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Inhibition of Na

+
/K
+
-ATPase and cytotoxicity of a few selected
gold(III) complexes
Voin Petrovi
a
, Sandra Petrovi
a
, Gordana Joksi
a
, Jasmina Savi
a
, Mirjana olovi
a
, Maria Agostina Cinellu
b
,
Lara Massai
c
, Luigi Messori
c
, Vesna Vasi
a,

a
Department of Physical Chemistry, Vina Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia
b
Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari, Italy
c
Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 4 April 2014
Received in revised form 16 July 2014
Accepted 16 July 2014
Available online 1 August 2014
Keywords:
Sodium pump
Gold(III) complexes
Human blood cells
Inhibition
Cytotoxicity
Na
+
/K
+
-ATPase is in charge of maintaining the ionic and osmotic intracellular balance by using ATP as an energy
source to drive excess Na
+
ions out of the cell inexchange for K
+
ions. We explored whether three representative
cytotoxic gold(III) compounds might interfere withNa
+
/K
+
-ATPase and cause its inhibition at pharmacologically
relevant concentrations. The tested complexes were [Au(bipy)(OH)
2
][PF
6
] (bipy = 2,2-bipyridine), [Au(py
dmb
-
H)(CH
3
COO)
2
] (py
dmb
-H = deprotonated 6-(1,1-dimethylbenzyl)-pyridine), and [Au(bipy
dmb
-H)(OH)][PF
6
]
(bipy
dmb
-H = deprotonated 6-(1,1-dimethylbenzyl)-2,2-bipyridine). We found that all of them caused a pro-
nounced and similar inhibition of Na
+
/K
+
-ATPase activity. Inhibition was found to be non-competitive and re-
versible. Remarkably, treatment with cysteine resulted in reversal or prevention of Na
+
/K
+
-ATPase inhibition.
It is very likely that the described effects may contribute to the overall cytotoxic prole of these gold complexes.
2014 Elsevier Inc. All rights reserved.
1. Introduction
Gold complexes, either gold(III) or gold(I), are attracting growing at-
tention in the eld of medicinal inorganic chemistry as candidate agents
for the treatment of a variety of diseases [14]. In particular, during the
last two decades, the interest for gold(III) complexes has greatly
increased because of preliminary but very promising results in cancer
treatment [59]. Being isoelectronic (d
8
) with platinum(II) and having
the same square-planar geometries as cisplatin [10], gold(III) com-
plexes may be expected to manifest reduced side effects in comparison
to the severe ones caused by established platinum(II) anticancer drugs,
such as gastrointestinal and hematological toxicity, or drug-resistance
phenomena [11,12]. However, gold(III) complexes are still highly reac-
tive and characterized by large positive redox potentials. They show a
relatively poor stability prole under physiological conditions [13,14],
which hampers their further development as drugs. In spite of that, a
far greater stability of gold(III) complexes could be achieved by using
appropriate ligands with nitrogen atoms as donors [7]; in most cases
multidentate ligands such as polyamines (cyclams, terpyridines,
phenanthrolines and damp (N-benzyl-N,N-dimethylamine)) were the
most effective in stabilizing the gold(III) center [8,9,15].
Subsequent studies demonstrated that profoundly different molecu-
lar mechanisms are exploited by platinum(II) and gold(III) complexes
in exerting their cytotoxic effects against cancer cells [68,16]. Unlike
platinum drugs, it was found that proteins, rather than DNA, are the
maintarget for the biological actions of goldcomplexes. Thus it was pro-
posed that the molecular basis for the biological action of gold(III) com-
plexes could be the modication of surface protein residues and
associate loss of protein function [2,15]. Since gold(III) complexes man-
ifest the strong afnity towards S-donor ligands such as glutathione
(GSH) and L-cysteine, and a limited reactivity against nucleosides and
their bases, exposed cysteine residues of proteins might be proper tar-
gets for gold(III) complexes. They can also cleave the disulde bond of
cystine [1719], and oxidize methionine [2022] and glycine [23], sug-
gesting that amino terminus of peptides and proteins could be deami-
nated by gold(III).
