Kinesin Eg5 Selective Inhibition by Newly Synthesized Molecules as an Alternative Approach to Counteract Breast Cancer Progression: An In Vitro Study

Simple Summary Breast cancer is the most widespread and diagnosed cancer in women. This study tested newly synthesized Eg5 inhibitors to find valid therapeutic alternatives to common drugs able to counteract breast cancer progression. Our results evidence that thiadiazole-based Eg5 inhibitors, 2 and 41, appear to negatively modulate proliferation, invasiveness and migration of cancer cells, affecting also the angiogenesis process and the remodeling of the extracellular matrix, thus representing a new promising drug strategy to control breast cancer. Abstract Breast cancer (BC) is one of the most diagnosed cancers in women. Recently, a promising target for BC treatment was found in kinesin Eg5, a mitotic motor protein that allows bipolar spindle formation and cell replication. Thus, the aim of this work was to evaluate the effects of novel thiadiazoline-based Eg5 inhibitors, analogs of K858, in an in vitro model of BC (MCF7 cell line). Compounds 2 and 41 were selected for their better profile as they reduce MCF7 viability at lower concentrations and with minimal effect on non-tumoral cells with respect to K858. Compounds 2 and 41 counteract MCF7 migration by negatively modulating the NF-kB/MMP-9 pathway. The expression of HIF-1α and VEGF appeared also reduced by 2 and 41 administration, thus preventing the recruitment of the molecular cascade involved in angiogenesis promotion. In addition, 2 provokes an increased caspase-3 activation thus triggering the MCF7 apoptotic event, while 41 and K858 seem to induce the necrosis axis, as disclosed by the increased expression of PARP. These results allow us to argue that 2 and 41 are able to simultaneously intervene on pivotal molecular signaling involved in breast cancer progression, leading to the assumption that Eg5 inhibition can represent a valid approach to counteract BC progression.


Introduction
Breast cancer (BC) is one of the most widespread cancers among women [1] and in 2020, 2.3 million new cases were recorded (11.7% of incidence and 6.9% of mortality), surpassing lung cancer (11.4% of incidence) as the main cause of cancer death worldwide [2]. This type of tumor shows a high grade of variability and based on molecular and histological characteristics, different types of BC can be identified: BC positive for both estrogen and progesterone receptors (ER + , PR + ), named Luminal A, BC positive for ER, PR and for human epidermal receptor 2 (HER2 + ) (named Luminal B), BC positive only for HER2 (named HER2-overexpression) and triple-negative BC (TNBC, negative for ER, PR, and HER2 receptors), also called Basal-like [3,4]. There are different approaches to treat patients with BC such as surgical resection of the tumor and regional lymph node (axillary lymph node) and postoperative radiation for patients with non-metastatic BC, systematic therapy specific Newly thiadiazoline Eg5 inhibitors, analogs of their parent compound K858, were synthesized, purified (≥96% purity), and characterized, as already reported [26]. The compounds were kept as a 50 mM stock solution in dimethyl sulfoxide (DMSO) at −20 • C.
Human gingival fibroblasts (HGFs) were obtained from 10 donors which were not affected by periodontal and systemic diseases. Before undergoing third molar surgical extraction, donors signed the informed consent in accordance with the Italian Legislation and with Ethical Principles for Medical Research including the Human Subjects code of the World Medical Association (Declaration of Helsinki). The project received the Local Ethical Committee of the University of Chieti approval on 31 March 2016 (Chieti, Italy; protocol number 1173). After withdrawal, gingiva samples were placed in DMEM, rinsed in phosphate saline buffer (PBS), cut into smaller pieces, and cultured in DMEM, with 10% FBS, 1% penicillin/streptomycin, and 1% fungizone (all purchased from EuroClone, Milan, Italy). Cells were maintained in a humidified incubator at 37 • C with 5% CO 2 . After 10 days fungizone was removed from culture medium and cells were used for biological experiments (passage [7][8][9][10][11][12][13][14], as elsewhere reported [27].
HGFs were seeded in a 96-well cell culture plate at a density of 7000 /well; their metabolic activity was evaluated after 24 and 48 h of treatment with 2 at 1.5 µM, 41 at 5 µM, and K858 at 10 µM. At the established experimental times, the culture medium was replaced by a new one added with 0.5 mg/mL MTT and incubated in the presence of cells at 37 • C for 5 h. The plate was then probed with DMSO for 30 min to allow violet formazan salt dissolution, formed after the reduction of MTT by viable cells. The colored solution was read at 540 nm by a GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Metabolically active cell percentage was obtained by normalizing obtained values with values of DMSO-treated cells.

