Unfolding biological properties of a versatile dicopper(II) precursor and its two mononuclear copper(II) derivatives

Journal of Inorganic Biochemistry 174 (2017) 25–36

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Unfolding biological properties of a versatile dicopper(II) precursor and its two mononuclear copper(II) derivatives

MARK

Anup Paula,1, Susanta Hazraa,⁎, Gunjan Sharmab,1, M. Fátima C. Guedes da Silvaa,⁎, Biplob Kochb,⁎, Armando J.L. Pombeiroa a b

Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Sulfonated Schiff base copper(II) Cytotoxicity in vitro Fluorescence activated cell sorting Mitochondrial distribution Reactive oxygen species

Synthesis, inter-conversions and biological study of the dichloro bridged dicopper(II) compound [CuLCl]2 (1) and its two mononuclear derivatives [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3) (HL = 2-(2-pyridylmethyleneamino)benzenesulfonic acid) are described. The dimeric compound 1 collapses into monomers 2 and 3 in the presence of coordinating solvents, water and pyridine, respectively, and 1 is regenerated upon simple stirring of 2 or 3 in methanol. The reactions of 1 with neutral (present study) and charged (earlier studies) ligands result in monomeric and multimeric compounds, respectively, attesting that it is a versatile dicopper(II) precursor. The anticancer activity of these copper complexes (1–3) was screened against lung (A-549) and breast (MDA-MB-231) human cancer cell lines. The IC50 (half maximal inhibitory concentration) value for one (3) of the compounds suggests preferential cytotoxicity against breast cancer MDA-MB-231 cell line. Furthermore, the IC50 value obtained for complex 3 is found to be almost two-fold times cytotoxic than the standard drug cisplatin. In addition, the underlying possible mechanism of its apoptosis-inducing efficacy in MDA-MB-231 cells has been rationalized by using flow cytometry (FACS) and Hoechst 33342/propidium iodide (PI) fluorescence staining. The stimulation of apoptotic induction for complex 3 has further been affirmed by reactive oxygen species (ROS) generation and mitochondrial aggregations studies.

1. Introduction Cisplatin, the first inorganic anti-cancer therapeutic drug still preserves a main role in the treatment of cancer [1–4]. In spite of severe side effects, it is among the most widely used platinum(II) anti-cancer drugs, which also include carboplatin, oxaliplatin and nedoplatin [5–10]. The outburst triumph of platinum(II) compounds has spurred a surge of investigations not only for other platinum drugs but also for non-platinum metallodrugs involving Ti, Au, Sn, Ru, Pd, etc. metal ions for use in chemotherapy with improved specificity and decreased toxic side effects [11–46]. Moreover, a notable limitation of platinum-based anti-cancer drugs is that only a restricted number of tumors can be treated with them, which inspires the search for non-platinum metalbased antitumor agents which are also cytotoxic (cell-killing) interfering in some way with the operation of the DNA cells. In this respect, attention has been paid to copper(II) complexes as

these are regarded as promising antitumor agents and have attracted considerable importance owing to their capability of interacting directly with DNA/nuclear proteins [47–50]. On the other hand, Schiff base metal complexes are relevant for their diverse solid state structures and physical properties [51–77], but sulfonic acid containing Schiff base metal complexes [68–77] have rarely been investigated. A few copper complexes [68–77] have been synthesized and applied in catalysis, namely in oxidations (of alkanes or alcohols) [69–73] or nitro-aldol [68] reactions. Interestingly, such a limited number of copper complexes have shown a variety of solid state structure, such as monomeric, dimeric, tetrameric and polymeric (1D or 2D) ones [68–77], which encouraged us to explore further into this field. Very recently we have reported two Sn(IV) complexes derived from a sulfonated ligand, displaying a great potential as anticancer drugs [73]. This has prompted us to investigate related systems using a different metal ion [copper(II)] which might improve dissolution

Abbreviations: DNA, deoxyribonucleic acid; IC50, half maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FT-IR, fourier transform infrared; FBS, fetal bovine serum; FACS, fluorescence activated cell sorting; PI, propidium iodide; ROS, reactive oxygen species; DMSO, dimethyl sulfoxide; DMEM, dulbecco's modified eagle's medium; PBS, phosphate buffered saline; DCFH-DA, dichloro-dihydro-fluorescein diacetate ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Hazra), [email protected] (M.F.C. Guedes da Silva), [email protected] (B. Koch). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.jinorgbio.2017.05.013 Received 22 March 2017; Received in revised form 26 May 2017; Accepted 30 May 2017 Available online 31 May 2017 0162-0134/ © 2017 Elsevier Inc. All rights reserved.