Na
+
/K
+
-ATPase is a heterodimeric transmembrane protein
consisting of two types of subunits ( and ). This enzyme regulates
many cellular functions, including those associated with tumor cell
growth [2426]. The main function of Na
+
/K
+
-ATPase is to maintain
the ionic and osmotic balance by using ATP as an energy source to
drive excess Na
+
out of cells in exchange for K
+
. The catalytic subunit
of the enzyme is responsible for binding Mg
2+
, ATP, Na
+
, K
+
, and cardi-
ac glycosides, since the subunit is a glycoprotein that regulates gap
junction proteins. For the past ten years, published studies have
suggested a role for Na
+
/K
+
-ATPase in the regulation of cell growth
and expression of particular subunits of Na
+
/K
+
-ATPase in some kinds
Journal of Inorganic Biochemistry 140 (2014) 228235
Corresponding author at: P.O. Box 522, 11 001 Belgrade, Serbia. Tel.: +381 11 3408
287; fax: +381 11 6453 967.
E-mail address: evasic@vin.bg.ac.rs (V. Vasi).
http://dx.doi.org/10.1016/j.jinorgbio.2014.07.015
0162-0134/ 2014 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ j i nor gbi o
of cancers [27]. In addition, alterations in overall Na
+
/K
+
-ATPase activ-
ity and relative subunit abundance were observed in carcinoma cell
lines obtained froma variety of tissues. As a consequence, many studies
were directed towards the seeking for modulators of Na
+
/K
+
-ATPase
which selectively target these cellular abnormalities. Recently,
it has been found that some specic modulators of Na
+
/K
+
-ATPase
activity induced the antitumor effects through the inhibition of
Na
+
/K
+
-ATPase [25].
The inhibition of Na
+
/K
+
-ATPase activity by platinum containing
drugs was usually related both to the efcacy of anti-cancer therapy as
well as to nephrotoxicity and ototoxicity [28,29]. The drug binding
mode of some Pt(II) group complexes and the reactivation of inhibited
enzyme activity by thiols were studied using various techniques
[3033] but the inhibition mechanism still remains to be elucidated.
Recently, we found also that [Au(DMSO)
2
Cl
2
]
+
and [Au(bipy)Cl
2
]
+
induced the non-competitive, reversible inhibition of Na
+
/K
+
-ATPase
activity, which was prevented and/or recovered in the presence of
reduced glutathione (GSH) and L-cysteine [34]. This paper represents
the continuation of our work concerning the inuence of Au(III) com-
plexes with higher stability on sodium pump. The selected complexes,
[Au(bipy)(OH)
2
][PF
6
] (where bipy = 2,2-bipyridine) designated
AubipyOH in further text, [Au(py
dmb
-H)(CH
3
COO)
2
] (where py
dmb
-H =
deprotonated 6-(1,1-dimethylbenzyl)-pyridine) designated AupyOAc in
further text, and [Au(bipy
dmb
-H)(OH)][PF
6
] (where bipy
dmb
-H =
deprotonated 6-(1,1-dimethylbenzyl)-2,2-bipyridine) designated
Aubipy
c
in further text, are characterized by the presence of a bipyridyl li-
gand within a square planar arrangement of the gold(III) center [35] and
satisfy the strict requirements for stability under the physiological condi-
tions. Besides being stable, these complexes all bare a structurally related
and potentially cytotoxic group of ligands based on bipyridine which
may provide better cytotoxic properties [36]. Notably the three com-
pounds differ in that [Au(bipy)(OH)
2
][PF
6
] is a simple gold(III) complex
whereas [Au(bipy
dmb
-H)(OH)][PF6] and[Au(py
dmb
-H)(CH
3
COO)
2
] are or-
ganometallic compounds with a carbongold bond. Furthermore, al-
though there is a high level of homology between porcine Na
+
/K
+
-
ATPase and human Na
+
/K
+
-ATPase, we wished to denitively prove the
inhibitory effect of the investigated complexes on the human enzyme.
The study is extendedto the invitro investigationof sodiumpumpactivity
in human blood, as well as to the comparison of the present results with
the in vitro cytotoxic effects of these complexes on human lymphocytes
as a model system.
