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Water soluble heterometallic potassium-dioxidovanadium(V) complexes as potential antiproliferative agents
Journal of Inorganic Biochemistry 155 (2016) 17–25
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Water soluble heterometallic potassium-dioxidovanadium(V) complexes as potential antiproliferative agents Manas Sutradhar a,⁎, Alexandra R. Fernandes a,b,⁎, Joana Silva b, Kamran T. Mahmudov a,c, M. Fátima C. Guedes da Silva a, Armando J.L. Pombeiro a,⁎ a b c
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal Department of Chemistry, Baku State University, Z. Xalilov Str. 23, Az, 1148 Baku, Azerbaijan
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 1 October 2015 Accepted 10 November 2015 Available online 11 November 2015 Keywords: Dioxidovanadium(V) Heterometallic complex X-ray structure Antiproliferative agent
a b s t r a c t Two water soluble heterometallic potassium–dioxidovanadium polymers, [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) [H2L1 = (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide and H2L2 = (2,3-dihydroxybenzylidene)benzohydrazide], have been synthesized and characterized by IR, NMR, elemental analysis and single crystal X-ray diffraction. The antiproliferative potentials of 1 and 2 were examined towards human colorectal carcinoma (HCT116), and lung (A549) and breast (MCF7) adenocarcinoma cell lines. 1 exhibits a high cytotoxic activity against colorectal carcinoma cells (HCT116), with IC50 lower than those for cisplatin. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The use of Schiff bases and their transition metal complexes in biological, clinical and analytical applications has been the object of an increased interest [1–3]. Among the various transition metal complexes used in biological and pharmacological studies, vanadium compounds exhibit important roles as antitumor, antimicrobial agents, reducers of hyperlipidemia, hypertension and obesity, and enhancers of oxygen affinity by hemoglobin and myoglobin, as well as of the metabolic effects of insulin [4–10]. Particularly, the development of medicinal applications of oxidovanadium(IV) and (V) complexes has achieved a considerable interest in drug discovery. Emphasis has been given to the insulin mimetic activity of bis(maltolato)oxidovanadium(IV) (BMOV) and bis(ethylmaltolato)oxidovanadium(IV) (BEOV), the only two vanadium compounds tested in clinical trials to date [11]. Frontline investigations to find the molecular targets for these compounds have been carried out in order to understand how they interfere in the insulin pathway and in glucose and lipid metabolisms [11,12]. Besides the insulin-mimetic activity, these compounds have also gained attention towards their antimicrobial activity, tumor growth inhibition and prophylaxis against carcinogenesis [13–24]. Successful in vitro studies over the past few decades have advanced the anticancer research on vanadium into the preclinical stage [3]. The understanding of the ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Sutradhar), [email protected] (A.R. Fernandes), [email protected] (A.J.L. Pombeiro).
http://dx.doi.org/10.1016/j.jinorgbio.2015.11.010 0162-0134/© 2015 Elsevier Inc. All rights reserved.
molecular effects of vanadium complexes in cancer cells has been a concern and, in this regard, cytotoxicity, apoptosis induction and biological targets such as DNA and proteins have been investigated [25–27]. These modes of action have also been demonstrated for most cytostatic vanadium compounds although some of them, for example vanadocenes, may directly intercalate with DNA [3,7]. In this study, we have used two azine-type Schiff base ligands [28– 32] for the synthesis of two water soluble heterometallic potassium– dioxidovanadium(V) complexes. Such a type of ligands renders a great stability to the V(IV) and V(V) complexes [33–37]. The antiproliferative potential of the two complexes was examined towards human colorectal carcinoma (HCT116) and lung (A549) and breast (MCF7) adenocarcinoma cell lines. The cytotoxicity of both complexes against normal human fibroblasts was also assessed. 2. Experimental 2.1. General materials and procedures All synthetic work was performed in air. The reagents and solvents were obtained from commercial sources and used as received, i.e., without further purification or drying. [VO(acac)2] (acac− = acetylacetonate) was used as the metal source for the synthesis of complexes 1 and 2. C, H, and N elemental analyses were carried out on Perkin Elmer PE 2400 Series II by the Microanalytical Service of the Instituto Superior Técnico. Infrared spectra (4000–400 cm− 1) were recorded on a BRUKER VERTEX 70 or Jasco FT/IR-430 instrument in KBr pellets,
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Scheme 1. Synthesis of polymers 1 and 2.
wavenumbers are in cm−1. Mass spectra were run in a Varian 500-MS LC Ion Trap Mass Spectrometer equipped with an electrospray (ESI) ion source. For electrospray ionization, the drying gas and flow rate were optimized according to the particular sample with 35 psi nebulizer pressure. Scanning was performed from m/z 100 to 1200 in methanol solution. The compounds were observed in the negative mode (capillary voltage = 80–105 V). The 1H NMR spectra were recorded at room temperature on a Bruker Avance II + 300 (UltraShield™ Magnet) spectrometer operating at 300.130 MHz for proton. The chemical shifts are reported in ppm using tetramethylsilane as the internal reference. 51V NMR spectra were recorded on a Bruker 400 UltraShield spectrometer at ambient temperature (297 K) in DMSO–d6. The vanadium chemical shifts are quoted relative to external [VOCl3]. UV spectra were recorded in an Evolution 300 UV–Vis spectrophotometer (Thermo Scientific).
Table 1 Crystal data and structure refinement details for complexes 1 and 2.
Empirical formula Formula weight Crystal system Space group a/Å b/Å c/Å β/° V (Å3) Z Dcalc (g cm−3) μ(Mo Kα) (mm−1) Rfls. collected/unique/observed Rint Final R1a, wR2b (I ≥ 2σ) Goodness-of-fit on F2 a b
1
2
C14H10KN2O6V 392.28 Tetragonal I−4 22.475 22.475 5.9163(2) 90 2988.45(10) 8 1.744 0.977 13,534/2574/1910 0.0652 0.0471, 0.1012 1.055
C14H12KN2O6V 394.30 Monoclinic C 2/c 29.5903(7) 6.1898(2) 17.7502(4) 109.935(1) 3056.29(14) 8 1.714 0.956 21,827/2787/2244 0.0424 0.0436, 0.1089 1.079
R = Σ||Fo | − |Fc ||/Σ|Fo |. wR(F2) = [Σw(|Fo |2 − |Fc |2)2 / Σw|Fo |4]1/2.
2.2. Synthetic procedures 2.2.1. Synthesis of the pro-ligands H2L1 and H2L2 The Schiff base pro-ligands (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide (H2L1) and (2,3-dihydroxybenzylidene)benzohydrazide (H2L2) (Scheme 1) were prepared by following a reported method [38–41] which involves the condensation of the corresponding aroylhydrazides with 2,3-dihydroxybenzaldehyde.
Table 2 Selected features, bond distances (Å) and angles (°) in polymers 1 and 2.