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properties and thereby influence cytotoxicity. Herein, we present the in vitro cytotoxic effect of the newly synthesized Cu-mononuclear complexes [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3) (HL = 2-(2-pyridylmethyleneamino)benzenesulfonic acid) and the previously reported [68–71] dichloro bridged dicopper(II) compound [CuLCl]2 (1) against human lung (A-549) and breast (MDA-MB-231) cancer cell lines, along with a possible mechanism of action involving the antiproliferative efficacy.

at room temperature. After a day, the dark green crystalline precipitate containing single crystals of 3 suitable for X-ray diffraction was collected by filtration. Yield: 0.347 g (79%). Anal. Calcd. for C17H14N3O3SClCu (439.37): C, 46.47; H, 3.21; N, 9.56. Found: C, 46.55; H, 3.25; N, 9.51. FT-IR (cm− 1, KBr): ν(C = N), 1593s; ν(sulfonate), 1371 m, 783 m.

2. Experimental section

In a 20 mL beaker, a suspension of 0.100 g of 2 or 3 in 10 mL of methanol was vigorously stirred for six hours at room temperature. The green precipitate was then separated by filtration and washed with methanol. FT-IR and elemental analyses of the final compound matched those of the compound 1. However, the compound 3, which was prepared from the compound 1 (see syntheses Section 2.2), can be synthesized from 2 following a similar procedure. To a hot and stirred CH3CN suspension of compound 2 (0.100 g) was added a dropwise CH3CN solution of pyridine until a clear solution was obtained. The hot green solution was then kept for slow crystallization and after a day the green solid containing diffractive single crystals was collected by filtration and washed with cold CH3CN. FT-IR, unit cell dimensions and elemental analyses of the final compound matched those of the compound 3. Nonetheless, attempt to convert 3 into 2 was not successful.

2.3. Interconversions of 1–3

2.1. Materials and physical methods All the reagents and solvents were purchased from commercial sources and used as received. Water used in all the syntheses was double distilled. Elemental (C, H, and N) analyses were performed by the Microanalytical Service of the Instituto Superior Técnico. The infrared spectra (4000–400 cm− 1) were recorded on a Bruker Vertex 70 instrument in KBr pellets; abbreviations: s = strong, m = medium and w = weak. Human lung (A-549) and breast (MDA-MB-231) cancer cell lines were procured from National Centre for Cell Science (NCCS), Pune, India. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), streptomycin, penicillin, L-glutamine, and Trypsin-EDTA were purchased from CELL clone™ Genetix Biotech Asia Pvt. Ltd. Tissue culture flask (surface area 25 cm2, canted neck, vented cap) and plates (96 and 6 well, flat bottom) were procured from Eppendorf. 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Hi-Media Laboratories Pvt. Ltd. Mumbai, India. DMSO (dimethyl sulphoxide) and RNAse A, DNase free obtained from Merck Millipore. MitoTracker red (MitoRed) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) procured from Sigma-Aldrich. Alexa Fluor 488 Annexin V/dead cell apoptosis kit was purchased from Molecular Probe™ by Life technology, Thermo Fisher Scientific. Propidium iodide (PI) and Hoechst 33342 were purchased from calbiochem. The remaining chemicals were purchased from local firms and were of highest purity grade. Mass spectra of sample solution in methanol were acquired on a Bruker HCT quadrupole ion trap equipped with an electrospray ion source using the following typical instrumental parameters: solution flow rate, 2.5 μL/min; ESI needle spray voltage, + 4 kV; capillary exit voltage, − 129 V; nebulizer gas pressure, 8 psi; dry gas flow rate, 4 L/ min; dry gas temperature, 250 °C; octopole RF amplitude, 187 Vpp. The spectra were recorded in the range 100–1500 Da. Spectra typically correspond to the average of 20–35 scans.

