Maltol Complexes of Vanadium (IV) and (V) Regulate In Vitro Alkaline Phosphatase Activity and...
Transcript of Maltol Complexes of Vanadium (IV) and (V) Regulate In Vitro Alkaline Phosphatase Activity and...
Journal of
J. Trace Elements Med. BioI. Vol. 11. pp. 110-115 (1997) Trace Elements In Medicine and Biology
© 1997 by Gustav Fischer Verlag
Maltol Complexes of Vanadium (IV) and (V) Regulate In Vitro Alkaline Phosphatase Activity and Osteoblast-like Cell Growth
D. A. BARRIO*, M. D. BRAZIUNAS*, S. B. ETCHEVERRY*** andA. M. CORTIZO*I
"C,ltedra de Bioqufmica Patol6gica. **CEQUINOR. Facultad de Ciencias Exactas.
Universidad ~acional de la Plata. 47 y 115 (1900) La Plata. Argentina
(Received January/May 1997)
Summary
Vanadium compounds have been found to possess insulin- and growth factor-mimetic etlects. In conse
quence. these derivatives are potentially useful as effective oral therapeutic agents in diabetic patients. Howev
er. their use has been limited by various toxic side-effects and by the low solubility of different derivatives. Re
cently. vanadium complexes with maltol, a sugar used as a common food additive. have been synthesised and
investigated in animals. showing possible insulin-mimetic effects with low toxic side-effects. In the present
study we have investigated the effect ofbis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato )dioxova
nadium (V) (B\!IV) on bone cells in culture as well as their direct effect on alkaline phosphatase in I'itro. A
comparison was also made with the action of vanadate and vanadyl cation. Vanadium compounds regulated cell
proliferation in a biphasic manner with similar potencies. Osteoblast differentiation. assessed by alkaline phos
phatase activity. was found to be dose-dependent. with the inhibitory effect being stronger for vanadate and
BMOV than for vanadyl and BMY. All vanadium compounds directly inhibited bovine intestinal ALP with a
similar potency. Thus. maltol vanadium derivatives behave in a similar way to vanadate and vanadyl in osteob
last-like UMR 106 cells in culture.
Kcnmrds:Vanadium-maltol complexes. proliferation. differentiation. osteoblasts. alkaline phosphatase.
Introduction
Vanadium. a trace transition element. promotes a
number of cellular events. both ill ritro and ill rim (1-3).
It regulates several enzymatic activities crucial to inter
mediate metabolism. nucleic acid synthesis. protein ex
pression. growth promotion and cell differentiation.
These observations have led to the suggestion that vana
dium acts both as an insulin- and growth factor-mimetic
compound. Oral vanadium administration in rodent mod
eb of diabetes was found to improve hyperglycacmia (4-
'To whom correspondence should be addressed.
6). Thus. vanadium has been proposed as a potential ther
apeutic agent for diabetic patients. However. extended
treatment with vanadate induces toxic effects in animals
as well as in cell culture systems (3.7-9). It is for this rea
son that the bioactivity and toxicity of new vanadium de
rivatives are being investigated.
Previous studies in our laboratory have shown that
low doses of different vanadium compounds promote the
proliferation of fibroblasts and osteoblast-like cells (10-
12). However. this effect was biphasic in our culture
models. Both an inhibition of cell growth and an induc
tion of morphological transformation were observed at
concentrations over 50 ~M for most of the vanadium (V)
compounds. On the other hand. the vanadium (IV) deriv
atives appeared to be less toxic. which is in agreement
with previous results (13.14). Using two osteoblastic
lines. U MR I 06 and MC3T3E I. we have also shown that
vanadium regulates the osteoblast phenotype as assessed
by alkaline phosphatase (ALP) specific activity (10.12).