Cytotoxicity Assay (LDH Assay)
To estimate membrane integrity and to quantify MCF7 cytotoxicity occurrence after treatment with 2 1.5 µM, 41 5 µM, and K858 10 µM, lactate dehydrogenase (LDH) leakage within the medium was measured by means of "CytoTox 96 non-radioactive cytotoxicity assay" (Promega, Madison, WI, USA), following the manufacturer's instructions. The assay was carried out after 24 and 48 h of treatment and the measured LDH leakage in each well was normalized with the optical density values obtained from MTT test.

Transwell Invasion and Migration Assay
Both MCF7 cell invasion and migration assays were carried out by using a 24-well Transwell Boyden chamber consisting of inserts with 8 µm pore size membranes (Corning, Lowell, MA, USA).
For MCF7 cell invasion assay, a thin layer of ECM Gel (Merck Life Science, Milan, Italy), with a final concentration of 0.4 mg/mL (stock solution: 8-12 mg/mL), was used to coat the pores of membranes insert, mimicking the extracellular matrix. An amount of 150 µL of 0.4 mg/mL of ECM gel diluted in ice-cold serum-free DMEM was placed in the upper chamber compartment of the insert and incubated for 2 h at 37 • C to allow gel to solidify. After that, MCF7 were treated with 2 1.5 µM, 41 5 µM, and K858 10 µM in a serum-free DMEM at cell density of 65,000/150 µL and then added to the upper chamber of insert. Medium with 10% of FBS was added to the lower chamber as a chemoattractant and the assay was performed after 48 h of treatment.
For cell migration assay, cells were treated and seeded directly in the upper chamber of insert, as previously described for invasion assay, and the medium with 10% of FBS was added to the lower chamber as a chemoattractant allowing cells to migrate; the assay was performed after 24 h of treatment.
At the established experimental times, remaining cells on the membrane top side were removed using a cotton swab, whereas invading or migrating cells were fixed with 5% of glutaraldehyde for 15 min and stained with crystal violet for 10 min at room temperature. Images were acquired with an inverted light microscope Leica Dmi1 (Leica Cambridge Ltd., Cambridge, UK Leica) provided with a camera Leica MC120 HD (Leica Cambridge Ltd., Cambridge, UK) for the acquisition of digital images. Images were analyzed by Leica Application Suite-X (LAS-X) image analysis software. The invasion and migration levels percentages were established by measuring the surface covered by crystal violet-stained cells.

Wound Healing
When MCF7 cells, seeded in a 6-well tissue culture plate, reached 70% confluence, cell monolayer was scraped with a p200 pipet tip and treated with 2 1.5 µM, 41 5 µM, and K858 10 µM. After 0, 24 and 48 h of treatment the images were taken with an inverted light microscope Leica Dmi1 (Leica Cambridge Ltd., Cambridge, UK Leica) provided with a camera Leica MC120 HD (Leica Cambridge Ltd., Cambridge, UK). The cut width was quantified by LAS-X image software (Leica Cambridge Ltd., Cambridge, UK) and expressed as Pixel.

Protein Extraction and Western Blot analysis
MCF7 cells were seeded in a 6-well plate at 250,000 cells/well density and treated with 2, 41, and K858, as mentioned before. After 24 and 48 h of treatment, cells were trypsinized, centrifuged, and harvested in cold PBS. Pellets were lysed in RIPA buffer containing inhibitors (Phenylmethylsulfonyl fluoride (PMSF) 100 µg/mL, Aprotinin 10 µg/mL, Leupeptin 50 µg/mL and Sodium Orthovanadate 1 mM (all purchased from Merck Life Science, Milan, Italy). A bicinchoninic acid assay (QuantiPro™ BCA Assay kit, Merck Life Science, Milan, Italy) was used to determine protein concentration following the manufacturer's indications.

Detection of Apoptosis and Necrosis by Flow Cytometry
MCF7 cells were cultured in 6-well plates and underwent treatments as previously described, for 48 h. After having harvested supernatants, cells were washed once with PBS at room temperature. Then, cells were detached by trypsin, and supernatants were collected after centrifugation. Staining with annexin V and propidium iodide (PI) (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) was carried out in order to discriminate apoptotic and necrotic cells, according to the manufacturer's instructions. Samples were probed in binding buffer and of annexin V FITC (197 µL +3 µL, respectively), in the dark, for 15 min at room temperature. After that, the volumes were doubled, and the cells were centrifuged and resuspended in binding buffer with PI. A CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) was used to determine the fluorescence. A 530/30 bandpass filter was used to analyze FITC fluorescence (FL-1), and a 650 nm long pass filter for PI fluorescence (FL-3). Data were acquired (2 × 10 4 events/sample) and analyzed by means of the CytExpert Software (Beckman Coulter, Brea, CA, USA). Viable cells percentage (annexin V − ; PI − ) were revealed in the lower left quadrant (unstained) of density plots, cells in apoptosis in lower right quadrant (annexin V + /PI − ), cells in late apoptosis in upper right quadrant (annexin V + /PI + ) and cells in necrosis in upper left quadrant (annexin V − /PI + ).