Involving L−2 N–N C–Oenolate C–Ophenolate C–Ophenol N–Cenolate–C–C N–N–C–Oenolate Involving vanadium Coord. number Polyhedral volume (Å3) V–Ooxido V–Ophenolate V–Oenolate V–N ∠ O–V–N (5-memb. Ring) ∠ O–V–N (6-memb. Ring) Involving potassium Coord. number Polyhedral volume (Å3) K–O
Minimum M…M distances V…V V…K K…K
Minimum Maximum
1
2
1.394(7) 1.298(8) 1.332(8) 1.365(7), 1.367(9) 163.2(7) 4.3(9)
1.392(3) 1.319(3) 1.341(3) 1.365(4) −175.0(3) −0.4(4)
5 (τ5 = 0.22) 4.966 1.593(5) 1.637(4) 1.926(5) 1.968(5) 2.134(5) 74.3(2) 82.2(2)
5 (τ5 = 0.08) 4.847 1.6098(19) 1.6485(19) 1.9109(19) 1.9598(19) 2.134(2) 73.52(8) 82.93(9)
7 30.537 2.653(5) 3.098(5)
8 12.475 2.734(2) 3.275(2)
5.636(1) 3.6647(17) 4.781(2)
5.6634(6) 3.6842(10) 4.9787(12)
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2.2.2. Syntheses of the dioxidovanadium(V) complexes Complexes [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) have been synthesized by using a common general method. To a 30 mL ethanolic suspension of H2L1 (0.272 g, 1.00 mmol for 1) or H2L2 (0.256 g, 1.00 mmol for 2), 0.265 g (1.00 mmol) of [VO(acac)2] was added and the reaction mixture refluxed. After 1 h the mixture was cooled down to room temperature and an aqueous solution of 0.1 M KOH was added with constant stirring until a pH of ca. 8
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was obtained. The reflux was taken up for another hour. The resultant dark yellow solution was filtered and the filtrate was kept in air. After ca. 2 d, X-ray quality yellow crystals were isolated, washed 3 times with cold ethanol and dried in open air. [KVO2(L1)]n (1) — Yield 74% (0.290 g, based on vanadium). Anal. Calcd for C 14 H 10 KN 2 O 6 V: C, 42.86; H, 2.57; N, 7.14. Found: C, 42.79; H, 2.54; N, 7.09. IR (KBr; cm− 1 ): 3172 ν(OH), 1600 ν(C = N), 1261 ν(C–O)enolic, 1078 ν(N–N), 954, 913 ν(V_O). ESI-
Fig. 1. Molecular structure of polymers 1 (top) and 2 (bottom) with partial atom labeling schemes. Symmetry codes: i) x,y,1 + z ii) 1/2-x,1/2-y,1/2 + z iii) 1/2-x,1/2-y,1.5 + z iv) y,-x,-z v) 1/2-x,1/2-y,-1/2 + z vi) 1/2-x,1/2-y,-1.5 + z vii) -y,x,-z viii) x,y,-1 + z (for 1) and i) x,-1 + y,z; ii) 1/2-x,-1/2 + y,1/2-z; iii) 1/2-x,1/2 + y,1/2-z; iv) 1/2-x,1/2-y,1-z; v) x,1 + y,z; vi) 1/2x,-1/2 + y,1/2-z (for 2).
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MS(−): m/z 353 [VO2 (L1 )] − (100%). 1 H NMR (DMSO-d6, δ): 11.10 (s, 1H,OH), 8.69 (s, 1H, − CH_N), 7.82–6.78 (m, 8H, C 6 H 4 ). 51 V NMR (DMSO-d6, δ) — 526. [KVO2(L2)(H2O)]n (2) — Yield 71% (0.279 g, based on vanadium). Anal. Calcd for C14H12KN2O6V: C, 42.65; H, 3.07; N, 7.10. Found: C, 42.68; H, 2.86; N, 7.09. IR (KBr; cm−1): 3210 ν(OH), 1602 ν(C_N), 1258 ν(C–O)enolic, 1074 ν(N–N), 960, 918 ν(V_O). ESI-MS(−): m/z 337 [VO2(L2)]− (100%). 1H NMR (DMSO-d6, δ): 11.16 (s, 1H,OH), 8.57 (s, 1H, −CH_N), 7.97–6.87 (m, 8H, C6H4).51V NMR (DMSO-d6, δ) — 532.
same conditions as MCF7 cell line [45–47]. All cell lines were purchase from ATCC (www.atcc.org). 2.4.2. Compound exposure for dose–response curves Cells were plated at 5000 cells/well in 96-well plates. Media was removed 24 h after platting and replaced with fresh media containing: 0.1–200 μM of 1, 2, H2L1 and H2L2 or the precursor [VO(acac)2] or 0.1% (v/v) DMSO (vehicle control). All the previous solutions were prepared from concentrated stock solutions (in DMSO) of the complexes and ligands.
2.3. X-ray measurements X-ray single crystals of complexes 1 and 2 were immersed in cryo-oil mounted in Nylon loops and measured at a temperature of 150 (1) or 296 K (2). Intensity data were collected using a Bruker AXS-KAPPA APEX II or a Bruker APEX-II PHOTON 100 with graphite monochromated Mo-Kα (λ 0.71073) radiation. Data were collected using phi and omega scans of 0.5° per frame and a full sphere of data was obtained. Cell parameters were retrieved using Bruker SMART [42] software and refined using Bruker SAINT [42] on all the observed reflections. Absorption corrections were applied using SADABS [42]. Structures were solved by direct methods by using the SHELXS-97 package [43] and refined with SHELXL-2014 [43]. Calculations were performed using the WinGX System—Version 1.80.03 [44]. The hydrogen atoms of hydroxide groups were found in the difference Fourier map and the isotropic thermal parameters were set at 1.5 times the average thermal parameters of the belonging oxygen atoms, with their distances restrained (in 2) by using the DFIX commands. Coordinates of hydrogen atoms bonded to carbon atoms were included in the refinement using the riding-model approximation with the Uiso(H) defined as 1.2Ueq of the parent aromatic atoms. Crystal data and refinement parameters are presented in Table 1 and selected features, bond distances and angles are given in Table 2. 2.4. Biological assays 2.4.1. Cell culture Human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corp., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Invitrogen Corp.) and maintained at 37 °C in a humidified atmosphere of 5% (v/v) CO2 [45–46]. MCF7 cell line derived from pleural effusion of breast adenocarcinoma from a female patient was grown in similar conditions, supplemented with 1% MEM non-essential amino acids (Invitrogen Corp.) [47]. Normal Human fibroblasts were grown in the
2.4.3. Viability assays After 48 h of cell incubation in the presence or absence of each complex or ligand, cell viability was evaluated with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described [45–49]. In brief, this is a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96® AQueous Assay is composed of solutions of MTS and an electron coupling reagent (phenazinemethosulfate, PMS). MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product was measured in a Bio-Rad microplate reader Model 680 (Bio-Rad, Hercules, CA, USA) at 490 nm, as absorbance is directly proportional to the number of viable cells in culture. 2.4.4. Assessment of apoptosis through Hoechst 33,258 staining HCT116 cells grown as described above were plated at 7500 cells/mL and incubated for 48 h in culture medium containing the compound 1 or 0.1% (v/v) DMSO (vehicle control). Hoechst staining was used to detect apoptotic nuclei as previously described [46]. Briefly, medium was removed, cells were washed with phosphate-buffered saline 1X (PBS) (Invitrogen), fixed with 4% (v/v) paraformaldehyde in PBS 1× (10 min in the dark) and incubated with Hoechst dye 33,258 (Sigma, Missouri, USA; 5 μg/mL in PBS 1×) for another 10 min. After being washed with PBS 1×, cells were mounted using 20 μL of PBS:glicerol (3:1; v/v) solution. Fluorescent nuclei were sort out according to the chromatin condensation degree and characteristics. Normal nuclei showed noncondensed chromatin uniformly distributed over the entire nucleus. Apoptotic nuclei showed condensate or fragmented chromatin. Some cells formed apoptotic bodies. Plates were photographed in an AXIO
Fig. 2. Cytotoxicity of 1 in HCT116, A549 and MCF7 cell lines. HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
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Fig. 3. Cytotoxicity of 2 in HCT116, A549 and MCF7 cell lines. HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
