1. Invest New Drugs (2009) 27:503–516
DOI 10.1007/s10637-008-9202-8
PRECLINICAL STUDIES
Regression of Dalton’s lymphoma in vivo via decline
in lactate dehydrogenase and induction of apoptosis
by a ruthenium(II)-complex containing 4-carboxy
N-ethylbenzamide as ligand
Raj K. Koiri & Surendra K. Trigun & Lallan Mishra &
Kiran Pandey & Deobrat Dixit & Santosh K. Dubey
Received: 23 September 2008 / Accepted: 12 November 2008 / Published online: 29 November 2008
# Springer Science + Business Media, LLC 2008
Summary A novel ruthenium(II)-complex containing 4- inducing mitochondrial dysfunction–apoptosis pathway
carboxy N-ethylbenzamide (Ru(II)-CNEB) was found to without producing any toxicity to the normal tissues.
interact with and inhibit M4-lactate dehydrogenase (M4-
LDH), a tumor growth supportive enzyme, at the tissue Keywords Dalton’s lymphoma . Ruthenium .
level. The present article describes modulation of M4-LDH Ru(II)-CNEB . Lactate dehydrogenase (LDH) . Apoptosis .
by this compound in a T-cell lymphoma (Dalton’s Lym- Anticancer agent
phoma: DL) vis a vis regression of the tumor in vivo. The
compound showed a dose dependent cytotoxicity to DL
cells in vitro. When a non toxic dose (10 mg/kg bw i.p.) of Introduction
Ru(II)-CNEB was administered to DL bearing mice, it also
produced a significant decline in DL cell viability in vivo. Amongst non-platinum anti-cancer metal compounds, Ru-
The DL cells from Ru(II)-CNEB treated DL mice showed a thenium complexes are of much current interest due to their
significant decline in the level of M4-LDH with a low toxicity, effective bio-distribution, reproducible bio-
concomitant release of this protein in the cell free ascitic activities [1, 2] and in some cases their selective anti-
fluid. A significant increase of nuclear DNA fragmentation metastatic properties [3]. The DNA, once considered as the
in DL cells from Ru(II)-CNEB treated DL mice also main target of metallo-drugs [1, 4, 5], is now evident to be
coincided with the release of mitochondrial cytochrome c unselective [6]. Therefore, instead of targeting compounds
in those DL cells. Importantly, neither blood based to interact with DNA, directing them to attenuate certain
biochemical markers of liver damage nor the normal biochemical steps which are over expressed in the tumors is
patterns of LDH isozymes in other tissues were affected an evolving concept [6–8]. Ru-complexes are found to be
due to the treatment of DL mice with the compound. These more versatile in this respect [3], as Ru metal centre can
results were also comparable with the effects of cisplatin interact with and organize different ligands which can
(an anticancer drug) observed simultaneously on DL mice. modulate cellular functions differentially [9].
The findings suggest that Ru(II)-CNEB is able to regress The glycolytic phenotype of tumor cells, popularly
Dalton’s lymphoma in vivo via declining M4-LDH and known as Warburg effect, is now evident to be a near
universal trait of all the growing tumors [10]. This ensures
adequate production of cellular energy through a non-
R. K. Koiri : S. K. Trigun (*) : K. Pandey : D. Dixit
mitochondrial route even when O2 supply is not a limiting
Biochemistry & Molecular Biology Laboratory, Centre
of Advanced Studies in Zoology, Banaras Hindu University, factor and thus, protects tumor cells from oxidative stress
Varanasi 221005, India [11–13]. Therefore, to restrict tumor growth, inhibition of
e-mail: sktrigun@sify.com tumor glycolysis could be a logical target for the novel
L. Mishra : S. K. Dubey
anticancer compounds [11, 12, 14, 15].
Department of Chemistry, Banaras Hindu University, Inhibition of glycolysis by 2-deoxy-D-glucose [16] and
Varanasi 221005, India inactivation of hexokinase II (HKII), the first committed

2. 504 Invest New Drugs (2009) 27:503–516
enzyme of glycolytic pathway, by 3-bromopyruvate [17], tissue level [24]. The present article investigates whether
have been reported to kill certain tumors in hypoxic this compound is (a) able to decline the level of active M4-
condition. Importantly, in colon cancer cells with mito- LDH and to induce apoptosis in the tumor cells in vivo and
chondrial defects, it has been demonstrated that inactivation (b) effective in restricting tumor growth without being toxic
of tumor glycolysis activates glycolysis–apoptosis pathway to the normal tissues.
with a concomitant increase in tumor cell death [18].
Nonetheless, effectiveness of 2-deoxy-D-glucose is signifi-
cantly masked by the presence of normal glucose in Materials and methods
circulation [17]. In addition, if inactivation of HKII is not
tumor specific, it is likely to affect normal cell energy Chemicals
metabolism also by restricting substrate supply to mito-
chondrial oxidative phosphorylation. Therefore, it is im- Ru(II)-CNEB was synthesized and characterized as de-
portant to identify certain enzymatic proteins which are scribed in an earlier report [24]. The ligand 4-carboxy N-
over expressed selectively in the cancer cells, and thus can ethylbenzamide (CNEB) was characterized by elemental
be targeted by the novel compounds. analysis and single crystal X-ray crystallography. On the
In growing tumors, a hypoxia induced factor (HIF1α) is basis of elemental analysis and mass spectroscopy data
known to activate the genes of glycolytic enzymes under a [24], empirical composition of the final Ru(II)-CNEB com-
variety of oncogenic stimulations [12, 19]. HIF1α also plex was assigned as [Ru(CNEB-H)2(bpy)2]2PF6·0.5NH4PF6.
restricts entry of pyruvate to TCA cycle by inhibiting The structure of the complex has been presented in Fig. 1,
pyruvate dehydrogenase (PDH) complex. As a result, which suggests coordination of ligand with the metal through
mitochondrial function gets attenuated and pyruvate is its amide N due to the presence of electron releasing ethyl
channeled to produce lactate by LDH [12]. Thus, enhanced group attached to it. The crystallographic data has been
production of lactate becomes a survival factor for deposited to the Cambridge Crystallographic Data Center,
malignant tumors [20]. Contrary to this, in normal cells, CCDC No. 618507.
due to the less activity of HIF1α, pyruvate pool is β-NADH (β-nicotinamide adeninedinucleotide, reduced),
channeled to mitochondria for oxidative phosphorylation NAD, Na-pyruvate, trypan blue, agarose, 4-carboxybenzalde-
without implicating LDH. Therefore, selective inactivation hyde, RuCl3·3H2O, ammonium hexafluorophosphate and
of LDH is less likely to hamper energy metabolism in anti β-actin were purchased from Sigma-Aldrich Co., USA.
