1. Biometals (2008) 21:117–126
DOI 10.1007/s10534-007-9098-3
Metal Cu(II) and Zn(II) bipyridyls as inhibitors of lactate
dehydrogenase
Raj Kumar Koiri Æ Surendra Kumar Trigun Æ
Santosh Kumar Dubey Æ Santosh Singh Æ
Lallan Mishra
Received: 19 February 2007 / Accepted: 24 April 2007 / Published online: 2 June 2007
Ó Springer Science+Business Media B.V. 2007
Abstract Metal complex–protein interaction is an As compared to the control sets, Cu-bpy caused a
evolving concept for determining cellular targets of significant decline (P < 0.05–0.001) in the activity of
metallodrugs. Lacatate dehydrogenase (LDH) is crit- LDH in all the tissues studied. However, Zn-bpy
ically implicated in tumor growth and therefore, showed inhibition of LDH only in liver (P < 0.01),
considered to be an important target protein for anti- kidney (P < 0.001) and heart (P < 0.01), but with no
tumor metal complexes. Due to efficient biocompat- effect in spleen, brain and skeletal muscle tissues.
ibility of copper (Cu2+) and zinc (Zn2+), we synthe- PAGE analysis suggested that all the five LDH
sized CubpyAc2 Á H2O (Cu-bpy) and ZnbpyAc2 Á H2O isozymes are equally sensitive to both the complexes
(Zn-bpy; where bpy = 2,20 bipyridine, in the respective tissues. The results suggest that Cu-
Ac = CH3COOÀ) complexes and evaluated their and Zn-bpy are able to interact with and inhibit LDH,
interaction with and modulation of LDH in mouse a tumor growth supportive target protein at tissue
tissues. The increasing concentration of both the level.
complexes showed a significant shift in UV–Vis
spectra of LDH. The binding constant data Keywords Lactate dehydrogenase Á Metal bipyridyl
(Kc = 1 · 103 MÀ1 for Cu-bpy and 7 · 106 MÀ1 complexes Á Glycolysis Á Tumor growth Á LDH
for Zn-bpy) suggested that Zn-bpy-LDH interaction isozymes
is stronger than that of Cu-bpy-LDH. LDH modulat-
ing potential of the complexes were monitored by
perfusing the mice tissues with non-toxic doses of
Cu-bpy and Zn-bpy followed by activity measure- Introduction
ment and analysis of LDH isozymes on non-dena-
turing polyacrylamide gel electrophoresis (PAGE). Metal ions and metal coordination compounds are
known to affect cellular processes in a dramatic way
(Bertini et al. 1994, 2001). During recent past, a
R. K. Koiri Á S. K. Trigun Á S. Singh
number of metal complexes have been formulated
Biochemistry & Molecular Biology Section, Centre of
Advanced Studies in Zoology, Banaras Hindu University, and evaluated for their anti-tumor properties (Clarke
Varanasi 221005, India 2003; Keppler et al. 1993). Cytotoxicity of most of
the metal complexes has been mainly correlated with
S. K. Dubey Á L. Mishra (&)
their ability to bind with and damage DNA (Keppler
Department of Chemistry, Banaras Hindu University,
Varanasi 221005, India et al. 1993; Novakova et al. 2003). However, the
e-mail: lmishrabhu@yahoo.co.in dictum that DNA is the primary target for metallo-
123

2. 118 Biometals (2008) 21:117–126
drugs is rapidly declining (Dyson and Sava 2006), However, information is scanty on protein based
and therefore, defining additional pharmacological anti-neoplastic activity of Cu- and Zn-complexes
targets for metal complexes at cellular level is of except some reports, wherein, a 8-hydroxyquinoline-
much current interest. Cu(II) and 5,7-dichloro-8-hydroxyquinoline-Cu(II)
Metal–protein interaction is of prime importance have been shown to inhibit proteasome and to induce
for the biodistribution, mechanism of action and for tumor cell apoptosis (Daniel et al. 2004a, b) and
the toxic effects of anti-tumor metal complexes certain Zn metals that inhibit some tumor associated
(Trimerbaev 2005; Dyson and Sava 2006). In this extracellular enzymes (Cox and McLendon 2000;
respect, formulating complexes of biologically Berg and Shi 1996; Jiang and Guo 2004). Moreover,
important metal ions is of special merit as they are most of these findings are based on in vitro studies on
likely to be better biocompatible and hence, can be cell lines and thus, it is important to define pharma-
delivered to the cells easily. In addition to iron, cological targets of Cu- and Zn-complexes at cellular
copper (Cu), and zinc (Zn) are the most abundant level in animal system. Therefore, examining and
metal ions implicated in a number of biochemical defining tumor associated protein targets for exoge-
reactions and as structural determinant of several nous Cu- and Zn-complexes at tissue level in animal
proteins (Theophanides and Anastassopoulou 2002; models are of much relevance.
