Interaction of Zn with Losartan. Activation of Intrinsic Apoptotic Signaling Pathway in Lung Cancer Cells and Effects on Alkaline and Acid Phosphatases

A new losartan [2-butyl-5-chloro-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol zinc(II) complex [Zn(Los)Cl], was synthesized and characterized. The crystal structure was determined by x-ray diffraction methods. When aqueous solutions of the ligand and the metal were mixed, the known and more soluble powder [Zn(Los)2].3H2O (ZnLos) complex has been obtained. The interactions with phosphatases showed a concerted mechanism displayed by the Zn ions and ZnLos up to 500 μM concentration: a decrease of the acid phosphatase (AcP) associated with an increase in the alkaline phosphatase (ALP) activities. The complex and ZnSO4 showed a cytotoxic behavior on human lung A549 cancer cell line at concentrations higher than 75 μM with reactive oxygen species (ROS) generation and GSH (and GSH/GSSG ratio) depletion. Apoptotic cells were observed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method, a mechanism accompanied by upregulation of BAX protein, downregulation of Bcl-XL and release of caspase-3. The BAX/Bcl-XL ratio was found to be significantly higher in cells exposure to ZnLos than cells treated with ZnSO4, in agreement with the higher apoptotic percentage of cells found for the complex. Cell death was found to be produced by apoptosis and no necrosis has been observed. On the contrary, losartan exerted low effects on phosphatases, produced some reduction of cancer cell viability (concentrations > 250 μM, number of apoptotic cells similar to the basal) with low ROS depletion, without alteration of the GSH/GSSG and low BAX/Bcl-XL ratios. In the MRC-5, normal lung fibroblasts cell line only ZnSO4 at concentrations higher than 200 μM displays cytotoxic effects. Graphical abstract Interaction of Zn with losartan. Activation of intrinsic apoptotic signaling pathway in lung cancer cells and effects on alkaline and acid phosphatases Interaction of Zn with losartan. Activation of intrinsic apoptotic signaling pathway in lung cancer cells and effects on alkaline and acid phosphatases


Introduction
Lung cancer is one of the leading causes of death around the world and non-small cell lung cancer (NSCLC) accounts for about 85% of this type of cancer [1]. On the other hand, the renin-angiotensin system regulates blood pressure and the angiotensin receptor blockers (ARBs or sartans) have been designed to selectively binding and blocking to the angiotensin II type 1 (AT1) receptor [2], reducing the vasoconstrictor action of the angiotensin II peptide. Recent studies showed that the AT1 receptors are expressed in the A549 human lung adenocarcinoma cells and that the treatment with an AT1 receptor antagonist could inhibit the proliferation of lung cancer cell lines [3]. Therefore, medications may provide synergistic effects to existing chemotherapies by reducing Ang IImediated mitogenesis and angiogenesis [4]. To introduce structural modifications to the sartans (whose functional groups bear multiple binding sites and strong coordinating ability), we have synthesized coordination complexes with the biometal copper(II) that allow the improvement of some of the biological properties or the ARBs such as the anticancer effects [5].
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12011-018-1334-x) contains supplementary material, which is available to authorized users.
Zinc is a trace essential element in which deficiency includes growth retardation and cell-mediated immune dysfunctions. Zinc deficiencies in diets can also contribute in the damage of DNA that increase risk for cancer development [6]. Zinc treatment enhances cell-mediated immunity and displays protective mechanisms against DNA damage which may be a possible mechanism for its anticancer activity [7]. Moreover, zinc supplementation decreases angiogenesis and the induction of inflammatory cytokines hence increasing apoptosis in cancer cells [8]. The anticancer effects of Zn(II) ions have been determined in several cancer cell lines including, for instance, non-small-cell lung cancer cells [9].
Considering that sartans have the potential to reduce zinc levels in hypertensive patients, yielding an increase in urinary Zn excretion and a decrease of serum Zn levels [10], we started the study of the synthesis of a new Zn-sartan complexes and the determination of some of the biological effects. Therefore, we have introduced newer therapeutic strategies to supply the biometal Zn together with the sartan drug, and avoid the alteration of the metal concentrations in specific body organs and/or entire body by the generation of a metal complex with sartan. The strategy of the combination of Zn(II) and sartan in a coordination compound has been developed to bring additional biological properties during the administration of the antihypertensive drugs. In fact, the Zn/azilsartan complex previously prepared in our group [11] produced an improvement of the anticancer effect of the parent drug, azilsartan.
One of the orally effective pharmaceutical drugs used for the treatment of arterial hypertension (ARB) is losartan potassium, the potassium salt of [2-butyl-5-chloro-3-[[4-[2-(2H-tetrazol-5yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol (from now on, losartan, Los) (Fig. 1). The structure of losartan consisted in a biphenyltetrazole ring system attached to a substituted imidazole ring through a methylene spacer (Fig. 1). The thermal, spectroscopic characterization, antioxidant evaluation (against 1,1diphenyl-picrylhydrazyl, DPPH) and the pyrolysis [12] and the complex formation equilibria of losartan with Zn(II) [13], [Zn(Los) 2 ].3H 2 O (ZnLos), have recently been reported. Here, we report some spectroscopic characterizations and the biological studies of this solid complex. In an attempt to obtain single crystals suitable for structural determinations using gel diffusion techniques, a new Zn(II) complex with losartan and chloride ions in its coordination sphere, [Zn(Los)Cl] has been obtained. Due to the insolubility of the [Zn(Los)Cl] complex, the biological determinations were performed using the known [Zn(Los) 2 ].3H 2 O complex.
Phosphoregulation is involved in many biological events and often occurs as a network-like cascade, in which the activity of one phosphatase or kinase is dependent on the upstream activity of another. A considerable rise in alkaline phosphatase (ALP) activity is produced in various types of malignant tumors and is used as a tumor marker [14]. While ALP contains Zn ions, the addition of concentrations lower than 1 mM Zn(II) could increase its enzymatic activity. However, acid phosphatases (AcP) are being inhibited by Zn(II) [15]. Herein, we report our findings on the Zn(II) complex, the metal and the ligand on their action exerted on AcP and ALP. Additionally, we determined their possible anticancer activities in a human lung A549 cell line and the mechanism of action: cellular reactive oxygen species (ROS) generation and glutathione (GSH) depletion with the concomitant oxidation of GSH to GSSG and presence of apoptotic or necrotic cells (terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL), ratio of the apoptotic regulators proteins BAX to Bcl-XL, caspase-3 activation). Data are presented and discussed above. The cytotoxic effect of the compounds against human normal lung fibroblast (MRC-5) cell line has also been determined.

