Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s12011-009-8538-z
Abstract The dihydrated potassium salt of the complex anion [VO(O2)NTA]2− (NTA=
nitrilotriacetate anion, [N(CH2-COO)3]3−) was thoroughly characterized by electronic and
vibrational (infrared and Raman) spectroscopies. The bioactivity of the complex on the cell
proliferation was tested on three cell lines in culture (UMR106 rat osteosarcoma-derived
cells, Caco-2 derived from a human colon adenocarcinoma, and RAW 264.7, a macrophage
murine cell line).
Introduction
Studies on the pharmacological activity of vanadium compounds have awaked great interest
during the last years, and different interesting and promising results have been recently reported
[1–6]. In this context, some vanadium(V) peroxo complexes have shown important anti-
tumoral activity against different experimental systems [1, 4–7], and it is supposed that their
mechanism of action may be different from that of other metallic antitumoral systems [4].
G. Arrambide : D. Gambino
Cátedra de Química Inorgánica, Departamento Estrella Campos, Facultad de Química,
Universidad de la República, 11800 Montevideo, Uruguay
As a part of our studies devoted to the search and characterization of new vanadium
complexes with pharmacological activity, in a previous paper, we have investigated in detail
the physicochemical and biological behavior of the K3[VO(O2)2CO3]·H2O complex [8].
These studies have shown that this diperoxocomplex seems to be the most toxic compound
that has so far been tested on osteoblast-like cells in culture. In order to verify if this toxic
action is, eventually, related to the presence of two peroxo groups in the complex, we have
now investigated the activity of K2[VO(O2)NTA]·2H2O, containing only one peroxo group,
and which has been reported as unusually stable [9] and presenting low toxicity [7, 9].
Materials Vanadium pentoxide and KOH were products from Merck, and nitrilotriacetic
acid (H3NTA, N(CH2–COOH)3) was from Aldrich. Tissue culture materials were purchased
from Trading New Technologies (Buenos Aires, Argentina). Dulbecco’s Modified Eagles
Medium (DMEM) and fetal bovine serum (FBS) were from GBO, Argentina S.A.; trypsin–
EDTA was provided by Gibco (Gaithersburg, MD, USA); crystal violet, glycine, MgCl2,
and all the other chemicals used were of analytical grade from Sigma.
Synthesis of the Compound The synthesis and crystal structure of the investigated complex
were reported by Djordjevic et al. [9]. It can be obtained in the form of red orange crystals
by reacting V2O5, dissolved in KOH, with nitrilotriacetic acid and 30% H2O2. Finally, the
pH is adjusted to about 5–6, and after filtration of the solution, precipitation is initiated by
addition of ethanol [9]. Analysis: Calculated for K2[VO(O2)NTA]·2H2O (MW=401.0) C,
17.9; H, 2.5; N, 3.5; V, 12.7%. Found C, 17.8; H, 2.6; N, 3.5 V, 12.6%.
Cell Culture Two tumoral cell lines, UMR106 rat osteosarcoma-derived cells and the
Caco-2 cell line derived from a human colon adenocarcinoma were grown in DMEM
supplemented with 100 U/ml penicillin, 100 μg/mL streptomycin, and 10% (v/v) FBS at
37°C, 5% CO2. A third line included in this study was the macrophage cell line RAW
264.7 from murine origin that was cultivated under conditions similar to those already
detailed. When the cells growing in monolayer reach 70–80% confluence, they were
subcultured using 0.1% trypsin+1 mM EDTA in Ca2+/Mg2+-free phosphate buffered
saline (PBS; 11 mM KH2PO4, 26 mM Na2HPO4, 115 mM NaCl, pH7.4) [10–12]. For
the experiments, the cells were grown in multiwell plates. When the cells reached
Spectroscopic Behavior and Biological Activity of K2[VO(O2)NTA]·2H2O 243
70% confluence, the monolayers were washed twice with DMEM and were incubated
with different concentrations of aqueous solutions of the vanadium complex for the
proliferation assays.