Dudev 2003). The clinical use of 18fluorodeoxyglucose posi-
Increased level of Cu in cells is known to bind tron-emission tomography has demonstrated that the
DNA (Bar-Or et al. 2001; Theophanides and Anas- glycolytic phenotype is observed in most human
tassopoupou 2002) and also to certain proteins cancers (Gatenby and Gillies 2004). The cells
(Halliwell and Gutteridge 1990). However, DNA genetically engineered to become cancerous have
damage potential of Cu is attributed to Cu dependent also shown significant increase in its glycolytic
production of reactive oxygen species (ROS) in the pathway (Ramanathan et al. 2005). This is mainly
cells (Theophanides and Anastassopoupou 2002). due to the increased energy demand for angiogen-
The relevance of Cu-complexes as anti-neoplastic esis (neovascularization) in the cancerous cells
agent got attention after a Cu-phenanthroline com- (Palmer et al. 1990; Biskupiak and Krohn 1993).
plex was found to show nuclease activity (Downey There are some reports on direct involvement of
et al. 1980; Marshall et al. 1981). Later, Cu-bipyridyl glycolytic enzymes in cellular immortalization (Kim
[Cu(bpy)2+] complexes were found more effective, as
2 et al. 2005; Kondoh et al. 2005), including the
they showed strong affinity for DNA (Yang et al. increased expression of hexokinase and lactate
2004). Recently, a Cu-1,10-phenanthroline has been dehydrogenase, the first and the last enzyme of
reported to induce apoptosis by increasing ROS load glycolytic pathway, respectively (Mathupala et al.
in a liver carcinoma (Cai et al. 2007). However, little 1997; Dang et al. 1997).
is known about modulation of metabolic enzymes by Lactate dehydrogenase (LDH, EC 1.1.1.27) is a
exogenous Cu-complexes except inhibition of certain tetrameric protein consisting of two types of subunits,
enzymes that do not require Cu for their catalytic the M type (pre-dominantly expressed in skeletal
functions, like NADH oxidase and lactate dehydro- muscle) and the H type (pre-dominantly expressed in
genase by Cu(I) & Cu(II) ions (Zwart et al. 1991). heart) contributed by the two genes (Cahn et al. 1962;
Zinc is the second most abundant transition metals Markert 1963). Combination of these two subunits in
in animal tissues implicated in a number of physio- different ratio gives rise five isozymic forms (M4,
logical functions (Berg and Shi 1996; Prasad 1998; M3H, M2H2, MH3 & H4), which are expressed in a
Frederickson et al. 2000; Kikuchi et al. 2004). It tissue specific manner in most of the mammalian
shows strong interaction with and modifies a variety tissues. The differential expressions of LDH iso-
of cellular proteins (Cox and McLendon 2000) zymes in different types of tumors have been reported
including certain key enzymes (Trigun and Singh (Koukourakis et al. 2003), and therefore, inhibition of
1989) and several transcription factors (Prasad 1998). LDH mediated step is now considered to be one of
A Zn-thiosemicarbazone complex has also been the effective mechanisms to control tumor growth
shown to act as anti-tumor agent in vitro and is as (Gaber 2006). Abnormally high LDH activity in
cytotoxic as cisplatin (Beraldo and Gambino 2004). cancerous tissues vis-a-vis decreased LDH activity
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3. Biometals (2008) 21:117–126 119
with tumor regression have been demonstrated in methanol and analyzed on IR and 1H-NMR (for Zn
animal models (Koukourakis et al. 2003; Niakan only).