Materials and Methods
All chemicals were of analytical grade and used without further purification. Losartan, potassium salt was purchased from Parafarm and Zinc(II) nitrate (Merck) was used as supplied. A sodium silicate solution (14% NaOH-27% SiO 2 , SiO 2 .NaOH, Sigma-Aldrich) was used for the diffusion method. Infrared spectra of powdered samples were measured with a Bruker IFS 66 FTIR-spectrophotometer from 4000 to 400 cm −1 using the KBr pellet technique. FT-Raman spectra were measured using the FRA 106 Raman accessory with germanium detector operating at liquid nitrogen temperature. A continuouswave Nd/YAG laser working at 1064 nm was employed for Raman excitation. Elemental analyses for carbon, hydrogen and nitrogen were performed using a Carlo Erba EA 1108 analyzer. Thermogravimetric analysis (TGA) were measured with a Shimadzu system (model TG-50), working in an oxygen flow of 50 mL/min and at a heating rate of 10°C/min. Sample quantities ranged between 5 and 10 mg. Electronic absorption spectra were recorded on a Hewlett-Packard 8453 diode-array spectrophotometer, using 1-cm quartz cells. Fluorescence spectra were measured using a Shimadzu RF-6000 spectrophotometer. The molar conductance of the complex was measured on a Conductivity TDS Probe -850,084,

Synthesis of [Zn(Los)Cl]
To obtain the crystal structure of the ZnLos, complex single crystals suitable for x-ray determinations were grown in Utubes by the gel diffusion method. The bottom of the U-tube was filled with silicate gel (sodium silicate 5% aqueous solution with the addition of 50% HCl up to pH 7). One side-arm of the tube was filled with an aqueous solution of losartan (1 mmol, 10 mL) and the other with a Zn nitrate aqueous solution (0.5 mmol, 5 mL

Single-Crystal X-ray Diffraction Data
The structural determinations of the [Zn(Los)Cl] complex were performed on an Oxford Xcalibur Gemini, Eos CCD diffractometer with graphite-monochromated CuKα (λ = 01.54184 Å) radiation. X-ray diffraction intensities were collected (ω scans with ϑ and κ-offsets), integrated and scaled with CrysAlisPro [16] suite of programs. The unit cell parameters were obtained by least-squares refinement (based on the angular settings for all collected reflections with intensities larger than seven times the standard deviation of measurement errors) using CrysAlisPro. Data were corrected empirically for absorption employing the multi-scan method implemented in CrysAlisPro. The structure was solved by the intrinsic phasing method implemented in SHELXT of the SHELX suit of programs [17] and refined by full-matrix least-squares with SHELXL of the same package. The hydrogen atoms were located in a difference Fourier map and all but the ones of -CH 3 group were refined at their found positions with isotropic displacement parameters. The methyl H atoms were refined with the riding model as a rigid group allowed to rotate around the C-CH 3 bond such as to maximize the sum of the residual density at the calculated positions. The methyl group converged to a staggered rotational conformation. Crystal data, data collection procedure, structure determination methods, and refinement results are summarized in Table 1.