2.4. Crystal structure determinations The X-ray diffraction data of 1–3 were collected using a Bruker APEX-II PHOTON 100 with graphite monochromated Mo-Kα radiation. Data were collected using omega scans of 0.5° per frame, and a full sphere of data was obtained. Cell parameters were retrieved using Bruker SMART software and refined using Bruker SAINT [78] on all the observed reflections. Absorption corrections were applied using SADABS [78]. Structures were solved by direct methods by using the SHELXS-97 package [79] and refined with SHELXL-97 [79]. Calculations were performed using the WinGX System-Version 1.80.03 [80]. All the hydrogen atoms were inserted in calculated positions. Least square refinements with anisotropic thermal motion parameters for all the non-hydrogen atoms and isotropic for most of the remaining atoms were employed. The final refinement converged to R1 (I > 2σ(I)) values 0.0297, 0.0362 and 0.0333 for 1, 2 and 3, respectively. Selected crystallographic data for 1, 2 and 3 are given in Table S1, Supplementary Information (SI). 2.5. Cell culture and proliferation/MTT assays The cytotoxicity and antiproliferation activity of complexes were determined in human lung adenocarcinoma (A-549) and breast cancer (MDA-MB-231) cell lines by MTT colorimetric assay based on the conversion of the soluble yellow colored tetrazolium salt (MTT) to insoluble purple colored formazan crystals after reaction with mitochondrial dehydrogenase of metabolically active or live cells [81,82]. Stock solutions of complexes 1–3 were prepared in DMSO (100 mM) and further dilution was made in complete DMEM where the final concentration of DMSO did not exceed 0.1% v/v. Both human cancer cell lines were maintained in complete DMEM (culture medium consisting 10% fetal bovine serum, 20 mM L-glutamine, 100 units/mL Penicillin and 100 μg/mL Streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. At 80 to 90% confluence, A-549 and MDA-MB-231 cells were trypsinised, counted and 1 × 104 cells were seeded in 96 well tissue culture plates separately and incubated for 24 h in humidified CO2 incubator. After 24 h, the spent medium was replaced with fresh medium containing various concentrations of complexes 1–3 and incubated for further 24 h in a 5% CO2 humidified atmosphere at 37 °C. In the next step, the medium was discarded and 100 μL of fresh medium containing 10 μL of 5 mg/mL MTT was added and incubated for additional 2 h. Finally, DMSO was added to dissolve

2.2. Syntheses [CuLCl]2 (1) was synthesized following a reported procedure [68–71]. Anal. Calcd. for C24H18N4O6S2Cl2Cu2 (721): C, 40.01; H, 2.52; N, 7.78. Found: C, 40.35; H, 2.49; N, 7.71. FT-IR (cm− 1, KBr): ν(C = N), 1594s; ν(sulfonate), 1359 m, 783 m. 2.2.1. Synthesis of [CuLCl(H2O)]·H2O (2) 1.0 mmol (0.720 g) of [CuLCl]2 (1) was dissolved in water (20 mL) and the resulted green solution was kept at room temperature. After a few days, a green precipitate containing single crystals of 2 suitable for X-ray diffraction was collected by filtration. Yield: 0.587 g (74%). Anal. Calcd. for C12H13N2O5SClCu (396.31): C, 36.33; H, 3.31; N, 7.07. Found: C, 36.27; H, 3.36; N, 7.02. FT-IR (cm− 1, KBr): ν(water), 3451 m (br); ν(C = N), 1604s; ν(sulfonate), 1362 m, 782 m. 2.2.2. Synthesis of [CuLCl(py)] (3) To a hot and stirred CH3CN solution (25 mL) of [CuLCl]2 (1) (0.360 g, 0.5 mmol) was added dropwise a CH3CN solution (5 mL) of pyridine (0.081 g, 1 mmol). The resulted dark green solution was kept 26

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Scheme 1. Syntheses of 2 and 3 from 1 and their inter-conversion.

Fluor 488 conjugate) and component B i.e., PI was added to a final concentration of 1 μg/mL and left for 15 min in the dark at 37 °C. After staining, 200 μL Annexin binding buffer were further added to each sample and analyzed under FACS Scan by the CELL Quest software (Becton Dickinson Facs Calibur, USA) for the evaluation of cell apoptosis.

the insoluble formazan crystals followed by incubation for 30 min. The absorbance of each well of 96 well plates was measured at 570 nm with ELISA plate reader. Cytotoxic effect was expressed as the percentage of treated cells relative to control cells. 2.6. Morphological analysis of apoptotic cells with Hoechst/PI staining

2.8. Cell cycle analysis

To observe the changes in nuclear morphology (nuclear condensation and fragmentation) of treated cells, a microscopic study with Hoechst/PI staining was done. Briefly, 1 × 104 MDA-MB-231 cells were seeded in 12 well plates and after 24 h the cells were treated with different concentrations (2.5, 5 and 7.5 μM/mL) of complex 3 for 24 h. Subsequently, cells were washed with PBS and stained with Hoechst 33342 (10 μg/mL) and propidium iodide (10 μg/mL) solutions. Cells were then washed again with PBS and images were taken with a fluorescent microscope (Evos FL, Life technologies) in phase contrast, red and blue channels.

