Characterization and cytotoxic effect of aqua-(2,2′,2′′-nitrilotriacetato)-oxo-vanadium salts on human osteosarcoma cells

The use of protonated N-heterocyclic compound, i.e. 2,2′-bipyridinium cation, [bpyH+], enabled to obtain the new nitrilotriacetate oxidovanadium(IV) salt of the stoichiometry [bpyH][VO(nta)(H2O)]H2O. The X-ray measurements have revealed that the compound comprises the discrete mononuclear [VO(nta)(H2O)]− coordination ion that can be rarely found among other known compounds containing nitrilotriacetate oxidovanadium(IV) moieties. The antitumor activity of [bpyH][VO(nta)(H2O)]H2O and its phenanthroline analogue, [phenH][VO(nta)(H2O)](H2O)0.5, towards human osteosarcoma cell lines (MG-63 and HOS) has been assessed (the LDH and BrdU tests) and referred to cis-Pt(NH3)2Cl2 (used as a positive control). The compounds exert a stronger cytotoxic effect on MG-63 and HOS cells than in untransformed human osteoblast cell line. Thus, the [VO(nta)(H2O)]− containing coordination compounds can be considered as possible antitumor agents in the osteosarcoma model of bone-related cells in culture.


Introduction
Despite numerous attempts to define the role of vanadium in biological processes its impact on the functioning of higher organisms remains to be elucidated. During the last 10-15 years, progress in the chemistry of vanadium, namely in the search of its therapeutic applications has been exponential and several reviews have been published (Rehder 2013;Willsky et al. 2011;Pessoa and Tomaz 2010;Jakusch et al. 2011;Gambino 2011;Pessoa et al. 2015a, b;Kioseoglou et al. 2015;Leon et al. 2016a, b;Rehder 2017). In particular, much attention has been paid on insulin-mimetic (-enhancing) properties (Srivastava and Mehdi 2005;Marzban and McNeill 2003;Thompson et al. 2009). Among the compound tested as small molecule insulin-mimetics, or insulin-enhancers, VO(maltolato) 2 (BMOV) (McNeill et al. 1992; Levina and Lay 2011) and VO(Etmaltolato) 2 Electronic supplementary material The online version of this article (doi:10.1007/s10534-017-0001-6) contains supplementary material, which is available to authorized users.
(BEOV) (Thompson et al. 2009) have been extensively studied (Fig. 1). BMOV and BEOV may be taken orally and both lower plasma glucose levels in streptozotocin-induced (STZ) diabetic rats (Thompson and Orvig 2006), BEOV having completed Phase I and IIa of clinical trials.
The vanadocene(IV) compound, [VCp 2 Cl 2 ], was extensively studied in preclinical testing against both animal and human cancer cell lines, exhibiting a high in vitro activity (Havelek et al. 2012;Vinklarek et al. 2004;Gleeson et al. 2009;Palackova et al. 2007). Metvan induces apoptosis in different tumoral cell lines of human origin such as leukemia cells, breast cancer, ovarian, prostate and testicular cancer patients (Evangelou 2002;D'Cruz and Uckun 2002;Dong et al. 2000). The broad spectrum of anticancer activity of Metvan together with favorable pharmacodynamic features and a lack of toxicity emphasizes that this V 4? -compound has a potential to be the first vanadium coordination compound as an alternative to the platinum-based chemotherapy (D'Cruz and Uckun 2002).
Another interesting group of vanadium compounds are complexes of oxidovanadium(IV) with ligands that hold multiple donor atoms able to coordinate with metal centers. Binary and ternary oxodiacetate (oda) coordination compounds of VO 2? , VO(oda), VO(oda)(bpy) and VO(oda)(phen), display important effects in bone related cells in culture (Fig. 3) (Rivadeneira et al. 2010).
All these compounds were tested on two osteoblastlike cell lines in culture (MC3T3E1 derived from mouse calvaria and UMR106 derived from rat osteosarcoma cells). VO(oda) caused an inhibition of a cellular proliferation in both cell lines, but the cytotoxicity was stronger in the normal (MC3T3E1) than in the tumoral (UMR106) osteoblasts. VO(oda)(phen) in the osteoblastic model caused the inhibition of the cellular proliferation in both cell lines (MC3T3E1 and UMR106), but the cytotoxicity was stronger in the normal than in the tumoral osteoblasts (León et al. 2012b). On the contrary, VO(oda)(bpy) was statistically stronger in the tumoral cells (León et al. 2012b). A nuclease activity of the three compounds ( Fig. 3) revealed that the DNA cleavages caused by VO(oda)(bpy) and VO(oda) were similar, while VO(oda)(phen) showed a stronger effect. VO (oda)(phen) presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)(bpy) and then by VO(oda) according to the number of intercalating heterocyclic moieties (Yodoshi et al. 2007).
The subject of our continuous interest are polycarboxylate vanadium coordination compounds since it has been found that they are able to scavenge superoxide free radicals (O 2 Á-) as well as protect the HT22 hippocampal neuronal cell line against an oxidative damage (Tesmar et al. 2015;Wyrzykowski et al. 2013Wyrzykowski et al. , 2015a. The participation of the oxidovanadium(IV) compounds in leveling of reactive oxygen and nitrogen species (RONS) suggests that vanadium compounds can be beneficial in the  (Pessoa et al. 2015b). However, the main concern as regards the application of vanadium compounds as drugs is to minimalize their adverse side effects (Shukla et al. 2006). It is the crucial issue for the future use of vanadium-based drugs in medicine. For these reasons the studies on structure, physicochemical and biological properties of the vanadium compounds with a potential pharmacological ability are the subject of interest to many research groups. Strong chelating ligands are very important in aqueous systems since they are models for trapping, transport and storage of different metallic species in living organisms (Harding et al. 1993). For this reason we have used nitrilotriacetate ions (nta) as they are known to form fairly stable complexes with oxidovanadium(IV) ions (Felcman and Fraústo da Silva 1983). In this paper, the crystal structure and physicochemical properties of the new VO 2? -compound, namely 2,2 0bipyridinium aqua-(2,2 0 ,2 00 -nitrilotriacetato)-oxo-