Scope (Carl Zeiss, Oberkochen Germany), and three random microscopic fields per sample with ca. 50 nuclei were counted. Mean values were expressed as the percentage of apoptotic nuclei [46]. 2.4.5. Stability of complexes 1 and 2 in biological conditions In order to assess complexes stability in biological conditions phosphate buffered saline (PBS) (pH 7.0) was used. Compounds (33 μM) were incubated in PBS for 0, 24 and 48 h at 37 °C and UV spectra were recorded in 240 to 440 nm range. 2.4.6. Statistical analysis All data were expressed as mean ± SEM from at least three independent experiments. Statistical significance was evaluated using the Student's t-test; p b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Synthesis and X-ray structural characterization It is well known that [VO(acac)2] undergoes aerial oxidation in solution (preferably in alcohol medium) in the presence of hydrazone Schiff bases [28,31,33,50,51]. Herein, we used aqueous solution of KOH which
stabilize the dioxidovanadium(V) centre at ~pH 8. Compounds 1 and 2 (Fig. 1, and Figs. S1 and S2 in ESI) crystallized as 3D and 2D polymers, respectively, and were synthesized by following the procedure described in Scheme 1. They were prepared by reaction of [VO(acac)2] in refluxing ethanol with the Schiff base pro-ligands (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide (H2L1) and (2,3-dihydroxybenzylidene)benzohydrazide (H2L2), respectively. They were isolated as yellow crystalline solids and characterized by IR, NMR, elemental analysis and single crystal X-ray diffraction. The dianionic Schiff base ligands (L2−) in both 1 and 2 coordinate to vanadium(V) in the enolate form, the metal exhibiting distorted (in 1) or almost perfect (in 2) square pyramidal O4N coordination geometries (τ5 = 0.22 or 0.08, respectively, Table 2) [52]. The V(V) cation is situated 0.491 (in 1) and 0.479 Å (in 2) away from the basal plane and towards the atom in the apical position. The V–Ooxido distances in the 1.593(5)–1.6485(19) Å range (Table 2) are consistent with vanadium–oxygen double bonds and are close to those commonly found in five- and six-coordinate vanadium(IV) and (V) complexes [22,28,30, 47,48]. As previously found [28,31,33,50,51], this distance is shorter than the V–Ophenolate length (avg. 1.9185 Å, Table 2) which, in turn, is slightly shorter than the V–Oenolate (avg. 1.9639 Å, Table 2). The potassium cations are in all-O coordination environment being hepta- (in
Fig. 4. Cytotoxicity of complexes 1 and 2 in normal human fibroblasts. Fibroblasts cells were treated with increasing concentrations of complex 1 (dark gray) and 2 (light gray) for 48 h and cell viability was determined by the MTS assay. The data were normalized against the control treated only with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each complex).
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Fig. 5. Cytotoxicity of ligand H2L1 in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (Black bar), HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
1) and octa-coordinated (in 2), organized as three Ooxido, two Ophenolic, one Ocarbonyl and one Ophenolate (in 1), or three Ooxido, one Ophenolic, one Ocarbonyl, one Ophenolate and two Owater (in 2). The K–O bond distances range from 2.653(5) to 3.275(2) Å, the latter slightly exceeding the limiting distance (3.2 Å) usually considered for a significant K–O interaction [53]. The shortest K–O lengths [2.653(5) in 1 and 2.734(2) in 2, Table 2] involve the Ooxido atoms while the longest [3.098(5) in 1 and 3.275(2) in 2] include the Ocarbonyl ones. The L2 − ligand in 1 deviates slightly from planarity as indicated by the N–Cenolate–C–C torsion angles of 163.2(7) °, which is −175.0(3)° in 2. Accordingly, the N–N–C–Oenolate torsion angle assume values of 4.3(9)° and − 0.4(4)° in 1 and 2, respectively. 3.2. Spectroscopic characterization of the complexes The IR spectra of 1 and 2 contain all the characteristic bands of the coordinated tridentate anionic ligand L2− in the enol form, viz., 3172, 1600, 1261 and 1078 cm−1 for 1 and 3210, 1602, 1258 and 1074 cm−1for 2. In addition, two ν(V_O) bands are observed at 954 and 913 (for 1) and 960 and 918 cm−1 (for 2), respectively. The 1H NMR spectra of 1 and 2 in DMSO-d6 show the characteristic resonances of the ligand [38,39]: the proton of the azomethine nitrogen
at ca. 8.6 ppm, the aromatic protons as multiplets in the 7.97–6.78 ppm range and the phenolic OH proton at 11.1 ppm. The 51V NMR spectra of 1 and 2 in DMSO-d6 show the characteristic resonances of dioxidovanadium(V) species at −526 and −532 ppm, respectively. ESI-MS data indicate that both 1 and 2 exist as a mononuclear species in solution. 3.3. Cytotoxic potential The in vitro antiproliferative activities of the dioxidovanadium(V) complexes 1 and 2 were analyzed in three human tumor cell lines, HCT116, A549 and MCF7, by the application of the MTS colorimetric assay. This methodology relies on the reduction of MTS into a brownish formazan product by the mitochondrial dehydrogenases in metabolically active, viable cells [56]. A decrease of the cell viability in a dose-dependent manner was observed for the three tumor cell lines after 48 h exposure to complex 1 or 2 (Figs. 2 and 3). Concerning complex 1, at 48 h the IC50 (concentration that inhibits the proliferation of 50% of the cell population) values of 2.75 ± 0.33 μM, 112.9 ± 1.23 μM and N200 μM were determined for HCT116, A549 and MCF7 cells, respectively. These values revealed a completely different cytotoxic effect of 1 in these three tumor cell lines, being the
Fig. 6. Cytotoxicity of ligand H2L2 in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (Black bars), HCT116 cells (dark gray bars), A549 (light gray bars) and MCF7 (white bars) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
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Fig. 7. Cytotoxicity of the precursor [VO(acac)2] in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (black bar), HCT116 cells (dark gray bars), A549 (light gray bars) and MCF7 (white bars) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
HCT116 cell line much more sensitive to the complex cytotoxic effects in comparison with the other two cell lines. The IC50 values for 2 in the three tumor cell lines after 48 h of incubation (75.32 ± 1.57 μM for HCT116, N200 μM and N 200 μM for A549 and MCF7 respectively; Figs. 2 and 3) are much higher than for complex 1, demonstrating that complex 2 do not exhibit an antiproliferative activity for the three cell lines tested (Fig. 3). Complexes 1 and 2 do not exhibit an antiproliferative activity against normal human fibroblasts at the concentrations tested (IC50 higher than 200 μM for both complexes) (Fig. 4). In particular, complex 1 demonstrates a high specificity and cytotoxicity towards the HCT116 tumor cells. Also as observed in Fig. S3 (ESI) both complexes are stable when incubated for 48 h in PBS at 37 °C (to mimic biological assays conditions). In order to understand if the viability loss observed for 1 in HCT116 cells was due to the complex or a result of the ligand per se, cell viability was also assessed for both ligands H2L1 and H2L2 and the precursor [VO(acac)2] at 48 h (Figs. 5–7). In Table 3 an IC50 comparison for complexes 1, 2, precursor and ligands is shown (at 48 h). The IC50 values for the ligands H2L1, H2L2 (Table 3 and Figs. 5 and 6) and for the precursor [VO(acac)2] in HCT116 cells was 7.69 ± 0.04 μM, 19.86 ± 0.25 μM and 118.2 ± 1.10, respectively (Table 3). Although H2L1 demonstrates to be highly cytotoxic for this type of tumor cells, the cytotoxicity of complex 1 is almost 3 × higher (Figs. 2 and 5, and Table 3). Interestingly the precursor is much more cytotoxic for normal cells compared to tumor cells (Fig. 5 and Table 3). The H2L2 ligand is cytotoxic for HCT116 (19.86 ± 0.25 μM) but with a higher IC50 as compared to H2L1 (7.69 ± 0.04 μM) or even to complex 1 (2.75 ± 0.33 μM). In this regard we can conclude that complex 1 is more
Table 3 IC50 values for complexes 1 and 2, ligands H2L1 and H2L2 and the vanadium(IV) precursor.