2 × 105 MDA-MB-231 cells/well were seeded in 6 well plates as described above and then treated with various concentrations (2.5, 5 and 7.5 μM/mL) of complex 3 for 24 h. After treatment, cells were washed with cold PBS, trypsinised, fixed in ice-cold 70% ethanol and kept overnight at − 20 °C. The cells were then washed twice in PBS, 500 μL of Triton X/PI/RNase solution [960 μL of 0.1% (v/v) Triton X100, 20 μL of PI (1 mg/mL) and 20 μL of RNase (10 mg/mL in PBS)] were added to each sample followed by incubation at 37 °C for 30 min in dark and analyzed under FACS Scan by the CELL Quest software (Becton Dickinson FacsCalibur, USA) for cell cycle phase distribution [28,77].

2.7. Detection of apoptotic induction with annexin V/PI staining Quantitative analysis of apoptosis induction in MDA-MB-231 cells was assessed by flow cytometry using Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit. It contains three components, component A [Alexa Fluor 488 Annexin V solution in 25 mM HEPES, 140 mM NaCl, 1 mM EDTA, pH 7.4, 0.1% bovine serum albumin (BSA)], component B [propidium iodide (PI) 1 mg/mL (1.5 mM) solution in deionized water] and component C [5 × Annexin-binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2) pH 7.4]. In brief, MDA-MB-231 cells were seeded in 6-well plates (5 × 105 cells/well), after 24 h cells were treated with different concentrations (2.5, 5 and 7.5 μM/mL) of complex 3 for 24 h. After treatment, cells were washed twice with ice-cold PBS, trypsinised and then centrifuged. The pellet was made single cell suspension by gentle agitation and then cells from each sample were suspended separately in 50 μL of component C (Annexin binding buffer). 3 μL of component A (Annexin V Alexa

2.9. Detection of reactive oxygen species To detect the intracellular ROS level in treated cells, intracellular redox status as compared to control were measured with an oxidationsensitive fluorescence probe, 2′,7′-dichlorofluorescine diacetate (DCFHDA). DCFH-DA was hydrolyzed by intracellular esterase of the cells and converted into a non-fluorescent DCF-H product. Further, this DCF-H becomes oxidized in the presence of intracellular ROS and converted into highly fluorescent DCF product [83]. 2 × 105 cells/well were seeded in 6-well culture plates, treated with three different concentrations (2.5, 5 and 7.5 μM) of the complex 3 and incubated at 37 °C with 5% CO2 for 24 h. After incubation with drug, cells were stained with 10 μM DCFH-DA solution and incubated at 37 °C for 30 min. The cells were washed twice with PBS and images were captured with a 27

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Fig. 1. Idealized ball and stick presentation of the crystal structure of [CuLCl]2 (1), [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3). Symmetry: 1–x, − y, 1–z (1).

fluorescence microscope (Evos FL, Life technologies) in phase contrast and green channel.

crystalline form with good yield (74–79%). In their IR spectra, compounds 2 and 3 exhibit an intense for ν(C = N) band at 1604 and 1593 cm− 1, respectively, which is comparable to that (1594 cm− 1) of the mother compound (1). The presence of the sulfonate group is evidenced by the medium intense bands at 1362 and 1371 cm− 1 for 2 and 3, respectively, being similar to that (1359 cm− 1) of 1. A broad medium intense band at 3451 cm− 1 in the IR spectrum of 2 indicates the presence of water molecule. They were also characterized by single crystal X-ray diffraction analysis. The crystal structure of 1 was known [85] but the current structure, obtained at a low temperature, is of a better quality (see below and Table S2, SI).

2.10. Detection of mitochondrial distribution pattern In brief, 2 × 105 MDA-MB-231 cells/well were seeded in a 6 well plate and allowed to adhere for 24 h in a humidified CO2 incubator. The medium was discarded and cells were then treated with three different concentrations (2.5, 5 and 7.5 μM) of complex 3 and incubated further for 24 h in a 5% CO2 and humidified atmosphere. Subsequently, cells were washed with PBS and stained with MitoTracker Red (1 μM) and Hoechst (10 μg/mL) solution for 30 min. Finally, cells were washed twice with PBS and images were taken with a fluorescent microscope (EVOS FL cell imaging system) in phase contrast, red and blue channels [84].