Statistical Analysis
Data were statistically analyzed by GraphPad 7 software (San Diego, CA, USA) using ordinary one−way ANOVA followed by post hoc Tukey's multiple comparison test.

Effect of K858 and Its Analogues on MCF7 Cell Viability
With the aim of evaluating the capability of newly synthetized Eg5 inhibitors, namely 2, 4, 26, 30, 31, 41 and 44, to affect MCF7 viability, an initial screening, by means of MTT assay, was performed. These compounds are analogues of K858 presenting the same thiadiazoline core nucleus of K858 with different substituents at C5 (Figure 1).

Detection of Apoptosis and Necrosis by Flow Cytometry
MCF7 cells were cultured in 6-well plates and underwent treatments as previously described, for 48 h. After having harvested supernatants, cells were washed once with PBS at room temperature. Then, cells were detached by trypsin, and supernatants were collected after centrifugation. Staining with annexin V and propidium iodide (PI) (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) was carried out in order to discriminate apoptotic and necrotic cells, according to the manufacturer's instructions. Samples were probed in binding buffer and of annexin V FITC (197 μL +3 μL, respectively), in the dark, for 15 min at room temperature. After that, the volumes were doubled, and the cells were centrifuged and resuspended in binding buffer with PI. A CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA) was used to determine the fluorescence. A 530/30 bandpass filter was used to analyze FITC fluorescence (FL-1), and a 650 nm long pass filter for PI fluorescence (FL-3). Data were acquired (2 × 10 4 events/sample) and analyzed by means of the CytExpert Software (Beckman Coulter, Brea, CA, USA). Viable cells percentage (annexin V − ; PI − ) were revealed in the lower left quadrant (unstained) of density plots, cells in apoptosis in lower right quadrant (annexin V + /PI − ), cells in late apoptosis in upper right quadrant (annexin V + /PI + ) and cells in necrosis in upper left quadrant (annexin V − /PI + ).

Statistical Analysis
Data were statistically analyzed by GraphPad 7 software (San Diego, CA, USA) using ordinary one−way ANOVA followed by post hoc Tukey's multiple comparison test.

Effect of K858 and Its Analogues on MCF7 Cell Viability
With the aim of evaluating the capability of newly synthetized Eg5 inhibitors, namely 2, 4, 26, 30, 31, 41 and 44, to affect MCF7 viability, an initial screening, by means of MTT assay, was performed. These compounds are analogues of K858 presenting the same thiadiazoline core nucleus of K858 with different substituents at C5 (Figure 1).  The compounds were tested on MCF7 at 0.5, 5, 50, and 100 µM, while K858 was tested at 0.5, 5, and 50 µM, all for 24 h (Figure 2A The compounds were tested on MCF7 at 0.5, 5, 50, and 100 μM, while K858 was tested at 0.5, 5, and 50 μM, all for 24 h (Figure 2A  After 24 h of treatment at the highest dose administered (100 μM) for compounds 4 and 26, the recorded cell viability percentage is of 80%, approximately. After 48 h of treatment, the viability rate recorded at 50 μM still is higher with respect to that recorded with other compounds and the cell viability percentage reaches 50% only after 72 h of treatment. Compounds 31 and 44 lead to record viability levels higher than 50% after 24 h of treatment (approximately 77% at 50 μM), while after 48 and 72 h they significantly affect cell viability leading to measure percentages lower than 50%. Compound 2 shows a different profile as, after 24 h of treatment and at the lower dose (5 μM) administered it determines After 24 h of treatment at the highest dose administered (100 µM) for compounds 4 and 26, the recorded cell viability percentage is of 80%, approximately. After 48 h of treatment, the viability rate recorded at 50 µM still is higher with respect to that recorded with other compounds and the cell viability percentage reaches 50% only after 72 h of treatment. Compounds 31 and 44 lead to record viability levels higher than 50% after 24 h of treatment (approximately 77% at 50 µM), while after 48 and 72 h they significantly affect cell viability leading to measure percentages lower than 50%. Compound 2 shows a different profile as, after 24 h of treatment and at the lower dose (5 µM) administered it determines a reduction of cell viability at approximately 65%, reaching 39% and 35% after 48 h and Based on these preliminary screening results, 2, 30, and 41 were selected for further analyses on MCF7. Firstly, a new viability test was performed in order to establish a subtoxic concentration to be applied in future experiments. MCF7 were treated with 2, 30, and 41 at doses ranging from 1.560 µM to 100 µM, whereas K858 was administered at 0.05, 0.5, 5, and 50 µM, all for 48 and 72 h ( Figure 3). These three compounds were also characterized by good gastrointestinal absorption, discrete aqueous solubility, and no attitude to act as PAINS (Pan-Assay INterfering assayS) according to in silico evaluation using commercially available web tools. whereas 10 µM as subtoxic concentration is chosen for K858, as reported by De Iuliis and co-workers [20] and confirmed by our cell viability results.

