe. Such a method is widely used to monitor the occurrence and progress of autophagy. Among the investigated thiosemicarbazide derivatives, only compound 1 induced the autophagy process in MCF-7 and MDA-MB-231 breast cancer cells in a statistically significant manner. In the population of MCF-7 cells pretreated with 10 and 20 µg/mL of compound 1, 2.5 ± 0.2% and 11.5 ± 1.2% of cells were observed to beundergoing autophagy, respectively ( Figure 5). In the case of MDA-MB-231 cells, these values ranged from 3.0 ± 0.2% to 5.5 ± 0.9%. Regarding both untreated and etoposide-treated breast cancer cells, as low as about 1% of the cell population formed autophagosomes and/or autolysosomes. side-treated breast cancer cells, as low as about 1% of the cell population formed autophagosomes and/or autolysosomes.

Effect of Compounds 1-3 on the Activity of ATP-Binding Cassette (ABC) Transporters
ABC transporters play a pivotal role in the development of resistance to anticancer treatment. Therefore, the effect of thiosemicarbazide derivatives 1-3 on the activity o three major ATP-binding cassette (ABC) transporter proteins, i.e., MDR1 (also known as p-glycoprotein), MRP 1/2 (multidrug resistance-associated protein) and BCRP (breas cancer resistance protein), was evaluated. The quantitative analyses of ABC transporter activity wereexpressed using multidrug-resistance activity factor (MAF) values. Studies comparing MAF values with clinical responses to chemotherapy have suggested tha

Effect of Compounds 1-3 on the Activity of ATP-Binding Cassette (ABC) Transporters
ABC transporters play a pivotal role in the development of resistance to anticancer treatment. Therefore, the effect of thiosemicarbazide derivatives 1-3 on the activity of three major ATP-binding cassette (ABC) transporter proteins, i.e., MDR1 (also known as p-glycoprotein), MRP 1/2 (multidrug resistance-associated protein) and BCRP (breast cancer resistance protein), was evaluated. The quantitative analyses of ABC transporter activity were expressed using multidrug-resistance activity factor (MAF) values. Studies comparing MAF values with clinical responses to chemotherapy have suggested that agents with an MAF of <20 can be regarded as multidrug-resistance negative, while MAF values of >25 are indicative of multidrug-resistance positive specimens. The current studies revealed that the investigated compounds inhibited the activity of MDR1, MRP1/2 and BCRP transporters in a dose-dependent manner. Compounds 2 and 3, stronger than compound 1, decreased the activity of the examined protein transporters in MCF-7 cells. Even at the lower concentrations tested (i.e., 10 µg/mL), the former thiosemicarbazide derivatives impaired the ABC transporter activity, with MAF values much lower than 20 (for compound 2:

Screening of ADME-Tox Properties of Thiosemicarbazide Derivatives 1-3
CYP3A4 and CYP2D6 enzymatic activitieswereevaluated using Vivid™ CYP3A4 and CYP2D6 screening kits with ketoconazole and guanidine as positive controls, respectively. None of the tested compounds influenced the activity of CYP3A4 and CYP2D6 enzymes at a concentration of 10 µ g/mL in a statistically significant manner. At the same time, ketoconazole (10 µ M) decreased the activity of CYP3A4 to 6.49 ± 0.38% (vs. the negative control), while guanidine significantly impaired the activity of CYP2D6 (11.08 ± 0.81% vs. negative control) (Figure 8 A, B). The hemolytic effect of 1-3 varied from 0.86 ± 0.18% (compound 2) to 1.08 ± 0.26% (compound 1); however, there were no statistically significant differences between those results and hemolysis observed in untreated red blood cells (0.81 ± 0.11%) (Figure 8 C).

Screening of ADME-Tox Properties of Thiosemicarbazide Derivatives 1-3
CYP3A4 and CYP2D6 enzymatic activities were evaluated using Vivid™ CYP3A4 and CYP2D6 screening kits with ketoconazole and guanidine as positive controls, respectively. None of the tested compounds influenced the activity of CYP3A4 and CYP2D6 enzymes at a concentration of 10 µg/mL in a statistically significant manner. At the same time, ketoconazole (10 µM) decreased the activity of CYP3A4 to 6.49 ± 0.38% (vs. the negative control), while guanidine significantly impaired the activity of CYP2D6 (11.08 ± 0.81% vs. negative control) ( Figure 8A,B). The hemolytic effect of 1-3 varied from 0.86 ± 0.18% (compound 2) to 1.08 ± 0.26% (compound 1); however, there were no statistically significant differences between those results and hemolysis observed in untreated red blood cells (0.81 ± 0.11%) ( Figure 8C).

