Pro-Apoptotic and Anti-Migration Properties of a Thiazoline-Containing Platinum(II) Complex in MDA-MB-231 Breast Cancer Cells: The Role of Melatonin as a Synergistic Agent

Triple-negative breast cancer (TNBC) is an aggressive cancer insensitive to hormonal and human epidermal growth factor receptor 2 (HER2)-targeted therapies and has a poor prognosis. Therefore, there is a need for the development of convenient anticancer strategies for the management of TNBC. In this paper, we evaluate the antitumoral potential of a platinum(II) complex coordinated with the ligand 2-(3,5-diphenylpyrazol-1-yl)-2-thiazoline (DPhPzTn), hereafter PtDPhPzTn, against the TNBC cell line MDA-MB-231, and compared its effect with both cisplatin and its less lipophilic counterpart PtPzTn, the latter containing the ligand 2-(pyrazol-1-yl)-2-thiazoline (PzTn). Then, the putative potentiating actions of melatonin, a naturally occurring antioxidant with renowned antitumor properties, on the tumor-killing ability of PtDPhPzTn were also checked in TNBC cells. Our results show that PtDPhPzTn presented enhanced cytotoxicity compared to both the classical drug cisplatin and PtPzTn. In addition, PtDPhPzTn was able to induce apoptosis, being more selective for MDA-MB-231 cells when compared to non-tumor breast epithelial MCF10A cells. Likewise, PtDPhPzTn produced moderate S phase arrest and greatly impaired the migration ability of MDA-MB-231 cells. Most importantly, the co-stimulation of TNBC cells with PtDPhPzTn and melatonin substantially enhanced apoptosis and markedly improved the anti-migratory action compared to PtDPhPzTn alone. Altogether, our findings provide evidence that PtDPhPzTn and melatonin could be potentially applied to breast cancer treatment as powerful synergistic agents.


Introduction
Triple-negative breast cancer (TNBC) is the least common type of breast cancers, accounting for 10-15% of all cases, but is often more aggressive and difficult to treat due to the lack of efficient molecular targets, such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [1]. Chemotherapy is one of the most widely used therapeutic modalities in cancer treatment, which includes a wide variety of drugs. Chemotherapeutic agents induce apoptotic cell death not only in tumor cells but also in normal non-tumor cells, thus producing both therapeutic effects (i.e., killing tumor cells) and undesirable side effects (e.g., nephrotoxicity and cardiotoxicity).
Most metal-based antitumor therapies, such as cis-diamminedichloroplatinum(II) (cisplatin), have been successfully employed for more than 40 years. However, the use of cisplatin may be limited by the intrinsic or acquired resistance of various types of cancer and its toxic side effects. With the idea of overcoming limitations of cisplatin therapy, platinum Diamminedichloroplatinum(II) (cisplatin), melatonin, hydrocortisone, insulin, epidermal growth factor (EGF), and cholera toxin were bought from Sigma Aldrich (Madrid, Spain). All other reagents were of analytical/commercial grade and were used without further purification. The ligands PzTn and DPhPzTn [23,24] and the precursor [PtCl 2 (DMSO) 2 ] [25] were synthesized as described in the literature.
For the elemental analysis, a Leco CHNS-932 microanalyzer was used. IR spectra were recorded on a Perkin-Elmer 100 FTIR spectrophotometer from KBr pellets in the 4000-400 cm −1 range. 1 H NMR spectra were obtained with a Bruker Avance III 500 instrument at 500 MHz in DMF-d7. 1 H NMR signals were referenced to residual proton resonances in deuterated solvents.

General Preparation of Platinum(II) Complexes
To perform the synthesis (Scheme 1), (300.0 mg, 0.71 mmol) the [PtCl 2 (DMSO) 2 ] precursor was dissolved in 30 mL of ethanol in an Elenmeyer flask. On the other hand, the ligand was dissolved in 20 mL of ethanol. Once dissolved, the ligand corresponding was added to the precursor and it was refluxed and stirred for 24 h in the dark. The powder formed was vacuum filtered off and then recrystallized in DMF (for PtPzTn) or acetonitrile (for PtDPhPzTn). After that, crystals were separated by filtration, washed with distilled water and cold diethyl ether, and air-dried.