2001; Pathak and Vinayak 2005). There is a report on IR of both complexes (KBr pelletes) showed
designing active site inhibitors of human LDH for tpyridyl 750 cmÀ1 tc = o 1417 cmÀ1 where as 1H-
therapeutic applications also (Yu et al. 2001). NMR (DMSO d6) of corresponding Zn(II) compound
Recently, down regulation of LDH-A activity has showed a signal at d = 7.65(2H, s), 8.32(2H, s),
been reported to affect mitochondrial bioenergetics 8.67(4H, s). The structure of both the complexes has
and to retard proliferation of Neu 4145 tumor cells in been presented in Fig. 1.
culture (Fantin et al. 2006). And thus, LDH seems to
be a relevant and suitable candidate to study anti- Binding of complexes Cu-bpy and Zn-bpy with
cancer potential of newly formulated metallodrugs. LDH
We have demonstrated that a cytotoxic Ru(II)
complex containing flavones inhibits LDH revers- Binding study of Cu-bpy and Zn-bpy complexes with
ibly when administered orally to mice in a tissue LDH was done using UV–Vis spectral analysis. To a
specific manner (Mishra et al. 2004b). A recent fixed concentration of LDH (1 · 10À6 M) prepared in
report from our lab described that a cytotoxic methanol:water (1:1) mixture, increasing concentra-
Ru(II) complex containing 4-carboxy N-ethylbenza- tion of both the complexes (1 · 10À6–1 · 10À8 M),
mide [Ru(II)-CNEB] strongly interacts with and prepared in the same solvent, were incubated sepa-
inhibits LDH non-competitively (Trigun et al. rately for 30 min at room temperature followed by
2007). We could also formulate cytotoxic Cu- and measurement of optical density at 200–800 nm. To
Zn-bipyridyl complexes with the potential to estab- calculate association constant (Kc) between the
lish weak interactions with the proteins. In order to complexes and LDH, differences in OD observed
evaluate protein based therapeutic potential of these between the untreated and treated samples at 280 nm
complexes, the present study deals with whether were tested on Benesi–Hilde Brand (BH) equation
complexes Cu-bpy and Zn-bpy are able to interact (Mishra et al. 2004a). The formulae adopted was
with and modulate LDH in different tissues of
mouse. ½AŠ Á ½BŠ0 =d0 ¼ ½BŠ0 = þ 1=Kc
ð1Þ
with
Materials and methods:
d’ ¼ d À d0 A À d0 B ð2Þ
Chemicals
Here, [A]0 and [B]0 are the initial concentrations
Cu(II) acetate dihydrate and Zn(II) acetate dihydrate of the complex and LDH, respectively, d is the
and 2,20 bipyridine, purified LDH, b-NADH (b- absorbance of the mixture at 270 nm and d0A d0B
nicotinamide adeninedinucleotide, reduced) and so- are those of complex and LDH at the same
dium pyruvate were purchased from Sigma-Aldrich wavelength. Linear plot according to Eq. 1 was
Corporation, USA. Other chemicals and solvents obtained for each complex and association constant
used were purchased from SISCO Research Labora- (Kc) was calculated accordingly.
tory, Mumbai, India.
Animals
Synthesis and characterization of Cu- and Zn-bpy
Adult male mice (AKR strain) of same age group
Following the method reported earlier (Mishra et al. (18 weeks old) were used for the experiments.
2004a), Cu(II) acetate dihydrate (2.17 g, 1 mmol) and Animals were maintained under a constant 12 h light
Zn(II) acetate dihydrate (2.2 g, 1 mmol) were added and dark cycle at 25–308C. They were provided
separately to the solution of 2,20 bipyridine (1.56 g, standard mice feed and water ad libitum. The mice
1 mmol) in 20 ml methanol with constant stirring up were maintained as per the guidelines of institutional
to 4 h. The resultant solids were filtered, washed with (BHU) animal ethical committee.