cytotoxic in HCT116 cell line than VO(acac)2 (its precursor) and H2L1 and induce lower cytotoxicity in non-tumor cells. In order to confirm the cytotoxic effect of complex 1, the most promising towards HCT116 cells, IC50 was also calculated for different incubation periods (24 h and 72 h) in the presence of increasing concentrations of the complex 1 (Table 4). As expected there is a decrease in IC50 with time reinforcing the high cytotoxicity of this complex 1 towards colorectal carcinoma cells. All together these results demonstrate the advantage of coordination of H2L1 to vanadium since it allowed a higher selective cytotoxicity towards colorectal carcinoma cells. 3.4. Apoptotic potential The reduction of cell viability promoted by complex 1 in HCT116 cells (Fig. 8) prompted us to evaluate the underlined mechanisms of cell death. A preliminary analysis was performed by staining with Hoechst 33,258 dye due to its high affinity for DNA allowing the detection of nuclear alterations like chromatin condensation and nuclear fragmentation, typical features of apoptotic cells [54–57]. Hoechst 33,258 staining of HCT116 cells after 48 h of exposure to 2.75 μM (IC50) and 5.5 μM of complex 1 allowed us to observe a reduction in the number of stained cells (in a dose dependent manner) compared to control cells and also the nuclear condensation and fragmentation characteristics of apoptosis (Fig. 8). 4. Conclusions The antiproliferative potential of the water soluble heterometallic potassium–dioxidovanadium(V) polymers [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) was examined towards human colorectal carcinoma (HCT116), lung adenocarcinoma (A549) and breast adenocarcinoma (MCF7) cell lines. Complex 1 was found to have a high antiproliferative effect particularly when compared with complex 2 with no antiproliferative effect. Indeed, complex 1 exhibits a high
IC 50 (μM) Complex 2
H2L1
H2L2
[VO(acac)2]
Fibroblasts N200 HCT116 2.75 ± 0.33
N200 75.32 ±
N200 7.69 ±
N200 19.86 ±
54.89 ± 5.43 118.2 ± 1.10
A549
112.9 ±
1.57 ~200
0.04 0.25 162 ± 1.06 N200
MCF7
1.23 N200
N200
N200
Complex 1
N200
Table 4 IC50 values for complex 1 at 24 h, 48 h and 72 h. Time (h)
78.7 ± 0.42 156.3 ± 2.2
24 48 72
IC50 (μM) 6.74 ± 0.46 2.75 ± 0.33 0.95 ± 0.03
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concentration), making this a positive feature for further development particularly towards colorectal carcinoma. Acknowledgments Authors are grateful to the Foundation for Science and Technology (FCT) (projects PTDC/EQU-EQU/122025/2010 and UID/QUI/00100/ 2013), Portugal, for financial support. M.S. acknowledges the FCT, Portugal for a postdoctoral fellowship (SFRH/BPD/86067/2012). We thank the project Silence is golden (siAu) — silencing the silencers via multifunctional gold nanoconjugates towards cancer therapy (PTDC/ BBB-NAN/1812/2012) and UID/Multi/04378/2013 for financial support. We are also thankful to the Portuguese NMR Network (IST-UL Centre) for access to the NMR facility. The authors thank Fabiana Paradinha and Pedro V. Baptista for technical and scientific support concerning the stability data of complexes 1 and 2, respectively. Appendix A. Supplementary data CCDC 1413467 (for 1) and CCDC 1413468 (for 2) contain the supplementary crystallographic data for this paper. This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2015. 11.010. References
Fig. 8. Apoptotic morphological changes in HCT116 cells exposed to 1. Cells were treated with water (vehicle control) (A) and with 2.75 μM (IC50) (B), 5.5 μM (2 × IC50) (C) of the complex 1 for 48 h and stained with Hoechst 33,258. Typical morphologic features of apoptosis like chromatin condensation and nuclear fragmentation (circles) were identified.