ALP inhibition was observed after 24 h incubation with
vanadium (V) compounds. On the other hand. a weak
regulation of cell differentiation was observed with vana
clium (IV) derivatives. In addition. using il1l'itro cell-free
systems. we previously demonstrated that both vanadium
(IV) and (V) directly inhibited alkaline and acid phos
phatases in a dose-dependent manner (15.16). Thus. the
ohserved vanadium inhibition of osteoblast differentia
tion may be due to a direct effect on ALP activity. On the
other hand. vanadium compounds might specifically reg
ulate ALP gene expression. Our studies suggest that dif
ferent vanadium compounds might be able to exert selec
tive/specific effects on different cell and tissue models.
Further studies are required. therefore. to determine the
usefulness of vanadium compounds as therapeutic tools.
In the present study. we investigated the effects of bis
(maltolato) oxovanadium (IV) and bis (maltolato) diox
ovanadium (V) compounds on osteoblast-like UMR 106
cell growth. In addition. their direct effect on bovine in
testinal ALP was also studied. These results were com
pared with the effects of vanadate and vanadyl on the
same parameters.
Material and Methods
Materials
Vanadium (IV) oxide sulfate (vanadyl sulfate) (VO)
was obtained from Merck. Vanadate (V). p-nitrophenyl
phosphate (pNPP). ethylenediaminetetraacetic acid
(EOTA). maltol (3-hydroxy-2-methyl-4-pyrone). fast
blue RR salt. -naphthyl phosphate and bovine intestinal
alkaline phosphatase were obtained from Sigma. Tissue
culture material was provided by Corning or Falcon: Oul
becco's modified Eagle's medium (OMEM) and trypsin
were supplied by Gibco. and fetal bovine serum (FBS) by
Gen. Argentina.
\(/J1adi[(m Compounds and Solutio/1 Preparation
Fresh stock solutions (100 mM) of vanadate and va-
Maltol vanadium complexes in ostcoblasts III
nadyl were prepared hy dissolving the crystalline materi
al in distilled water. Bis (maltolato) oxovanadium (IV)
(BMOV) rC1,H",o7Vj was provided by Dr. E.1. Baran
and prepared according to McNeill et al. (17). Aqueous
solutions of maltol and vanadyl sulfate were mixed in a
2: I ratio. The pH of the solution was raised to 8.5 and the
solution was retluxed overnight; the green compound
was collected upon cooling. The solid product was char
acterized by IR spectroscopy. Bis (maltolato) dioxovana
dium (V) (BMV) was prepared according to Elvingson et
al. (18). Brictly. 10 mM vanadate and 20 mM maltol in
water were mixed (I :2 molar ratio) and the pH of the so
lution was kept between 6.5 and 7.0. The solution was
protected from light and was used within 48 h.
Cell clIltllre
Rat osteosarcoma cell line UMR 1 06 was grown in
OMEM supplemented with 10 o/c FBS and antibiotics
(100 U/ml penicillin - 100 ~g/ml streptomycin) in a hu
midified atmosphere of 95o/c air / 5o/c CO, ( 10). Cells
were grown to near confluence (70-800/c) when they were
subcultured using 0.19', trypsin - lmM EOTA in Ca'+
Mg'+ - free phosphate huffered saline (PBS). For experi
ments. about 2.5 • I O~ cells / well were plated into 24-well
plates. After the culture reached 700/, contluence. the
cells were washed with OMEM without serum and incu
bated in 0.5 ml OMEM plus different vanadium com
pounds for 24 h. We chose this cell line since it has been
reported to respond to insulin and vanadium compounds
(3.10.12).
C el/lJroliferation assay
A mitogenic bioassay was carried out as described by
Okajima et al. (19) with some modifications. Brietly.
cells in 24-well plates were washed with PBS and fixed
with 5clc glutaraldehyde / PBS at room temperature for 10
min. Then they were stained with 0.5o/c crystal violet /
2SC,1r methanol for 10 min. after which 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 mi/well 0.1 M glycine/HCl butler. pH=3.0 /30O/C
methanol and transferred to test tubes. Absorbance was
read at 540 nm after a convenient sample dilution. We
previously showed that under these conditions this color
imetric bioassay strongly correlates with cell count (10).