normal cells, however, it can inhibit energy yielding HRP-conjugated anti rabbit IgG and cisplatin [cis-diammi-
pathway of tumor cells. Thus, LDH could be a target of nedichloroplatinum (II)] were obtained from Genei, and
therapeutic intervention for restricting tumor growth. Cipla respectively. Anti cytochrome c, hydroxylamine
Lactate dehydrogenase (LDH; EC: 1.1.1.27) is a tetra- hydrochloride and ECL super signal western pico kit were
meric protein consisting of two types of subunits, the M/A purchased from Santa Cruz, Fluka and Pierce respectively.
type (preferentially catalyzes conversion of pyruvate to cis-Ru(bpy)2Cl2·2H2O was prepared by a reported procedure
lactate) and the H/B type (pre-dominantly expressed in the [25]. SGOT (serum glutamate oxaloacetate transaminase)
aerobic tissues and catalyzes conversion of lactate to and SGPT (serum glutamate pyruvate transaminase) assay
pyruvate). Combination of these two sub-units in different kits were purchased from Span Diagnostics Ltd, India. Nitro
ratio gives rise five LDH isozymes (M4, M3H, M2H2, blue tetrazolium (NBT), phenazine methosulfate (PMS), Li-
MH3 and H4), which are expressed in a tissue specific
manner in most of the animals. M4-LDH (LDH-5, LDH-A)
has been found to be over expressed in tumor cells to
2+
O
support increased production of lactate from pyruvate [21].
N N
The tumorogenic potential of M4-LDH deficient cells was
found to be diminished drastically, however, it was found to N N
Ru R=
be recovered by complementation with the human ortholog
of M4-LDH [11]. Also, there are some reports on decrease
in the growth of certain tumor cells in vitro due to
inhibition of LDH activity by certain chemotherapeutic C2H5 NH HN C2H5 HO O
agents [22, 23]. R R
Recently, we have synthesized and characterized a Ru
[Ru(CNEB-H)2(bpy)2 ]2PF6.0.5NH4PF6
(II)-CNEB complex which was found to be highly
biocompatible to mice in vivo and could interact with and Fig. 1 Structure of Ru(II)-CNEB: [Ru(CNEB-H)2(bpy)2] 2PF6·0.5
inhibit M4-LDH non-competitively both in vitro and at the NH4PF6

3. Invest New Drugs (2009) 27:503–516 505
lactate and other general chemicals were purchased from both the treated groups were analyzed by Kaplan–Meier
SISCO Research Laboratory, Mumbai, India. curve.
Induction of Dalton’s lymphoma (DL) in mice Preparation of samples for biochemical studies
Inbred AKR strain mice of 16–18 weeks age weighing 24– For biochemical studies, three to four mice from each group
26 g were used for the experiments. Mice were maintained were sacrificed, volume of the collected tumor ascite from
under standard laboratory conditions, as per the guidelines each group was measured and DL cells were pelleted by
and approval from the institutional animal ethical commit- centrifugation of ascites at 2,000×g for 10 min at 4°C.
tee, with free access to commercially available food pellets Other tissues like liver, kidney and brain were dissected
and water. As described earlier [26, 27], Dalton lymphoma out, washed in ice-cold physiological saline, and stored at
(DL) was induced by intraperitoneal (ip) serial trans- −70°C.
plantations of 1×107 viable tumor cells (assayed by trypan For blood based studies, blood samples from three to
blue method) per mice with 100% success each time. four mice in each group were collected into sterilized tubes
Development of DL was confirmed by abnormal belly containing heparin (15–20 IU/ml). For collecting serum, the
swelling and increased body weight which were visible on blood was collected in unheparinized tubes, allowed to clot
10–12 days of implantation. The untreated DL mice at room temperature (22°C) and centrifuged at 1,000×g for
survived for 18±2 days. 15 min.
Treatment schedule and study on survival time Short term cytotoxicity assay
Ru(II)-CNEB was first dissolved in the minimum volume Through pilot experiments it was determined that 1×106–
of 0.01% methanol followed by its further dilution in 107 DL cells collected from ascitic fluids could be
Kreb’s ringer buffer (KRB) composed of 9 mM D-glucose, maintained in sterilized KRB medium at 37°C up to
0.23 mM MgCl2, 4.5 mM KCl, 20 mM NaCl, 0.7 mM >24 h without any loss of cell viability. Accordingly, for
Na2HPO4, 1.5 mM NaH2PO4 and 15 mM NaHCO3 in vitro cytotoxicity assay of Ru(II)-CNEB, 1×106 viable
(pH 7.3). Different concentrations of the compound were DL cells were suspended in 0.25 ml KRB and were
also prepared in KRB for ip injections. Through pilot incubated with the increasing concentrations of Ru(II)-
experiments, a dose of 10 mg/kg bw of Ru(II)-CNEB, CNEB (0.005–10 mg/ml) at 37°C for 30 min and 20 h
given intraperitoneally, was found to be a sub-lethal dose to duration separately. After respective time intervals, the
normal mice and could increase the life span of DL bearing number of viable DL cells was determined in each set by
mice significantly. Therefore, this dose of Ru(II)-CNEB trypan blue exclusion method. A 10 μl sample of cell
was selected for the present study. The DL mice were suspension was mixed with an equal volume of 0.4%
randomly divided into three groups with nine to ten mice in trypan blue and the cells were counted using hemocytom-
each. The first group DL mice were treated with Ru(II)- eter. Similar method was adopted to determine the number
CNEB complex (10 mg/kg bw/day, ip), second group with of viable DL cells pelleted from the ascites of different
cisplatin (2 mg/kg bw/day, ip) and the third group, experimental groups. The DL cell viability was recorded as
designated as DL control, were similarly injected with % DL cell viability=(Total no of cells − trypan blue-stained
equal volume of KRB. As DL becomes visible on day 10– cells)/total no of cells)×100.