2. Experimental
2.1. Chemicals
All commercially available chemicals were of analytical grade.\
SH group donors L-cysteine and GSH were obtained from Fluka
(Switzerland). Gold(III) complexes [Au(bipy)(OH)
2
][PF
6
], [Au(py
dmb
-
H)(CH
3
COO)
2
] and [Au(bipy
dmb
-H)(OH)][PF
6
] were synthesized as
described in the literature [36,37]; analytical and spectroscopic data are
provided in Supplementary material. Structural formulas of these com-
plexes are giveninScheme 1. 1 10
3
Mand110
2
Mstock solutions
of the complexes and\SHdonors, respectively, in DMSOwere prepared
shortly before use. Working solutions were prepared by diluting the
stock solutions with deionized water to desired concentrations. Na
+
/
K
+
-ATPase from porcine brain cortex (specic activity of 0.1 IU/mg
solid), tetrabutylammonium hydroxide, 2,5-dihydroxybenzoic acid as
well as some chemicals for medium assay (adenosine triphosphate
(ATP), adenosinediphosphate (ADP), NaCl, KCl, MgCl
2
, and TrisHCl)
were purchased fromSigma-Aldrich (Germany). Chemicals for determi-
nation of Na
+
/K
+
-ATPase activity (stannous chloride and ammonium
molybdate) were fromMerck (Germany). PB-max karyotyping medium
for cell culturing was obtained from Invitrogen-Gibco, Paisley, UK. For
micronucleus assay, cytochalasin B and Giemsa stain were purchased
fromSigma-Aldrich(Germany). Methanol andacetic acid were obtained
from VWR (Germany). Deionized water was used throughout.
2.2. Stability studies by UVvisible (UVVis) spectroscopy
The UVVis spectra were recorded by diluting small amounts of
freshly prepared solutions of the individual complexes in DMSO in the
standard buffer (NaCl, KCl, MgCl
2
, and TrisHCl, pH 7.4). The concentra-
tion of each gold compound in the nal sample was 10
5
M. The
resulting solutions were monitored collecting the spectra over 72 h at
room temperature.
2.3. Human blood cell membrane preparation
Aliquots of heparinized human blood (0.5 mL) were put into 4.5 mL
of PB-max karyotyping medium. The cultures were treated with
increasing concentrations of gold(III) complexes (nal concentrations
from 10
8
M to 10
4
M) and incubated for 72 h at 37 C. Untreated
cell cultures served as controls. Upon incubation the cultures were
centrifuged at 2000 rpm for 10 min to separate the cells. The superna-
tant was discarded and replaced with 5 mL of ice cold lysis buffer
(20 mM TrisHCl, 20 mM MgCl
2
, pH = 8). Samples were then centri-
fuged again for 15 min at 4000 rpm. The supernatant was discarded
and more lysis buffer was added. This washing was repeated 34
times until a whitish pellet of cell membranes was obtained. Total
protein content was measured using Lowry's method [38]. Finally, the
pellet was separated and stored at 70 C until it was used in the
enzyme assays at a concentration of 1 mg protein/mL.
2.4. Na
+
/K
+
-ATPase activity assay
Na
+
/K
+
-ATPase activity was determined in a standard incubation
medium (200 L), containing 50 mM TrisHCl (pH 7.4), 100 mM NaCl,
20 mM KCl, 5 mM MgCl
2
, 2 mM ATP and protein (2 mg solid/mL) in
the absence (control) or presence of the desired concentration of
Au(III) complex. Incubation mixtures were preincubated for 15 min at
37 C in the presence of inhibitor or water (control). The reaction was
started by the addition of ATP, allowed to proceed for 15 min, and
interrupted by the addition of the ice cold HClO
4
and immediate cooling
on ice. The concentration of inorganic orthophosphate (Pi) liberated
from the hydrolysis of ATP was measured using a modied
Scheme 1. Structural formulas of the investigated Au(III) complexes: 1 [Au(bipy)(OH)
2
][PF
6
], 2 [Au(py
dmb
-H)(CH
3
COO)
2
], and 3 [Au(bipy
dmb
-H)(OH)][PF
6
].
229 V. Petrovi et al. / Journal of Inorganic Biochemistry 140 (2014) 228235
spectrophotometric procedure [39] based on the stannous chloride
method, by reading the absorbance at 690 nm. The results were
expressed as the meanpercentage of enzyme activity relative to the cor-
responding control value. All experiments were performed in triplicate.
The effect of L-cysteine and GSH on the prevention of the gold(III)
induced inhibition was measured under the same conditions as
described above, with the ligand added to the assay medium before
the exposure to the metal complex. The reactivating effect was mea-
sured by adding L-cysteine and GSH after a 15 minute preincubation
with the Au(III) complexes.
The activity of Na
+
/K
+
-ATPase from human blood cell membranes
was obtained in the standard incubation medium as above, by measur-
ing the concentration of ADP liberated due to the hydrolysis of ATP
using the ultra performance liquid chromatography (UPLC) method
[40]. ATP and ADP standards were used for method optimization. A
mobile phase composed of 2 mM tetrabutylammonium hydroxide in 4
mM phosphate buffer pH 7.2 (A) and methanol (B) (A:B = 79:29)
was used. Flow rate was 0.25 mL/min, while sample compartment
and column temperatures were 7 C and 40 C, respectively. Chromato-
grams were recorded at 254 nm. Samples used for chromatographic
analysis were prepared from standard incubation mixture by adjusting
its pH to 7 and ltration before injection. 2 L of each sample was
injected. ADP concentrations were determined from the area of chro-
matographic peak corresponding to ADP, by using calibration curves
constructed as the dependence of peak area on ADP concentrations.