II:: D. A. Barrio. M. D. Braziunas. S. B. Etcheverry and A. M. Cortizo
130 140,----------------------,
120
'iij r U>
.. ~ '" 110 ID ;;l:: I
100
90 ~ __ ~ __ ~~ __ L-__ L-__ ~_~
o 20 40 60 80 100 12C
Vanadium b.tM]
Figure I. Effect of vanadium compounds on UMR 106 osteoblastlike cell proliferation. Symbols are: .V .• VO .• BMV ..... BMOY. Data are expressed as '/, of the basal value with mean±SEM (n=9) being given.
C ell differentiation assay
Cells were incubated at 37°C / 24h in serum-free
DMEM containing different agents. The cell layer was
then washed with PBS and solubilized in 0.5 ml 0.1°11' Tri
ton X-100. Aliquots of the total cell extract (lO-200/d
were saved for protein determination using the Bradford
technique (20) and for measurement of alkaline phos
phatase activity as has been described (10). This assay
has been used as a marker of osteoblast phenotype ex
pression in cell cultures (3.10.12.21).
120
100
-' 80 « en « ID ;;l:: 60
40
20
o 20 40 60 80 100
Vanadium [liM]
Figure I. Effect of vanadium compounds on UMR I 06 differenciation. Symbols arc: • V .• YO .• BMV ..... BMOV. Data are expressed as '1c of the basal value with mean±SEM (n=9) being given.
Intestinal alkaline phosphatase acti"i!.'>'
The effect of vanadium compounds on ALP from bo-
vine intestinal mucosa was investigated. The initial rate
of ALP activity was determined using I Ilg/ml bovine in
testinal ALP - 5 mM pNPP in the incubation buffer (55
mM Glycine - 0.55 mM MgCI, buffer. pH=10.5) at 37°C
for 7 min. Under these experimental conditions. the prod
uct formation was linear until 10 min. The effect of vana
dium complexes was assessed by adding different con
centrations of each compound in the ALP assay. The pro
duction of pNP was determined measuring absorbance at
405 nm.
We chose this assay as a model of an in 1'itro cell-free
Alkaline phosphatase histochemistry system to test the direct effect of vanadium compounds
on alkaline phosphatase activity. as we have previously
UMR 106 osteoblasts were cultured into 6 well plates described ( 15).
at a density of 2.4 • I O~ cell/m!. For the experiments. cul
tures were washed with PBS and fixed with citrate-buff
ered acetone-formaldehyde. according to Gay et al (22).
After rinsing with water. cells were incubated with fresh
ly made -naphthyl phosphate and fast blue RR (O.Olo/c and 0.06 o/e. respectively). in 0.1 M Tris-HCI pH=8.5 with
or without different vanadium compounds for 15 min.
After this incubation period. eells were extensively
washed with water. The alkaline phosphatase staining
distribution on the cells was observed and photographs of
each dish were made at 40 x Obj magnification with a
black and white Kodak 125 ASA film.
Statistical methods
At least three experiments were performed in trip
licate for each experimental condition. Data were ex
pressed as mean±SEM. Statistical differences were ana
lysed using Student's t-test or analysis of variance. when
suitable. Linear correlation analysis was performed by
Pearson's correlation coefficient.
Results
Figure I shows the effect of different vanadium com
pounds on the proliferation of UMRI06 osteoblast-like
A
B
Figure 3. Alkaline phosphatase activity determined by histochemistry. Cells were incubated at 37 for 15 min in absence (A) or presence (B) of 100 11M BMOV. Obi 40x.
cells. All compounds significantly stimulated cell growth
in a biphasic manner. A maximal potency, similar for all
vanadium derivatives, was found at doses between 20 to
SO 11M (ranging from 110 - 120 'k over basal, p<0.02 - p<
0.001).