11 and DL bearing mice survived up to 18–20 days post
transplantation, the treatments of DL mice with the DNA fragmentation assay
compounds were started from day 11 of tumor transplant
and continued up to day 17th. The normal control group Quantitative determination of fragmented DNA was carried
mice were also treated simultaneously with KRB. To study out as described earlier [28] with slight modifications.
biochemical/molecular parameters, three to four mice from Briefly, DL cells were lysed in 0.5 ml of Tris–EDTA buffer
each group were sacrificed on day 18th. The remaining (pH 7.4) containing 0.2% (v/v) triton X-100 and centrifuged
mice in each group were allowed to be maintained on at 13,000×g at 4°C for 10 min. The pellets containing total
normal diets to study their survival time after the treatment. intact DNA (designated P) and the supernatants containing
In order to assess the effects of compounds on general smaller fragments of DNA (designated S) were treated
appearance of DL mice, body weight of mice was recorded separately with 0.5 ml of 25% trichloroacetic acid (TCA).
at an interval of 3 days starting from the day of Both the sets were left overnight at 4°C and DNA
transplantation up to 21 days. The mortality was noted in precipitated were collected by centrifugation. Each sample
each group and increases in the survival time of mice of was treated with 80 μl of 5% TCA followed by heat

4. 506 Invest New Drugs (2009) 27:503–516
treatment at 90°C for 15 min. Freshly prepared 1 ml quantified using gel densitometry software AlphaImager
diphenylamine (DPA) reagent was added in each sample, 2200.
tubes were allowed to stand overnight at room temperature
and OD was recorded at 600 nm. Percent DNA fragmen- Analysis of LDH isozymes by non-denaturing
tation was calculated as: polyacrylamide gel electrophoresis (PAGE)
% DNA fragmentation ¼ ½S=ðS þ PÞŠ Â 100
Non-denaturing PAGE is a preferred method to analyze
expression pattern of LDH isozymes. It employs substrate
Agarose gel electrophoresis of fragmented DNA specificity based detection of all the isozymes of LDH
distinctly in the same gel, and thus, it is considered highly
For electrophoretic analysis of fragmented DNA, the total relevant for correlating a change in the level of a specific
nuclear DNA was isolated from the DL cells according to LDH isozyme with that of a metabolic alteration at cellular
the method of Kuo et al. [29]. Briefly, 5×106 cells were level. This method has been successfully applied to
lysed in 1 ml of lysis buffer [20 mM Tris–Cl (pH 7.5), understand the implications of critical enzymes like SOD
0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X- and LDH in cancer and neuropathology [31–33].
100 and 25 mM Na2 pyrophosphate] at 37°C for 1 h. To Tissue extracts were prepared as described earlier from
precipitate out proteins, 0.4 ml of saturated NaCl was added this lab [30]. LDH isozymes in DL cell lysates and other
in each set of cell lysate, tubes were left on ice for 5 min tissues were analyzed on non-denaturing 10% PAGE
and centrifuged at 3,000×g for 30 min. To separate DNA following the method described earlier [32]. Briefly, the
from the intact chromatin, RNase (20 μg/ml) was added to extracts containing 60 μg protein were loaded in each lane
the supernatants collected and allowed to stand at 37°C for and electrophoresis was performed at 4°C. Gels were
15 min. DNA was then precipitated by adding two times subjected to substrate specificity based detection of LDH
chilled ethanol (v/v). Samples were frozen at −70°C bands [32] followed by scanning and quantification of the
overnight. The DNA precipitated was collected by centri- bands as described earlier. The different isozymes of LDH
fugation and dissolved in TAE buffer (40 mM Tris-acetate + in the gel were characterized by comparing their relative
1 mM EDTA). migration (from cathodic to anodic) with those of the tissue
The DNA samples were prepared in a loading solution specific standard LDH bands [2, 24, 32].
(0.25% bromophenol blue, 0.25% xylene cyanol FF and
30% glycerol) in the ratio of 1:5. The samples containing Analysis of superoxide dismutase (SOD: Mn-SOD)
10 μg DNA were loaded in each well of 1% agarose gel by non-denaturing PAGE
containing 0.5 μg/ml ethidium bromide. The electrophore-
sis was carried out in TAE buffer for 2–3 h. The DNA As described in case of analysis of LDH isozymes, 12%
bands in gel were observed under UV transilluminator and non-denaturing PAGE was performed to determine the level
photographed. of active fraction of SOD2 in different DL cell extracts. The
active SOD bands were developed following the method
Western blotting for cytochrome c release reported recently from our lab [33]. The gel was scanned
from the mitochondria and SOD bands were quantified as described in the
previous text. The Mn-SOD (SOD2) in the gel was
DL cells were lysed in the lysis buffer containing 1 mM identified by its greater cathodic migration than that of
PMSF. The cell lysate was incubated on ice for 15 min, SOD1 (Cu/Zn-SOD) which migrates faster towards anode
vortexed and centrifuged at 700×g for 10 min. To obtain [33].
mitochondria free cytosolic fraction, supernatant obtained
was centrifuged at 10,000×g for 30 min. Following the Other biochemical measurements
method described earlier [30], the samples containing
60 μg protein, prepared in Laemmli buffer, were subjected Protein concentrations in tissue extracts and in the blood/
to 15% SDS-PAGE (sodium dodecyl sulphate-polyaryla- serum were measured following the method of Bradford
mide gel electrophoresis). Proteins were transferred to [34]. The activity of LDH in cell extracts was measured as
nitrocellulose membrane followed by detection of cyto- described in an earlier report [24] and oxidation of 1 μmol
chrome c against a polyclonal anti-cytochrome c (1:1,000). of NADH per min at 25°C was defined as 1 U of the
The ECL super signal west pico kit was used to develop the enzyme and values were presented as unit per milligram
bands on X-ray films. Using monoclonal anti-β-actin protein.
peroxidase antibody (1:10,000), level of β-actin was The levels of SGOT and SGPT were determined
probed as a loading control. The bands were analyzed and following the manual of the kits used. Non-denaturing

5. Invest New Drugs (2009) 27:503–516 507
120
PAGE was performed to analyze LDH isozymes in the 30 min
Viabilty of DL cells
serum also. 100 ** ** 20 h
(% of control)
80
***
Statistical analysis 60
40 *** ***
Kaplan–Meier survival curves for the treated and untreated 20
group of DL mice were compared by using the log-rank ***
0
test. Other experimental data were expressed as mean±SD 0.0 0.05 0.5 5.0 10.0
and wherever required, Student’s t test was applied for Ru(II)-CNEB (mg/ml)
determining the level of significance. p<0.05 was consid-
Fig. 2 Effect of increasing concentrations of Ru(II)-CNEB on DL
ered significant. cells in vitro. DL cells (1×106) for each set were maintained in KRB
medium and incubated with the indicated concentrations of the
compound for different time intervals. Viability of DL cells after
Results 30 min and 20 h incubation was determined by trypan blue exclusion
method. The data represent mean±SD from three to four experimental
repeats. **p<0.01; ***p<0.001 (control versus respective experimen-
To evaluate anticancer potential of Ru(II)-CNEB in vivo, tal sets)
we selected a transplantable T cell lymphoma (Dalton’s
lymphoma: DL) as a tumor model, because, DL can be
induced in rodents within a short period of time with >95% decrease the DL cell viability in mice, ascitic fluid collected
reproducibility and with clear visible symptoms which can from untreated and treated DL bearing mice were analyzed.