Synthesis of [bpyH][VO(nta)(H 2 O)]H 2 O
The synthesis was carried out by a method similar to that previously used for the preparation of the phenanthrolinium salt (Tesmar et al. 2015). Thus, the mixture of VO(acac) 2 (2.65 g) and H 3 nta (1.91 g) in water (40 mL) was refluxed for ca. 0.5 h. The hot solution was filtered and cooled. To this solution, the methanolic solution of 2,2 0 -bipyridyl (1.56 g) was added. Then, the mixture was concentrated (in order to eliminate Hacac by an evaporation) and left for a crystallization at the room temperature. After 14 days a blue precipitate of the compound fell out. The recrystallization from hot water gave blue crystals after 7 days.  H,4.3%,N,9.4%,Found: C,42.7%,H,4.3%,N,9.3%. Aqueous solutions of the investigated compounds have shown a high stability, e.g. being resistant to the oxidation in air, i.e. remain unaltered (UV-Vis control) for at least 3 days.

X-ray measurements
The blue hexagonal prism crystal of [bpyH][VO(nta) (H 2 O)]H 2 O was sealed in a glass capillary filled with helium and next it was mounted on the Bruker APEXII automatic diffractometer equipped with the CCD detector, and used for a data collection. X-ray intensity data were collected with the graphite monochromated CuK a (k = 1.54178 Å ) radiation at temperature 100.0(1) K, with the x scan mode. The 27 s exposure time was used and reflections inside the Ewald sphere were collected up to h = 72.4°. The unit cell parameters were determined from 124 strongest reflections. Details concerning the crystal data and refinement are given in Table 1. Examination of reflections on two reference frames monitored after each 20 frames measured showed no loss of the intensity during measurements. During the data reduction the Lorentz, polarization and empirical absorption (Sheldrick 2003) corrections were applied. The structure was solved by the dual-space algorithm implemented in the XT software (Sheldrick 2015a). All the non-hydrogen atoms were refined anisotropically using the fullmatrix, least-squares technique on F 2 . All the hydrogen atoms were found from the difference Fourier synthesis after four cycles of an anisotropic refinement, and refined as ''riding'' on the adjacent atom with a geometric idealisation after each cycle of refinement and individual isotropic displacement factors equal 1.2 times the value of equivalent displacement factor of the parent methyl carbon atoms, and 1.5 times of parent oxygen or nitrogen atoms. The XL software (Sheldrick 2015b) was used for the refinement of the structure model. Atomic scattering factors were those incorporated in the computer programs. Tables of crystal data and structure refinement, anisotropic displacement coefficients, atomic coordinates and equivalent isotropic displacement parameters for non-hydrogen atoms, H-atom coordinates and isotropic displacement parameters, bond lengths and interbond angles have been deposited with the Cambridge Crystallographic Data Centre under No. CCDC1483068.