cytotoxic activity against colorectal carcinoma cells, with IC50 of one lower order of magnitude compared to cisplatin (15.2 ± 0.55 μM) [41]. The IC50 for cisplatin was performed under the same experimental conditions and using the same cell line [41]. The viability loss induced by complex 1 was in agreement with Hoechst 33,258 staining and the typical morphological apoptotic characteristics like chromatin condensation and nuclear fragmentation. Lung and breast adenocarcinoma cells are not sensitive to both complexes (Figs. 2, 3; Table 3). Taken together these results demonstrate that in comparison to its precursor, VO(acac)2, and its ligand, H2L1, complex 1 provides several advantages namely its high cytotoxic potential in HCT116 cell line with the simultaneously low cytotoxicity in healthy cells (at its IC50
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Water soluble heterometallic potassium-dioxidovanadium(V) complexes as potential antiproliferative agents Manas Sutradhar a,⁎, Alexandra R. Fernandes a,b,⁎, Joana Silva b, Kamran T. Mahmudov a,c, M. Fátima C. Guedes da Silva a, Armando J.L. Pombeiro a,⁎ a b c
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal Department of Chemistry, Baku State University, Z. Xalilov Str. 23, Az, 1148 Baku, Azerbaijan
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 1 October 2015 Accepted 10 November 2015 Available online 11 November 2015 Keywords: Dioxidovanadium(V) Heterometallic complex X-ray structure Antiproliferative agent
a b s t r a c t Two water soluble heterometallic potassium–dioxidovanadium polymers, [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) [H2L1 = (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide and H2L2 = (2,3-dihydroxybenzylidene)benzohydrazide], have been synthesized and characterized by IR, NMR, elemental analysis and single crystal X-ray diffraction. The antiproliferative potentials of 1 and 2 were examined towards human colorectal carcinoma (HCT116), and lung (A549) and breast (MCF7) adenocarcinoma cell lines. 1 exhibits a high cytotoxic activity against colorectal carcinoma cells (HCT116), with IC50 lower than those for cisplatin. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The use of Schiff bases and their transition metal complexes in biological, clinical and analytical applications has been the object of an increased interest [1–3]. Among the various transition metal complexes used in biological and pharmacological studies, vanadium compounds exhibit important roles as antitumor, antimicrobial agents, reducers of hyperlipidemia, hypertension and obesity, and enhancers of oxygen affinity by hemoglobin and myoglobin, as well as of the metabolic effects of insulin [4–10]. Particularly, the development of medicinal applications of oxidovanadium(IV) and (V) complexes has achieved a considerable interest in drug discovery. Emphasis has been given to the insulin mimetic activity of bis(maltolato)oxidovanadium(IV) (BMOV) and bis(ethylmaltolato)oxidovanadium(IV) (BEOV), the only two vanadium compounds tested in clinical trials to date [11]. Frontline investigations to find the molecular targets for these compounds have been carried out in order to understand how they interfere in the insulin pathway and in glucose and lipid metabolisms [11,12]. Besides the insulin-mimetic activity, these compounds have also gained attention towards their antimicrobial activity, tumor growth inhibition and prophylaxis against carcinogenesis [13–24]. Successful in vitro studies over the past few decades have advanced the anticancer research on vanadium into the preclinical stage [3]. The understanding of the ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Sutradhar), [email protected] (A.R. Fernandes), [email protected] (A.J.L. Pombeiro).
http://dx.doi.org/10.1016/j.jinorgbio.2015.11.010 0162-0134/© 2015 Elsevier Inc. All rights reserved.
molecular effects of vanadium complexes in cancer cells has been a concern and, in this regard, cytotoxicity, apoptosis induction and biological targets such as DNA and proteins have been investigated [25–27]. These modes of action have also been demonstrated for most cytostatic vanadium compounds although some of them, for example vanadocenes, may directly intercalate with DNA [3,7]. In this study, we have used two azine-type Schiff base ligands [28– 32] for the synthesis of two water soluble heterometallic potassium– dioxidovanadium(V) complexes. Such a type of ligands renders a great stability to the V(IV) and V(V) complexes [33–37]. The antiproliferative potential of the two complexes was examined towards human colorectal carcinoma (HCT116) and lung (A549) and breast (MCF7) adenocarcinoma cell lines. The cytotoxicity of both complexes against normal human fibroblasts was also assessed. 2. Experimental 2.1. General materials and procedures All synthetic work was performed in air. The reagents and solvents were obtained from commercial sources and used as received, i.e., without further purification or drying. [VO(acac)2] (acac− = acetylacetonate) was used as the metal source for the synthesis of complexes 1 and 2. C, H, and N elemental analyses were carried out on Perkin Elmer PE 2400 Series II by the Microanalytical Service of the Instituto Superior Técnico. Infrared spectra (4000–400 cm− 1) were recorded on a BRUKER VERTEX 70 or Jasco FT/IR-430 instrument in KBr pellets,
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Scheme 1. Synthesis of polymers 1 and 2.
wavenumbers are in cm−1. Mass spectra were run in a Varian 500-MS LC Ion Trap Mass Spectrometer equipped with an electrospray (ESI) ion source. For electrospray ionization, the drying gas and flow rate were optimized according to the particular sample with 35 psi nebulizer pressure. Scanning was performed from m/z 100 to 1200 in methanol solution. The compounds were observed in the negative mode (capillary voltage = 80–105 V). The 1H NMR spectra were recorded at room temperature on a Bruker Avance II + 300 (UltraShield™ Magnet) spectrometer operating at 300.130 MHz for proton. The chemical shifts are reported in ppm using tetramethylsilane as the internal reference. 51V NMR spectra were recorded on a Bruker 400 UltraShield spectrometer at ambient temperature (297 K) in DMSO–d6. The vanadium chemical shifts are quoted relative to external [VOCl3]. UV spectra were recorded in an Evolution 300 UV–Vis spectrophotometer (Thermo Scientific).
Table 1 Crystal data and structure refinement details for complexes 1 and 2.
Empirical formula Formula weight Crystal system Space group a/Å b/Å c/Å β/° V (Å3) Z Dcalc (g cm−3) μ(Mo Kα) (mm−1) Rfls. collected/unique/observed Rint Final R1a, wR2b (I ≥ 2σ) Goodness-of-fit on F2 a b
1
2
C14H10KN2O6V 392.28 Tetragonal I−4 22.475 22.475 5.9163(2) 90 2988.45(10) 8 1.744 0.977 13,534/2574/1910 0.0652 0.0471, 0.1012 1.055
C14H12KN2O6V 394.30 Monoclinic C 2/c 29.5903(7) 6.1898(2) 17.7502(4) 109.935(1) 3056.29(14) 8 1.714 0.956 21,827/2787/2244 0.0424 0.0436, 0.1089 1.079
R = Σ||Fo | − |Fc ||/Σ|Fo |. wR(F2) = [Σw(|Fo |2 − |Fc |2)2 / Σw|Fo |4]1/2.
2.2. Synthetic procedures 2.2.1. Synthesis of the pro-ligands H2L1 and H2L2 The Schiff base pro-ligands (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide (H2L1) and (2,3-dihydroxybenzylidene)benzohydrazide (H2L2) (Scheme 1) were prepared by following a reported method [38–41] which involves the condensation of the corresponding aroylhydrazides with 2,3-dihydroxybenzaldehyde.
Table 2 Selected features, bond distances (Å) and angles (°) in polymers 1 and 2.