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4. 120 Biometals (2008) 21:117–126
Fig. 1 Structural
representation of Cu-bpy
and Zn-bpy complexes
N N N N
Cu Zn
Where AcO = CH3COO- Where AcO = CH3COO-
AcO OAc AcO OAc
Cu - b p y Z n - bp y
Treatment of mice with Cu-bpy and Zn-bpy Assay of lactate dehydrogenase (LDH)
complexes
LDH activity was measured spectrophotometrically
Pilot experiments showed that both Cu- and Zn- following the method of Kornberg (1955) and as
bpy complexes are moderately cytotoxic to Dal- described in our earlier report (Trigun et al. 2006).
ton’s lymphoma cells (unpublished results) in vitro, The reaction mixture (3 ml) was composed of 20 mM
however, they are non-toxic/lethal to adult mice Tris–Cl (pH 7.4), 6 mM NADH, suitably diluted
even at a concentration of 2 mg/kg body weight. tissue extract and 1 mM sodium-pyruvate. The
Accordingly, Cu-bpy and Zn-bpy (2 mg each) decrease in absorbance at 340 nm was recorded up
were first dissolved in 500 ml of methanol and the to 10 min. The oxidation of 1 mmol of NADH per min
final volume was made 10 ml with Krebs–Ringer at 258C was defined as 1 unit of the enzyme and
Buffer (KRB: pH 7.2) so that final concentration values were presented as unit mgÀ1 protein.
of the solution becomes 0.2 mg/ml (non-toxic/ Protein content in all the samples was measured
lethal dose). Perfusion experiment was done using Folin method of Lowry et al. (1951).
´
according to Clemence et al. (2002) with some Statistical analysis, wherever required, was done
modifications as reported recently (Trigun et al. following Bruning and Kintz (1977). Student ‘t’ test
2007). Briefly, mice were anesthetized by thiopen- was performed to find the level of significant
tone injection and a canula was implanted between changes between the control and the experimental
ventricle and atrium in the heart so that it feeds groups.
the ventral aorta and perfusate reaches to tissues
directly through systemic circulation. Perfusion Non-denaturing polyacrylamide gel
was done at a rate of 0.5 ml/min and at constant electrophoresis (PAGE)
pressure by keeping the solution at 1 m height
above the heart for 20 min. Experimental group LDH isozymes were analyzed on non-denaturing
mice were perfused with KRB containing 0.2 mg/ 10% PAGE following the method described in our
ml of the complexes, whereas, control group of earlier report (Trigun et al. 2006). Briefly, polymer-
mice were perfused similarly with KRB alone. ization of gel and preparation of samples were done
3–4 mice were treated in each group. in a non-SDS buffer. Suitable amount (30 mg) of
protein samples were loaded in each lane and
Preparation of tissue extracts electrophoresis was performed using non-SDS run-
ning buffer (Tris–Glycine pH 8.3). After electropho-
Mice were sacrificed, tissues were dissected out and resis was over, gels were subjected to LDH specific
washed in ice-cold mammalian saline. Tissue extracts activity stain. Staining solution consisted of 0.125 M
(10% w/v) were prepared in 0.02 M Tris–Cl buffer Tris–Cl (pH 7.4), 0.5 mM MgCl2, 0.1 mM Li-Lactate,
(pH 7.4) containing protease inhibitors as described 1 mg/ml NAD, 0.01 M NaCl, 0.25 mg/ml nitro blue
earlier from this lab (Pandey et al. 2005). Extracts tetrazolium (NBT), and 0.025 mg/ml phenazine
were centrifuged at 40,000g and the supernatant methosulfate (PMS). After development of LDH
collected were used for non-denaturing PAGE and bands, gels were washed, LDH bands were scanned
other enzymatic studies. and quantitated by gel spectrometry using alpha
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5. Biometals (2008) 21:117–126 121
imager 2200 gel documentation software. The isozy- Zn-bpy though significantly inhibited LDH in liver
mic bands in the gel were characterized by comparing (P 0.01), kidney (P 0.001) and heart (P 0.001),
their migration with the pattern shown by the tissue but with no effect on the activity of LDH in spleen,
specific characteristic LDH bands as described earlier brain and skeletal muscle tissues.
(Mishra et al. 2004b; Trigun et al. 2006, 2007).