IR spectra
The IR spectra were recorded on the BRUKER IFS 66 spectrophotometer in a KBr pellet over the 4400-650 cm -1 range.

TG analysis
Thermogravimetric (TG) analyses in argon (Ar 5.0) were run on the Netzsch TG 209 apparatus (range 298-973 K, Al 2 O 3 crucible, empty crucible as a reference, a sample mass 8-10 mg, a heating rate 10 K min -1 , a flow rate of the carrier gas 20 mL min -1 ).

Potentiometric titrations
Potentiometric titrations were performed at 298.15 K by using Cerko Lab System microtitration unit fitted with 5-mL Hamilton's syringe and pH-combined electrode (Schott-BlueLine 16 pH type) All details for the measuring devices and the experimental setup were described in   (Alderighi et al. 1999).
Cell culturing (hFOB, MG-63, HOS) The cell lines: two human osteosarcoma cell lines (MG-63 and HOS) and untransformed hFOB were used to assess an anti-proliferative and cytotoxic effect, respectively. hFOB cells were grown in a mixture of Dulbecco's Modified Eagle's Medium and Ham F12 medium (1:1 ratio), MG-63 and HOS in Eagle's Minimum Essential Medium also containing sodium pyruvate 110 mg/L and supplemented with 10% fetal bovine serum, 6 lg/mL penicillin-G, and 10 lg/mL streptomycin.
Cell treatment (hFOB, MG-63, HOS) Cultured cancer cells with 80-90% confluence were used for plating. The adherent cells were trypsinized to detach cells. 100 lL of cells were seeded into each well of the 96-wells plates (1-5 9 10 4 cells per well). The plate was maintained at 37°C in a incubator for 48 h until 80-90% confluence. Then, old media were discarded and the cells were treated with tested compounds and cisplatin as positive control. The concentrations of investigated compounds used in experiments were carefully selected according to the results obtained from a preliminary concentrationresponse study (data not shown). A stock solution of cisplatin was prepared by dissolving solid cisplatin in phosphate buffered saline (PBS; water solubility: 0.253 g/100 mL at 25°C). Fresh solutions of cisplatin were made up for each experiment owing to its instability in water. The concentrations of cisplatin were selected based on published data found in the literature (Baharuddin et al. 2016;Křikavová et al. 2014). Both tested compounds and cisplatin were suspended in the SF cell culture and diluted to appropriate concentrations ex tempore every time before adding the cells. The dilutions of investigated compounds and cisplatin were filtered through a 0.22 lm membrane filter. Controls (negative) were treated with the serum free (SF) cell-culture medium.