Involving L−2 N–N C–Oenolate C–Ophenolate C–Ophenol N–Cenolate–C–C N–N–C–Oenolate Involving vanadium Coord. number Polyhedral volume (Å3) V–Ooxido V–Ophenolate V–Oenolate V–N ∠ O–V–N (5-memb. Ring) ∠ O–V–N (6-memb. Ring) Involving potassium Coord. number Polyhedral volume (Å3) K–O
Minimum M…M distances V…V V…K K…K
Minimum Maximum
1
2
1.394(7) 1.298(8) 1.332(8) 1.365(7), 1.367(9) 163.2(7) 4.3(9)
1.392(3) 1.319(3) 1.341(3) 1.365(4) −175.0(3) −0.4(4)
5 (τ5 = 0.22) 4.966 1.593(5) 1.637(4) 1.926(5) 1.968(5) 2.134(5) 74.3(2) 82.2(2)
5 (τ5 = 0.08) 4.847 1.6098(19) 1.6485(19) 1.9109(19) 1.9598(19) 2.134(2) 73.52(8) 82.93(9)
7 30.537 2.653(5) 3.098(5)
8 12.475 2.734(2) 3.275(2)
5.636(1) 3.6647(17) 4.781(2)
5.6634(6) 3.6842(10) 4.9787(12)
M. Sutradhar et al. / Journal of Inorganic Biochemistry 155 (2016) 17–25
2.2.2. Syntheses of the dioxidovanadium(V) complexes Complexes [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) have been synthesized by using a common general method. To a 30 mL ethanolic suspension of H2L1 (0.272 g, 1.00 mmol for 1) or H2L2 (0.256 g, 1.00 mmol for 2), 0.265 g (1.00 mmol) of [VO(acac)2] was added and the reaction mixture refluxed. After 1 h the mixture was cooled down to room temperature and an aqueous solution of 0.1 M KOH was added with constant stirring until a pH of ca. 8
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was obtained. The reflux was taken up for another hour. The resultant dark yellow solution was filtered and the filtrate was kept in air. After ca. 2 d, X-ray quality yellow crystals were isolated, washed 3 times with cold ethanol and dried in open air. [KVO2(L1)]n (1) — Yield 74% (0.290 g, based on vanadium). Anal. Calcd for C 14 H 10 KN 2 O 6 V: C, 42.86; H, 2.57; N, 7.14. Found: C, 42.79; H, 2.54; N, 7.09. IR (KBr; cm− 1 ): 3172 ν(OH), 1600 ν(C = N), 1261 ν(C–O)enolic, 1078 ν(N–N), 954, 913 ν(V_O). ESI-
Fig. 1. Molecular structure of polymers 1 (top) and 2 (bottom) with partial atom labeling schemes. Symmetry codes: i) x,y,1 + z ii) 1/2-x,1/2-y,1/2 + z iii) 1/2-x,1/2-y,1.5 + z iv) y,-x,-z v) 1/2-x,1/2-y,-1/2 + z vi) 1/2-x,1/2-y,-1.5 + z vii) -y,x,-z viii) x,y,-1 + z (for 1) and i) x,-1 + y,z; ii) 1/2-x,-1/2 + y,1/2-z; iii) 1/2-x,1/2 + y,1/2-z; iv) 1/2-x,1/2-y,1-z; v) x,1 + y,z; vi) 1/2x,-1/2 + y,1/2-z (for 2).
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MS(−): m/z 353 [VO2 (L1 )] − (100%). 1 H NMR (DMSO-d6, δ): 11.10 (s, 1H,OH), 8.69 (s, 1H, − CH_N), 7.82–6.78 (m, 8H, C 6 H 4 ). 51 V NMR (DMSO-d6, δ) — 526. [KVO2(L2)(H2O)]n (2) — Yield 71% (0.279 g, based on vanadium). Anal. Calcd for C14H12KN2O6V: C, 42.65; H, 3.07; N, 7.10. Found: C, 42.68; H, 2.86; N, 7.09. IR (KBr; cm−1): 3210 ν(OH), 1602 ν(C_N), 1258 ν(C–O)enolic, 1074 ν(N–N), 960, 918 ν(V_O). ESI-MS(−): m/z 337 [VO2(L2)]− (100%). 1H NMR (DMSO-d6, δ): 11.16 (s, 1H,OH), 8.57 (s, 1H, −CH_N), 7.97–6.87 (m, 8H, C6H4).51V NMR (DMSO-d6, δ) — 532.
same conditions as MCF7 cell line [45–47]. All cell lines were purchase from ATCC (www.atcc.org). 2.4.2. Compound exposure for dose–response curves Cells were plated at 5000 cells/well in 96-well plates. Media was removed 24 h after platting and replaced with fresh media containing: 0.1–200 μM of 1, 2, H2L1 and H2L2 or the precursor [VO(acac)2] or 0.1% (v/v) DMSO (vehicle control). All the previous solutions were prepared from concentrated stock solutions (in DMSO) of the complexes and ligands.
2.3. X-ray measurements X-ray single crystals of complexes 1 and 2 were immersed in cryo-oil mounted in Nylon loops and measured at a temperature of 150 (1) or 296 K (2). Intensity data were collected using a Bruker AXS-KAPPA APEX II or a Bruker APEX-II PHOTON 100 with graphite monochromated Mo-Kα (λ 0.71073) radiation. Data were collected using phi and omega scans of 0.5° per frame and a full sphere of data was obtained. Cell parameters were retrieved using Bruker SMART [42] software and refined using Bruker SAINT [42] on all the observed reflections. Absorption corrections were applied using SADABS [42]. Structures were solved by direct methods by using the SHELXS-97 package [43] and refined with SHELXL-2014 [43]. Calculations were performed using the WinGX System—Version 1.80.03 [44]. The hydrogen atoms of hydroxide groups were found in the difference Fourier map and the isotropic thermal parameters were set at 1.5 times the average thermal parameters of the belonging oxygen atoms, with their distances restrained (in 2) by using the DFIX commands. Coordinates of hydrogen atoms bonded to carbon atoms were included in the refinement using the riding-model approximation with the Uiso(H) defined as 1.2Ueq of the parent aromatic atoms. Crystal data and refinement parameters are presented in Table 1 and selected features, bond distances and angles are given in Table 2. 2.4. Biological assays 2.4.1. Cell culture Human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corp., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Invitrogen Corp.) and maintained at 37 °C in a humidified atmosphere of 5% (v/v) CO2 [45–46]. MCF7 cell line derived from pleural effusion of breast adenocarcinoma from a female patient was grown in similar conditions, supplemented with 1% MEM non-essential amino acids (Invitrogen Corp.) [47]. Normal Human fibroblasts were grown in the
2.4.3. Viability assays After 48 h of cell incubation in the presence or absence of each complex or ligand, cell viability was evaluated with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described [45–49]. In brief, this is a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96® AQueous Assay is composed of solutions of MTS and an electron coupling reagent (phenazinemethosulfate, PMS). MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product was measured in a Bio-Rad microplate reader Model 680 (Bio-Rad, Hercules, CA, USA) at 490 nm, as absorbance is directly proportional to the number of viable cells in culture. 2.4.4. Assessment of apoptosis through Hoechst 33,258 staining HCT116 cells grown as described above were plated at 7500 cells/mL and incubated for 48 h in culture medium containing the compound 1 or 0.1% (v/v) DMSO (vehicle control). Hoechst staining was used to detect apoptotic nuclei as previously described [46]. Briefly, medium was removed, cells were washed with phosphate-buffered saline 1X (PBS) (Invitrogen), fixed with 4% (v/v) paraformaldehyde in PBS 1× (10 min in the dark) and incubated with Hoechst dye 33,258 (Sigma, Missouri, USA; 5 μg/mL in PBS 1×) for another 10 min. After being washed with PBS 1×, cells were mounted using 20 μL of PBS:glicerol (3:1; v/v) solution. Fluorescent nuclei were sort out according to the chromatin condensation degree and characteristics. Normal nuclei showed noncondensed chromatin uniformly distributed over the entire nucleus. Apoptotic nuclei showed condensate or fragmented chromatin. Some cells formed apoptotic bodies. Plates were photographed in an AXIO