Behavior on Phosphatases Activities
Alkaline Phosphatase The effect of the Zn(II) cation, losartan, and the ZnLos complex on ALP activity was determined spectrophotometrically. The reaction was started by the addition of the substrate (p-NPP) and the generation of p-nitrophenol was monitored by the absorbance changes at 405 nm [18]. Briefly, the experimental conditions for ALP-specific activity measurement were as follows: 1 μg/mL of bovine intestinal ALP and 5 mM of p-NPP were dissolved in the incubation buffer (55 mM glycine + 0.55 mM MgCl 2 , pH = 10.5) and held for 4 min. The effects of the compounds were determined by addition of different concentrations (1-500 μM) of each one to the pre-incubated mixture. The compounds were dissolved in DMSO and the stock solutions were diluted in the buffer giving a final concentration of DMSO less than 1%. The effect of each concentration was tested in three independent experiments at least by triplicated. The initial rate, in absence of any compound (V 0 ), was determined as the rate of p-NPP hydrolysis at 37°C and pH = 10.5. V i values were determined like V 0 but in the presence of the different concentrations of each of the investigated systems. Data were expressed as mean ± SEM. An analysis of variance (one-way ANOVA) was applied to compare the means of multiple groups of measured data. Significance was defined as p < 0.05.

Acid Phosphatase
Acid phosphatase (AcP) inhibition test was performed according to Blum and Schwedt procedures [19] using acid phosphatase (AcP from potato, product number P-3752, Sigma Chemical Co. (St. Louis, MO). The stock solution of the enzyme was prepared by mixing 12.5 mg of the 0.25 U/mL acid phosphatase powder in 2.0-mL acetate buffer (pH 5.60). The stock solution of the compounds (100 μL in DMSO) was diluted in 1.9 mL of the buffer and 0.170 g of the substrate p-NPP were dissolved in distilled water (2.5 mL). A volume of 0.50 mL of each compound solution was mixed with 0.10 mL of the enzyme solution and 1.00 mL of buffer. The mixture was kept at 25°C for 20 min (incubation time). Then, the substrate (0.10 mL) has been added. The reaction was stopped with the addition of 0.50 mL of a 0.5 M sodium hydroxide solution. The final concentration of DMSO resulted less than 1%. The enzymatic activity was calculated by the measurement of the absorbance of 4-nitrophenolate at 405 nm against a blank prepared without the enzyme. Three independent replicates of each point were measured. In both experiments, 100% of the enzyme activity is assigned to a basal measurement containing all the reaction media including the same volume of DMSO in all the tests. It is worthy to mention that the presence of that very low quantity of DMSO did not affect the enzyme activity. Data were expressed as mean ± SEM. An analysis of variance (one-way ANOVA) was applied to compare the means of multiple groups of measured data. Significance was defined as p < 0.05.

Cellular Determinations
Cell Culture Human alveolar carcinoma cell line (A549) was used in cytotoxicity studies. Cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin, at 37°C in a humidified incubator with 5% CO 2 . At 85% confluence, cells were harvested using PBS-EDTA and suspended at a final concentration of 2 × 10 6 cells/mL into 6-and 48-well plates and 35-mm dishes, respectively, according to the selected experiment. To examine the effect of the compounds, cells were treated with ZnSO 4 , losartan, and ZnLos at different concentrations and then they were assessed in several assays.

Reactive Oxygen Species Assay
Intracellular reactive oxygen species (ROS) generation by the compounds was evaluated using dihydrorhodamine 123. This probe passively enters the cell and is oxidized to cationic rhodamine 123. The ROS level was measured by a fluorometric quantitative assay [21]. Briefly, 2 × 10 4 cells/mL were seeded in 48-well plates and were incubated with ZnSO 4 , losartan, and ZnLos (0-500 μM). Cultured cells were washed with PBS before being incubated in 200 μL of a solution of 10 μM DHR 123 for 30 min at 37°C under light protection. Afterwards, cells were lysed with 0.1% Triton X-100. The fluorescence spectra were recorded at 485-nm excitation and 520-nm emission wavelengths. The proteins were measured according to Bradford technique [22]. Results were presented as the percentage of fluorescence intensity relative to the basal measurements ± SD.

Estimation of Cell GSH and GSSG Content
A modification of Hissin and Hilf's method [23] was used for the determination of reduced (GSH) and oxidized glutathione (GSSG) content in treated A549 cells. The amounts of both compounds were estimated fluorometrically after the reaction with o-phthalaldehyde (OPT). Cells exposed to 0-500 μM of ZnSO 4 , losartan, and ZnLos were treated with 0.1% Triton X-100, and 100 μL of the lysates were used to determine the GSH, GSSG, and protein content. GSH selectively reacted with OPT (10 mg/mL in methanol) at pH 8.0 (ice-cold 0.1 M Na 2 HPO 4 -0.005 M EDTA buffer), whereas after the addition of 0.04 mM N-ethyl maleimide (NEM) in the lysates fractions, only GSSG reacted with OPT at pH 12.0 (0.1 N NaOH). Protein contents in each cellular extract were quantified using the Bradford assay. Standard concentrations of GSH and GSSG (0.05-1.0 μg/mL) were used. The fluorescence spectra were recorded at 350-nm excitation and 420-nm emission wavelengths. The concentrations in micrograms per milligrams were calculated from the respective calibration curves. The ratio GSH/GSSG was expressed as a percentage of the basal for all the experimental conditions.