be used for monitoring the progression as well as regression As compared to the untreated DL mice, there was a
of the DL throughout the period of experimentation. In significant decrease (p<0.01) in the volume of ascite
addition, homogeneous DL cells can be precipitated from collected from the DL mice treated with the both, Ru(II)-
the ascitic fluid for studying the biochemical and molecular CNEB and cisplatin (Fig. 3a). Moreover, there was a drastic
changes associated with development/regression of the decline (~80%) in the number of viable DL cells in the
tumor [26, 35]. Importantly, some mechanistic aspects of samples of ascites collected from both, the Ru(II)-CNEB
anticancer activity of cisplatin have been worked out using and cisplatin treated DL mice (Fig. 3b).
this model [26, 36, 37]. We could also induce DL in mice Release of LDH in cell free medium indicates cell
with 100% success each time and used DL bearing mice for damage in vitro as well as in vivo. In comparison to the
in vivo evaluation of Ru(II)-CNEB as an anticancer agent. level of M4-LDH in the cell free ascitic fluid from
untreated DL mice, there was a significant increase in the
Cytotoxicity of Ru(II)-CNEB on DL cells in vitro level of M4-LDH in those from Ru(II)-CNEB and cisplatin
treated DL mice (Fig. 3c). These findings clearly suggest
Cytotoxicity assay was done to ascertain whether Ru(II)- that both the compounds tested are able to restrict DL
CNEB is able to kill DL cells in vitro. For this, DL cells development and also to induce death of DL cells in vivo.
were maintained in a physiological buffer medium and their
viability was assayed after incubating them with increasing Effect of Ru(II)-CNEB on the level of M4-LDH in DL cells
concentrations of the compound for 30 min and 20 h. As
shown in Fig. 2, in 30 min set, though DL cell viability was Increased LDH activity is associated with tumor develop-
not affected up to 0.5 mg/ml Ru(II)-CNEB, a significant ment. To ascertain whether Ru(II)-CNEB and cisplatin are
decline in the number of viable DL cells was observed at able to decrease the activity of this enzyme in DL cells,
higher concentrations of the compound (at 5 mg/ml; p<0.01 LDH activity was compared in DL cells from untreated and
and at 10 mg/ml; p<0.001). Moreover, when incubation treated DL mice. According to Fig. 4a, as compared to the
period was increased to 20 h, a linear decline in the number LDH activity observed in DL cells from the untreated
of viable DL cells was observed starting from 0.05 mg/ml group, there was a significant decrease (p<0.01) in the
(p<0.01) to 10 mg/ml (p<0.001) of the compound. Thus, it activity of this enzyme in DL cells from Ru(II)-CNEB
was evident that Ru(II)-CNEB is able to kill DL cells in vitro treated DL mice. However, activity of LDH was found to
in a dose and incubation time dependent manner. be increased significantly in DL cells from the cisplatin
treated DL mice.
Effect of Ru(II)-CNEB on regression of DL cells in vivo In order to ascertain whether Ru(II)-CNEB affects the
level of active M4-LDH and/or the level of other LDH
In order to confirm whether administered dose of Ru(II)- isozymes in DL cells, non-denaturing PAGE analysis was
CNEB is able to restrict the development of DL and/or to preferred over the immunostaining methods. Though this

6. 508 Invest New Drugs (2009) 27:503–516
a
Vol. of ascitic fluid (ml)
16
14
12 c DL DL+Rc DL+Cpt
10
8
6 ** **
4 M4-LDH
2
0
DL DL+Rc DL+Cpt
b
Densitometry of LDH bands
120 350
Viability of DL cells
***
(% of DL control)
(% of DL control)
100 300
80 250
200
60 ***
150
40 ***
100
20 *** 50
0 0
DL DL+Rc DL+Cpt DL DL+RC DL+CPT
Fig. 3 Effects of Ru(II)-CNEB and cisplatin treatment on ascitic fluid substrate specific LDH bands were developed in the gel. The gel
volume (a), viability of DL cells (b) and on the release of M4-LDH in photograph is a representative of the three PAGE repeats. In lower
the cell free ascitic fluid (c) of DL bearing mice. In case of (a) and (b), panel of (c), relative densitometric values of LDH bands from
the data represents mean±SD where n=4. In (c), pooled cell free experimental group, as percent of the control DL lane, have been
ascitic fluid from three to four mice containing 60 μg protein was presented as mean±SD from three PAGE repeats. *p<0.05; **p<
loaded in each lane, 10% non-denaturing PAGE was performed and 0.01; ***p<0.001 (DL control versus treated DL groups)
method is relatively less sensitive than that of immunode- group mice showed the smears of fragmented DNA. Whereas,
tection, however, it is more relevant for interpreting the DNA isolated from DL cells of untreated mice showed a
metabolic changes associated with the enzymatic altera- single DNA band at higher molecular weight (MW) range.
tions. Because, this method utilizes substrate specificity These results clearly suggest that both the compounds tested
based detection of only active fraction of the enzyme were able to induce apoptosis in DL cells in vivo.
excluding inactive/denatured proteins, which otherwise can Oxidative stress and mitochondrial dysfunction are
not be excluded by immunostaining method. known to initiate final steps of apoptosis. Superoxide
Figure 4b shows that DL cells from the untreated DL dismutase (SOD) is the first committed enzyme that
mice expressed high amount of only M4-LDH which was neutralizes oxygen free radical (O2)− based oxidative stress
found to be decreased significantly (p<0.01) in the DL cells in the cells. Therefore, a decrease in the level of Mn-SOD
from Ru(II)-CNEB treated DL mice. However, there was (SOD2: mitochondrial isoform) may be considered as an
no change in the level of M4-LDH in the DL cells from indicator of oxidative stress in mitochondria. As shown in
cisplatin treated DL mice. This suggest that among the two Fig. 6a, there was a significant increase (p<0.05) in the
compounds tested, only Ru(II)-CNEB caused a decline in level of active SOD2 in DL cells from Ru(II)-CNEB treated
the level of M4-LDH in DL cells in vivo. DL mice than that from the untreated DL group. However,
the DL cells from cisplatin treated DL mice showed a
Apoptosis of DL cells in vivo by Ru(II)-CNEB significant decrease (p<0.01) in the level of active SOD2.