Discussion
Small molecule inhibitors still constitute an integral part of chemotherapy regimens. Among such molecules, special attention is given to dual-or multi-targeting agents, whose single molecules are able to act on multiple molecular targets [15,16]. One of their main advantages, when compared to anticancer agents that have a large molecular weight, is that they can pass through the cell membranes and interact with the intracellular targets. The use of dual-or multi-targeting anticancer agents allows to overcome the problems oflimited efficacy or chemoresistance, observed when single-targeting agents are applyied. Thiosemicarbazide derivatives 1-3 (Figure 1), recently designed and synthesized in our laboratory, constitute examples of dual-targeting cytotoxic agents acting on human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase. Previously, it was shownthat these compounds significantly reduced the viability of both ER/PR-positive MCF-7 cells and triple-negative MDA-MB-231 cells [4]. The anti-BC activitiesof 1-3 werehigher than that of etoposide, which is a marketed anticancer drug targeting human topoisomerase IIα. Importantly, current studies revealed that BC cells were more sensitive to 1-3 than normal breast epithelial cells (MCF-10A), with SI values of up to 5.6. Another beneficial effect of the investigated compounds, apart from their cytotoxic activity and selectivity towards BC cells, is associated with the reduction in the ABC

Discussion
Small molecule inhibitors still constitute an integral part of chemotherapy regimens. Among such molecules, special attention is given to dual-or multi-targeting agents, whose single molecules are able to act on multiple molecular targets [15,16]. One of their main advantages, when compared to anticancer agents that have a large molecular weight, is that they can pass through the cell membranes and interact with the intracellular targets. The use of dual-or multi-targeting anticancer agents allows to overcome the problems of limited efficacy or chemoresistance, observed when single-targeting agents are applied. Thiosemicarbazide derivatives 1-3 (Figure 1), recently designed and synthesized in our laboratory, constitute examples of dual-targeting cytotoxic agents acting on human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase. Previously, it was shown that these compounds significantly reduced the viability of both ER/PR-positive MCF-7 cells and triple-negative MDA-MB-231 cells [4]. The anti-BC activities of 1-3 were higher than that of etoposide, which is a marketed anticancer drug targeting human topoisomerase IIα. Importantly, current studies revealed that BC cells were more sensitive to 1-3 than normal breast epithelial cells (MCF-10A), with SI values of up to 5.6. Another beneficial effect of the investigated compounds, apart from their cytotoxic activity and selectivity towards BC cells, is associated with the reduction in the ABC transporter activity. This is crucial, because despite the clinical efficacy of anticancer agents acting as topoisomerase II poisons, their use in cancer treatment has significant limitations associated with the development of drug resistance [17]. Activation or overexpression of the ATP-binding cassette proteins, associated with the pumping of drugs from cancer cells, result in a decreasedresponse to chemotherapy [18][19][20]. The main role in the development of chemoresistance in BC cells is played by MDR1 (p-glycoprotein) and subsequently by MRP (multidrug resistanceassociated proteins) and BCRP (breast cancer resistance protein). Their improved activity correlates with cancer aggressiveness, progression of the disease and a poor prognosis [21]. The dose-dependent inhibition of the investigated ABC transporters (MDR1, MRP1/2 and BCRP) in MCF-7 and MDA-MB-231 cells by compounds 1-3 impliesalower risk of drug resistance. Interestingly, the compounds withthe lowest MAF values (i.e., compound 3 against MCF-7 cells and compound 2 against MDA-MB-231 cells) were also characterized by lower IC 50 values established in MTT assays.
Bearingin mind that topoisomerase inhibitors belong to the most effective inducers of apoptosis, the possible pro-apoptotic effect of 1-3 in BC cells was investigated. Using MCF-7 and MDA-MB-231 cells, it was demonstrated that the investigated thiosemicabazide derivatives promoted apoptosis in a manner correlated to their IC 50 values. In the case of topoisomerase II poisons, such a phenomenon can be explained as a result of intracellular accumulation of covalent topoII-DNA complexes. So-called "cleavable complexes" act as cellular toxins that block DNA replication and stimulate apoptotic pathways [22]. The data from the current study show that the pro-apoptotic effect produced by compounds 1-3 is mediated via activation of