Effect of 2, 41, and K858 on non-Tumoral Cells Viability
With the aim of evaluating the effect of Eg5 inhibitors at the selected concentrations on non-tumoral cells, 2, 41, and K858 were administered to HGFs and cell viability percentages, obtained from the MTT test, were read. After 24 h of treatment, K858 at 10 µM affects HGF viability in a significant manner: 60% approximately compared to untreated cells (100%), to 2 at 1.5 µM (100%), and to 41 at 5 µM (94%) ( Figure 4A).  [20] and confirmed by our cell viability results.

Effect of 2, 41, and K858 on non-Tumoral Cells Viability
With the aim of evaluating the effect of Eg5 inhibitors at the selected concentrations on non-tumoral cells, 2, 41, and K858 were administered to HGFs and cell viability percentages, obtained from the MTT test, were read. After 24 h of treatment, K858 at 10 μM affects HGF viability in a significant manner: 60% approximately compared to untreated cells (100%), to 2 at 1.5 μM (100%), and to 41 at 5 μM (94%) ( Figure 4A). After 48 h of treatment, a similar trend is evidenced: K858 decreases cell viability in a statistically significant manner (61%) compared to DMSO (100%), to 2 (92%), and to 41 (77%). Compounds 2 and 41 induce a significant cell viability reduction compared to DMSO ( Figure 4B).

Effect of 2, 41, and K858 on MCF7 Cytotoxicity
The MCF7 cytotoxicity was then evaluated by measuring the released LDH level within the culture medium, after 24 and 48 h of treatment with 2, 41, and K858 at the established concentrations. After 24 h, K858 determines a statistically significant increase in LDH release with respect to 2, 41 and to untreated cells (LDH released: DMSO = 35.78%, 2 = 57.60%, 41 = 62.14%, K858 = 97.13%) ( Figure 5A). further investigations, excluding compound 30, in order to explore similarities or differences in the effect exerted on MCF7 cell line. Thus, based on the results obtained from the MTT test, 1.5 μM for 2 and 5 μM for 41 are chosen as subtoxic concentrations for future experiments, whereas 10 μM as subtoxic concentration is chosen for K858, as reported by De Iuliis and co-workers [20] and confirmed by our cell viability results.

Effect of 2, 41, and K858 on non-Tumoral Cells Viability
With the aim of evaluating the effect of Eg5 inhibitors at the selected concentrations on non-tumoral cells, 2, 41, and K858 were administered to HGFs and cell viability percentages, obtained from the MTT test, were read. After 24 h of treatment, K858 at 10 μM affects HGF viability in a significant manner: 60% approximately compared to untreated cells (100%), to 2 at 1.5 μM (100%), and to 41 at 5 μM (94%) ( Figure 4A). After 48 h of treatment, a similar trend is evidenced: K858 decreases cell viability in a statistically significant manner (61%) compared to DMSO (100%), to 2 (92%), and to 41 (77%). Compounds 2 and 41 induce a significant cell viability reduction compared to DMSO ( Figure 4B).

Effect of 2, 41, and K858 on MCF7 Cytotoxicity
The MCF7 cytotoxicity was then evaluated by measuring the released LDH level within the culture medium, after 24 and 48 h of treatment with 2, 41, and K858 at the established concentrations. After 24 h, K858 determines a statistically significant increase in LDH release with respect to 2, 41 and to untreated cells (LDH released: DMSO = 35.78%, 2 = 57.60%, 41 = 62.14%, K858 = 97.13%) ( Figure 5A). After 48 h of treatment, the trend is maintained as K858 induces a higher significant LDH release (100%) with respect to untreated cells (16.6%), 2-treated (51.6%), and 41-treated (61.5%) cells; moreover, after 48 h cells exposed to 2 and to 41 disclose a significant increase in LDH release with respect to untreated cells ( Figure 5B).

Effect of Eg5
Inhibitors on MCF7 Cell Invasion, Migration and on NF-kB, MMP-9, HIF-1α, and VEGF Proteins Expression A specific assay was carried out to investigate MCF7 invasion capability after 48 h of treatment with 2, 41, and K858. K858 discloses a statistically significant reduction in the cell invasion percentage (28.7%) compared to untreated cells (44%), while 2 and 41 do not lead to significant cell invasion % reduction with respect to DMSO (36% and 39%, respectively) ( Figure 6). After 48 h of treatment, the trend is maintained as K858 induces a higher significant LDH release (100%) with respect to untreated cells (16.6%), 2-treated (51.6%), and 41treated (61.5%) cells; moreover, after 48 h cells exposed to 2 and to 41 disclose a significant increase in LDH release with respect to untreated cells ( Figure 5B).