Release of mitochondrial cytochrome c in the cytosol
The DNA fragmentation assay is a reliable tool to ascertain indicates for induction of mitochondrial dysfunction–
apoptotic cell death. In the present context, using a standard apoptotic pathway in the cells. Therefore, the level of
method, the percentage of fragmented DNA in DL cell cytochrome c in the cytosol of DL cells from untreated and
extracts was measured followed by its analysis on agarose treated DL groups was compared. As compared to the DL
gel electrophoresis. There was a significant increase (p< cells from untreated DL group, cytosolic fractions of DL
0.01) in the level of fragmented DNA in DL cells from cells from Ru(II)-CNEB and cisplatin treated DL mice
both, the Ru(II)-CNEB and cisplatin treated DL mice showed a significant increase (p<0.05) in the level of
(Fig. 5a). This was further confirmed by the results of cytochrome c (Fig. 6b). The results suggest induction of
agarose gel electrophoresis (Fig. 5b) wherein, DNA mitochondrial dysfunction–apoptotic pathway in the DL
samples from the DL cells collected from both the treated cells in vivo by both the compounds tested.

7. Invest New Drugs (2009) 27:503–516 509
a 6 ** mice treated with Ru(II)-CNEB, which was comparable
5 with the effect of cisplatin treatment also. Thus, it was
(U/mg protein)
LDH Activity
4 evident that like cisplatin, Ru(II)-CNEB is also able to
3 cause an increase in the survival period of DL bearing mice
** with remarkable improvements in DL associated symptoms.
2
1
0 Effect of Ru(II)-CNEB treatment on normal tissues of DL
DL DL+Rc DL+Cpt mice
b DL DL+Rc DL+Cpt
Liver metabolizes most of the drugs and kidney filters out
M4-LDH all the unwanted exogenous substances. Therefore, these
two organs are likely to be affected up to a greater extent by
Densitometry of LDH bands
140 the drug treatment. Also, it is important to ensure that an
anticancer compound does not cross the blood brain barrier
(% of DL control)
120
and central nervous system remains protected during the
100
** treatment. Therefore, these three tissues were selected to
80
assess toxicity of Ru(II)-CNEB on the normal tissue.
60
40
20 a
0
160
140
* *
Fragmented DNA
(% of DL control)
DL DL+Rc DL+Cpt 120
100
Fig. 4 Effects of Ru(II)-CNEB and cisplatin treatment on the activity
(a) and the level of M4-LDH (b) in the DL cells of DL bearing mice. 80
The values in (a) represent mean±SD where n=4 and each experiment 60
done in duplicate. In case of (b), pooled DL cell extracts from four 40
mice containing 60 μg protein in each lane was electrophoresed on 20
10% non-denaturing PAGE followed by substrate specific develop- 0
ment of LDH bands. The gel photograph is a representative of the DL DL+Rc DL+Cpt
three PAGE repeats. In lower panel of (b), relative densitometric
values of LDH bands from experimental group, as percent of the
control DL lane, have been presented as mean±SD from three PAGE b
repeats. **p<0.01 (DL control versus treated DL groups)
Improvements in the survival parameters of Ru(II)-CNEB
treated DL mice
Development of Dalton’s lymphoma in mice is character-
ized by the abdominal swelling and increased body weight.
Therefore, these parameters were measured to assess
whether Ru(II)-CNEB was able to bring recovery in the
DL associated symptoms in mice. As compared to the
control mice, DL implanted mice showed a significant
increase in their body weight (p<0.01) from day 1 to 14th,
which was found to be static thereafter (Fig. 7). However,
after the treatment with both, Ru(II)-CNEB and cisplatin DL DL+Rc DL+Cpt
from day 11 to 17, a significant decrease (p<0.01) in the
Fig. 5 Effects of Ru(II)-CNEB and cisplatin treatment on DNA
body weight of DL mice was observed. In addition, as
fragmentation in DL cells of DL bearing mice. The values in (a)
compared to the mean survival time of untreated DL mice represent mean±SD of three experimental repeats from the pooled DL
(18 days), the DL mice treated with Ru(II)-CNEB and cell extracts collected from four DL mice. In case of (b), 10 μg DNA
cisplatin could survive up to 24 and 26 days respectively. extracted from the pooled DL cells from three to four DL mice was
loaded in each lane and subjected to 1% agarose gel electrophoresis
Comparison of the survival time data on Kaplan–Meier
followed by detection of ethidium bromide stained DNA bands under
survival curves (Fig. 8) using log-rank statistics suggests a UV transilluminator. The photograph is a representative of three
significant increase (p<0.001) in the survival time of DL repeats. *p<0.05 (untreated versus treated groups)

8. 510 Invest New Drugs (2009) 27:503–516
a b DL DL+Rc DL+Cpt
DL DL+Rc DL+Cpt Cyto C
SOD 2
β Actin
Densitometry of SOD bands
140 *
Densitometric value
120 2.0
(% of DL control)
(Cyt C/β actin)
100 1.5 *
80 ** *
60 1.0
40 0.5
20
0 0.0
DL DL+Rc DL+Cpt DL DL+Rc DL+Cpt
Fig. 6 Effects of Ru(II)-CNEB and cisplatin treatment on the level of wherein, pooled DL cell extracts from three to four DL mice
active SOD2 (a) and on cytochrome c release in the cytosol of DL containing 60 μg protein in each lane was subjected to 15% SDS-
cells (b) of DL bearing mice. In (a), pooled DL cell extracts from four PAGE followed by western transfer on nitrocellulose membrane and
mice containing 60 μg protein in each lane were electrophoresed on detection of cytochrome c bands using a polyclonal anti cytochrome c.
12% non-denaturing PAGE followed by substrate specific develop- The level of β actin was probed as the loading control. The
ment of SOD2 bands in the gel. The gel photograph is a representative photograph is a representative of the three western blot repeats. In
of the three PAGE repeats. In lower panel of (a), relative lower panel of (b), normalized values of cytochrome c/β actin have
densitometric values of SOD2 bands from the treated group, as been presented as mean ± SD from three western blot repeats.
percent of the control lane (untreated DL group), have been presented *p<0.05; **p<0.01; (untreated versus treated groups)
as mean±SD from three PAGE repeats. b Immunoblotting results
Increased level of serum LDH indicates for vital tissue Ru(II)-CNEB as well as cisplatin treated DL group mice
damage and the levels of SGOT and SGPT are used as (Fig. 10a,b). Though there was a significant increase in the
blood based markers of liver damage. The serum of control level of M4-LDH (p<0.01) in the liver of control DL mice
as well as all the DL group mice showed the presence of than that of the normal mice, no change in the level of this
mainly M4-LDH with a minor fraction of M3H isozyme enzyme was observed in the liver of DL mice treated with
(Fig. 9). As compared to their levels in the serum of normal Ru(II)-CNEB (Fig. 10c). However, as compared to the
mice, both these isozymes were significantly increased (p< untreated DL group, a significant decline in the level of
0.05) in the serum of untreated DL mice. However, both of M4-LDH could be seen in the liver of cisplatin treated DL
them remained unchanged, as compared to the normal mice.