caspases-8 and -9, and thus involves both extrinsic and intrinsic apoptotic pathways in MCF-7 and MDA-MB-231 cells. Additionally, the autophagy process was observed in both types of investigated BC cells after incubation with compound 1. Autophagy itself is considered to be a double-edged sword. Depending on the type of cancer, its stage, genetic factors and the treatment applied, autophagy can suppress tumorigenesis or promote cancer cell survival and proliferation [23,24]. In our studies, the rate of autophagy in MCF-7 cells was nearly twice as high as in MDA-MB-231 cells. Importantly, some research suggests the existence of the antagonism between autophagy and apoptosis [25]. This may explain why compound 1, which turned out to be an autophagy inducer in MCF-7 and MDA-MB-231 cells, also had the weakest promoting effect on apoptosis out of the three thiosemicarbazide derivatives tested. However, the possible involvement of autophagy in compound 1-induced BC cell death requires further investigation and cannot be claimed at this stage of the study. Apart from the contribution of apoptosis and ABC transporter inhibition to the anticancer effect of 1-3, our studies also showed that these dual-targeting agents significantly inhibited the proliferation of BC cells by blocking cell cycle in the S-phase in a dose-dependent manner. The observed impairment in cell cycle progression was significantly stronger in MCF-7 cells. Thus, it seems clear that the disruption of so many intracellular processes results in efficient suppression of BC cells.
Administration of cancer therapy includes several routes, e.g., intravenous, oral, intraperitoneal, intrathecal, subcutaneous, intradermal, etc. However, chemotherapy is most commonly given intravenously. Although very rarely, anticancer agents may cause direct (toxic) or delayed (immunological) hemolytic effects. Therefore, it is of great importance to test the possible hemolytic activity of drug candidates at the earliest possible stage of their development. Drug-induced hemolysis can be mediated either by immune-or non-immune mechanisms. The latter mechanismis more likely in patients with glucose-6phosphate dehydrogenase (G6PD)deficiency, since this enzyme protects red blood cells from oxidative stress-related damages. The investigated thiosemicarbazides 1-3 did not result indirect hemolysis mediated by non-immune mechanisms after incubation with red blood cells in vitro. As a limitation of this study, it should be pointed out that the possible immunerelated hemolytic effects of 1-3 could only be observed in in vivo conditions. Therefore, additional experiments are required to completely exclude the hemolytic potential of the tested drug candidates.
During further testing of ADME-Tox properties of 1-3, their effects on specific cytochrome P450 (CYP) enzymes (i.e., CYP3A4 and CYP2D6) were evaluated. Preliminary safety assessments of drug candidates in terms of their possible risk of CYP induction/inhibition remains pivotal in drug discovery and development. CYP enzymes are responsible for biotransformation of drugs and other xenobiotics. Their inhibition or overactivation may lead to drug-drug interactions, causing toxic effects and/or therapeutic failure. Among the CYP enzymes, CYP3A4 and CYP2D6 isoforms are the most essential. Taken together, they are responsible for the metabolism of approximately 80% of the available drugs (CYP3A4 metabolizes approx. 50% of drugs, while CYP2D6 metabolizes approx. 30%) [26]. After incubation of CYP3A4 and CYP2D6 in the presence of compounds 1-3 (10µg/mL), no statistically significant changes in the activity of the tested enzymes were observed. Therefore, it can be concluded that the possible use of thiosemicarbazide derivatives 1-3 will be associated with a low risk of drug-drug interactions.

Effect of Compounds 1-3 on the Viability of MCF-10A Cells
MTT assays were performed to evaluate the cytotoxic activity of the investigated compounds (1-3) against MCF-10A cells. Asuspension of cells (1 × 10 5 cells/mL) was distributed onto 96-well plates at a volume of 100 µL/well. After attachment, the cells were treated with increased concentrations of the tested compounds in medium containing 2% FBS, and incubated for 24 h. Then, 15 µL of MTT working solution (5 mg/mL in PBS) was added to each well and the plates were incubated for 3 h. Subsequently, 100 µL of 10% SDS solution was added to each well in order to dissolve the formazan formed. After overnight incubation at 37 • C, the absorbance of the obtained solution was measured at λ= 570 nm using a microplate reader (Epoch, BioTek Instruments, Inc., Winooski, VT, USA). At least two independent experiments were performed in triplicate. IC 50 values of the tested compounds were calculated using the IC 50 online calculator [27].