3.2. Description of crystal structures The idealized ball and stick presentations of the crystal structures of 1–3 are depicted in Fig. 1, while the selected bond length and angles are listed in Table S2 (SI). The single-crystal X-ray diffraction studies revealed that 2 and 3 are mononuclear complexes, crystallizing in monoclinic P21/c and Cc space groups, respectively, whereas the reported compound 1 is a dinuclear one, crystallized in monoclinic P21/n space group. Both the compounds 2 and 3 contain a metal centre coordinated by the deprotonated Schiff base ligand (L−) acting solely as an N,N,O donor. The ligands in the crystal structures of 1–3 are slightly distorted. Indeed, angles between the pyridyl and the aniline rings are comparable and are in the order 2 > 1 > 3 (19.67° in 1, 28.98° in 2 and

3. Results and discussion 3.1. Synthesis and characterization The dichloro bridged dinuclear compound [CuLCl]2 (1) is known and was prepared following our reported procedure [68–71]. The new compounds [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3) are derived from 1 by dissolving it in different solvents. As shown in Scheme 1, dissolving the compound 1 in water produces 2 while the addition of pyridine in stirring acetonitrile solution of 1 results in the other mononuclear compound 3. Compounds 2 and 3 were isolated in 28

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12

Fig. 2. Two dimensional supramolecular associate in 1.

18.05° in 3). However, the second pyridyl ring (co-ligand) in 3 is virtually perpendicular to other two rings (86.64° and 89.65°, Fig. 1). The coordination geometries (ON2Cl2 in 1, O2N2Cl in 2 and ON3Cl in 3) of the copper centres are square pyramidal (τ5 descriptor of 0.056, 0.154 and 0.025 for 1, 2 and 3, respectively). The basal planes are defined by the Npyridyl, the Nimine, a Osulfonate and the bridged or coordinated Cl-atom or Npyridyl mutually trans to Nimine atom. The apical positions are occupied by the bridging Cl-atom and the Owater atom for 1 and 2 respectively, while the coordinated Cl-atom does that role for 3. Although the geometric distortion around the copper atom in 2 is greater than that in 1 and 3, as expressed by the τ5 descriptor (see above), the bond distances in the basal plane in 1 and 2 do not differ significantly (see Table S2, SI). However, the metal–Nimine and metal–Npyridyl distances (Table S2, SI) in 1 and 2 are considerably larger than the metal–Osulfonate bonds what can be due to the considerable trans effect from the chloride ligand. The difference in van-der-Waals radius of chloride and oxygen accounts for the larger apical distance (Cu–Cl = 2.6478(11) Å) in 1 than that (Cu–O = 2.244(2) Å) in 2. The bond lengths (Table S2, SI) in the basal N3O plane in 3 are not much different to each other but much shorter than the apical one (Cu–Cl = 2.4525(6) Å). Bond distances and angles surrounding the copper(II) centres of all compounds are in the ranges observed in our recent reported cuboid copper(II)-tetramer, a couple of copper(II)-dimers and a few copper(II)polymeric structures derived from the same or similar ligands [68–77]. The crystal structures of 1–3 are stabilized by a few non-covalent interactions, such as hydrogen bonding (in 1–3) or π·π stacking (in 1 only) to build two-dimensional associates (2D). Relevant parameters of

these interactions are presented in Table S3, SI. As shown in Fig. 2, single CeH·O interaction between imine CeH (C11eH11) and one (O13) of the sulfonate oxygens and the π·π stacking between pyridyl and phenyl rings of the ligand result in the formation of a two-dimensional sheet which along the b-axis appears as a ladder. In 2, two water molecules (coordinated or weakly bonded) interact with the chloride (Cl1) and two non-coordinated sulfonate oxygen (O2 and O3) atoms to provide total four H-bondings, three OeH·O and one OeH·Cl interactions. However, these additional donor or acceptor atoms in 2 do not provide a further dimension, producing, as in 1, a two-dimensional network, which along the a-axis presents a zigzag chain (Fig. 3). In contrast to 1 and 2, the crystal structure of 3 is stabilized by only two weak CeH·O (C2eH2·O3 and C14eH14·O2) interactions involving both pyridyl-C–H and the non-coordinated sulfonate oxygen atoms to form a 2D associate (Fig. 4). 3.3. [CuLCl]2 (1) as a versatile precursor The dicopper complex [CuLCl]2 (1) presents two Cl–Cu–Cl bridges with one of the CueCl bonds being very weak (see CueClapical bond distance, Table S2, SI). It is liable to collapse into the monomeric form [CuLCl] but a further neutral ligand is required to preserve a stable penta coordinated square pyramidal geometry. Thus, offering a common coordinating solvent like water or pyridine results in a monomer with a coordinating solvent. Since pyridine is a much bettercoordinating solvent than acetonitrile, it coordinates preferably to the latter one in a pyridine-acetonitrile solution. On the other hand, the neutral ligands break only the dimeric species 1 into monomers, such as 29

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Fig. 3. Two dimensional supramolecular associate in 2.