mice, in the serum of Ru(II)-CNEB and cisplatin treated DL
group mice. Also, the levels of SGOT and SGPT were 100
found to be unaltered among the normal, untreated DL and Logrank p=0.0007
80
Survival rate (%)
N DL DL+Rc DL+Cpt
60
40
Start of treatment
Body weight (g)
## ## DL
40
35 DL+Rc
#
* 20
** DL+Cpt
30
0
25 0 10 20 30 40
1 7 14 21 Days after DL transplantation
Period (days)
Fig. 8 Kaplan–Meier survival curve for untreated DL mice and the
Fig. 7 Effects of Ru(II)-CNEB and cisplatin treatment on the body DL mice treated with Ru(II)-CNEB (DL + Rc) and cisplatin (DL +
weight of DL mice. The data represents mean±SD where n=5–6. # p< Cpt). The log-rank analysis was performed to examine the level of
0.05; ## p<0.01 (normal control versus untreated DL group). *p<0.05; significance and a p value of <0.001 was obtained in case of both the
**p<0.01 (DL control versus treated DL groups) treated group of DL mice versus untreated DL mice

9. Invest New Drugs (2009) 27:503–516 511
NC DL DL+Rc DL+Cpt cisplatin treated DL group mice (Fig. 11a). Unchanged
patterns of all the five LDH isozymes were also observed in
the brain of control and all the DL (untreated and treated)
LDH
group mice (Fig. 11b). These results clearly suggest that the
M4
administered dose of Ru(II)-CNEB was nontoxic to the
M3H vital tissues of the DL bearing mice.
Densitometry of LDH bands
140
Discussion
120
(% of DL control)
100
# Low toxicity and efficient bio-distribution of Ru-complexes
80
are of great advantage over other metal complexes for
60
evaluating their anticancer potential in vivo [2, 3]. This was
40
found to be true in case of Ru(II)-CNEB also. When a
20
nontoxic dose of the compound, determined for normal
0
NC DL DL+Rc DL+Cpt
mice, was administered to DL bearing mice, it resulted in a
significant decrease in the number of viable DL cell in vivo
Fig. 9 Effects of Ru(II)-CNEB and cisplatin treatment on the release (Fig. 3b) without producing any toxic effect on the other
of LDH in the serum of DL mice. Pooled serum from three to four normal tissues (Figs. 9, 10, and 11).
mice containing 60 μg protein in each lane was electrophoresed on
10% non-denaturing PAGE followed by substrate specific develop-
The in vitro studies provide primary level information on
ment of LDH bands. The gel photograph is a representative of the cytotoxic potentials of a novel compound. We could also
three PAGE repeats. In lower panel, relative densitometric values of observe a dose and time dependent decrease in the number
LDH bands from experimental group, as percent of the control lane, of viable DL cells by Ru(II)-CNEB in vitro (Fig. 2).
have been presented as mean±SD from three PAGE repeat experi-
ments. #p<0.05 (normal control versus untreated DL group)
However, a more pronounced decrease observed in the
viability of DL cells from Ru(II)-CNEB treated DL mice
(Fig. 3b) clearly indicated a greater anticancer activity of
Results in Fig. 11a and b re-confirm that mice kidney this compound in vivo than ex vivo. Some other Ru-
and brain express all the five LDH isozymes. However, as complexes have also been shown to be less toxic in vitro
compared to the LDH pattern observed in the kidney of but could cause potent anti tumor activity in vivo [38].
normal mice, there was a significant decrease (p<0.05) in It has been suggested that different Ru-complexes show
the level of all the five isozymes in that of untreated DL their anticancer activities via distinctly different mecha-
mice, but with no change in case of Ru(II)-CNEB and nisms such as by interacting with DNA and some serum
a c
200
SGOT (IU/L)
150
NC DL DL+Rc DL+Cpt
100
M4-LDH
50
0
NC DL DL+Rc DL+Cpt
Densitometry of LDH bands
120
100 ## **
(% of DL control)
b 80
8
SGPT (IU/L)
6 60
4 40
2 20
0 0
NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+Cpt
Fig. 10 Effects of Ru(II)-CNEB and cisplatin treatment on the levels ment of LDH bands. The gel photograph is a representative of the
of SGOT (a), SGPT (b) and M4-LDH in the liver (c) of DL bearing three PAGE repeats. In lower panel of (c), relative densitometric
mice. The values in (a) and (b) represent mean±SD where n=4 and values of LDH bands from experimental group, as percent of the
each assay done in triplicate. In (c), pooled liver extracts from four normal control lane, have been presented as mean±SD from three
mice containing 60 μg protein in each lane was electrophoresed on PAGE repeats. **p<0.01 (untreated DL versus treated DL groups);
10% non-denaturing PAGE followed by substrate specific develop- ##p<0.01 (normal control versus untreated DL group)

10. 512 Invest New Drugs (2009) 27:503–516
a b
NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+Cpt
LDH
LDH
M4
M4
M3H
M3H
M2H2 M2H2
MH3 MH3
H4 H4
Densitometry of LDH bands
Densitometry of LDH bands
200 120
100
(% of DL control)
#
(% of DL control)
150
80
100 60
40
50 20
0 0
NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+Cpt
Fig. 11 Effects of Ru(II)-CNEB and cisplatin treatment on the level PAGE repeats for each tissue. In lower panel of (a) and (b), relative
of LDH isozymes in kidney (a) and brain (b) of DL mice. In upper densitometric values of LDH bands from experimental group, as
panels of (a) and (b), the pooled tissue extracts from four mice percent of the control DL lane, have been presented as mean±SD
containing 60 μg protein in each lane was electrophoresed on 10% from three PAGE repeats. ##p<0.01 (normal control versus untreated
non-denaturing PAGE followed by substrate specific development of DL group)
LDH bands. The gel photographs are the representative of the three
proteins and also by inhibiting certain enzymes like future interest to determine the actual levels of this protein
cytochrome c, protein kinase C, topoisomerase II etc [3]. in DL cells from treated and untreated mice, in the present
Being highly unselective, DNA is considered an unsuitable context, a significant decline in the level of active M4-LDH
target for anticancer agents [7]. Alternatively, selecting a in DL cells of Ru(II)-CNEB treated mice suggests decrease
protein as pharmacological target sounds better, however, it in energy metabolism of the cancerous cell due to the
is important to first ensure that inactivation of a cellular treatment with this compound. The tumor cells rely much