Apoptosis Assay
The terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL assay) were used to detect DNA strand breaks during apoptosis [24]. A549 cells were seeded in slides into 35-mm dishes. After 24 h of treatment with 500 μM of ZnSO 4 , losartan, and ZnLos, slides were fixed in 4% paraformaldehyde and permeabilized with proteinase K 15 μg/mL in 10 mM Tris-buffer. TUNEL assay was performed using an in situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. For positive controls, sections were treated with 0.7 mg/mL DNAse I (Sigma-Aldrich) for 15 min before treatment with terminal deoxynucleotidyl transferase (TdT). This enzyme was replaced with the same volume of distilled water in negative controls. Labeled cells were observed and photographed under a fluorescence microscope (Olympus CX-35 equipped with a Coolpix Digital camera) at ×40 magnification. Cells were considered TUNEL-positive when nuclei cells exhibit bright green fluorescence. The apoptotic index (AI) was expressed as the percentage of TUNEL-positive cells per 1000 examined A549 cells.
Immunocytochemistry A549 cells were grown over coverslips into 35-mm dishes for 24 h. Cells were treated with ZnSO 4 , losartan, and ZnLos (500 μM) for 24 h were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were quenched for 10 min with 3% hydrogen peroxide in methanol. Then, they were permeabilized with 1% Triton X-100 and stirred during 10 min. Slides were rinsed with 0.5% PBS and PBS-Tween.
The following primary antibodies were used: Bcl-XL, BAX, and caspase 3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). They were diluted 1:50 in PBS and incubated overnight at humidified atmosphere. Immunostaining was performed using a Dako linked streptavidin-biotin + horseradish peroxidase (LSAB+/HRP) kit (Dako Cytomation) followed by the application of a chromogen 3,3′-diaminobenzidine (DAB) (Dako kit) according to manufacturer's instructions. All negative controls were obtained by excluding the primary antibody from the reaction. Samples were then counter-stained with hematoxylin and visualized under a light microscope. Images were taken using an Olympus Coolpix-microdigital camera fitted on a CX-35 microscope (Olympus, Japan) [25].  [26]. Protein content in lysates was determined by Bradford method. Proteins (25 μg) were resolved by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, CA, USA). Membranes were blocked and then were incubated overnight at 4°C with the following primary antibodies: anti-BAX (1: 750) and anti-Bcl-XL (1: 1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-β actin (Sigma-Aldrich). The technique was followed with incubation for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Inc., USA). β actin detection was used to normalize inmunoblottings. Immunocomplexes were detected by an Opti4CN (4-chloro-1-naphthol) kit (Bio-Rad, CA, USA). Band optical density (OD) was analyzed using Scionbeta 2 image software and results were expressed as the ratio: (protein of interest OD/β-actin OD) ×100. All experiments have been performed by triplicate and a representative immunoreactive band of one experiment is shown.

AO/EtBr Staining
Acridine orange/ethidium bromide (AO/EtBr) staining was carried out to detect morphological evidence of apoptosis and necrosis. While AO is a vital dye that stains both live and dead cells and makes the nuclei appear green, EtBr only stains cells that have lost membrane integrity (nucleus in red). Early apoptotic cells stain green and contain bright dots in the nuclei. Late apoptotic cells also incorporate EtBr and show condensed and often fragmented nuclei. Necrotic cells also stain in orange, but present nuclear morphology resembling that of viable cells [27]. The stock solution of AO was prepared dissolving 5 mg in 1 mL of ethanol and stored in the refrigerator. The same procedure was made for EtBr. To prepare the staining solution 20 μL of AO and 20 μL of EtBr were added to 960 μL of PBS. Briefly, A549 cells were seeded into 100-mm dishes. After 24 h of treatment with ZnSO 4 , losartan, and ZnLos (500 μM), the supernatant (medium and floating A549 cells) were transferred to 15-mL tubes. The rest of the adherent cells were detached with PBS-EDTA. The supernatant and the detached cells from the same sample were pooled together in the 15-mL tubes. A549 cells were pelleted by centrifugation at 1000 RPM for 5 min and washed with 1 mL of cold PBS. Cell pellets were then re-suspended with 1 mL of the staining solution. The cells were then washed with PBS and examined under a fluorescence microscope (Olympus CX-35) at ×100 magnification.

Statistical Analysis
Data are expressed as the mean ± standard error (SE). The Sigma plot software package was used for statistical analysis. An analysis of variance (one-way ANOVA) was applied to compare the means of multiple groups of measured data. Significance was defined as p < 0.05.