Fig. 2. Cytotoxicity of 1 in HCT116, A549 and MCF7 cell lines. HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
M. Sutradhar et al. / Journal of Inorganic Biochemistry 155 (2016) 17–25
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Fig. 3. Cytotoxicity of 2 in HCT116, A549 and MCF7 cell lines. HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
Scope (Carl Zeiss, Oberkochen Germany), and three random microscopic fields per sample with ca. 50 nuclei were counted. Mean values were expressed as the percentage of apoptotic nuclei [46]. 2.4.5. Stability of complexes 1 and 2 in biological conditions In order to assess complexes stability in biological conditions phosphate buffered saline (PBS) (pH 7.0) was used. Compounds (33 μM) were incubated in PBS for 0, 24 and 48 h at 37 °C and UV spectra were recorded in 240 to 440 nm range. 2.4.6. Statistical analysis All data were expressed as mean ± SEM from at least three independent experiments. Statistical significance was evaluated using the Student's t-test; p b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Synthesis and X-ray structural characterization It is well known that [VO(acac)2] undergoes aerial oxidation in solution (preferably in alcohol medium) in the presence of hydrazone Schiff bases [28,31,33,50,51]. Herein, we used aqueous solution of KOH which
stabilize the dioxidovanadium(V) centre at ~pH 8. Compounds 1 and 2 (Fig. 1, and Figs. S1 and S2 in ESI) crystallized as 3D and 2D polymers, respectively, and were synthesized by following the procedure described in Scheme 1. They were prepared by reaction of [VO(acac)2] in refluxing ethanol with the Schiff base pro-ligands (2,3-dihydroxybenzylidene)-2-hydroxybenzohydrazide (H2L1) and (2,3-dihydroxybenzylidene)benzohydrazide (H2L2), respectively. They were isolated as yellow crystalline solids and characterized by IR, NMR, elemental analysis and single crystal X-ray diffraction. The dianionic Schiff base ligands (L2−) in both 1 and 2 coordinate to vanadium(V) in the enolate form, the metal exhibiting distorted (in 1) or almost perfect (in 2) square pyramidal O4N coordination geometries (τ5 = 0.22 or 0.08, respectively, Table 2) [52]. The V(V) cation is situated 0.491 (in 1) and 0.479 Å (in 2) away from the basal plane and towards the atom in the apical position. The V–Ooxido distances in the 1.593(5)–1.6485(19) Å range (Table 2) are consistent with vanadium–oxygen double bonds and are close to those commonly found in five- and six-coordinate vanadium(IV) and (V) complexes [22,28,30, 47,48]. As previously found [28,31,33,50,51], this distance is shorter than the V–Ophenolate length (avg. 1.9185 Å, Table 2) which, in turn, is slightly shorter than the V–Oenolate (avg. 1.9639 Å, Table 2). The potassium cations are in all-O coordination environment being hepta- (in
Fig. 4. Cytotoxicity of complexes 1 and 2 in normal human fibroblasts. Fibroblasts cells were treated with increasing concentrations of complex 1 (dark gray) and 2 (light gray) for 48 h and cell viability was determined by the MTS assay. The data were normalized against the control treated only with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each complex).
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Fig. 5. Cytotoxicity of ligand H2L1 in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (Black bar), HCT116 cells (dark gray), A549 (light gray) and MCF7 (white) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
1) and octa-coordinated (in 2), organized as three Ooxido, two Ophenolic, one Ocarbonyl and one Ophenolate (in 1), or three Ooxido, one Ophenolic, one Ocarbonyl, one Ophenolate and two Owater (in 2). The K–O bond distances range from 2.653(5) to 3.275(2) Å, the latter slightly exceeding the limiting distance (3.2 Å) usually considered for a significant K–O interaction [53]. The shortest K–O lengths [2.653(5) in 1 and 2.734(2) in 2, Table 2] involve the Ooxido atoms while the longest [3.098(5) in 1 and 3.275(2) in 2] include the Ocarbonyl ones. The L2 − ligand in 1 deviates slightly from planarity as indicated by the N–Cenolate–C–C torsion angles of 163.2(7) °, which is −175.0(3)° in 2. Accordingly, the N–N–C–Oenolate torsion angle assume values of 4.3(9)° and − 0.4(4)° in 1 and 2, respectively. 3.2. Spectroscopic characterization of the complexes The IR spectra of 1 and 2 contain all the characteristic bands of the coordinated tridentate anionic ligand L2− in the enol form, viz., 3172, 1600, 1261 and 1078 cm−1 for 1 and 3210, 1602, 1258 and 1074 cm−1for 2. In addition, two ν(V_O) bands are observed at 954 and 913 (for 1) and 960 and 918 cm−1 (for 2), respectively. The 1H NMR spectra of 1 and 2 in DMSO-d6 show the characteristic resonances of the ligand [38,39]: the proton of the azomethine nitrogen
at ca. 8.6 ppm, the aromatic protons as multiplets in the 7.97–6.78 ppm range and the phenolic OH proton at 11.1 ppm. The 51V NMR spectra of 1 and 2 in DMSO-d6 show the characteristic resonances of dioxidovanadium(V) species at −526 and −532 ppm, respectively. ESI-MS data indicate that both 1 and 2 exist as a mononuclear species in solution. 3.3. Cytotoxic potential The in vitro antiproliferative activities of the dioxidovanadium(V) complexes 1 and 2 were analyzed in three human tumor cell lines, HCT116, A549 and MCF7, by the application of the MTS colorimetric assay. This methodology relies on the reduction of MTS into a brownish formazan product by the mitochondrial dehydrogenases in metabolically active, viable cells [56]. A decrease of the cell viability in a dose-dependent manner was observed for the three tumor cell lines after 48 h exposure to complex 1 or 2 (Figs. 2 and 3). Concerning complex 1, at 48 h the IC50 (concentration that inhibits the proliferation of 50% of the cell population) values of 2.75 ± 0.33 μM, 112.9 ± 1.23 μM and N200 μM were determined for HCT116, A549 and MCF7 cells, respectively. These values revealed a completely different cytotoxic effect of 1 in these three tumor cell lines, being the
Fig. 6. Cytotoxicity of ligand H2L2 in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (Black bars), HCT116 cells (dark gray bars), A549 (light gray bars) and MCF7 (white bars) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
M. Sutradhar et al. / Journal of Inorganic Biochemistry 155 (2016) 17–25
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Fig. 7. Cytotoxicity of the precursor [VO(acac)2] in HCT116, A549, MCF7 tumor cell lines and normal human fibroblasts. Fibroblasts (black bar), HCT116 cells (dark gray bars), A549 (light gray bars) and MCF7 (white bars) cells were treated with increasing concentrations of the complex for 48 h and cell viability was determined by MTS assay. The data were normalized against the control treated with 0.1% (v/v) DMSO. The results showed are expressed as mean ± SEM of three independent assays. The symbol * in the figure means that the results are statistically significant with a p b 0.05 (as compared to control for each cell line).