Flow Cytometry Assessment of Annexin V and Propidium Iodide Binding
Flow cytometry analyses for apoptosis induction by the compounds 1-3 and etoposide wereperformedusing an FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, San Diego, CA, USA). The rationale of the assay is based on the externalization of phosphatidylserine on the surface of apoptotic cells. In early and late apoptotic cells, the phosphatidylserine translocates into the outer layer of the membrane, which allows annexin V-FITC to attach. Propidium iodide (PI) is, in turn, a fluorescent dye that penetrates into the late apoptotic and necrotic cells with impaired cell membrane integrity. Therefore, using annexin V and PI, it is possible to distinguish four groups of cells: living cells, early apoptotic cells, late apoptotic cells and necrotic cells. MCF-7 and MDA-MB-231 cells were incubated with 10 and 20 µg/mL of compounds 1-3 and etoposide (reference drug) for 24 h. After incubation, the cells were dyed with FITC-labeled annexin V and PI. The analysis was performed on a BD FACS Canto II flow cytometer using FACSDiva software (version 6.1.3, BD Biosciences Systems, San Diego, CA, USA). The equipment was calibrated with BD Cytometer Setup and Tracking Beads (BDBiosciences, San Diego, CA, USA). resuspended in 500 µL of PBS and run on the flow cytometer immediately. Measurements were conducted on a BD FACSCanto II flow cytometer using FACSDiva software (version 6.1.3, BD Biosciences, San Diego, CA, USA). The multidrug resistance activity factor (MAF) was calculated from the difference between the mean fluorescence intensity (MFI) of cells with and without the highly selective inhibitors. The calculations followed these formulas: The theoretical range of the MAF values is between 0 and 100. Studies comparing MAF values with the clinical response to a chemotherapeutic treatment suggest that a specimen with an MAF value of <20 can be regarded as multidrug resistance negative, while MAF values of >25 are indicative of multidrug resistance positive specimens.

Hemolytic Activity of Compounds 1-3
Human red blood cell (RBC) concentrate was obtained from the Regional Blood Donation and Transfusion Centre (Lublin, Poland). The RBC concentrate (5 mL) was washed three times with sterile PBS at 37 • C and centrifuged at 500 g for 3 min. The obtained pellet was resuspended using warm, sterile PBS in order to obtain a 2% RBC suspension. Next, 1 mL of the RBC suspension was mixed with 1 mL of a solution of the investigated compounds. The final concentration of the tested compounds was 10 µg/mL. The mixtures were incubated at 37 • C for 30 min and centrifuged at 1500× g for 10 min. Finally, avolume of 100 µL of supernatant from each sample was transferred into a 96-well plate and the amount of free hemoglobin was measured spectrophotometrically at 405 nm. PBS alone (negative control) and 0.1% Triton-X (positive control) were examined using similar conditions. The experiments were run in triplicate.

Effect of Compounds 1-3 on CYP3A4/CYP2D6 Enzymatic Activity
CYP3A4 and CYP2D6 enzymatic activities were evaluated using a Vivid™ CYP3A4 Green Screening Kit and a Vivid™ CYP2D6 Cyan Screening Kit (both from Thermo Fisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer's protocol. According to these assays, the examined compounds are analyzed by their ability to inhibit the production of a fluorescent signal in the reaction between recombinant CYP3A4 or CYP2D6 isozymes and specific Vivid substrates. The experiments were performed in the endpoint mode of the assay, in which solutions of examined compounds were first combined with the Master Pre-mix. After a short incubation, the background fluorescence of the Master Pre-mix and the examined compounds was measured. Next, a mix of the respective Vivid substrate and NADP + was added in order to initiate the enzymatic reaction. After 20 min of incubation, the reaction was terminated using a stop solution and the fluorescence was measured using the recommended (by the manufacturer) excitation and emission wavelengths. Ketoconazole and quinidine (both at 10 µM) were used as positive controls (i.e., inhibitors of CYP3A4 and CYP2D6, respectively). A reaction mixture without tested compounds was used as a negative control and its fluorescence was designated as 100%. The experiments were run in triplicate and the results were presented as means ± SD.

Conclusions
This paper provides an insight into the anticancer effects of three topoIIα/IDO 1 inhibitors against breast cancer cell lines. The investigated compounds 1-3 selectively