Results and Discussion
Description of the Structure of [Zn(Los)Cl] Figure 2 shows an ORTEP [28] drawing of the supra-molecular zinc(II) complex and the corresponding bond distances and angles around the metal are given in Table 2. The losartan ligand is negatively charged by loss of the proton at the tetrazole CN 4

Vibrational Spectroscopy
Some of the vibrational FTIR bands of the spectrum of ZnLos have previously been assigned and compared with the  [29,30]. According to the results shown in Table 3, the N-H stretching band (at ca. 3500 cm −1 ) is absent due to the deprotonation of the losartan potassium salt and the O-H stretching bands of the ligand and of the water molecules of the complex are located in the 3380-3180 cm −1 range. Small modifications are found in the bending COH and stretching CO (COH) modes upon coordination. The symmetric CN and CC aromatic stretching bands (ca. 1615-1524 cm −1 ) show higher intensities in the Raman spectra, as expected. The infrared and Raman vibrational modes of the C-C bridge bond biphenyl of losartan (1260 and 1206 cm −1 ) have been shifted in positions and show different intensities upon complexation. The main changes in the vibrational spectra of the complex are due to the modes of the tetrazole group (from 1100 to 954 cm −1 ) indicating that the coordination of the zinc cation occurred through this anionic moiety of the ligand. The changes observed in the vibrational spectra of the OH modes may be indicative that this group is also involved in the coordination to the metal center.
The vibrational spectrum of the crystal [Zn(Los)Cl] showed some changes with respect to the spectrum powder complex [Zn(Los) 2 ].3H 2 O. The OH stretching appeared as a very strong band, shifted to low energy because the H atom is bonded to the N atom of tetrazole (H bond, see Fig. 2). Bands related to the COH bending were also shifted (1154 cm −1 ) with respect to the ligand and the spectrum of the powder complex. The coordination of the Zn ion to the imidazole group in this complex produced a shift of the bands assigned to νCN (1460 and 1426 cm −1 ) to higher energies. The resolved structure of the complex showed the presence of a tetrazole bridge and then these vibrational modes must appear shifted with respect to the powder complex bands. In effect, the new band at 1032 and the 1013 cm −1 band that shifted to the blue, are indicative of a different coordination mode of the tetrazole group to the Zn ion.

Biological Studies
The [Zn(Los) 2 ].3H 2 O complex has been selected for the biological determinations due to its higher solubility. Considering previous reports that showed that the cytotoxic effects of ZnSO 4 were most likely due to the Zn ions rather than the sulfate ions and that ZnCl 2 exhibited pronounced cytotoxic effects, even stronger than those caused by Zn sulfate, we have selected the latter salt for the biological studies [31]. Stability studies have been performed measuring the variation of the ZnLos electronic absorption spectra and the molar conductivities with time. For the electronic absorption spectra measurements, the dissolution of the complex was performed in EtOH 96%. There was no observable variation in the electronic spectra of the ethanolic solution of the complex at least during 90 min (Fig. S1). The same behavior has been observed for DMSO solutions (data not shown). These results demonstrated that during the manipulation time of the samples for the biological determinations, a significant amount of the complex remained without decomposition. Conductivity measurements of 1-mM individual solutions of ZnSO 4 , losartan potassium salt in water, and ZnLos in DMSO, and a 0.5% DMSO-H 2 O mixture were carried out at 25°C. The molar conductivity values for solutions of ZnSO 4 and the losartan potassium salt resulted 171 and 68 Ω −1 cm 2 /mol −1 , respectively, typical for 2:2 and 1:1 electrolytes and similar to previous reported data [32,33]. These values are practically unchanged up to 4 h (data not shown). The molar conductivity of the complex either in water or in a 0.5% DMSO-H 2 O mixture (14 and 13 Ω −1 cm 2 /mol −1 , respectively) is indicative or an undissociated neutral Zn(II) complex. This molar conductivity values remain unchanged up to 4 h (see Table 4). Hence, it has been determined using two experimental assays that the complex in solution stands without decomposition at least during the first 4 h of manipulation.