2 and 3, which, however, are convertible into 1 upon stirring in methanol, a weekly coordinating solvent. However, recently we have reported a few polymeric systems [68–71] which were derived by replacing the chloride ligand of 1 with charged ligands, such as pseudohalides (azide, thiocyanate and dicyanamide) [68] or carboxylates (cyclohexane-1,4-dicarboxylate, benzene1,3,5-tricarboxylate and benzene-1,2,4,5-tetracarboxylate) (Scheme 2) [69–71]. Thus, 1 is a versatile dicopper(II) precursor forming mono or multinuclear species in presence of neutral or charged ligands, respectively (Scheme 2).

in breast cancer cell line is almost two-fold times better than that of the standard drug cisplatin under similar experimental conditions (Table 1). The cytotoxicity and the inhibitory effect of 3 obtained from the MTT assay clearly reveal its higher apoptotic activity than the other complexes against the aforementioned human cancer cell lines. Thus, complex 3 was found to be more potent cytotoxic and antiproliferative agent for breast cancer cells. Pyridyl based metal complexes are well known for their anticancer activities [26,86–88]. An additional pyridine co-ligand in 3, in comparison to 1 and 2, increases its lipophilicity as indicated by its higher solubility in DMF or DMSO [77], what conceivably can be related to its higher activity. In fact, antitumor activity is dependent on lipophilicity of the ligands [34,77,89]. However, the ligand system (sulfonated Schiff base with pyridyl moiety) used in the current study has never been investigated in biological study and thus, further investigation of related systems would be required to understand the better biological activity of 3.

3.4. Cytotoxicity and IC50 values The effectiveness of the dicopper(II) complex 1 and its two-mononuclear copper(II) derivatives (2 and 3) to induce cytotoxicity and inhibit cell proliferation has been evaluated by MTT colorimetric assay. The cytotoxicity activities (IC50 values) of these copper complexes were investigated against two different human cancer cell lines consisting of human lung cancer (A-549) and breast cancer (MDA-MB-231) cell lines. The IC50 values of all the complexes were found to be (57 ± 3, 1; 77 ± 3, 2; and 51.5 ± 0.3 μM, 3) against A-549 lung cancer and (100, 1; > 100, 2; and 5.0 ± 0.3, 3) against MDA-MB-231 breast cancer cell lines (Table 1; Fig. S1, Supplementary Information). Among the three complexes, complex 3 exhibited the highest cytotoxicity and antiproliferative activity on MDA-MB-231 breast cancer cell line. It is also noteworthy to mention that the IC50 value obtained for complex 3

3.5. Effect of complex 3 on cells and nuclear morphology Chromatin condensation and fragmentation of nucleus is one of the major properties of apoptotic cell death. Thus, fluorescence microscopy with DNA binding dyes Hoechst 33342 and PI was performed to observe the alteration in nuclear morphology of cells caused by 3 (Fig. 5). In untreated cells, uniform blue fluorescence nuclei were observed which indicates that most of the cells were alive and healthy. However, 30

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Fig. 4. Two dimensional supramolecular associate in 3. Scheme 2. Formation of monomeric and multimeric compounds from 1, depending on the types of ligands.

complex 3 treated cells appeared with condensed nuclei with deep blue fluorescence in lower concentration which indicates early apoptotic cells (indicated by yellow arrows). For cells treated at the IC50 value of complex 3, the frequency of early and late apoptotic cells with fragmented nuclei (indicated by blue arrows) were increased. However, at

higher concentration, most of the cells appeared to undergo with bright blue colored nuclei and some necrotic cells fluorescence (indicated by red arrows) were also observed. this observation, it can be concluded that complex 3 favors mode of cell death in breast cancer (MDA-MB-231) cells. 31

apoptosis with red Based on apoptotic

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of various copper complexes are potentially due to induced apoptosis in different types of cancer cells [90,91]. It was also reported that some of the Cu(II) complexes directly interact at the molecular level and induce DNA fragmentation in a pUC19 plasmid, lymphocytes and in HeLa cells [92]. Interestingly, our studies demonstrate that the compound under investigation potentially induces apoptosis as analyzed by fluorescence microscopy and flow cytometry in MDA-MB-231 cells in a dose-dependent manner. Based on these observations (Fig. 6), it may be inferred that the complex 3 may have interacted directly or indirectly at the molecular level of breast cancer cells and favored the apoptotic mode of cell death.