protein is cancer cell specific and does not hamper normal on the energy pathway lead by M4-LDH dependent
cell metabolism. In this respect, inhibiting glycolytic production of lactate from pyruvate [11, 20, 21]. Thus,
efficiency of tumor cells seems to be the most relevant inactivation of this isozyme can severely affect only tumor
target, as all tumor cells switch over to enhanced aerobic cell energy metabolism. Contrary to this, as normal cells
glycolysis [10] for their additional energy needs [11, 12]. utilize pyruvate for mitochondrial oxidative phosphoryla-
Also, the two key glycolytic enzymes, PKM2 (a fetal tion rather than to produce lactate by M4-LDH, decline of
isoform of pyruvate kinase) and M4-LDH, have been found this isozyme in normal tissues is less likely to affect their
to be over expressed selectively in most of the tumors and energy metabolism. This argument also justifies a greater
therefore, both of these enzymes are argued to be the potential decrease in the number of viable DL cells in vivo than in
targets for novel anticancer compounds [8, 11, 21, 24]. vitro due to the treatment with Ru(II)-CNEB (Figs. 2 and
Based upon our recent findings on inhibition of M4- 3b). Isolated tumor cells maintained in vitro are devoid of
LDH by Ru(II)-CNEB [24] and modulation of this enzyme true hypoxia and they can exploit aerobic pathway for
by other metal complexes [32], we selected M4-LDH as a energy production even if LDH activity is declined
target protein for evaluating anticancer activity of Ru(II)- significantly and thus, can survive better. Contrary to this,
CNEB. It has been reported that like most of the tumors, due to greater hypoxia faced by the tumor cells in vivo,
DL cells also over express M4-LDH [26, 27]. A highly they rely much on anaerobic glycolysis [12] and thus, as a
intense band of M4-LDH in the DL cell extracts from consequence of diminished M4-LDH activity, they can be
untreated DL mice (Fig. 4b; lane 1) also corroborate these deprived of adequate energy production resulting into poor
earlier findings and accordingly, a significant decline in the survival. In addition, it has been reported [20] that tumor
level of M4-LDH in DL cells from Ru(II)-CNEB treated stroma associated fibroblasts help in the survival of tumor
mice (Fig. 4b; lane 2) suggests that this compound was able cells via recycling of lactate produced in excess by the
to decline the active level of this enzyme in DL cells in tumor cells. However, the blockage of tumor LDH-5 (M4-
vivo. Though immunostaining of M4-LDH would be of LDH) suppresses this additional route of metabolic supple-

11. Invest New Drugs (2009) 27:503–516 513
mentation and thus, can render tumor cells susceptible to oxidative stress [47]. We have observed a direct relation-
death [20]. ship between the release of cytochrome c and increased
Tissue damage causes leakage of LDH in body fluids level of DNA fragmentation in the DL cells from Ru(II)-
[39, 40]. Thus, a significant increase in the level of M4- CNEB treated DL mice (Figs. 5 and 6b). A similar pattern
LDH in the cell free ascitic fluid from Ru(II)-CNEB treated of DNA fragmentation and increased level of cytochrome c
DL mice, than that from the untreated DL mice (Fig. 3c; release in the DL cells from cisplatin treated DL mice were
lane 1 vs lane 2), indicates for DL cell death caused by this also observed and thus, suggesting that both, Ru(II)-CNEB
compound in vivo. A more pronounced increase in the level and cisplatin, have been able to induce apoptosis in DL
of M4-LDH in cell free ascitic fluid from cisplatin treated cells in vivo via release of cytochrome c.
DL mice (Fig. 3c; lane 3) further strengthened this There could be more than one mechanism for inducing
argument, as cisplatin induced regression of DL cells has apoptosis by chemotherapeutic agents. Anticancer drug
been shown to accompany the release of LDH in ascitic causing induction of apoptosis via inhibition of glycolysis
fluid [26]. Moreover, since cisplatin did not alter the level in tumor cells is a relatively new concept [18, 48]. Though
of M4-LDH in DL cells, which was observed to be declined the link between inhibition of glycolysis and tumor cell
significantly by Ru(II)-CNEB, it may be speculated that the apoptosis is yet to be defined, it may be speculated that
mechanism of cell death caused by both the compounds are depletion of energy and growth promoting substrates due to
different from each other. decline in glycolytic efficiency could act as an inducer of
Development of Dalton’s lymphoma is characterized by apoptosis in the tumor cells. Tumor cells show aberrant
the increments in the body weight and volume of the ascitic NADH/NAD shuttle of mitochondria resulting into in-
fluid and thus, measurement of both these parameters are creased level of NADH in the cytosol [49]. This may alter
used to determine the development of DL and its regression redox state of the cells and can induce final apoptotic
in vivo [41, 42]. In comparison to the untreated DL mice, pathway in those cells [50]. Decline in the level of M4-
~50% decrease observed in the ascitic volume in case of Ru LDH, which utilizes NADH as substrate, may further
(II)-CNEB treated DL mice (Fig. 3a) suggest that this contribute for the accumulation of NADH in cytosol. This
compound was able to restrict DL development in mice. argument gets support from ~2 times increase in NADH/
The range of reduction observed in ascitic volume is NAD ratio observed in M4-LDH deficient tumor cells [11].
comparable with a ∼2 times reduction caused by the extract Thus, it may be argued that the resultant increase in NADH/
of a macrofungus [42] and ∼50% reduction in tumor weight NAD ratio, due to a significant decrease in the level of M4-
by the extract of Withania somnifera [43]. Reports are LDH, might be implicated as a biochemical mediator to
scanty on Ru-complex induced regression of lymphoma in induce apoptosis in DL cells in Ru(II)-CNEB treated DL
vivo. Therefore, ∼80% decline in the number of viable DL mice. Mitochondrial dysfunction in DL cells of treated
cells (Fig. 3b) in Ru(II)-CNEB treated DL mice is of great group mice has also been suggested by a significant
relevance. The reductions in ascitic volume and DL cell increase in the release of mitochondrial cytochrome c
viability by Ru(II)-CNEB treatment were also comparable (Fig. 6b).