Effect on LDH isozyme
Results To ascertain whether the effects of Cu-bpy and Zn-
bpy are LDH isozyme specific, the untreated and
Interaction of Cu-bpy and Zn-bpy with LDH treated tissue extracts were subjected to PAGE
analysis of LDH isozymes. Results in Figs. 3–5B
In order to assess whether Cu-bpy and Zn-bpy and C re-confirm that liver, spleen and skeletal
complexes are able to bind with LDH protein, UV– muscle express mainly M4-LDH (LDH-5) whereas
Vis spectral studies of purified LDH was performed kidney, heart and brain tissues show the expression of
in the presence of increasing concentration of both all the five isozymes. Nonetheless, a comparison of
the complexes separately. According to Fig. 2A and intensities of LDH bands from untreated and the
B, as compared to the untreated LDH sample, there is corresponding treated tissues apparently corroborate
a significant leftward shift in kmax (from 280 nm to the activity measurements data (Figs. 3–5A) in the
270 nm) of LDH treated with the increasing concen- respective tissues. Also, it was observed that wher-
tration of both the complexes. A linear increase in the ever there was inhibition in LDH activity due to Cu-
OD due to treatment with both these complexes also bpy and Zn-bpy, all the isozymes expressed in the
suggests the possibility of significant charge transfer concerned tissue were equally sensitive to both these
between LDH protein and both the complexes. The complexes.
treatment of absorption data on Benesi–Hilde Brand
equation produced linear plots for both the complexes
and the association constant calculated was Discussion
1 · 103 MÀ1 for Cu-bpy-LDH and 7 · 106 MÀ1 for
that of Zn-bpy–LDH interactions. A mechanistic understanding of how metal com-
plexes act at cellular level is crucial to their clinical
Effect on LDH activity success. Metal centers are positively charged and
therefore, likely to bind with negatively charged
According to Figs. 3–5A, in comparison to the biomolecules like nucleic acids and proteins, and thus
control group of mice, perfusion of non-toxic dose offer a predictable mechanism for their use as
of Cu-bpy caused a significant decline in the activity pharmacological agents. Since, Cu and Zn metal ions
of LDH in liver (P 0.001), kidney (P 0.001), heart are known to modulate the catalytic functions of
(P 0.001), spleen (P 0.001), brain (P 0.05) and several enzymatic proteins, it is speculated that the
skeletal muscle (P 0.01). However, perfusion of complexes containing such biocompatible metal ions
Fig. 2 UV–Vis spectral
pattern of purified LDH
(1 · 10À6 M) at 280 nm in
the presence of different
concentration (a = LDH
alone, b = 1 · 10À6 M,
c = 2 · 10À6 M,
d = 4 · 10À6 M,
e = 8 · 10À6 M ) of Cu-bpy
(A) and Zn-bpy (B)
complexes
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6. 122 Biometals (2008) 21:117–126
Fig. 3 Effect of Cu-bpy A
and Zn-bpy complexes on
A
Liver Kidney
Specific activity
(U/mg protein)
Specific activity
the activity (A) and
(U/mg protein)
3 .0 1 .0
*** ***
isozymic pattern (B) of 2 .0
*** **
0 .5
LDH in liver and kidney. 1 .0
Data in panel A represents 0 .0 0 .0
mean ± SD from 3 to 4 Control Cu-bpy Zn-bpy Control Cu-bpy Zn-bpy
observations. ** P 0.01,
*** P 0.001 (Control B B
versus treated samples). In
panel B, 30 mg protein from C o n t r o l C u- b p y Zn - b p y
the pooled tissue extracts M4
from 3 to 4 mice from the
M3H
untreated and treated groups M4
were loaded in the M2H2
corresponding lanes. Panel MH3
C represents relative H4
intensity of LDH band
taking total of the three C
C
Relative density
lanes as 100%) 30
Relative density
60
20
(%)
40
(%)
10
20
0
Control Cu-bpy Zn-bpy 0
Co ntro l Cu-bpy Zn-bpy
Fig. 4 Effect of Cu-bpy A A
and Zn-bpy complexes on Heart Brain
Specific activity
Specific activity
(U/mg protein)
(U/mg protein)
3 1 .2
the activity (A) and *** 0 .9 *
2 ***
isozymic pattern (B) of 0 .6
1 0 .3
LDH in heart and brain.