General Preparation of Platinum(II) Complexes
To perform the synthesis (Scheme 1), (300.0 mg, 0.71 mmol) the [PtCl2(DMSO)2] precursor was dissolved in 30 mL of ethanol in an Elenmeyer flask. On the other hand, the ligand was dissolved in 20 mL of ethanol. Once dissolved, the ligand corresponding was added to the precursor and it was refluxed and stirred for 24 h in the dark. The powder formed was vacuum filtered off and then recrystallized in DMF (for PtPzTn) or acetonitrile (for PtDPhPzTn). After that, crystals were separated by filtration, washed with distilled water and cold diethyl ether, and air-dried.

Cell Culture and Treatments
The cell lines displayed in Table 1 were cultured into 25 cm 2 culture flasks with the indicated media and supplements. Cells were kept under a humidified atmosphere containing 5% CO 2 at 37 • C, and their viability was routinely tested by the Trypan-blue exclusion method. Cells were challenged with the platinum(II) complexes PtDPhPzTn and PtPzTn, the reference drug cisplatin, melatonin, or vehicle for 24 h at the indicated

Cytotoxicity Assay
To examine the cytotoxic effects of the drugs on the different cell lines, the MTS method was performed by means of the CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega, Madrid, Spain), as previously reported [6,7]. The experiments were run in triplicate and cells were challenged with the different drugs for 24 h.

Determination of Apoptotic Cells
The population of apoptotic cells was studied by analyzing phosphatidylserine externalization via annexin V-FITC/propidium iodide (PI) assay (ThermoFisher Scientific, Barcelona, Spain), as previously described [14]. The percentages of live, early apoptotic, late apoptotic, and secondary necrotic cells were computed.

Caspase-3 and -9 Analysis
Caspase-3 and -9 activation was measured with CaspGLOW fluorescein active caspase-3 staining kit and Caspase-9 staining kit (Red), respectively, as published elsewhere [4]. The analysis of caspase activation was restricted to live cells, which were detected by using the vital dye Hoechst 33258 (10 µg/mL). Data are presented as the percentage of caspase-positive cells.

Measurement of Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) detection was performed by incubating cells (30 min, 37 • C) with 0.4 µM 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA), as previously reported [4]. Again, the analysis of ROS production was restricted to live cells, which were detected by using the vital dye Hoechst 33258 (10 µg/mL). Data are portrayed as the percentage of DCF-stained cells.

Cell Cycle Analysis
To investigate changes in cell cycle progression upon treatment, a cell cycle distribution analysis was carried out in ethanol-fixed, PI-stained cells, as published elsewhere [4]. The Antioxidants 2022, 11,1971 5 of 14 populations of cells in G0/G1 transition, S phase, and G2/M transition, as well as sub-G1 (hypodiploid) cells are reported.

Wound-Healing Assay
Cells were seeded into 24-well plates and incubated at 37 • C until they reached 75-80% confluency. To evaluate cell migration, cells were equally wounded with a sterile 100 µL pipette tip and were subsequently challenged with the different treatments. The experiments were run in duplicate. The relative cell migration rates over 12 and 24 h were quantified based on the changes in wound area at time 0 h and after 12 or 24 h using an epifluorescence microscope (Nikon Eclipse TS100). The images were analyzed with the plugin "Wound healing size tool" available in Fiji/ImageJ software version 1.49 (https://github.com/AlejandraArnedo/Wound-healing-size-tool/wiki, accessed on 1 July 2022) [28].