Cell Proliferation Assay A mitogenic bioassay was carried out as described by Okajima et
al. [13], with some modifications. Briefly, the cells were grown in 48-well plates. When the
cells reached 70% confluence, the monolayers were washed twice with serum-free DMEM
and incubated with different concentrations of aqueous solutions of the complex (2.5–
100 μM) at 37°C for 24 h. Then, the monolayers were washed with PBS and fixed with 5%
glutaraldehyde per PBS at room temperature for 10 min. After this treatment, they were
stained with 0.5% crystal violet per 25% methanol for 10 min. Finally, the dye solution was
discarded, and the plate was washed with water and dried. The dye taken up by the cells
was extracted using 0.5 mL per well 0.1 M glycine per HCl buffer, pH 3.0, in 30%
methanol and transferred to test tubes. Absorbance was read at 540 nm after a convenient
sample dilution. We have previously shown that under these conditions, the colorimetric
bioassay strongly correlated with the cell proliferation measured by cell counting in a
Neubauer chamber [10].
As shown by Djordjevic et al. [9], the complex crystallizes in the orthorhombic space group
Pna21 with Z=4. Vanadium(V) presents a distorted pentagonal bipyramidal coordination,
and the NTA acts as a tetradentate ligand. As shown in Fig. 1, the peroxo group, together
with the N atom and two carboxylato O atoms, conforms the equatorial plane, whereas the
oxo group and a third carboxylato oxygen atom occupy the axial positions.
C O
N O
C V O
C O
O C O
O
C
O
244 Arrambide et al.
Spectroscopic Behavior
Electronic Spectrum
Vibrational Spectra
The FTIR spectrum of the complex is presented in Fig. 2, and the proposed assignments,
supported also by Raman data, are shown in Table 1. These assignments were performed on
the basis of some standard references [18, 19] as well as on our previous studies of vanadium
peroxocomplexes [8, 17, 20]. Their principal aspects are briefly commented as follows:
– The two O–H stretching vibrations of the water molecules (not shown in Fig. 2) are present
as very strong and well-defined infrared (IR) bands, whereas not signals for these modes
were found in the Raman spectrum. Unfortunately, it is not possible to perform a better
analysis of these vibrations due to the fact that the structural peculiarities of the water
Fig. 2 FTIR spectrum of K2[VO(O2)NTA]·2H2O in the spectral range between 2,000 and 400 cm−1
Spectroscopic Behavior and Biological Activity of K2[VO(O2)NTA]·2H2O 245
IR Raman Assignments
molecules were not reported in the structural analysis of the complex [9]. Besides, the
deformational mode of these molecules, δ(H2O), is evidently overlapped by the strong
1,664–1,615 cm−1 IR doublet.
– Regarding the carboxylate bands, in the free acid, the ν(C=O) vibration is seen as a
very strong IR band at 1,726 cm−1 whereas the corresponding ν(C–O) mode appears as
a medium intensity band at 1,334 cm−1. After complex formation, both bands are
displaced, one to lower and the other one to higher frequencies. Interestingly, in the
complex, both the νas(COO−) and the νs(COO−) vibrations appear clearly splitted, in
agreement with the presence of two structurally different carboxylato ligands (two of
them involved in the equatorial plane bonding and the remaining one occupying an
axial position) [9]. On the other hand, the energy difference between these two
carboxylate stretching vibrations (250 cm−1) is clearly in agreement with the fact that
all the COO− groups of the ligand are coordinated as unidentate moieties [21].
– For the analysis of the vibrations of the peroxo groups and of the related VO2 motions,
we have employed the usual model, admitting that the metal peroxo grouping behaves
as an equilateral triangle with C2v symmetry [8, 17, 22].