Effect of Eg5 Inhibitors on MCF7 Cell Invasion, Migration and on NF-kB, MMP-9, HIF-1α, and VEGF Proteins Expression
A specific assay was carried out to investigate MCF7 invasion capability after 48 h of treatment with 2, 41, and K858. K858 discloses a statistically significant reduction in the cell invasion percentage (28.7%) compared to untreated cells (44%), while 2 and 41 do not lead to significant cell invasion % reduction with respect to DMSO (36% and 39%, respectively) ( Figure 6). A migration and a wound healing assay, after 24 h and after 24 h and 48 h, respectively, were performed on MCF7 to evaluate 2, 41, and K858 capability in controlling cell migration. After administration of 2, 41, and K858 at the established concentrations for 24 h, untreated MCF7 migration is of 35% approximately, while after 41 and K858 treatment the migration is significantly reduced with respect to DMSO (22% and 17%, respectively). Further, 2 shows a strong significant capability to suppress cell migration with respect to untreated cells (3%); both new Eg5 inhibitors 2 and 41 are able to reduce MCF7 cell migration in a statistically significant manner compared to the parent compound K858 (Figure 7). A migration and a wound healing assay, after 24 h and after 24 h and 48 h, respectively, were performed on MCF7 to evaluate 2, 41, and K858 capability in controlling cell migration. After administration of 2, 41, and K858 at the established concentrations for 24 h, untreated MCF7 migration is of 35% approximately, while after 41 and K858 treatment the migration is significantly reduced with respect to DMSO (22% and 17%, respectively). Further, 2 shows a strong significant capability to suppress cell migration with respect to untreated cells (3%); both new Eg5 inhibitors 2 and 41 are able to reduce MCF7 cell migration in a statistically significant manner compared to the parent compound K858 (Figure 7). These results are confirmed by wound healing assay: after 24 h, untreated cells show a reduction of cut width, with respect to 0 h, and after 48 h this cut is almost completely closed. A similar reduction of cut width is recorded in MCF7 treated with 2, 41, and K858 after 24 h of treatment. Conversely, after 48 h the cut width of MCF7 exposed to all Eg5 inhibitors does not show any narrowing compared to the cut width of cells exposed to DMSO (Figure 8).  These results are confirmed by wound healing assay: after 24 h, untreated cells show a reduction of cut width, with respect to 0 h, and after 48 h this cut is almost completely closed. A similar reduction of cut width is recorded in MCF7 treated with 2, 41, and K858 after 24 h of treatment. Conversely, after 48 h the cut width of MCF7 exposed to all Eg5 inhibitors does not show any narrowing compared to the cut width of cells exposed to DMSO (Figure 8). These results are confirmed by wound healing assay: after 24 h, untreated cells show a reduction of cut width, with respect to 0 h, and after 48 h this cut is almost completely closed. A similar reduction of cut width is recorded in MCF7 treated with 2, 41, and K858 after 24 h of treatment. Conversely, after 48 h the cut width of MCF7 exposed to all Eg5 inhibitors does not show any narrowing compared to the cut width of cells exposed to DMSO (Figure 8).  To better clarify the signaling cascades recruited by 2, 41, and K858 in cell invasion and migration processes, nuclear factor kappa-B (NF-kB) and matrix metalloproteinase-9 (MMP-9) expression were evaluated by means of western blot analysis. As NF-kB is a nuclear transcription factor, its expression was measured earlier (24 h) than the downstream molecule. After 24 h of treatment, all tested Eg5 inhibitors reduce NF-kB expression with respect to untreated cells; furthermore, 2 and 41 determine a statistically significant protein expression decrease compared to the parent compound K858 ( Figure 9A). To better clarify the signaling cascades recruited by 2, 41, and K858 in cell invasion and migration processes, nuclear factor kappa-B (NF-kB) and matrix metalloproteinase-9 (MMP-9) expression were evaluated by means of western blot analysis. As NF-kB is a nuclear transcription factor, its expression was measured earlier (24 h) than the downstream molecule. After 24 h of treatment, all tested Eg5 inhibitors reduce NF-kB expression with respect to untreated cells; furthermore, 2 and 41 determine a statistically significant protein expression decrease compared to the parent compound K858 ( Figure 9A). A similar trend is recorded for MMP-9: after 24 h of treatment 2, 41 and K858 significantly reduce the MMP-9 expression compared to DMSO; conversely, 41 decreases MMP-9 expression levels compared to K858. After 48 h of treatment, 2 and K858 decrease MMP-9 expression in a statistically significant manner compared to untreated cells and, in addition, K858 induces a significant reduction in protein expression compared to 41 ( Figure  9B).
Considering that the invasion and migration capabilities of cancer cells are closely related to the angiogenic process, hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins expression was also estimated after 24 h of treatment with Eg5 inhibitors by means of western blot analysis. All compounds provoke a strong and significant reduction of HIF-1α expression with respect to control; moreover, 2 and 41 significantly decrease HIF-1α expression with respect to K858 (Figure 10). A similar trend is recorded for MMP-9: after 24 h of treatment 2, 41 and K858 significantly reduce the MMP-9 expression compared to DMSO; conversely, 41 decreases MMP-9 expression levels compared to K858. After 48 h of treatment, 2 and K858 decrease MMP-9 expression in a statistically significant manner compared to untreated cells and, in addition, K858 induces a significant reduction in protein expression compared to 41 ( Figure 9B).
Considering that the invasion and migration capabilities of cancer cells are closely related to the angiogenic process, hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins expression was also estimated after 24 h of treatment with Eg5 inhibitors by means of western blot analysis. All compounds provoke a strong and significant reduction of HIF-1α expression with respect to control; moreover, 2 and 41 significantly decrease HIF-1α expression with respect to K858 (Figure 10). After 24 h of treatment VEGF expression levels appear significantly reduced in the presence of 2 and 41 with respect to control, while no significant modification is evidenced after exposure to K858; 41 significantly decreases VEGF expression with respect to K858 ( Figure 10B).