Behavior on Alkaline Phosphatase Activity
Alkaline phosphatase (ALP) is a homodimeric enzyme widely distributed and it has been isolated from eukaryotes as well as from prokaryotes. It hydrolyzes non-specifically phosphate monoesters at alkaline pH to produce inorganic phosphate and an alcohol. It is present in a number of tissues including liver, bone, intestine, and placenta. Serum ALP is of interest in the diagnosis of hepato and bone diseases. A considerable rise in ALP activity is produced in various types of malignant tumors and is used as a tumor marker [14] and also in different injuries such as brain and cerebrovascular diseases [34]. The inhibition of ALP has been related to the improvement of some diseases and for instance, the inhibitory effect of vanadate anion has directly been associated with the insulin-enhancing activity of vanadate complexes [35]. All highly purified alkaline phosphatases have proved to be Zn(II)metalloenzymes and this metal behaved as activator due to the saturation of Zn(II) binding  (3) Cl (2) Symmetry transformations used to generate equivalent atoms: sites. Alkaline phosphatase from bovine intestinal mucosa is an homodimeric metalloenzyme, containing in each active site one Mg ion (for structural stabilization) and two Zn ions involved in catalysis and a novel fourth metal site is occupied by Ca(II). The addition of Zn increase the enzymatic activity but an excess of Zn could replace Mg at binding sites in the ALP hence producing an inhibitory effect [36]. The effects of the compounds on ALP activity are shown in Fig. 3. Zn ion produced enzymatic activation up to a concentration of 0.5 mM.
Losartan potassium salt showed low inhibitory effect that can be correlated to the chelating ability of losartan to the active sites of the enzyme. On the contrary, the ZnLos complex barely enhanced the activity of ALP, modulating the activating action of the Zn(II) ions. Behavior on Acid Phosphatase Activity Acid phosphatases are ubiquitous and abundant enzymes in plants, animals, fungi, and bacteria. Their action includes production, transport, and recycling inorganic phosphate which is crucial for cellular metabolism and bioenergetics. Intracellular and secreted AcPs are believed to play a major role in inorganic phosphate scavenging and the utilization and turnover of inorganic phosphate-rich sources occurring in either animal lysosomes or plant vacuoles [37]. In mammals, a role of AcPs is ascribed to iron transport (non-enzymatic process), bone resorption, and generation of reactive oxygen species (ROS) as an immune response. AcPs are abundantly expressed in osteoclasts, activated macrophages (in which the enzyme may play a role in the immune defense system increasing ROS production) and dendritic cells. A variety of biological roles have been proposed for plant AcPs due to its bifunctionality (hydrolysis and peroxidation). The major function of these enzymes is the mobilization of inorganic phosphates from organophosphates in the soil and the participation in phosphate metabolism [38]. AcP from potato is a nonspecific phosphomonoesterase with a Fe 2+ /Fe 3+ catalytic site together with the co-catalytic sites of Mn 2+ , Zn 2+ , and trace copper [39]. The enzyme displayed a pH optimum of 5.8, was activated by Mg 2+ , and was potently inhibited by molybdate, vanadate, and Zn 2+ [15]. From Fig. 4, it can be seen that while losartan exerted low effects on AcP, the Zn ion inhibited the enzymatic activity in a dose-response manner and the ZnLos complex behaved like the metal at concentrations higher than 75 μM. A pattern of low and high activity has been observed in bony tissues of rats, as well as a pattern of low alkaline phosphatase activity during acid phosphatase activity peaks and vice versa [40]. Phosphoregulation is involved in many biological events and often occurs as a network-like cascade, in which activity of one phosphatase or kinase is dependent on the upstream activity of another. The same concerted mechanism has been displayed by the Zn ion and the ZnLos complex at the tested concentrations: a decrease of the AcP activity has been associated with an increase in the ALP activity.

MTT Assay
The cytotoxic effects of ZnSO 4 , losartan, and ZnLos complex on A549 cell line were determined by the MTT assay. The viability of the normal human lung fibroblasts cell line MRC-5 incubated with the different compounds has also been  Fig. 3 Effect of ZnLos (circles), losartan (squares), and ZnSO 4 (triangles) on ALP activity from bovine intestinal mucose. The results are expressed as the percentage of the basal level and represent the mean ± SEM (n = 9). Asterisk indicates significant differences versus basal p < 0.05. Pound sign indicates significant differences at the same concentration between ZnLos, losartan, and Zn, p < 0.05  . Asterisk indicate significant differences versus basal p < 0.05. Pound sign indicate significant differences at the same concentration between ZnLos, Los, and Zn, p < 0.05 studied. Cancer cells were treated with different concentration (0-500 μM) for 24 h. As shown in Fig. 5, no cytotoxicity was observed in losartan up to 250 μM concentration. A similar low effect of this antihypertensive drug at high concentrations has been measured for leukemic cell lines [41] but reports on A549 lung cancer cells (72-h incubation) were somewhat different [42]. Cell viability decreased upon cellular incubation with ZnSO 4 from 100 μM, as has previously been determined [43]. The complex ZnLos declined cell proliferation in a dosedependent manner and showed a significantly decrease from 50 μM. The cytotoxic effect of the compounds has been evaluated in a human normal lung fibroblasts MRC-5 cell line. It can be seen (Fig. 6) that the compounds did not exhibit significant cytotoxic activities up to 150-μM concentration. This behavior has also been reported for losartan in human normal lung fibroblasts, WI-38 [42]. From Fig. 6, a cytotoxic effect of an excess of Zn(II) ions at concentrations higher than 200 μM (see Section BROS Generation^) in this normal cell line can be seen. Then, it could be demonstrated that losartan complexation attenuates the toxic effect of Zn at elevated concentrations.

ROS Generation
Like an essential element, the excess of zinc is toxic, inducing apoptosis in cell lines that involve oxidative stress production [44]. Cancer cells display an intrinsic oxidative stress higher than normal cells. Hence, the exposure to ROS generating compounds will cause more injury in cancer cell lines due to a decrease of the antioxidant defenses. Zinc ions are able to induce oxidative stress in cancer cells by different molecular mechanisms. The enhancement of ROS production could be generated by the induction of mitochondrial respiration impairment, the inhibition of glutathione reductase activity or the inhibition of the lipoamide dehydrogenase multienzyme complex (LADH), which has a role in the preservation of the reducing environment of mitochondria [45]. Acidosis, an increase of ROS generation in several tumor cells and stimulation of apoptosis by zinc exposure has been reported [9]. ROS generation was detected and quantified in response of the incubation of the A549 cells with ZnSO 4 , losartan and ZnLos for 24 h (Fig. 7). Cells treated with increasing concentrations of ZnSO 4 (from 100 μM) showed an increase in the oxidative stress, as expected in comparison with reported data in A549 cell line [31]. Upon incubation with increasing doses of ZnLos and losartan a significant increase and a decrease in oxidative stress, respectively, has been detected. These results suggested that the production of ROS plays a key role in the ZnSO 4 and ZnLos-induced apoptosis (see below) and subsequent cell death.