– The strongest Raman line (942 cm−1), with its also very strong IR counterpart at
940 cm−1, can confidently be assigned to the characteristic ν(V=O) stretching
vibration. The energy of this vibration is practically coincident with that measured in
246 Arrambide et al.
Biological Activity
Figure 3 shows the effect of different concentrations of the vanadium complex on the three
cell lines in culture. As can be seen, at low doses (5 μM), the complex stimulated the
proliferation of UMR106 cells (115% over basal, p<0.05) while for concentrations higher
than 25 μM, the complex caused inhibition of mitogenesis in a dose-dependent manner (p<
0.001). For the tumoral osteoblasts, only ca. 10% of the basal (without complex addition)
cellular population could survive at 75 and 100 μM (p<0.001). The half maximal inhibitory
concentration (IC50) for K2[VO(O2)NTA].2H2O in the osteosarcoma cell line was 35 μM.
These results indicate that in the tumoral osteoblasts UMR106, the actual complex was less
toxic than the vanadium(V) K3[VO(O2)2CO3].H2O, a compound with two peroxo groups in
the coordination sphere of the vanadium atom, which caused no stimulation of mitogenesis
at low doses in this tumoral cell line [8]. Moreover, in Caco-2 and Raw 265.7 cell lines, the
complex K2[VO(O2)NTA]·2H2O exerted a dose–response inhibition in the full range of
tested concentrations as can be seen also from Fig. 3. Even though K2[VO(O2)NTA]·2H2O
was an inhibitory compound for the proliferation of these two cell lines in the whole range
of concentrations, the decrease observed for Caco-2 mitogenesis was minor than for the
macrophages (ca. 25% and 10% of metabolically active cells vs control at 100 μM,
respectively). The observed difference may be attributed to the fact that Caco-2 cells grow
forming groups of cells instead of monolayers as the macrophages. This feature might be a
protection factor for the former cell line. Nevertheless, the IC50 for these two lines is 10 μM
indicating a stronger sensitivity of these cells toward the deleterious action of the complex
when compared with the UMR106 osteoblast-like cells.
Usually, vanadium compounds exert a biphasic effect on mitogenesis; they promote
this event at low doses and inhibit cell growth at higher concentrations. Several
Proliferation
125
100
75
% Basal
50
25
0
0 20 40 60 80 100 120
Vanadium (µM)
Fig. 3 Effect of K2[VO(O2)NTA]·2H2O on cell proliferation. Cells were incubated in serum-free DMEM
alone (basal) or with different concentration of the complex at 37°C for 24 h. Results are expressed as
percentage basal and represent the mean±standard error of the mean (n=6)
Spectroscopic Behavior and Biological Activity of K2[VO(O2)NTA]·2H2O 247
mechanisms may underlay the observed biological effects. One of the strongest proposals
is that vanadium compounds activate different signaling pathways mainly through the
inhibition of tyrosine phosphatases (PTPases) [23]. It is known that UMR106 cells have a
cytosolic alkaline phosphatase that behaves, at least partially, as a PTPase [24]. Low
doses of K2[VO(O2)NTA]·2H2O might inhibit this PTPase, and in this way, it can
promote the activation of proliferation pathways which in turn allows the enhancement of
mitogenesis. Nevertheless, in the present paper, our preliminary results on the effect of
K2[VO(O2)NTA]·2H2O on cells in culture also show an antiproliferative action of the
compound on Caco-2 and RAW 264.7 cell lines. The increment in the oxidative stress or
the activation of signal pathways related to the cell death through PTPases inhibition
might explain the antiproliferative effect of the higher concentrations of the complex in
the UMR106 osteosarcoma line. Further investigations in animal models for different
types of tumors are needed to understand the potential antitumoral effect of K2[VO(O2)
NTA]·2H2O. Altogether, these results suggest the complexity of the cytotoxicity of metal
complexes on cells in culture since the observed results depend on the metal, the
coordination sphere, the nature of the ligands, the cell type features, and the cellular
transduction pathways underlying the toxic action.
Acknowledgments This work has been supported by the Consejo Nacional de Investigaciones Científicas y
Técnicas de la República Argentina, CONICET (PIP 5078 and 6366), and the National University of La
Plata. It is also a part of the PROSUL Project Nr. 490.600/2007-08 supported by CNPq (Brazil) and of the
CYTED network RIIDFCM (Red Temática 209 RT0380). D.A.B., S.B.E., and E.J.B. are members of the
Research Career from CONICET.
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