Evaluation of MCF7 Apoptosis and Necrosis Induction by 2, 41, and K858 Treatment
After exposure to 2, 41, and K858 for 48 h, apoptosis and necrosis occurrence were evaluated by flow cytometry. Untreated cells appear viable, being negative for both annexin V and PI, with about 75% of cell viability. Conversely, all compounds induce a statistically significant reduction in the percentage of viable cells with respect to DMSO, with a major extent for MCF7 treated with 41 (54%) and K858 (52%). The percentage of apoptotic cells is significantly augmented when MCF7 are exposed to 2 (16%), 41 (17%), and K858 (15%) with respect to untreated cells (6%); 41 and K858 also induce an increase in necrotic cells (28% and 31%, respectively), in a statistically significant manner with respect to control (Figure 11). After 24 h of treatment VEGF expression levels appear significantly reduced in the presence of 2 and 41 with respect to control, while no significant modification is evidenced after exposure to K858; 41 significantly decreases VEGF expression with respect to K858 ( Figure 10B).

Evaluation of MCF7 Apoptosis and Necrosis Induction by 2, 41, and K858 Treatment
After exposure to 2, 41, and K858 for 48 h, apoptosis and necrosis occurrence were evaluated by flow cytometry. Untreated cells appear viable, being negative for both annexin V and PI, with about 75% of cell viability. Conversely, all compounds induce a statistically significant reduction in the percentage of viable cells with respect to DMSO, with a major extent for MCF7 treated with 41 (54%) and K858 (52%). The percentage of apoptotic cells is significantly augmented when MCF7 are exposed to 2 (16%), 41 (17%), and K858 (15%) with respect to untreated cells (6%); 41 and K858 also induce an increase in necrotic cells (28% and 31%, respectively), in a statistically significant manner with respect to control ( Figure 11). Therefore, the expression of a pro-apoptotic and an anti-apoptotic protein, represented by Bax and Bcl-2, respectively, was measured through a western blot after 24 h of treatment. Bax expression is statistically increased after treatment with K858 compared to DMSO and to 2 and 41 ( Figure 12A); the same trend is recorded for Bax/Bcl-2 ratio ( Figure 12B). Therefore, the expression of a pro-apoptotic and an anti-apoptotic protein, represented by Bax and Bcl-2, respectively, was measured through a western blot after 24 h of treatment. Bax expression is statistically increased after treatment with K858 compared to DMSO and to 2 and 41 ( Figure 12A); the same trend is recorded for Bax/Bcl-2 ratio ( Figure  12B).  Therefore, the expression of a pro-apoptotic and an anti-apoptotic protein, represented by Bax and Bcl-2, respectively, was measured through a western blot after 24 h of treatment. Bax expression is statistically increased after treatment with K858 compared to DMSO and to 2 and 41 ( Figure 12A); the same trend is recorded for Bax/Bcl-2 ratio ( Figure  12B). Lastly, in order to shed light and better clarify the apoptosis and necrosis occurrence during treatment with novel Eg5 inhibitors, caspase-3, and poly(ADP-ribose) polymerase (PARP) expression levels were also quantified. Compound 2 statistically increases the caspase-3 cleaved/full-length ratio with respect to untreated cells and to K858. Compounds 41 and K858 provoke an augmentation of caspase-3 cleaved/full-length ratio compared to control, while there is not a statistically significant difference between 41 and K858 ( Figure 13A). independent experiments. Tubulin is used as a loading control. (A): The bar graph displays Bax densitometric values normalized on loading control. *** p < 0.0001 DMSO vs. K858; ### p < 0.0001 K858 vs. 2 and 41. (B): The bar graph displays densitometric values expressed as mean ± SD of Bax/Bcl-2 ratio normalized on loading control. * p < 0.05 DMSO vs. K858; ## p < 0.0074 K858 vs. 41; # p < 0.05 K858 vs. 2.
Lastly, in order to shed light and better clarify the apoptosis and necrosis occurrence during treatment with novel Eg5 inhibitors, caspase-3, and poly(ADP-ribose) polymerase (PARP) expression levels were also quantified. Compound 2 statistically increases the caspase-3 cleaved/full-length ratio with respect to untreated cells and to K858. Compounds 41 and K858 provoke an augmentation of caspase-3 cleaved/full-length ratio compared to control, while there is not a statistically significant difference between 41 and K858 ( Figure 13A). In contrast, compound 2 induces a statistically significant increase in PARP cleaved/full-length ratio in comparison to untreated cells, whereas 41 and K858 provoke a significant increase in PARP cleaved/full-length ratio compared to DMSO. In addition, the parent compound K858 increases the expression of PARP in a statistically significant manner with respect to 2 ( Figure 13B). All original images of western blot are available in the Supplementary material ( Figure S1-S11) In contrast, compound 2 induces a statistically significant increase in PARP cleaved/ full-length ratio in comparison to untreated cells, whereas 41 and K858 provoke a significant increase in PARP cleaved/full-length ratio compared to DMSO. In addition, the parent compound K858 increases the expression of PARP in a statistically significant manner with respect to 2 ( Figure 13B). All original images of western blot are available in the Supplementary material ( Figures S1-S11)