HCT116 cell line much more sensitive to the complex cytotoxic effects in comparison with the other two cell lines. The IC50 values for 2 in the three tumor cell lines after 48 h of incubation (75.32 ± 1.57 μM for HCT116, N200 μM and N 200 μM for A549 and MCF7 respectively; Figs. 2 and 3) are much higher than for complex 1, demonstrating that complex 2 do not exhibit an antiproliferative activity for the three cell lines tested (Fig. 3). Complexes 1 and 2 do not exhibit an antiproliferative activity against normal human fibroblasts at the concentrations tested (IC50 higher than 200 μM for both complexes) (Fig. 4). In particular, complex 1 demonstrates a high specificity and cytotoxicity towards the HCT116 tumor cells. Also as observed in Fig. S3 (ESI) both complexes are stable when incubated for 48 h in PBS at 37 °C (to mimic biological assays conditions). In order to understand if the viability loss observed for 1 in HCT116 cells was due to the complex or a result of the ligand per se, cell viability was also assessed for both ligands H2L1 and H2L2 and the precursor [VO(acac)2] at 48 h (Figs. 5–7). In Table 3 an IC50 comparison for complexes 1, 2, precursor and ligands is shown (at 48 h). The IC50 values for the ligands H2L1, H2L2 (Table 3 and Figs. 5 and 6) and for the precursor [VO(acac)2] in HCT116 cells was 7.69 ± 0.04 μM, 19.86 ± 0.25 μM and 118.2 ± 1.10, respectively (Table 3). Although H2L1 demonstrates to be highly cytotoxic for this type of tumor cells, the cytotoxicity of complex 1 is almost 3 × higher (Figs. 2 and 5, and Table 3). Interestingly the precursor is much more cytotoxic for normal cells compared to tumor cells (Fig. 5 and Table 3). The H2L2 ligand is cytotoxic for HCT116 (19.86 ± 0.25 μM) but with a higher IC50 as compared to H2L1 (7.69 ± 0.04 μM) or even to complex 1 (2.75 ± 0.33 μM). In this regard we can conclude that complex 1 is more
Table 3 IC50 values for complexes 1 and 2, ligands H2L1 and H2L2 and the vanadium(IV) precursor.
cytotoxic in HCT116 cell line than VO(acac)2 (its precursor) and H2L1 and induce lower cytotoxicity in non-tumor cells. In order to confirm the cytotoxic effect of complex 1, the most promising towards HCT116 cells, IC50 was also calculated for different incubation periods (24 h and 72 h) in the presence of increasing concentrations of the complex 1 (Table 4). As expected there is a decrease in IC50 with time reinforcing the high cytotoxicity of this complex 1 towards colorectal carcinoma cells. All together these results demonstrate the advantage of coordination of H2L1 to vanadium since it allowed a higher selective cytotoxicity towards colorectal carcinoma cells. 3.4. Apoptotic potential The reduction of cell viability promoted by complex 1 in HCT116 cells (Fig. 8) prompted us to evaluate the underlined mechanisms of cell death. A preliminary analysis was performed by staining with Hoechst 33,258 dye due to its high affinity for DNA allowing the detection of nuclear alterations like chromatin condensation and nuclear fragmentation, typical features of apoptotic cells [54–57]. Hoechst 33,258 staining of HCT116 cells after 48 h of exposure to 2.75 μM (IC50) and 5.5 μM of complex 1 allowed us to observe a reduction in the number of stained cells (in a dose dependent manner) compared to control cells and also the nuclear condensation and fragmentation characteristics of apoptosis (Fig. 8). 4. Conclusions The antiproliferative potential of the water soluble heterometallic potassium–dioxidovanadium(V) polymers [KVO2(L1)]n (1) and [KVO2(L2)(H2O)]n (2) was examined towards human colorectal carcinoma (HCT116), lung adenocarcinoma (A549) and breast adenocarcinoma (MCF7) cell lines. Complex 1 was found to have a high antiproliferative effect particularly when compared with complex 2 with no antiproliferative effect. Indeed, complex 1 exhibits a high
IC 50 (μM) Complex 2
H2L1
H2L2
[VO(acac)2]
Fibroblasts N200 HCT116 2.75 ± 0.33
N200 75.32 ±
N200 7.69 ±
N200 19.86 ±
54.89 ± 5.43 118.2 ± 1.10
A549
112.9 ±
1.57 ~200
0.04 0.25 162 ± 1.06 N200
MCF7
1.23 N200
N200
N200
Complex 1
N200
Table 4 IC50 values for complex 1 at 24 h, 48 h and 72 h. Time (h)
78.7 ± 0.42 156.3 ± 2.2
24 48 72
IC50 (μM) 6.74 ± 0.46 2.75 ± 0.33 0.95 ± 0.03
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concentration), making this a positive feature for further development particularly towards colorectal carcinoma. Acknowledgments Authors are grateful to the Foundation for Science and Technology (FCT) (projects PTDC/EQU-EQU/122025/2010 and UID/QUI/00100/ 2013), Portugal, for financial support. M.S. acknowledges the FCT, Portugal for a postdoctoral fellowship (SFRH/BPD/86067/2012). We thank the project Silence is golden (siAu) — silencing the silencers via multifunctional gold nanoconjugates towards cancer therapy (PTDC/ BBB-NAN/1812/2012) and UID/Multi/04378/2013 for financial support. We are also thankful to the Portuguese NMR Network (IST-UL Centre) for access to the NMR facility. The authors thank Fabiana Paradinha and Pedro V. Baptista for technical and scientific support concerning the stability data of complexes 1 and 2, respectively. Appendix A. Supplementary data CCDC 1413467 (for 1) and CCDC 1413468 (for 2) contain the supplementary crystallographic data for this paper. This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2015. 11.010. References
Fig. 8. Apoptotic morphological changes in HCT116 cells exposed to 1. Cells were treated with water (vehicle control) (A) and with 2.75 μM (IC50) (B), 5.5 μM (2 × IC50) (C) of the complex 1 for 48 h and stained with Hoechst 33,258. Typical morphologic features of apoptosis like chromatin condensation and nuclear fragmentation (circles) were identified.
cytotoxic activity against colorectal carcinoma cells, with IC50 of one lower order of magnitude compared to cisplatin (15.2 ± 0.55 μM) [41]. The IC50 for cisplatin was performed under the same experimental conditions and using the same cell line [41]. The viability loss induced by complex 1 was in agreement with Hoechst 33,258 staining and the typical morphological apoptotic characteristics like chromatin condensation and nuclear fragmentation. Lung and breast adenocarcinoma cells are not sensitive to both complexes (Figs. 2, 3; Table 3). Taken together these results demonstrate that in comparison to its precursor, VO(acac)2, and its ligand, H2L1, complex 1 provides several advantages namely its high cytotoxic potential in HCT116 cell line with the simultaneously low cytotoxicity in healthy cells (at its IC50
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