The IR spectroscopic characterization
The characteristic for the oxidovanadium(IV) compounds band at 981 cm -1 can be assigned to the V=O stretching mode (Pranczk et al. 2016;Banik et al. 2014). Two bands at 1586 and 1402 cm -1 correspond to the antisymmetric and symmetric vibrations of the ionized COOgroups, respectively. This finding confirms the contribution of the carboxylate groups in the coordination of V(IV) in a monomeric [VO(nta)(H 2 O)]coordination entity. The difference, Dm, between the frequencies of asymmetrical [m as (OCO -)] and symmetrical [m s (OCO -)] vibrations for carboxylate group in the compound (Dm = 1586-1402 = 184 cm -1 ) and in the nitrilotriacetate sodium salt, Na 3 nta, Dm = 1598-1406 = 192 cm -1 ) suggests the ionic character of the VO-nta interactions (Nakamoto 2009). The band at 488 cm -1 corresponds to the stretching vibration m(V-N) and agrees with the X-ray results showing that nta acts as a tetradentate ligand. The band at 1095 cm -1 that can be assigned to the stretching vibration m(C-N) of the nta ligand (Tomita and Ueno 1963) is shifted ca. 100 cm -1 in relation to m(C-N) in the free H 3 nta (1200 cm -1 ). It indicates that the N atom of the nta ligand coordinates to V atom. Most relevant infrared bands of bpyH ? are: 1474 cm -1 bpym ring , 1456 cm -1 bpy -m ring ? d ring-H , 1274 cm -1 , 1224 cm -1 and 1040 cm -1 bpy -d(CH) in plane . The Symmetry transformations used to generate equivalent atoms: (i) -x ? 1/2, y -1/2, -z ? 3/2; (ii) -x ? 3/ 2, y -1/2, -z ? 3/2; (iii) -x ? 2, -y ? 1, -z ? 1; (iv) x, y -1, z; (v)x ? 1, -y ? 2, -z ? 1; (vi) -x ? 1/2, y ? 1/2,z ? 3/2; (vii) x ? 1, y -1, z presence of the stretching vibration band at 3464 cm -1 indicates the attachment of a proton to the nitrogen atom of bpy. This is in line with the results obtained from the X-ray measurements. Moreover, the IR spectrum of the compound shows bands at 3300-3100 and 1660-1610 cm -1 that can be assigned to antisymmetric and symmetric OH stretching and HOH bending bands of the lattice and coordination water, respectively.
The thermal analysis  Table 5. The logarithm of the overall equilibrium constants of the complex species (Table 5) were refined by least-squares calculations using the Hyperquad2008 (ver. 5.2.19) computer program (Gans et al. 1996). Fig. 5. Due to the presence of an aqua ligand in the coordination sphere of VO 2? the competition of the [VO(nta)(H 2 O)]ion with the organic cation (bpyH ? or phenH ? ) for hydroxide takes place. The aqua complex is stable to the pH of 5. At a higher pH range it undergoes a hydrolysis and the resulting hydroxo complex species ([VO(nta)(OH)] 2-) reach the highest concentration at around pH 9.8. The ability of [VO(nta)(H 2 O)]to hydrolysis is a very important feature that has an impact on the susceptibility and a rate of the oxidation of (IV) to V(V) (Nishizawa et al. 1985). At the high concentration of the [VO(nta)(OH)] 2ions the dinuclear species of the [(VO) 2 (l 2 -O)(nta) 2 ] 4type are formed (Fig. 5). These type of oxidovanadium(IV) coordination entities have previously been reported in solid (Zhang et al. 2005). Thus, the similar coordination mode of the VO 2? cations can be expected in solutions. The oxo-bridged dioxidovanadium(IV) complexes ([(VO) 2 (l 2 -O)(nta) 2 ] 4-) exist at equilibrium with the mononuclear [VO(nta) (OH)] 2ions in aqueous solutions at pH above 10. Thus, physicochemical and biological properties of the nitrilotriacetate oxidovanadium(IV) ions are affected by the pH of the system under study.  (5) Cg (1)ÁÁÁCg (2) iii 3.7910 (9) 9.46 (7) 29.70 -3.5246 (5) Cg (2)ÁÁÁCg (1) iii 3.7909 (9) 9.46 (7) 21.61 -3.2928 (6) Cg (1), Cg(2) indicates the centroids of six-membered aromatic rings (R) containing N11, N22 atoms respectively, a is a dihedral angle between planes I and J, b is an angle between Cg(I) and Cg(J) vector and normal to plane I and d p is a perpendicular distance of Cg(I) on ring J plane Symmetry transformations as in Table 3 The cytotoxicity of oxidovanadium(IV) compounds in human osteoblast and osteosarcoma cell lines