0 0 .0
Data in panel A represents Co ntrol Cu-bpy Zn-bpy Control Cu-bpy Zn-bpy
mean ± SD from 3 to 4
observations. * P 0.05,
*** P 0.001 (Control B B
versus treated samples). In
panel B, 30 mg protein from
the pooled tissue extracts M4 M4
M3H
from 3 to 4 mice from the M3H
M2H2
untreated and treated groups M2H2 MH3
were loaded in the MH3 H4
corresponding lanes. Panel H4
C represents relative
intensity of LDH band
taking total of the three C C
Relative density
Relative density
lanes as 100%) 60 40
40 30
(%)
(%)
20
20 10
0 0
Co ntro l Cu-bpy Zn-bpy Co ntro l Cu-bpy Zn-bpy
could be of much use for protein based therapeutic 2004b; Trigun et al. 2007). We describe here that Cu-
applications. LDH, critically implicated in tumor bpy and Zn-bpy complexes, synthesized and charac-
growth (Koukourakis et al. 2003; Niakan 2001; terized in our laboratory, could be the other candi-
Pathak and Vinayak 2005), though does not need dates that can interact with and inhibit LDH at tissue
any metal ion for its activity, however, the enzyme level in mice.
has been described to be a potential target for certain A shift in the absorption pattern of a protein at
metal complexes in vivo and in vitro (Mishra et al. 280 nm in the presence of a metal complex suggests
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7. Biometals (2008) 21:117–126 123
Fig. 5 Effect of Cu-bpy A A
and Zn-bpy complexes on Spleen
Specific activity
Muscle
(U/mg protein)
0.6
Specific activity
(U/mg protein)
the activity (A) and 4
0.4 ** 3 **
isozymic pattern (B) of
0.2 2
LDH in spleen and muscle. 1
Data in panel A represents 0.0 0
mean ± SD from 3 to 4 Control Cu-bpy Zn-bpy Co ntro l Cu-bpy Zn-bpy
observations. ** P 0.01
(Control versus treated B B
samples). In panel B, 30 mg
protein from the pooled M4
tissue extracts from 3 to 4 M 3H
mice from the untreated and M4 M2H2
MH3
treated groups were loaded
in the corresponding lanes.
Panel C represents relative
intensity of LDH band C C
taking total of the three
Relative density
Relative density
60 60
lanes as 100%) 40
40
(%)
(%)
20 20
0 0
Co ntro l Cu-bpy Zn-bpy Co ntro l Cu-bpy Zn-bpy
for protein–metal interaction. Such an approach has Based on in vitro studies using cell lines, it has
been applied to study the interaction of certain metal- been reported that certain Cu-complexes act as anti-
complexes with serum proteins (Trynda-Lemiesz tumor (Gokhle et al. 2001), as anti-proliferative
et al. 2000; Piccioli et al. 2004; Kartz et al. 1994) (Collins et al. 2000) and pro-apototic (Daniel et al.
and also with LDH (Trigun et al. 2007). Using the 2004a, b; Cai et al. 2007) agents in selected tumor
similar parameter, results in Fig. 2A and B suggest cells. However, cellular targets of such complexes
significant binding of both these complexes with in vivo largely remain undefined. Enzymatic proteins
LDH. Nonetheless, binding constant data indicates are highly sensitive to ligand dependent allosteric
that Zn-bpy–LDH interaction is stronger than the Cu- modulations and therefore, considered to be the most
bpy–LDH. suitable targets for pharmacological agents (Groebe
The binding study data presented may be inter- 2006). An exogenous factor can modulate the enzyme
preted in terms of electrostatic interaction and/or H- activity in two ways. First, by altering the synthesis
bonding or a direct metal coordination of the complex of the enzyme at gene level and second, by direct
to certain surface residues of the protein. Based on interaction with the enzymatic proteins. Since, both
the absorption and spectrofluorometric data, the the complexes have shown their strong interaction
interaction of gold (III) with serum albumin has been with LDH in vitro, it was worth to first work out with
interpreted in similar way (Marcon et al. 2003). Cu the second possibility in animal system. In order to
and Zn metals have been found to bind with the assess specificity of both these complexes with
cellular proteins (Halliwell and Gutteridge 1990) and respect to the different isozymes of LDH, different
suggested to interact at His, Cys, Asp, and Glu side mice tissues, including those who express all the five
chains of most of the proteins studied in this context isozymes, were selected for the present study. Liver,
(Jernigan et al. 1994; Maynard and Covell 2001; skeletal muscle and spleen express M4-LDH,
Dudev and Lim 2002; Dudev et al. 2003). These whereas, kidney, brain and heart are known to
amino acid residues could be the putative sites on contain all the five LDH isozymes in different ratio.