Table 1 IC50 values of all complexes after 24 h exposure on A-549 (Human Lung cancer) and MDA-MB-231 (Breast cancer) cell lines (n = 3). Compounds

Incubation time – 24 h [IC50 value (in μM)] Cell lines

1 2 3 Cisplatin

Lung cancer (A-549)

Breast cancer (MDA-MB-231)

57 ± 3 77 ± 3 51.5 ± 0.3 8.1 ± 0.5

100 > 100 5.0 ± 0.2 9.5 ± 0.2

3.6. Detection of apoptotic cells by Annexin V/PI staining

3.7. Cell cycle analysis with PI

To further strengthen our speculation regarding the apoptotic mode of cell death in breast cancer (MDA-MB-231) cells by complex 3, quantitative assessment for detection of apoptotic cells using Annexin V/PI of complex 3 treated with MDA-MB-231 cells was analyzed using flow cytometry. The lower left quadrant of dot plot shows the live and healthy cells that excluded PI as well as Annexin V Alexa Fluor 488 binding (Annexin V−/PI− cells). Approximately, 92% of viable cells were observed in untreated MDA-MB-231 cells, whereas with increasing concentration the percentage of viable cells decreases and apoptotic cells increases. The lower right quadrant of dot plot represents the early apoptotic cells, Annexin V positive and PI negative cells (Annexin V+/PI− cells), with externalized phosphatidylserine. Annexin V+/PI+ cells in the upper right quadrant of dot plot demonstrate late apoptotic cells population, which were also observed to increase from 1.23% to 52.37% in treated groups (Fig. 6). The percentage of early apoptotic cells increased (from 2.7% in control to 45.37% in 7.5 μM) in treated cells from lower to higher concentration (Fig. 6). The results from this observation further claim the apoptotic mode of cell death induced by complex 3. Several research groups have reported that the anticancer activities

The DNA content of the cell can provide a great deal of information about the cell cycle and consequently the effect on the cell cycle of added stimuli, e.g., transfected genes or drug treatment, and this information can be gathered by performing fluorescence activated cell sorting (FACS) method using PI staining. For this, MDA-MB-231 breast cancer cells were stained with PI after 24 h of treatment with various concentrations of complex 3. Data of flow cytometry analysis showed that only 1.04% of dead cells were observed (sub-G1) in control group. However, on treatment with complex 3, the cell population principally shifted to sub G1 phase of cell cycle indicating that complex 3 exhibits its anticancer properties in MDA-MB-231 cell line mainly due to induction of cell death i.e., apoptosis. The percentage of dead cells increased with increasing concentration of complex 3 and up to 99.5% of dead cells were evaluated at 7.5 μM concentration of complex 3 (Fig. 7), while no cell cycle arrest/delay was observed in G1, S and G2/ M phases. This provides strong evidence that complex 3 exerts cytotoxic activity by selectively inducing apoptosis in breast cancer cell without influencing cell cycle progression.

Fig. 5. Images showing the effect of 3 on the morphology of MDA-MB-231 breast cancer cells detected with dual staining of Hoechst/PI. Yellow, blue and red arrows indicate early apoptotic, late apoptotic and necrotic cells respectively. Scale bars represent 100 μM.

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Fig. 6. Flow cytometry analysis of MDA-MB-231 breast cancer cells treated with different concentrations of complex 3 for 24 h. Treated cells were examined for apoptotic cell death using Annexin V-Alexafluor 488 co-stained with PI. Annexin V−/PI− cells (lower left quadrant) were alive and healthy cells, Annexin V+/PI− cells (lower right quadrant) were in early stages of apoptosis and double positive cells (upper right quadrant) were in late apoptosis, whereas Annexin V−/PI+ cells (upper left quadrant) were necrotic dead.

Fig. 7. Effect of complex 3 on the distribution of cell cycle phases through flow cytometry of PI-stained MDA-MB-231 breast cancer cells after 24 h incubation.

consequently cell death by ROS-mediated apoptosis [93,94,95]. To investigate the probable mechanism for the apoptotic inducing property of complex 3 by ROS-mediated pathway, intracellular ROS generation was determined in MDA-MB-231 breast cancer cells at different concentrations. Fluorescence microscopy revealed that complex 3 generates an increased production of reactive oxygen species in a

3.8. Detection of reactive oxygen species (ROS) production in MDA-MB231 cells Several studies have demonstrated that Cu(II) complexes generate overproduction of ROS through Fenton-like reaction and mitochondrial toxicity which might contribute to intensifying the redox imbalance and 33

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Fig. 8. MDA-MB-231 cells were treated with 3 for 24 h and quantitate for intracellular ROS generation by dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescence staining. Scale bars represent 100 μM.