We also evaluated the effect on total protein content
after 24 h incubation with different concentrations of va
nadium complexes. A dose-dependent effect was ob
served for the BM-vanadium complexes and va (range
110- 124 'I, of basal. p<O.02 - p<O.OO I ) (data not shown).
In contrast. V showed a biphasic effect with no stimula
tion of protein content at 100 11M concentration (data not
shown). Maximal induction of protein content for all va
nadium complexes was in the same concentration range
as for the cell proliferation effect (20-50 11M).
The effect of malto vanadium complexes on osteob
last-like cell differentiation was assessed by alkaline
phosphatase specific activity. Figure 2 shows that BMOV
and V strongly inhibited cell differentiation in a dose-de
pendent manner. The maximal effect was at 10- SO or 75
11M, for BMOV or V respectively. This effect showed a
tendency to disappear above 80 11M for both compounds.
On the other hand, BMV complex and va show only a
slight effect on cell differentiation « 20'k inhibition) in
all the concentration ranges investigated. These results
Maltol vanadium complexes in osteoblasts 113
Table I. Intensity of ALP staining associated with the membrane of osteoblast-like cells
Concentration BMV BMOV V VO
25 11M ++++ ++++ +++ ++++
50 +++ +++ +++ +++
100 ++ ++ ++ ++
500 + + + +
1000
Pluses indicate relative amounts of reaction product. minuses signify absence of reaction product. Basal value was determined in absence of vanadium compounds (++++)
are in agreement with our previous studies of vanadyl ac
tion on osteoblast-like cell differentiation (10,12).
We also analyzed the direct effect of BMOV and
BMV on alkaline phosphatase activity from osteoblast
like cells and bovine intestinc. Figure 3 shows ALP activ
ity in UMR I 06 osteoblast-like cells detected by histo
chemistry. The ALP reaction product was mainly associ
ated with the cell membrane. and to a lesser extent in the
cytoplasm. Control cells showed a strong brown staining
reaction (Fig 3A). ALP activity was inhibited by both
BMOV and BMV complexes to the same extent and in a
dose-response manner (Table I). Thus. there was a pro
gressive decrease in the histochemically detectable mem
brane-associated ALP activity (Fig 3B). Vanadate and
va also inhibited the ALP reaction depending on the
dose. Approximately 50 o/r. of the control staining intensi
ty was observed after incubation with 100 11M of any of
the vanadium compounds (Table I). These results seem
to indicate that osteoblast ALP is equally sensitive to all
the vanadium compounds tested.
The direct effect of BM-vanadium complexes on bo
vine intestinal alkaline phosphatase activity was investi
gated and compared with the effect induced by Vand va. The enzyme rates measured without inhibitor (Vo) and
with increasing concentrations of different vanadium
compounds (Vi) were calculated and plotted as a function
of the inhibitor concentration (Figure 4). The ratio VoNi
was found to be directly proportional to the vanadium
compound concentration (correlation coetTicients be
tween 0.935 - 0.996). Thus. all the vanadium complexes
tested behaved as active inhibitors under the conditions
of this assay. The EDeo (vanadium doses at which 50 o/c inhibition takes place) for each vanadium derivative were
also calculated. All vanadium compounds were found to
show similar potency. The ED)II were: 30, 27. IS and 20
11-1 D. A. Barrio. M. D. Br'lLiunas. S. B. Etcheverry and A. M. Corlizo
6 ,-------------------------------------,
5
4
:> o 3 >
2
o
ture system as did vanadate, vanadyl and othcr growth
factors (10). On the other hand. both malto vanadium
complexes and VO stimulated the total cell protein con
tent in a dose-dependent manner. The same effect was ob
served for vanadate only at low doses not over 75 11M.
suggesting a relative cytotoxicity.