Concentrations of ADP, liberated due to ATP hydrolysis, were propor-
tional to enzyme activity.
2.5. Lymphocyte cultures
Blood samples were obtained from three healthy, non-smoking
young male volunteer donors in accordance with the current Health
and Ethical regulations in Serbia [41]. Aliquots of heparinized whole
blood (0.5 mL) were placed in cultures containing PB-max karyotyping
mediumand treated with increasing concentration of Au(III) complexes
(nal concentrations from 10
7
M to 10
5
M). Untreated cell cultures
served as controls.
2.6. Micronucleus assay
For micronucleus preparation, the cytokinesis block methodof Fenech
[42] was used. Cytochalasin B at a nal concentration of 4 g/mL was
added to each blood culture after 44 h of incubation in order to inhibit
cytokinesis. The lymphocyte cultures were further incubated for 28 h.
Cells were collected by centrifugation and treated with hypotonic solu-
tion at 37 C. Hypotonic solution consisted of 0.56% KCl and 0.90% NaCl
(mixed in equal volumes). Cell suspension was xed in methanol/acetic
acid (3:1), washed three times with xative, and dropped onto a clean
slide. Slides were air dried and stained in alkaline Giemsa (2%). For each
sample, at least 1000 binucleated cells were scored and micronuclei
were recorded using a microscope.
2.7. Cell proliferation index
A cytokinesis-block proliferation index (CBPI) was calculated
according to the method of Surralls et al. [43] as follows: CBPI =
MI + 2MII + 3(MIII + MIV) N, where MIMIV represent the num-
ber of cells with one to four nuclei, respectively, and N is the number
of cells scored. Statistical analysis was performed using the statistical
software package Statistica 8.0, and OriginPro 8 for Microsoft Win-
dows, and was done using Student's t test and productmoment
and partial correlations. p values less than 0.05 were considered sig-
nicant and less than 0.001 were considered highly signicant. Re-
sults are expressed as percentage of control.
2.8. Instrumentation
The absorbance was measured using the Perkin Elmer Lambda 35
spectrophotometer with a thermostated 1.00 cm quartz cell, in the
wavelength range from 200 to 500 nm. The micronuclei were recorded
using an AxioImager A1 microscope (Carl Zeiss, Germany) with400or
1000 magnication. pH values of the solutions were measured by a
Methronom pH meter, Model 713. Chromatographic separations were
done by a UPLC system with a photodiode array (PDA) detector
(Waters, USA). A Waters BEH C
18
column (100 mm 2.1 mm; particle
size 1.7 m) was used as stationary phase. UVVis spectra were record-
ed on a Varian Cary 50 UVVis spectrophotometer.
3. Results
3.1. The selected gold(III) complexes
The Au(III) complexes that were selected for this study are schemat-
ically represented in Scheme 1. In all three complexes the gold(III)
center is tetracoordinatedina square planar fashion. Inthe coordination
compound AubipyOHthe gold atomis bound to the two nitrogen atoms
of the bipyridine ligand and to oxygen atoms of two hydroxo groups.
AupyOAc and Aubipy
c
are cyclometalated complexes with anionic C^N
and C^N^N donor ligands, respectively; in AupyOAc coordination is
completed by two acetate ligands and in Aubipy
c
by a hydroxide. The
Fig. 1. Solution stability kinetic the three gold compounds (1 10
5
M) were incubated
ina standardmedium(50 mMTrisHCl (pH 7.4), 100 mMNaCl, 20 mMKCl, 5 mMMgCl
2
)
for 72 h. Red line is starting time.
230 V. Petrovi et al. / Journal of Inorganic Biochemistry 140 (2014) 228235
multidentate ligand renders the Au(III) center resistant towards reduc-
tion. The presence of a direct goldcarbon bond in AupyOAc and
Aubipy
c
further stabilizes the Au(III) center. Au(III) tetracoordination
is completed by two hydroxo groups in AubipyOH, by two acetates in
AupyOAc and by a single hydroxo groupin Aubipy
c
. Notably, these com-
plexes show an acceptable solubility prole in aqueous buffers and a
remarkable stability over long observation times, and indeed all gold
complexes preserve their Au(III) center. Spectrophotometric proles
of their stability analyzed over 72 h are shown in Fig. 1. The three
gold(III) complexes may react with biological targets via -ligand
metathesis with HX species (X = O-, S-, N-donor). Indeed, the two
hydroxo complexes, and to a lesser extent the acetato complex, have
been proven to undergo exchange reaction with a variety of ROH, RSH
and RNH
2
to give alkoxo, thiolato and amido complexes [44].