Transwell Migration Assay
To assess cell migration, transwell migration assay was carried out in transwell chambers with non-coated membrane (24-well insert, pore size: 8 mm, Millipore, Madrid, Spain). Cells (1 × 10 5 ) were plated in serum free medium in the upper chamber. The medium containing 10% FBS was added in the lower chamber to act as chemoattractant. After 24 h, the cells that migrated through the pores to the lower surface of the inserts were fixed in 70% ethanol solution for 10 min and afterwards stained with crystal violet for additional 10 min. Inserts were then imaged and, subsequently, the membranes of the inserts were cut, placed in a 24-well plate, and incubated with 5% sodium dodecyl sulfate (SDS) to extract crystal violet (5 min on orbital shaker). Finally, the absorbance was read at 560 nm for quantification.

Statistical Analysis
Data represent the mean ± standard deviation (S.D.). To compare between treatments, statistical significance was calculated by a one-way analysis of variance (ANOVA) followed by Dunnett's test, unless otherwise specified. The half maximal inhibitory concentration (IC 50 ) values of the dose-response curves were calculated by using nonlinear regression. p < 0.05 was considered to indicate a statistically significant difference. The statistics software used was GraphPad Prism 7.04 for Windows (GraphPad Software, San Diego, CA, USA).

Results and Discussion
The biological activity of a thiazoline-containing platinum(II) complex PtDPhPzTn was first assessed. As depicted in Figure 1C, PtDPhPzTn exhibited a dose-dependent cytotoxic effect against the TNBC cell line MDA-MB-231 with an IC 50 value of 10.47 ± 1.02 µM ( Figure 1D), which was more than 5-fold lower than that of the reference drug cisplatin (56.80 ± 2.41 µM, Figure 1A,D) and more than 3-fold lower than that of its less lipophilic counterpart PtPzTn (33.51 ± 3.83 µM, Figure 1B,D). The same trend was observed when the metallodrugs were tested in other cell lines, namely U-937, HL-60, and HeLa ( Figure 1D), thereby demonstrating that PtDPhPzTn outperforms the reference drug cisplatin (and PtPzTn) in terms of cytotoxicity, though this fact was especially striking in the case of MDA-MB-231 cells, which were moderately resistant to cisplatin (IC 50 > 50 µM). These findings agree with our previous research [7], thus indicating that the incorporation of aromatic groups in the ligand can lead to the improved cytotoxicity of metallodrugs.
1D), thereby demonstrating that PtDPhPzTn outperforms the reference drug cisplatin (and PtPzTn) in terms of cytotoxicity, though this fact was especially striking in the case of MDA-MB-231 cells, which were moderately resistant to cisplatin (IC50 > 50 µM). These findings agree with our previous research [7], thus indicating that the incorporation of aromatic groups in the ligand can lead to the improved cytotoxicity of metallodrugs. Given that the platinum(II) complex PtDPhPzTn was the most effective drug in terms of cytotoxicity and the well-known anti-proliferative and pro-oxidant properties of the indoleamine melatonin in tumor cells, the apoptosis-promoting effect of the combinatory treatment with PtDPhPzTn and melatonin was determined in TNBC cells by analyzing phosphatidylserine externalization in the presence of PI. For this purpose, MDA-MB-231 cells were exposed at the fixed IC50 dose level of PtDPhPzTn (i.e., 10.4 µM) in the absence or presence of 1 mM melatonin. As shown in Figure 2, the platinum(II) complex caused a remarkable increase (33.25 ± 4.34% vs. 2.78 ± 1.04%; p < 0.05) in the population of secondary necrotic cells at the expense of a significant drop (32.13 ± 11.98% vs. 73.63 ± 8.38%; p < 0.05) in the proportion of live cells with respect to the control cells (Figure 2A,B), which is in line with our preliminary findings, indicating that the cytotoxic effect of PtDPhPzTn was associated with the induction of ROS-mediated apoptosis [7]. Previous studies have reported