Fig. 9. Effect of complex 3 at depicted concentrations on mitochondrial distribution pattern in MDA-MB-231 breast cancer cell line after 24 h of incubation. Scale bars represent 25 μM.

mitochondria were stained with the help of MitoRed and counterstained with Hoechst 33342. A nucleus staining dye was used to locate the mitochondrial aggregation position inside the cells. Mitochondria are crucial organelle to regulate the redox status of cells that play very important role in drug-induced apoptosis [97,98]. It was also reported that mitochondrial dynamics may have an important role in regulating apoptosis [99]. Therefore, the mitochondrial distribution pattern was analyzed by MitoTracker Red in complex 3 treated cells. Untreated cells showed uniform and organized mitochondria distribution pattern in the cytoplasm, whereas in complex 3 treated MDA-MB-231 cells showed disorganized mitochondrial distribution, which seemed to be aggregated near to nucleus or perinuclear

concentration-dependent manner in comparison to control (Fig. 8). A similar study has also been reported with some other Cu complexes which induced apoptosis by ROS-mediated pathway [96]. This study demonstrates that complex 3 efficiently increases ROS generation in breast cancer cells and consequently induces apoptosis.

3.9. Mitochondrial aggregation during apoptosis It was reported that some synthetic anticancer drugs induce apoptosis by interfering with the mitochondrial dynamics which can be related to pro-apoptotic signals [96]. Thus, to investigate the effect of complex 3 on mitochondrial distribution in MDA-MB-231 cells, 34

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locus of the cells and emitted bright red fluorescence (Fig. 9). This imaging data clearly indicate that altered mitochondrial distribution pattern might be the cause of pro-apoptotic stimulation in treated MDAMB-231 breast cancer cells that ultimately lead to apoptotic cell death.

the online version. These CIF data can also be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc. cam.ac.uk/data_request/cif. Supplementary data associated with this article can be found in the online version, at doi: http://dx.doi.org/10.1016/j.jinorgbio.2017.05. 013.

4. Conclusions

References

We have presented the synthesis, conversion, crystal structure and biological study of a known Schiff base dicopper complex [CuLCl]2 (1) and its two new mononuclear derivatives [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3). The introduction of the coordinating solvents, water and pyridine, to the dimeric compound 1 results in the breaking of the Cu–Cl–Cu bridges with the formation of the solvent coordinated monomeric complexes (2 and 3, respectively). Interestingly, 2 and 3 can easily be converted into 1 by stirring them in methanol. Our previous studies [68–71] proved that the chloride ligand of 1 is replaceable by charged ligands, e.g., pseudohalides (azide, cyanide and thiocyanate) and carboxylates (cyclohexane-1,4-dicarboxylic acid, benzene-1,3,5tricarboxylic acid and benzene-1,2,4,5-tetracarboxylic acid), to afford multinuclear complexes. Herein, we present the dynamic nature of the Cu–Cl–Cu bridges in 1 being breakable by neutral ligands like water and pyridine, but regenerated in the presence of another solvent (MeOH) without a strong coordinating ability. These interesting features indicate that the dimeric compound 1 is a versatile precursor for monomeric and polymeric compounds, deserving more attention for future investigation. In vitro studies have revealed that all three complexes were found to be modest to highly effective against A-549 (lung) and MDA-MB-231 (breast) cancer cell lines with IC50 values of (57 ± 3, 1; 77 ± 3, 2; and 51.5 ± 0.3 μM, 3) and (100, 1; > 100, 2; and 5.0 ± 0.3, 3), respectively. Notably, complex 3 shows a marked cytotoxic activity against MDA-MB-231 (breast cancer) cell line, with the IC50 value even better than that of the commercially and widely accepted drug cisplatin (IC50 = 9.5 ± 0.2 μM). Furthermore, the superior cytotoxicity activity of the complex 3 (in MDA-MB-231) was rationalized using Hoechst/PI fluorescence staining through microscopy and Annexin/PI staining through fluorescence activated cell sorting (FACS). Various studies including flow cytometry for the analysis of cell cycle phase distribution demonstrated that complex 3 exhibits cytotoxic activity by selectively inducing apoptosis in MDA-MB-231 cell line without influencing cell cycle progression. In addition, the possible key factors behind the mode of the apoptotic induction pathway were affirmed through redox status and mitochondrial distribution pathway which might have initiated or critically indulged in anticancer activity of complex 3 in breast cancer cells. Briefly, a simple synthetic approach was followed to produce highly biologically active copper(II) complexes, one of them being significantly active towards MDA-MB-231 cell line following apoptotic pathway. Further studies should be performed to conclude the potential of complex 3 as a cancer chemotherapeutic agent.

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