GSH Content and GSH/GSSG Ratio
To further analyze the involvement of ROS in ZnLos-induced cell death, the levels of reduced glutathione (GSH) and the GSH/GSSG ratio was estimated in the cells after ZnLos treatment for 24 h (Fig. 8). The antioxidant cellular levels were also measured for losartan and Zn(II) ions. A significant decrease of cellular GSH content after ZnLos treatment for 24 h was found. A concomitant depletion in the intracellular GSH Asterisk indicate significant differences versus basal p < 0.05. Pound sign indicates significant differences at the same concentration between ZnLos, losartan, and ZnSO 4 , p < 0.05 levels and the GSH/GSSG ratio has been observed with the enhancement in ROS levels. Besides, GSH levels and GSH/ GSSG ratio increased at low ZnSO 4 concentrations reaching maximum at ZnSO 4 concentration 25 μM corresponding with the hypoxia (low oxygen stress) state of A549 cells and decreased at higher concentrations. This decrease has previously been reported (up to 100 μM) [46] and explained on the basis that zinc transporters played an important role in the resistance to high concentrations of extracellular zinc. Losartan showed some decay of GSH content and had no changes in GSH/GSSG ratio. Then, it can be demonstrated that increased ROS production followed by mitochondrial GSH depletion represented a crucial event, which irreversibly produced cell death.

TUNEL Assay
A sub-cytotoxic concentration of 25 and 100 μM have been determined for ZnLos and ZnSO 4 , respectively. Therefore, 500 μM concentration of each compound was used for further analysis. To determine the mechanisms of cell death, the generation of apoptotic cells by the action of the compounds on the A549 cell line has been determined. Despite that exposure to exogenous zinc resulted in increased apoptosis, growth inhibition, and altered oxidative stress in cancer cells [9], from Fig. 9a, it can be seen that ZnLos and losartan induced a significant increment of the apoptotic index (AI) in A549 cells (p > 0.05). However, the apoptotic induced effect of ZnLos was double higher than the noticed for losartan (Fig. 9b). It can then be concluded that the decrease in cell viability could be achieved by a programmed cellular death or apoptosis and that both ROS generation and GSH depletion could act as regulators.

Immunocytochemical Staining
The morphological changes caused by incubation of the cells with the different compounds are shown in Fig. 10. Cells treated with ZnSO 4 and ZnLos became round and the volume shrank. Apoptotic characteristics such us apoptotic bodies, agglutination, and margination of chromatin and cytoplasm vacuolization could be seen.
Apoptosis is modulated by antiapoptotic and proapoptotic effectors, which involves a large number of proteins. The proapoptotic and antiapoptotic members of the Bcl-2 family regulate programmed cell death and are targets of anticancer therapy [47]. The cytosolic proapoptotic BAX protein modified its conformation when initiation of apoptotic signaling and becomes mitochondrial membrane-associated, producing the opening of the mitochondrial voltage-dependent anion channel. This process produced the loss in membrane potential and the release of cytochrome-c and caspase-3 activation. From Fig. 10, it can be seen that BAX and Caspase-3 protein expression were significantly higher in cells treated with 500 μM ZnSO 4 and ZnLos than for losartan at the same concentration.
Then, it can be seen that the antiapoptotic Bcl-XL protein was significantly higher in cells treated with losartan and was lower in cells treated ZnSO 4 and ZnLos. The apoptogenic effect of elevated concentration of Zn, associated with increased levels of BAX or decreased Bcl-XL has also been observed in prostate cancer cells [9,48].

Western Blotting
The ratio of proapoptotic proteins of Bcl2 family to antiapoptotic members that determines whether a cell responds to a programmed cell death stimulus has also been examined using Western blotting. The ratio between BAX and Bcl-XL that determines whether cells undergo apoptosis correlated well with the immunohistochemical expression of the proteins. This ratio was found to be significantly higher in A549 cells exposed to ZnLos than cells treated with ZnSO 4 . Losartan treatment caused low BAX/ Bcl-XL ratio. (Fig. 11).
The sequence of biochemical events occurring in human lung A549 cancer cells after modulation of the cellular redox state has been determined. The decline in cellular GSH, in response to ZnLos treatment, coincided with the induction of mediators of apoptotic signaling including mitochondrial BAX release and caspase-3 activation and an increase of the BAX/Bcl-XL ratio.

AO/EtBr Staining Assays
Because TUNEL assay fails to discriminate apoptotic from necrotic cells, AO/EtBr staining assays were performed to a  Fig. S2, it was clear that with the addition of increasing concentrations of the compounds no changes associated with necrosis were observed. Therefore, necrotic effects could be discarded even at concentrations of 500 μM (higher than sub-cytotoxic doses).