Discussion
Considering the necessity of discovering new molecules to overcome side effects and tumor resistance to common anticancer drugs and based on the possibility to find new molecular targets to treat patients, researchers spent efforts on the purpose of finding new therapies including innovative compounds. With this aim, new targets have been explored, among them, kinesin Eg5, which has been demonstrated to be involved in several widely diffused cancers [11][12][13][14][15].
Seven newly synthetized Eg5 inhibitors were already tested on gastric adenocarcinoma cell line and two of them, 2 and 41, were chosen for their promising in vitro effects in counteracting tumor cell viability and in negatively modulating angiogenic cascade [24,25]. Based on these results on gastric adenocarcinoma and since Eg5 has been reported to be overexpressed also in BC [17], kinesin Eg5 inhibitors, along with the parent compound K858, were tested on MCF7 breast adenocarcinoma cell line. As already found in our previous work [24], compounds 2 and 41 were selected from a wider panel for their capability to counteract MCF7 viability at lower concentrations, thus, 1.5 µM and 5 µM, for 2 and 41, respectively, were established as subtoxic concentrations. Conversely, based on both what was reported in the scientific literature [20] and on our results, the parent compound K858 required a higher concentration, 10 µM, to obtain the same effect in terms of MCF7 viability reduction. This concentration is probably also responsible for the higher MCF7 cytotoxicity and percentage of dead healthy cells registered for K858 and not for 2 and 41. These preliminary findings underline the possibility to administer 2 and 41 at lower doses to preserve healthy cell viability.
The main cause of BC mortality is metastasis formation: in this process cancer cells spread outside the primary tumor mass to form secondary tumoral sites, far from the origin point [28]. During this process cell invasion represents the first step, in which tumor cells penetrate the basement barrier and enter nearby tissues. After the invasion, cancer cells' migration through the extracellular matrix (ECM) represents the second critical stage [29]. Previous works already reported that in different in vitro models, Eg5 inhibition could represent a valid strategy to counteract cancer cell invasion and migration [30]. Our experimental model results disclose the K858 capability to inhibit MCF7 cell invasion, thus validating the same feature previously demonstrated against head and neck squamous cancer cells [31] highlighting an appreciable K858 ability to reduce cell migration. Compounds 2 and 41 have already evidenced a strong effect in counteracting gastric adenocarcinoma cell migration [24], especially when 2 was combined with the hesperidin polyphenol [25]. This aspect was corroborated in MCF7 in which, in addition, the capability of 2 to control cell migration markedly exceeds K858 confirming the active role of Eg5 inhibition in preventing the key passages of metastasis formation.
The activity of matrix metalloproteinases (MMPs) is known to be crucial for cancer cell spread, in particular, MMP-9 triggers the degradation of ECM essential proteins, such as type IV collagen, allowing the spreading of cancer cells [32]. Previous reports have evidenced that K858 inhibits MMP-9 expression in human glioblastoma cells [33]. This finding is supported by our experimental model results, in which K858 is shown to reduce MMP-9 expression. Interestingly, the downregulation of MMP-9 expression is enhanced in presence of 2 and 41 with respect to K858. Several papers demonstrated that, in experimental tumor models among which breast cancer is included, a correlation between NF-kB and MMP-9 exists [34][35][36]. In detail, the transcriptional factor NF-kB can translocate into the nucleus activating MMP-9 gene transcription. Considering that MMP-9 expression markedly reflects NF-kB levels it is likely to suppose that novel Eg5 inhibitors can counteract MMP-9 expression through NF-kB inhibition even if further studies of upstream NF-kB silencing are needed to confirm this hypothesis The degradation of ECM, led by MMP-9, speeds up the angiogenic process [37], essential for tumor mass growth, by promoting the release of downstream angiogenic factors in the tumor microenvironment, such as VEGF [38]. In fact, it is widely recognized that MMP-9 is synthesized by cancer cells regulating VEGF release promoting angiogenesis and spread of metastasis [39]. Conversely, MMP-9 inhibition leads to a reduction of endothelial cell invasion [40]. Furthermore, a correlation between VEGF and Eg5 has been already demonstrated: after human recombinant VEGF administration in endothelial cells, the gene encoding for Eg5 protein, namely KIF11, appeared upregulated [41]. Our results clearly confirm both these points. In fact, 2 and 41 negatively modulate VEGF expression, more than K858, as already demonstrated in the gastric adenocarcinoma model [25]. In parallel, the comparable levels of MMP-9 and VEGF lead to assume that a correlation between the two molecules could exist.