that pyrazole-platinum(II) complexes, especially those containing methyl substituents at the pyrazole ring, exhibited strong pro-apoptotic activity in MDA-MB-231 and MCF-7 breast cancer cell lines [29]. Likewise, dinuclear berenil-platinum(II) complexes were also described to trigger cellular oxidative modifications and, subsequently, apoptosis in MDA-MB-231 and MCF-7 breast cancer cell lines [30,31]. Melatonin per se did not Given that the platinum(II) complex PtDPhPzTn was the most effective drug in terms of cytotoxicity and the well-known anti-proliferative and pro-oxidant properties of the indoleamine melatonin in tumor cells, the apoptosis-promoting effect of the combinatory treatment with PtDPhPzTn and melatonin was determined in TNBC cells by analyzing phosphatidylserine externalization in the presence of PI. For this purpose, MDA-MB-231 cells were exposed at the fixed IC 50 dose level of PtDPhPzTn (i.e., 10.4 µM) in the absence or presence of 1 mM melatonin. As shown in Figure 2, the platinum(II) complex caused a remarkable increase (33.25 ± 4.34% vs. 2.78 ± 1.04%; p < 0.05) in the population of secondary necrotic cells at the expense of a significant drop (32.13 ± 11.98% vs. 73.63 ± 8.38%; p < 0.05) in the proportion of live cells with respect to the control cells (Figure 2A,B), which is in line with our preliminary findings, indicating that the cytotoxic effect of PtDPhPzTn was associated with the induction of ROS-mediated apoptosis [7]. Previous studies have reported that pyrazole-platinum(II) complexes, especially those containing methyl substituents at the pyrazole ring, exhibited strong pro-apoptotic activity in MDA-MB-231 and MCF-7 breast cancer cell lines [29]. Likewise, dinuclear berenil-platinum(II) complexes were also described to trigger cellular oxidative modifications and, subsequently, apoptosis in MDA-MB-231 and MCF-7 breast cancer cell lines [30,31]. Melatonin per se did not produce any significant pro-apoptotic effect on MDA-MB-231 cells (Figure 2A,B). Interestingly, the combinatory treatment with PtDPhPzTn and melatonin further promoted apoptosis in TNBC cells, as demonstrated by a notable rise (49.11 ± 10.86% vs. 33.25 ± 4.34%; p < 0.05) in the percentage of secondary necrotic cells compared to the PtDPhPzTn-treated cells (Figure 2A,B). In this sense, melatonin has been previously shown to synergistically enhance antitumor function in tunicamycin-challenged MDA-MB-231 cells [32], which is in line with the results reported in this paper. The potentiating actions of melatonin on apoptosis induction by classical drugs, such as doxorubicin, lapatinib, and arsenic trioxide, has been also documented in breast cancer cell lines [33][34][35][36] and may be related to its prooxidant capacity in tumor cells [36]. To corroborate the combinatorial effect of PtDPhPzTn and melatonin in MDA-MB-231 cells, 5, 10, and 25 µM of the platinum(II) complex and 0.5, 1, and 2 mM of the indoleamine were combined and cell death was determined after 24 h to calculate the CI values ( Figure S1). Our results show a synergistic effect of the combination of PtDPhPzTn and melatonin, especially when combining 1 mM melatonin and the three different concentrations of the platinum(II) complex (Table S1).
produce any significant pro-apoptotic effect on MDA-MB-231 cells (Figure 2A,B). Interestingly, the combinatory treatment with PtDPhPzTn and melatonin further promoted apoptosis in TNBC cells, as demonstrated by a notable rise (49.11 ± 10.86% vs. 33.25 ± 4.34%; p < 0.05) in the percentage of secondary necrotic cells compared to the PtDPhPzTntreated cells (Figure 2A,B). In this sense, melatonin has been previously shown to synergistically enhance antitumor function in tunicamycin-challenged MDA-MB-231 cells [32], which is in line with the results reported in this paper. The potentiating actions of melatonin on apoptosis induction by classical drugs, such as doxorubicin, lapatinib, and arsenic trioxide, has been also