3.2. Inhibition of Na
+
/K
+
-ATPase induced by Au(III) complexes
Two model systems were used for evaluation of the inuence of
Au(III) complexes on Na
+
/K
+
-ATPase activity. In the rst set of experi-
ments, the puried, commercially available Na
+
/K
+
-ATPase from
porcine brain cortex was exposed to increasing concentrations of the
studied complexes, in the range from1 10
9
Mto 1 10
3
M. Since\
SH containing molecules can interfere with gold binding to the enzyme
[3234,45,46], an experiment was carried out in simulated biological
conditions, in order to provide insights into the inhibitory potential of
Au(III) complexes towards Na
+
/K
+
-ATPase in a biological environment.
The second model systemfor estimation of enzyme activity was human
blood cell membranes previously cultivated for 72 h in the absence
(control) and presence of inhibitors in the same concentration range
as for puried enzyme.
Fig. 2 illustrates the dose-dependent inhibition curves obtained for
all investigatedcomplexes inthe same concentrationrange, constructed
for commercially available enzyme. The inhibition curves for the
enzyme fromhuman blood cells are available in Supplementary materi-
al (Fig. S1). In the case of human enzyme the UPLC method was used to
directly measure the concentration of the liberated ADP during the
enzyme hydrolysis of ATP [47].
The dependencies of relative enzyme activity (REA), expressed as a
percentage of the control value (Na
+
/K
+
-ATPase activity obtained
without inhibitor), on the concentration of Au(III) complexes t a sig-
moid function. Inhibitory parameters can be easily obtained using the
Hill analysis [48] of inhibition curves according to the relation (Fig. 2
inset):
log
REA
100REA

nlog I nlogIC
50
where [I] is the complex concentration, the IC
50
value represents the in-
hibitor concentration whichinduces 50% enzyme activity inhibition and
n is equal to the Hill coefcient of cooperativity. The values of IC
50
and
Hill coefcient for the investigated Au(III) complexes are presented in
Table 1.
The obtained inhibition parameters suggest no signicant difference
in potency among the investigated complexes towards Na
+
/K
+
-ATPase
activity in both model systems. The values of the Hill coefcient n N 1
indicate a positive cooperative binding of inhibitor to enzyme. This
means that the binding of one inhibitor molecule affects the binding
of subsequent molecules, i.e. the positive cooperativity increases the
afnity of the other binding sites for the inhibitor.
3.3. Kinetic analysis
For further understanding the type of inhibition (mode of interac-
tion between Na
+
/K
+
-ATPase and the investigated Au(III) complexes),
a kinetic study was carried out using the puried enzyme, as described
previously [34]. Typical MichaelisMenten kinetic curves in the pres-
ence of various inhibitor concentrations were obtained and are present-
ed in Fig. 3a. The LineweaverBurk linearization of the Michaelis
Menten curves was applied to determine the kinetic parameters of the
reaction, K
m
and V
max
. The double reciprocal plots (dependence of 1/
v
o
vs. 1/C
MgATP
) for all investigated complexes are presented in Fig. 3a
inset.
The results summarized in Table 2 suggest that V
max
decreased in
the presence of the inhibitors, while K
M
remained constant, comparing
to the control. The presence of inhibitor signicantly decreased enzyme
ability to convert substrate to product without changes in enzyme
afnity for substrate. This kind of enzyme behavior in the presence of
Au(III) complexes indicated the noncompetitive reversible type of
Na
+
/K
+
-ATPase inhibition. This mode of interaction suggests that the
inhibitor and the substrate bind randomly and independently of each
other at different enzyme sites. These results are in accordance with
our previously published data for some more simple Au(III) complexes
[34].
In order to evaluate Na
+
/K
+
-ATPase afnity for binding with Au(III)
complexes the inhibitor constant (K
I
), i.e., dissociation constant of the
enzymeinhibitor complex was determined. The secondary replot of
the LineweaverBurk graph, representing dependence of the primary
plot slope on inhibitor concentrations, is shown in Fig. 3b. The inhibitor
constants (K
I
) were obtained directly from intersection of replot with
abscissa and are presented in Table 2.