Discussion
We have previously determined that the antihypertensive drug losartan did not displayed anticancer effects in a bone cancer cell line (UMR106) at the maximum tested concentration value (500 μM) but copper(II) complexation enhanced the cell killing effect of the ligand [49]. Then, we could determine that the introduction of a structural modification of losartan by metal complexation enhanced its antiproliferative action. On the other hand, we have performed a structural modification of azilsartan, a modern angiotensin II receptor antagonist by Zn(II) complexation, considering that some studies suggested that the biometal Zn displayed anticancer effects. The complex ZnAzil also produced a significant decrease in the viability of the A549 lung cancer cell line in comparison to the antihypertensive drug through a mitochondrial apoptotic pathway [11]. We herein report the biological behavior of a complex formed between losartan and Zn(II). Like Cu(II) ions, the Zn(II) ions are also able to modify the structure and the anticancer properties of losartan, measured in the lung cancer cell line A549. The proposed mechanism for the anticancer effect of the ZnLos complex is shown in Fig. 12 cells treated with the aforementioned concentration of ZnLos complex, suggests that the changes in the BAX/Bcl-XL ratio, could allow BAX to be available to signal apoptosis through the intrinsic apoptotic pathway. Thus, the mitochondrial pores in the outer membrane with the depolarization of the transmembrane potential trigger the release of the proapoptotic factor BAX and the cytochrome-c to cytoplasm, which lead to the caspase-3 activation and the subsequent DNA cleavage. Table 5 displayed data of cell survivals and oxidative stress measured after cellular incubation with ZnLos and ZnAzil complexes. As it can be seen, ZnAzil complex produces high levels of ROS at each tested concentration thereby causing Fig. 11 BAX and Bcl-xL expression in A549 cells. a Representative Immunoblot images corresponding to cultures treated with 500 μM ZnSO 4 , losartan, ZnLos, and DMSO (negative control). Immunoblottings were performed by triplicate from a single sample of three independent experiences. The β actin was used as an internal loading control. b BAX/Bcl-XL ratio. Protein levels were analyzed by densitometry using Scion Image Software (NIH USA). Data were normalized to beta-actin. Pound sign indicates significant differences between ZnSO 4 , losartan, and ZnLos, p < 0.05 Fig. 12 Diagram of the proposed mechanism for the anticancer effect of the ZnLos complex oxidation of the main cellular antioxidant GSH. The depletion on the glutathione to oxidized glutathione ratio that potentially diminished the antioxidant defenses follows the same trend. As a consequence, although both complexes exerted anticancer effects, A549 cell line survival resulted lower upon ZnAzil treatment. Even though TUNEL, immunocytochemical staining and Western blotting assays were performed at different concentrations for ZnLos and Zn Azil, it has been demonstrated that both compounds exerted their anticancer effects by programmed cell death or apoptosis through the mitochondrial pathway, regulated by the Bcl-2 family of proteins.

Conclusions
Due to the flexible coordination ability and characteristic coordination behavior, such as folded conformation of the tetrazole and imidazole N atoms, two different coordination modes for Zn(II) ion coordination to losartan were obtained, working at different experimental conditions. Three-dimensional aggregation is achieved with the tetrazole groups bridging the Zn(II) ions and by the coordination of the remote imidazole groups of losartanate to the metal center giving a metal to ligand (1:1) stoichiometry, [Zn(Los)Cl]. A previous reported complex with two losartanate groups coordinated to the Zn(II) metal center has also been prepared as a powder. Structural determinations for this known complex, [Zn(Los) 2 ].3H 2 O (ZnLos), could not be performed because attempts to obtain single crystals failed. Both compounds were characterized by vibrational and diffuse reflectance spectroscopies but the latter compound has been selected for the biological tests because of its high solubility.
The complex ZnLos and the Zn(II) ions showed a concerted mechanism for the interaction with phosphatases at the tested concentrations: a decrease of the AcP activity has been associated with an increase in the ALP activity. A weak effect of losartan on phosphatases inhibition has been determined. The present study showed that ZnLos could induce oxidative stress correlating with cytotoxicity in the human alveolar carcinoma cell line A549. The programmed cell death involved a significant increase of ROS generation upon incubation with the ZnLos complex followed by depletion of GSH that induces apoptosis through the intrinsic pathway and no necrosis has been detected. The present report about chemical and anticancer effect of ZnLos on A549 cells, contribute to new knowledge about the potential use complex for inducing the mitochondrial apoptotic pathway in lung adenocarcinoma treatments. According to our measurements, ZnSO 4 produced lower levels of ROS and higher levels of the natural intracellular antioxidant system, GSH up to 50 μM concentrations, and then it did not show cytotoxic effects on the cancer A549 cell line. Then, supplementation of ZnLos complex instead of a ZnSO 4 salt in hypertensive patients could modulate the anticancer effect of the biometal at low concentrations. However, further molecular studies are required to know the benefits of the treatment with ZnLosartan as an antitumor therapeutic agent.