documented in breast cancer cell lines [33][34][35][36] and may be related to its pro-oxidant capacity in tumor cells [36]. To corroborate the combinatorial effect of PtDPhPzTn and melatonin in MDA-MB-231 cells, 5, 10, and 25 µM of the platinum(II) complex and 0.5, 1, and 2 mM of the indoleamine were combined and cell death was determined after 24 h to calculate the CI values ( Figure S1). Our results show a synergistic effect of the combination of PtDPhPzTn and melatonin, especially when combining 1 mM melatonin and the three different concentrations of the platinum(II) complex (Table S1).  On the other hand, the complex PtDPhPzTn turned out to be less harmful to MCF-10A human breast epithelial cells than to tumor cells, as the very same dose of PtDPhPzTn (10.4 µM) only provoked a moderate reduction in the proportion of live cells (64.58 ± 9.31% vs. 84.76 ± 8.14%; p < 0.05; Figure 2A,C) with respect to control cells. Other platinum(II) complexes, including cyclometalated complexes and metal complexes with berenil and nitroimidazole, have been informed to show lower toxicity towards normal MCF-10A cells as well [37,38]. In addition, melatonin per se did not affect apoptosis of MCF10A cells (Figure 2A,C), which agrees with previous findings documenting no effect on nontumoral breast epithelial MCF10A cells [39,40]. Concerning the combinatory treatment, and contrary to what happened on MDA-MB-231 breast cancer cells, it did not display any potentiating effect on the PtDPhPzTn-induced apoptosis of MCF10A cells (Figure 2A,C), thus indicating that this combined treatment exhibits lower apoptosis-promoting actions on normal mammary epithelial cells than on breast cancer cells.
To confirm that the main mode of cell death elicited by the platinum(II) complex was apoptosis, the activation of caspases, which are enzymes critically involved in the initiation and execution of such process, was investigated. When MDA-MB-231 cells were treated with 10.4 µM PtDPhPzTn for 24 h, a remarkable rise in the proportion of caspase-3positive (60.17 ± 4.27% vs. 14.57 ± 9.08%; p < 0.05; Figure 3A,D) and caspase-9-positive (70.63 ± 6.37% vs. 15.07 ± 4.67%; p < 0.05; Figure 3B,E) cells was noted, thus indicating that the platinum(II) complex triggered intrinsic apoptosis in the TNBC cell line. In this sense, earlier studies have informed that both pyrazole-platinum(II) complexes and dinuclear berenil-platinum(II) complexes also caused caspase-3 and caspase-9-dependent apoptosis in breast cancer cells [29][30][31]. Of note, the combinatorial treatment with PtDPhPzTn and melatonin moderately potentiated the activation of caspases, though this effect was not statistically significant compared to PtDPhPzTn alone (78.28 ± 10.30% vs. 60.17 ± 4.27% and 86.30 ± 8.43% vs. 70.63 ± 6.37% for caspase-3 and -9, respectively; Figure 3A,B,D,E). Again, melatonin per se produced negligible effect on caspase activation of MDA-MB-231 cells ( Figure 3A,B,D,E). On the other hand, the involvement of ROS in the apoptotic process evoked by the platinum(II) complex was also explored. Our findings show that PtDPhPzTn notably increased intracellular ROS production after 4 h (20.70 ± 3.66 vs. 7.35 ± 1.15% DCF-positive cells; p < 0.05; Figure 3C,F), which ultimately led to apoptotic cell death. These results agree with previous research describing that other platinum(II)-based also exerted their cytotoxic effect in breast cancer cells through oxidative stress-mediated apoptosis [30]. Additionally, the combined treatment with PtDPhPzTn and melatonin further augmented intracellular ROS production, this effect not being statistically significant compared to PtDPhPzTn-treated cells (26.29 ± 5.39% vs. 20.70 ± 3.66 DCF-positive cells; p < 0.05; Figure 3C,F). As for melatonin alone, it did not induce any change in the ROS production of MDA-MB-231 cells ( Figure 3C,F).