LDH also for the interaction of Cu- and Zn-bpy The results in Figs. 3–5A, B, and C suggested that
complexes. Moreover, with a pharmacological view- Cu-bpy inhibits LDH in all the tissues studied,
point, it was important to define whether these however, Zn-bpy could inhibit this enzyme only in
complexes after binding to LDH modulate the liver, kidney, and heart. Nonetheless, wherever LDH
activity of this enzyme at tissue level. was inhibited, all the five isozymes were found to be
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8. 124 Biometals (2008) 21:117–126
sensitive (PAGE results: panel B and C) for both urakis et al. 2003; Niakan 2001; Pathak and Vinayak
these complexes, suggesting that Cu-bpy and Zn-bpy 2005) even in the presence of oxygen (Kim et al.
are able to inhibit all the five isozymes of LDH. 2005). The use of anti-metabolite 2-deoxy-D-glucose
It is hard to explain why Zn-bpy is able to inhibit to antagonize the high glucose dependency of tumors
LDH only in selected tissues. Nonetheless, it has been has been evaluated in vitro as a novel chemothera-
described that tissue specific factors present in the peutic approach against cancer (Aft et al. 2002). The
cellular milieu generally determine cell responses to findings presented here, in addition, provide a metal-
the endogenously developed conditions and the based strategy to inhibit LDH whose activation is
exogenous factors (Singh and Kanungo 1968; Trigun critically implicated in meeting the increased energy
and Singh 1989). This could also be the reason in need of the tumor cells.
case of non-responsiveness of Zn-bpy to LDH in It has been demonstrated that many cancer cells
certain tissues. The argument gets support from a accumulate high amount of copper (Kuo et al. 2002;
significant inhibition of purified LDH (out side the Chan et al. 1993; Geraki et al. 2002; Jayadeep et al.
tissue) by Zn-bpy (unpublished data) and also from 1997). While the precise role of copper in tumors is
the greater sensitivity of LDH for Zn-bpy than the not yet clear, it is suggested to be required for
Cu-bpy in some of the tissues like kidney and heart angiogenesis (Daniel et al. 2004a, b) and can also
(Figs. 3, 4). It has also been reported that DNA generate ROS in tumor cells (Theophanides and
cleavage potential of a Cu-bpy is not uniform and Anastassopoupou 2002). In the present context, we
depends on the concentration of porphyrin and DTT speculate that property of Cu to get accumulated in
(Laine et al. 2004). tumor cells would be helpful in delivering exoge-
In general, binding of a ligand to an enzyme is nously given Cu-complexes preferentially to the
known to induce subtle changes in the enzyme tumor cells than the normal cells. Based up on the
conformation resulting into alterations in the enzyme similar approach, an organic Cu-complex has been
activity (Proud 1984; Trigun and Singh 1989; Groebe predicted to act as an anti-tumor agent in human
2006). So far modulations of enzymatic proteins by cancer cells (Daniel et al. 2004a, b). Thus, capability
metal complexes are concerned, inhibition of cyto- of Cu-bpy and Zn-bpy complexes to inhibit all the
chrome c activity by a Ru(III) complex (Trynda- five isozymes of LDH assumes great significance
Lemiesz 2004) and that of LDH by Ru(II)-CNEB with a viewpoint of their therapeutic applications.
complex (Trigun et al. 2007) have been tested on this
parameter. The data presented here provide evidence Acknowledgment This work was financially supported in
part by Department of Biotechnology, New Delhi joint project
that Cu-bpy Zn-bpy complexes are also able to to LM and SKT (BT/PR5910/BRB/10/406/2005), Govt. of
inhibit LDH by their interaction at protein level. India, and CAS Programme, Department of Zoology, BHU.
Keeping in account of biocompatibility of both these
complexes, our findings provide a relevant biochem- References
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