Positive cooperative binding of inhibitor to enzyme was conrmed
using the Scatchard analysis of inhibition curves [49,50]. The concentra-
tions of bound and free inhibitors were obtained frominhibition curves
and K
I
values. The dependence of bound/free vs. bound inhibitor
concentration ratio is presented in Fig. 4. A concave-down curve that
intersects the origin is indicative of positive cooperativity in all cases.
Fig. 2. Inhibition of commercially available Na
+
/K
+
-ATPase activity induced by Au(III)
complexes. Inset: Hill analysis of inhibition curves. The values given are the mean of at
least three experiments S.E.M.
Table 1
IC
50
values and Hill coefcients n for the inhibition of Na
+
/K
+
-ATPase, isolated from hu-
man blood cells and porcine cerebral cortex, induced by Au(III) complexes.
Complex IC
50
10
6
[M] n
Human blood cells Puried enzyme Puried enzyme
AubipyOH 2.5 0.5 3.5 0.1 1.1 0.2
AupyOAc 6.9 0.5 7.6 0.1 1.6 0.4
Aubipy
c
6.4 0.5 7.3 0.1 1.6 0.3
231 V. Petrovi et al. / Journal of Inorganic Biochemistry 140 (2014) 228235
3.4. Enzyme recovery and prevention of inhibition using L-cysteine and
glutathione
To investigate the possibility for the prevention of Na
+
/K
+
-ATPase
inhibitioninducedby Au(III) complexes andthe recovery of the inhibited
enzyme activity using ligands containing\the SH group, L-cysteine and
GSH were tested in different assays.
In the rst group of assays, the investigated ligands (the concentra-
tion range was from 1 10
6
M to 1 10
3
M) were pre-incubated in
the reaction mixture for 15 min before the addition of inhibitors. For
each inhibitor the concentrations were equal to their IC
20
, IC
50
and
IC
100
values, i.e. the concentrations of inhibitor which induce 20%, 50%
and 100% enzyme inhibition, respectively. Na
+
/K
+
-ATPase assay was
carried out 15 min after inhibitor addition. The results presenting the
dependence of relative enzyme activity on L-cysteine and GSH in the
presence of selected Au(III) complexes are shown in Figs. 5a and S2a
in Supplementary material, respectively. The complete prevention of
inhibition in the presence of\ SH donors was achieved for inhibitor
concentration corresponding to the IC
20
values of each of the three
investigated Au(III) complexes.
In the second set of experiments, the enzyme was incubated with
the inhibitors at IC
20
, IC
50
and IC
100
concentrations for 15 min. After
the addition of one of the investigated \SH donors in the concentra-
tion range as in the previous experiment, the medium was pre-
incubated for another 15 min before the enzymatic reaction was
started. This preincubation period was long enough to establish the
equilibrium between the complexes and the enzyme. In this way
any leftover inhibitor molecules would be neutralized, and some of
the inhibitor already bound to the enzyme would be removed, restor-
ing a portion of the enzyme activity. According to the results present-
ed in Figs. 5b and S2b in Supplementary material, it is obvious that the
applied\ SH donors exerted a dose dependent recovery effect on
inhibited enzyme activity.
Both thiols demonstrated a similar recovery capability. The complete
regeneration was possible only in the case of low enzyme inactivation
(inhibition of 20% enzyme activity) using higher concentrations of
reactivators \(SH donor concentration N 1 mM, Fig. 5b, Fig. S2b, line
group 1). As it can be seen in the graphs (Figs. 5 and S2), both prevention
and reactivation take place as expected, which indicates that the binding
of the inhibitor with the enzyme likely takes place via L-cysteine side
chains that are present and available in the structure of Na
+
/K
+
-ATPase.
3.5. The correlation of the cytotoxic effects of Au(III) complexes with Na
+
/
K
+
-ATPase inhibition
The cytotoxic effects of Au(III) complexes were evaluated by the
study of the incidence of micronuclei (MN/1000 binuclear cells) and
cytokinesis-block proliferation index (CPBI) in lymphocyte cultures.
The samples were treated with Au(III) complexes in the concentration
range from 1 10
7
M to 1 10
5
M, since these concentrations
were inthe decreasing range of the inhibitioncurves (Fig. S1). Fig. 6 pre-
sents the dependence of the micronucleus induction (a) and cell prolif-
eration (b) on the Au(III) complex concentration.