As the platinum(II) complex displayed pro-apoptotic actions against MDA-MB-231 cells, its impact on cell cycle progression was anticipated and, therefore, cell cycle distribution analysis was carried out. Thus, when TNBC cells were challenged with 10.4 µM PtDPhPzTn, a moderate S phase arrest (55.32 ± 4.28% vs. 38.30 ± 5.44% in control cells; p < 0.05; Figure 4) was observed at the expense of the percentage of cells in G2/M transition. Additionally, the platinum(II) complex caused a rise in the subpopulation of cells with hypodiploid DNA content (sub-G1 cells; Figure 4), which is indicative of DNA fragmentation. In this line, earlier studies have proven that cyclometalated platinum(II) complexes also affect S phase of the cell cycle in breast cancer cells [37]. Nonetheless, other investigations have demonstrated that the inhibition of cell survival induced by pyrazole-platinum(II) complexes occurs by arresting the G1 phase of the cell cycle [29]. The same trend was observed for the combinatory treatment, though no potentiating effect was noticed. In fact, when TNBC cells were treated with 10.4 µM PtDPhPzTn in the presence of 1 mM melatonin, a significant accumulation of MDA-MB-231 cells at S phase (53.18 ± 6.39% vs. 38.30 ± 5.44% in control cells; p < 0.05; Figure 4) was ascertained. Likewise, the co-incubation with the platinum(II) complex and the indoleamine evoked an increment in the percentage of cells with hypodiploid DNA content ( Figure 4). Nevertheless, melatonin per se did not induce any remarkable change in the cell cycle distribution of MDA-MB-231 cells (Figure 4), which is in line with previous research indicating no effect of indoleamine on the cell cycle of breast cancer cells [41].
1, x FOR PEER REVIEW 9 of 14 As the platinum(II) complex displayed pro-apoptotic actions against MDA-MB-231 cells, its impact on cell cycle progression was anticipated and, therefore, cell cycle distribution analysis was carried out. Thus, when TNBC cells were challenged with 10.4 µM PtDPhPzTn, a moderate S phase arrest (55.32 ± 4.28% vs. 38.30 ± 5.44% in control cells; p < 0.05; Figure 4) was observed at the expense of the percentage of cells in G2/M transition. Additionally, the platinum(II) complex caused a rise in the subpopulation of cells with hypodiploid DNA content (sub-G1 cells; Figure 4), which is indicative of DNA fragmentation. In this line, earlier studies have proven that cyclometalated platinum(II) complexes also affect S phase of the cell cycle in breast cancer cells [37]. Nonetheless, other investigations have demonstrated that the inhibition of cell survival induced by pyrazole-platinum(II) complexes occurs by arresting the G1 phase of the cell cycle [29]. The same trend was observed for the combinatory treatment, though no potentiating effect was noticed. In fact, when TNBC cells were treated with 10.4 µM PtDPhPzTn in the presence of 1 mM melatonin, a significant accumulation of MDA-MB-231 cells at S phase (53.18 ± 6.39% vs. 38.30 ± 5.44% in control cells; p < 0.05; Figure 4) was ascertained. Likewise, the co-incuba- Finally, the ability of the platinum(II) complex PtDPhPzTn to modulate cell migration capacity of TNBC cells was analyzed by wound-healing and transwell migration assays. As shown in Figure 5A,B, the PtDPhPzTn complex greatly impaired the ability of MDA-MB-231 cells to migrate after both 12 h (29.76 ± 13.11% vs. 66.79 ± 8.40% in control cells; p < 0.05) and 24 h post-scratch (66.19 ± 24.20% vs. 98.44 ± 3.26% in control cells; p < 0.05), thus suggesting that this compound may be useful in blocking the metastatic migration of breast cancer cells. The same trend was observed in the transwell migration assay, wherein the platinum(II) complex almost completely blunted the migration capacity of MDA-MB-231 cells (p < 0.05; Figure 5C). In this sense, other authors have also synthesized metal complexes with potential to suppress the cell migration ability of TNBC cells, including cis-diphenyl pyridineamine platinum(II) complexes and platinum(II) complexes with tridentate ligands [42,43]. As for the