The representative species distribution diagram for [bpyH][VO(nta)(H 2 O)]H 2 O is displayed in
The cytotoxicity of the compounds The concentration-dependent effects of investigated compounds on the normal, hFOB (hFOB 1.10) and human osteosarcoma cell line (MG-63) were tested at the plasma membrane level (the LDH leakage) after 48 h of an incubation (Figs. 6, 7). The results were referred to the aqueous soluble inorganic derivative of bi-valent platinum, i.e. cisplatin (cis-Pt(NH 3 ) 2 Cl 2 ) (Florea and Busselberg 2011;Prylutskyy et al. 2015).
Cisplatin is currently one of the most extensively used chemotherapeutic drugs for the cancer treatment (Leon et al. 2014a, b). In osteosarcoma cells cis-Pt(NH 3 ) 2 Cl 2 induces a selective inhibition of DNA synthesis and, as a consequence, a cell proliferation and reproduction.
In the concentration range of 10-50 lM no significant cytotoxic effects of the compounds on the untransformed hFOB (hFOB 1.19) were observed. The highest concentration of the compounds (100-500 lM) triggers a decrease in the viability of the hFOB 1.19 cells to about 20%. As far as the human osteosarcoma cell line (MG-63) is concerned, the concentration-dependent cytotoxic effect of the compounds was observed. It is interesting to note that both compounds exhibited a stronger cytotoxicity than cisplatin used as a positive control. The study has revealed that the compounds, in the low concentration range (10-50 lM), have a significant selectivity for malignant cells. These results point to the fact that the investigated compounds show promising properties to be further investigated as possible antitumor agents in this model of bone-related cells.  (Jakusch et al. 2002) and 4.93 (Duma and Hancock 1994), respectively  Table 5 The antiproliferative activity of the compounds Many attempts have been taken to determine the putative mechanisms of an action involved in the antitumoral effects of the oxidovanadium(IV) compounds (Rivadeneira et al. 2010;Leon et al. 2013Leon et al. , 2014aLeon et al. , 2015Leon et al. , 2016aFerrer et al. 2006). It has been reported that these compounds, depending Data are expressed as mean values ± SD from three experiments. **p \ 0.01; ***p \ 0.001 versus control on the cellular line, may promote the generation of the reactive oxygen species (ROS) mainly in the mitochondria (Leon et al. 2012a(Leon et al. , 2014bRivadeneira et al. 2009;Di Virgilio et al. 2011) leading to a decrease of glutathione (GSH) concentration. GSH is one of the mayor reducing agents responsible for maintaining the cellular redox status through the balance of the couple glutathione/glutathione disulphide (GSH/GSSG). The depletion of GSH concentration alters the intracellular redox balance (GSH/ GSSG) on an account of the accumulation of GSSG inside the cells. Furthermore, the oxidative stress causes a dissipation of the mitochondria membrane potential (MMP) that can lead the cells into apoptosis and necrosis (Mayer and Oberbauer 2003). The concentration-dependent (in the concentration range of 1-100 lM) effect of the compounds on the two human MG-63 and HOS osteosarcoma cell lines was investigated by the measurement of the BrdU (5bromo-2 0 -deoxyuridine) incorporation by actively dividing cells after 48 h of culture in the presence of different concentrations of compounds (Figs. 8,9). It has been found that all investigated compounds induce a significant reduction of the BrdU incorporation into cellular DNA indicating a concentrationdependent anti-proliferative effect. Importantly, the cytotoxic effect of the compounds against cancer cell line was already found at the concentrations which are non-toxic for untransformed hFOB. phen and bpy metal coordination compounds (Yodoshi et al. 2007;Chakravarty 2006).

Conclusions
When the inorganic cations (NH 4 ? , La 3? , Eu 3? , Nd 3? ) are present as the counter-ions, mononuclear [VO (nta)(H 2 O)]coordination units easily undergo a dimerization via an oxide bridge. We have previously reported that the use of the cation formed by protonation of N-heterocyclic compound (i.e. 1,10-phenanthrolinium cation, phenH ? ) enabled to obtain discrete mononuclear [VO(nta)(H 2 O)]coordination ions. This finding has been confirmed in the present study for other protonated N-heterocyclic compound, i.e. 2,2 0 -bipyridinium cation, [bpyH ? ]. The X-ray measurements have revealed that [bpyH][VO(nta)(H 2 O)]H 2 O comprises the discrete mononuclear [VO(nta)(H 2 O)]coordination ions linked through O-HÁÁÁO hydrogen bonds formed between the inner coordination sphere of water molecules and the oxygen atoms of the carboxylate groups. IR spectra as well as thermal analysis have confirmed the results obtained from the X-ray measurements.
The potentiometric titration method has successfully been applied to assess the stability of the complexes in aqueous solutions. The [VO(nta)(H 2-O)]ion undergoes a hydrolysis. At a higher pH range (above pH 10) the resulting hydroxo complexes form the oxo-bridged dioxidovanadium(IV) species of the [(VO) 2 (l 2 -O)(nta) 2 ] 4type. Thus, the physicochemical properties and biological activities of the complexes depend on the pH of the investigated system. Biological