According to the results presented in Fig. 6 it is obvious that all of the
investigated Au(III) complexes signicantly enhanced micronucleus
Fig. 3. a) Initial reaction rate (v
o
) vs. MgATP
2
concentration in the absence (control) and presence of 2.5 10
6
MAubipyOH, 5 10
6
MAupyOAc and 5 10
6
MAubipy
c
. The values
given are the mean of at least three experiments. Inset: LineweaverBurk linearization of the obtained results. b) Secondary replots of LineweaverBurk graphs: slope vs. the inhibitor
concentrations for Au(III) complexes.
Table 2
The values of kinetic parameters and inhibitory constants for Au(III) complexes deter-
mined from LineweaverBurk linearization of MichaelisMenten hyperbola.
Sample K
M
[mM] v
max
[mmol/h/mg] K
I
[M]
Control 0.69 0.07 0.094 0.005
AubipyOH 0.68 0.10 0.052 0.005 1.42 10
6
AupyOAc 0.68 0.12 0.050 0.006 5.97 10
6
Aubipy
c
0.67 0.09 0.051 0.004 6.02 10
6
Fig. 4. Scatchard analysis of the inhibition curves presented in Fig. 2.
232 V. Petrovi et al. / Journal of Inorganic Biochemistry 140 (2014) 228235
incidence (p b 0.05) and decreasedcell proliferationina dose dependent
manner. Maximum induction of micronuclei occurred at concentration
3 10
6
5 10
6
M. Afterwards, the incidence of micronuclei
decreased, but still was signicantly higher than that in control (p b
0.05). The strongest effect was noticed for the Aubipy
c
complex. Be-
sides, a signicant decrease of CBPI was observed for the same concen-
tration range in all cell cultures treated with Au(III) complexes (p b
0.001). The incidence of micronuclei and cytokinesis-block proliferation
index were correlated inversely and statistically signicant (p b 0.05).
In all cases, the maximal antiproliferative effects were observed for
the highest concentrations employed. The observed decline in the
micronuclei incidence at the highest concentrations employed is the
consequence of reduced number of cells entering mitosis.
Our results further demonstrate a strong correlation between pa-
rameters of cytotoxicity and Na
+
/K
+
-ATPase activity affected by the
same concentration of Au(III) complexes. As shown in Fig. 7a, incidence
of micronuclei inversely correlates with enzyme activity. Increased MN
inductionis followed by decreased enzyme activity up to the concentra-
tion of Au(III) complexes that induce cell death. On the other hand, CBPI
(Fig. 7b) and enzyme activity correlate positively, meaning that inhibi-
tionof cell proliferationis accompanied by inhibitionof enzyme activity.
These results at least in part suggest that Au(III) complexes inhibit cell
proliferation possibly via Na
+
/K
+
-ATPase activity inhibition.
4. Discussion
The results presented in this work showed that a fewselected Au(III)
complexes are potent Na
+
/K
+
-ATPase inhibitors. Although the literature
surveys indicate that the oxidation state 3+ is more stable in the case of
the selected organogold(III) complexes ([Au(py
dmb
-H)(CH
3
COO)
2
], and
[Au(bipy
dmb
-H)(OH)][PF
6
]) compared to [Au(bipy)(OH)
2
][PF
6
] [9,35,
36], the obtained inhibition parameters, i.e. IC
50
values, are quite similar.
The kinetic studies allowedus to establishthat Na
+
/K
+
-ATPase inhibition
is positively cooperative, reversible and non-competitive. These ndings
are inagreement withthe previously publishedresults concerning the in-
hibition of Na
+
/K
+
-ATPase induced by some complexes of noble metals
(e.g. Pd, Pt, and Au) [29,3234,46]. Moreover, complex formation
between some Pt(II) and Pd(II) compounds and Na
+
/K
+
-ATPase was
previously conrmed using various experimental techniques [30,32].
Spectroscopic evidence suggested that cisplatin binds mainly to the hy-
drophobic portion of the enzyme, through the polypeptide C_O and
C\N groups [30], since terpyridine platinum reacts with Cys452, which
is on the exterior of the nucleotide binding pocket [28]. Previous studies
showed that the interactionof Pt(II) complexes with proteins is for sever-
al orders of magnitude slower compared to Pd(II) and Au(III) complexes,
with a signicantly lower overall binding constant [28,33,34]. Au(III)
complexes investigated in this paper with the bulkier ligands compared
Fig. 5. Prevention of inhibition (a) and reactivation of inhibitedNa
+
/K
+
-ATPase (b) by L-cysteine inthe presence of gold(III) complexes at concentrations whichinduce 20% (line group 1),
50% (line group 2) and 100% (line group 3) enzyme inhibition. These characteristic concentrations were: IC
20
=6 10
6
M, IC
50
=2.5 10
6
M, and IC
80
=6 10
7
M, for AubipyOH;
IC
20
= 1.5 10
5
M, IC
50
= 7 10
6
M, and IC
80
= 2 10
6
M for AupyOAc and IC
20
= 1.5 10
5
M, IC
50
= 7 10
6
M, and IC
80
= 2 10
6
M = for Aubipy
c
.