During the tumor mass growth, the oxygen supply is often poor, especially at the center of the mass, thus generating hypoxic regions. Cell survival in hypoxic conditions requires the activation of survival pathways recruiting the HIF-1α transcription factor [42] which, in turn, is involved in the angiogenetic process by transcriptionally activating several factors and their receptors, such as VEGF [43]. Our model demonstrated that kinesin inhibitors are able to downregulate HIF-1α expression. Taken together these results revealed that 2 and 41 kinesin inhibitors, better than their parent compound K858, negatively control VEGF activation by simultaneously acting both on MMP-9 and on HIF-1α, thus significantly reducing the possibility of VEGF recruitment. The results regarding the Eg5 inhibitors' capability to counteract cell migration, invasion, and angiogenic pathways recruitment led us to focus our attention not only on the mitotic properties of Eg5 but also on the non-mitotic activities of this protein. In fact, among the non-mitotic functions, it has been proposed that Eg5 could also function as a movable molecular bridge between microtubules and ribosomes to improve the effectiveness of polypeptide synthesis [44] and also the transport of secretory proteins from the Golgi complex to the cell surface [45]. Thus, it could be argued that, through Eg5 inhibition, a block of the synthesis and of the transport of crucial proteins involved in cell migration and invasion occurs, as demonstrated also in our experimental model.
The ability of K858, 2, and 41 to induce apoptosis, because of Eg5 inhibition and mitotic process failure, has been already reported in the scientific literature and by our group [20,24,31,33,46]. This ability to induce apoptosis is not an exclusive prerogative of K858 and its analogs: several compounds, belonging to different chemical classes of Eg5 inhibitors, such as monastrol, ispinesib, filasenib, etc., are able to trigger apoptotic cascade in many types of cancer models [47][48][49]. All these classes of Eg5 inhibitors are able to bind to an allosteric site located in the motor domain, formed by helix α2, loop L5, and helix α3. The present results are in accordance with previous findings: Eg5 inhibitors appeared to induce MCF7 apoptosis, as demonstrated both by the annexin PI results and by the increased caspase-3 and PARP levels. The high levels of Bax expression recorded after K858 treatment lead to suppose that the parent compound could activate the intrinsic apoptotic pathway, whereas 2 and 41 recruit alternative molecular signaling. In particular, it is likely that 2 triggers the extrinsic apoptotic pathway, as it provokes a caspase-3 activation without Bax recruitment; conversely, 41 seems to activate the necrotic pathway, as suggested by annexin PI results and by the higher level of PARP.

Conclusions
These findings shed light on two points. Firstly, they underline that kinesin Eg5 represents a valid and alternative target for advanced cancer therapies. Secondly, among thiadiazoline-based Eg5 inhibitors, 2 and 41, simultaneously modulating several key processes of breast cancer spread and progression, such as cell proliferation, invasiveness, migration, ECM remodeling, and angiogenesis, along with apoptosis or necrosis occurrence, represent a new drug strategy to control breast cancer. As these are preliminary results derived from newly synthesized molecules, further detailed studies are needed to deepen the molecular mechanisms underlying Eg5 inhibitors administration and to aim at in vivo trials.
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by Local Ethical Committee of the University of Chieti.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are contained within the article and available from the corresponding author.