Osteoblast phenotype was assessed by the determina
tion of ALP specific activity. We have previously shown
that vanadium (V) derivatives are more effective inhibi
tors of osteoblast differentiation than vanadium(lV) com
plcxes (12). In the present study. BMOV strongly inhibit
ed cell differentiation. This effect was similar to that of
vanadate. and higher concentrations (> 80 11M) of BMOV
o 20 40 60 80 100 12C or vanadate did not affect the osteoblast phenotype. As in Vanadium [~Ml
Figure -I. Vo/Vi ratios for bovine intestinal alkaline phosphatase activity plotted as a function of the vanadium concentrations. Symbols arc: ev. .VO." BMV ..... BMOV. Data are expressed as 'I; of the basal value \v ith I11can±SEM (n=-,) being given.
11M for \. YO. BMV and BMOV respectivcly. Thus. all
vanadium compounds inhibited over SO '7r of ALP activi-
ty at concentrations above SO 11M.
Discussion
Recent studies have demonstrated the insulin-mimetic
properties of a newly synthesized vanadium compound.
bis(maltolato)oxovanadium (IV) (17. 23. 24). This com
pound effectively induccd the lowering of plasma glucose
concentrations. as well as the normalization of plasma
cholesterol and triglyceride levels. in rats with induced
diabetes. BMOV was found to be more potent and consid
erably less toxic than vanadyl sulfate. However. it failed
to correct the body weight gain to the values attained by
thc control non-diabetic animals. It was suggested that
this might be due to the failure of vanadium to normalize
amino acid metabolism (23).
To our knowledge the present study is the first investi
gation of the direct etfect of this new vanadium derivative
on primary or cultured cells. We have investigated the ef
fect of BMOV and BMV on the growth of osteoblast-like
cells and their relative potency in comparison with vanad
ate and vanadyl.
Bis(maltolato)oxovanadium (IV) and (V) were both
equally effective in stimulating cell proliferation. with a
maximal potency in the range 20-50 11M. However. these
compounds showed a weak mitogenic effect in this cul-
the case ofvanadyL BMV slightly affected cell differenti
ation. These results were inspected in the light of our pre
vious report which showed that the malto vanadium (IV)
derivative behaved like a vanadium (V) compound. The
reason for thcse results is not clear but they might indicatc
either that cells handle BMOV similarly to vanadate or
that it is intracellularly hydrolized to vanadate. Evidence
from ill l'itro studies indicated that while stable in the sol
id state. BMOV is oxidized in solution by ambient 0 .. in a
solvent-dependent reaction (25). Since we did not meas
ure the intracellular decomposition of the compounds. we
can only speculate about their intracellular stability. Our
results do not allow us to differentiate between BMOV
and vanadate on the basis of cell growth effects.
Alternatively. BMOV might interact with certain sys
tems which induce signals controling cell ditfcrentiation
in a similar way to vanadate. Further studies are being
carried out to test this hypothesis.
In addition. we tested the effect of BMV on cell
growth for the first time. This compound showed a be
haviour similar to that of VO in our cell differentiation
assay. despite its containing vanadium (V). ESR spectra
have demonstrated the spontaneous reduction of BMV in
a cell-free system (18), If this reduction took place in our
cell culture system. it could explain the observed data.
We have shown that vanadium compounds directly
inhibit alkaline and acid phosphatase (15.16). In order to
test the effect of malto vanadium complexes on alkaline
phosphatase activity, we chose as our study systems a
commercial intesitinal ALP on the one hand, and the his
tochemical detcrmination of ALP on UMR cell~. on the
other. In both systems, all vanadium compounds directly
inhibited ALP in a dose-dependent manner after a short
incubation period (7 to IS min). Taken together. our evi
dence indicates that the effects of the maltol-vanadium
derivatives are similar to those of their parent compounds
vanadate and vanadyl. However, they are not more potent
than their parent compounds in our osteoblast-like cell
system.
Acknowledgements
This work was supported by grants from Facultad de
Ciencias Exactas, UNLP; Universidad Nacional de La
Plata, Argentina. SBE is a member of the Carrera del In
vestigador, CONICET, Argentina; AMC is member of the
Carrera del Investigador, CICPBA, Argentina.
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