Fig. 6. Incidence of micronuclei (MN) (a) and cytokinesis-block proliferation index (CPBI) (b) in lymphocyte cultures treated with increasing doses of Au(III) complexes.
233 V. Petrovi et al. / Journal of Inorganic Biochemistry 140 (2014) 228235
to those studied in our previous work are isoelectronic with cisplatin-like
Pt(II) complexes. However, the mechanism of their action is different
from that of cisplatin [36,51]. The role of hydrolysis as an activation step
for this class of compounds is not clear yet; for these analogues, hydrolysis
may represent an activation step. As stated in our previous work, the
sodiumpumpinhibitionby selectedAu(III) complexes cannot be ascribed
only to the simple coordination of central metal ion with the\SHgroups
[34]. The cleavage of disulde bridges, required for enzyme functionality,
due to the redox reaction of Au(III) complexes with proteins must be also
taken into account [17], since it can lead to the signicant functional
alterations [52].
The interaction of Au(III) complexes with Na
+
/K
+
-ATPase can be
prevented and, more importantly, reversed by the addition of\ SH
donors. However, the complete prevention or recovery of the inhibited
activity was not achieved even in the presence of 100 fold higher
concentration of the thiol containing ligand. This can also be the conse-
quence of the interaction of the complexes with thiol donors since the
reduction of Au(III) to Au(I) can occur, and the two oxidation states of
gold react differently with a protein [53]. Also, the mentioned cleavage
of disulde bridges is not remedied by the addition of\SH donors.
In simulated biological environments such as cell cultures and
incubated human blood, these complexes have caused cell death in
experimental conditions and hindered cell growth in lower con-
centrations. Moreover, there is no signicant difference in cytotoxic
properties between organogold complexes ([Au(py
dmb
-H)(CH
3
COO)
2
],
[Au(bipy
dmb
-H)(OH)][PF
6
]) and [Au(bipy)(OH)
2
][PF
6
]. This nding is
in accordance with inhibition parameters determined in this work, indi-
cating that the introduction of the Au\C bond did not affect the biolog-
ical properties signicantly. Our previous work has already indicated
that bipyridine derivatives may have stronger cytotoxic properties and
the results shown here conrm it [34]. It is unclear whether this effect
comes from the ligands alone upon complex destruction in the cell, or
from the complex as a whole. Bipyridine and its derivatives are well
known to cause DNA damage that leads to cell death. A comparative
study would be required to see if these complexes have a synergic effect
of the inhibition of important enzymes and DNAdamage or is it the sim-
ple superposition of these two effects. However, nucleic acid damage
seen in micronucleus assay, combined with the results obtained from
the human blood Na
+
/K
+
-ATPase inhibition implies a mixed cytotoxic
effect.
5. Conclusion
In conclusion we can state that the here investigated complexes
interact with the Na
+
/K
+
-ATPase in much the same way as other
similar complexes. They show medium to high IC
50
values, and their
effect can be prevented or remedied by common biological molecules
that contain\ SH groups. The mechanism of their action is non-
competitive, which is in agreement with similar compounds and it
appears that they exhibit a more potent biological effect on living cells
than previously researched compounds of the same type. They mani-
fested the ability to inhibit the Na
+
/K
+
-ATPase in simulated human
blood medium, which is important when considering the fact that
these complexes are degraded by biological molecules containing\ SH
groups.
Abbreviations
bipy 2,2-bipyridine
bipy
dmb
-H deprotonated 6-(1,1-dimethylbenzyl)-2,2-bipyridine.
CBPI cytokinesis-block proliferation index
GSH glutathione
MN micronuclei
py
dmb
-H deprotonated 6-(1,1-dimethylbenzyl)-pyridine
REA relative enzyme activity
Acknowledgments
This work was nancially supported by the Ministry of Education,
Science and Technological Development of the Republic of Serbia, pro-
ject no. 172023. The generous nancial support by AIRC (IG-12085)
and Benecentia Stiftung Vaduz is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2014.07.015.
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