combined treatment, the exposure of MDA-MB-231 cells at 10.4 µM PtDPhPzTn in the presence of 1 mM melatonin markedly enhanced the antimigration effect of the platinum(II) complex after both 12 h (15.57 ± 7.53% vs. 29.76 ± 13.11% in PtDPhPzTn-treated cells; p < 0.05; Figure 5A,B) and 24 h post-scratch (37.85 ± 12.93% vs. 66.19 ± 24.20% in PtDPhPzTn-treated cells; p < 0.05; Figure 5A,B). Regarding the transwell migration assay, the combinatory treatment diminished (p < 0.05) the proportion of migrating cells to the same extent as the platinum(II) complex alone ( Figure 5C). Other studies has also proven that indoleamine synergistically improves the antimigration properties of apatinib, a tyrosine kinase inhibitor thought to inhibit angiogenesis, in breast cancer stem cells derived from MDA-MB-231 cells [44], whereas novel melatonin-tamoxifen drug conjugates were shown to effectively inhibit migration in different breast cancer cell lines, including tamoxifen-resistant cells [45]. Nonetheless, even though melatonin has been described to inhibit cell invasion potential in MDA-MB-231 and MCF-7 breast cancer cell lines [46], indoleamine per se only evoked a moderate, non-significant impairment in the ability of TNBC cells to migrate ( Figure 5A,B) and showed no effect in the transwell migration assay.
1, x FOR PEER REVIEW 10 of 14 ( Figure 4), which is in line with previous research indicating no effect of indoleamine on the cell cycle of breast cancer cells [41]. Finally, the ability of the platinum(II) complex PtDPhPzTn to modulate cell migration capacity of TNBC cells was analyzed by wound-healing and transwell migration assays. As shown in Figure 5A,B, the PtDPhPzTn complex greatly impaired the ability of MDA-MB-231 cells to migrate after both 12 h (29.76 ± 13.11% vs. 66.79 ± 8.40% in control cells; p < 0.05) and 24 h post-scratch (66.19 ± 24.20% vs. 98.44 ± 3.26% in control cells; p < 0.05), thus suggesting that this compound may be useful in blocking the metastatic migration of breast cancer cells. The same trend was observed in the transwell migration assay, wherein the platinum(II) complex almost completely blunted the migration capacity of MDA-MB-231 cells (p < 0.05; Figure 5C). In this sense, other authors have also synthesized Antioxidants 2022, 11, x FOR PEER REVIEW 11 of 14 melatonin has been described to inhibit cell invasion potential in MDA-MB-231 and MCF-7 breast cancer cell lines [46], indoleamine per se only evoked a moderate, non-significant impairment in the ability of TNBC cells to migrate ( Figure 5A,B) and showed no effect in the transwell migration assay.

Conclusions
Platinum(II)-based complexes are undoubtedly an interesting alternative to canonical chemotherapy agents as they can help avoiding drug resistance and off target toxicity by promoting the apoptosis of tumor cells via multiple mechanisms, including oxidative stress, mitochondrial injury, and DNA damage. In this paper, we described that the thiazolinecontaining platinum(II) complex PtDPhPzTn, which reportedly induces oxidative stress and apoptosis in tumor cells [7], outperformed, by far, the reference drug cisplatin and its less lipophilic counterpart PtPzTn in terms of cytotoxicity against MDA-MB-231 cells. More importantly, the combinatory treatment with PtDPhPzTn and the renowned antioxidant melatonin potentiated the pro-apoptotic, cell cycle arrest and anti-migration properties of the platinum(II) complex alone against MDA-MB-231 breast cancer cells. Therefore, our findings provide evidence that a combined therapy with PtDPhPzTn and melatonin could be potentially applied to breast cancer management as powerful synergistic agents.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11101971/s1, Figure S1. Synergistic effect of the combination of PtDPhPzTn and melatonin in MDA-MB-231 cells, Table S1. Calculation of the combination index (CI) data for nonconstant combination of PtDPhPzTn and melatonin in cell death induction.