with the data obtained with cisplatin treatment and thereby, Alternatively, DNA damage also induces apoptosis,
suggesting further for a potent anticancer activity of Ru(II)- however, such a possibility in this case was ruled out by
CNEB on DL in vivo. observing a poor DNA-Ru(II)-CNEB interaction in vitro
One of the major mechanisms in cancer therapy is to (unpublished data). Also, (O2)− based oxidative stress is
induce apoptosis in transformed cells by chemotherapeutic known to cause cytochrome c release and in turn induction
agents [44, 45]. Some Ru(II)-complexes derived organo- of apoptosis in the affected cells, however, under depleted
metallic compounds have been reported to mediate their antioxidant condition. SOD is the committed enzyme of
cytotoxicity on lymphoma cell lines in vitro via induction antioxidant pathway and Mn-SOD (SOD2) in particular
of apoptosis [46]. However, reports are scanty on the plays a critical role in protecting mitochondria from (O2)−
induction of apoptosis in tumor cells in vivo by Ru(II)- insult. We observed a significant increase in the level of
complexes. Increased fragmentation of DNA is an impor- SOD2 in the DL cells from Ru(II)-CNEB treated than those
tant parameter to suggest apoptotic death of a cell. Thus, a from the untreated DL mice (Fig. 6a). This suggests that
significant increase in the level of fragmented DNA in the antioxidant potential of DL cells was not depleted due to
DL cells from Ru(II)-CNEB treated DL mice (Fig. 5a, b) the treatment with Ru(II)-CNEB and hence, rules out
clearly suggests that Ru(II)-CNEB is able to induce possibility of a role of (O2)− based oxidative stress in Ru
apoptosis in DL cells in vivo. Release of cytochrome c (II)-CNEB induced apoptosis in the DL cell.
from mitochondria is an indicator of mitochondrial dys- Thus, the results presented here suggest that Ru(II)-
function and has been correlated with the induction of CNEB might be implicating the decline of M4-LDH and
apoptosis under a variety of metabolic derangements and mitochondrial dysfunction to induce apoptosis in DL cells

12. 514 Invest New Drugs (2009) 27:503–516
in vivo. The induction of glycolysis–apoptotic pathway in basis for standardizing the dose and the treatment schedule
tumor cells due to chemotherapeutic intervention is a of this compound against a variety of tumors in vivo.
relatively less explored area. Therefore, these findings are The major limitation of cancer therapy is the injury of
of much current interest with respect to identify novel Ru- normal tissues leading to multiple organ toxicity [54].
complexes which can inhibit a critical step of glycolytic Detection of increased level of LDH in serum is a widely
pathway resulting into induction of apoptosis in the tumor used parameter for blood based diagnosis of tissue damage
cells in vivo. as well as to characterize the rapid turnover of cancerous
Cisplatin is a well studied compound on a variety of cells in vivo [39]. Therefore, unaltered patterns of serum
tumors [51]. It was interesting to note that cisplatin also LDH observed in case of Ru(II)-CNEB and cisplatin treated
induced apoptosis in DL cells in vivo via release of DL mice (Fig. 9; lanes 3 and 4 versus lane 1) clearly
cytochrome c, however, without affecting the level of M4- suggest that no damage has occurred to the normal tissues
LDH. Thus, it is likely that cisplatin might be adopting due to the treatment with both these compounds. Accord-
LDH independent mechanism to induce apoptosis in DL ingly, a significant increase in the level of M4-LDH in
cells. DNA has been shown to be the major target of serum of untreated DL mice (Fig. 9; lane 2) may be
cisplatin induced cytotoxicity, wherein, cisplatin-DNA correlated with the rapid turnover of DL cells in the
adduct formation is known to induce oxidative stress and untreated DL mice.
finally to initiate tumor cell death [52]. There was a Most of the drugs given through systemic routes undergo
significant decline in the level of SOD2 with a concomitant their final metabolism in liver, and therefore, liver is likely
increase in the level of cytochrome c in the DL cells from to be affected adversely during chemotherapeutic treat-
cisplatin treated DL mice (Fig. 6a, b). Thus, it may be ments [54]. Increased levels of SGOT and SGPT are the
argued that, as against the role of Ru(II)-CNEB in DL cell most widely used blood based markers to ascertain liver
apoptosis, cisplatin adopts (O2)− dependent mitochondrial dysfunction. Therefore, unaltered patterns of SGOT and
dysfunction pathway to induce apoptosis in these cells. SGPT in the treated and untreated DL mice (Fig. 10a, b)
Caspase 9, an important component of oxidative stress indicate that doses of both the compounds tested are non
induced apoptosis, has also been reported to be implicated toxic to the liver. In addition, corroborating an earlier
in apoptotic death of certain tumor cells by cisplatin [45]. finding [27], though M4-LDH was slightly increased in the
Increase in the life span and improvement in overall liver of untreated DL mice, it remained unaltered in the
appearance of cancerous animal after the treatment are the liver of Ru(II)-CNEB treated DL mice (Fig. 10c) and thus,
ultimate criteria to ascertain anticancer potential of a suggesting further that liver of DL mice was unaffected due
chemotherapeutic agent. A significant decline (∼50% ) in to the treatment with this compound. Kidney is involved in
body weight (Fig. 7) of the Ru(II)-CNEB treated DL mice the filtration of blood born factors continuously. Though
and a significant increase in their survival period (Fig. 8) the levels of all LDH isozymes were found to be decreased
suggest that the molecular alterations induced by this in the kidney of DL bearing mice, they remained unaltered
compound has resulted into an overall improvement in the in the DL mice treated with Ru(II)-CNEB (Fig. 11a). Blood
life of the cancerous mice. The ranges of decrease in the brain barrier protects brain from most of the exogenous
body weight and increase in the survival period reported factors. It was evident in the present context also. All the
here are well correlated with the similar findings on DL five LDH isozymes were found to remain unaltered in the
bearing animal treated with the extracts of a macrofungus brain of both, the treated and the untreated group of DL
[42] and that on TLX5 lymphoma bearing mice treated with mice (Fig. 11b) and thus, suggesting that neither the
the different antimetastatic agents [38]. Also, the findings development of DL nor Ru(II)-CNEB treatment caused
on Ru(II)-CNEB treated DL mice were comparable and any alteration in the expression pattern of any of the LDH
very close to the data obtained from the DL mice treated isozymes in the mouse brain. Thus, it is evident that the
with cisplatin (Figs. 7 and 8). NAMI-A is the most widely dose of Ru(II)-CNEB used in this experiment did not
studied Ru-complex as an anticancer agent which was also produce any damage to the normal tissues in vivo. The dose
shown to reduce the increased body weight of the of cisplatin used also did not produce much change in the
cancerous animal maximum up to 50% that too when given level of LDH isozymes in the normal tissues. However, it
in combination with cisplatin [53]. The data on the caused a significant decline in the level of M4-LDH in liver
increased survival time reported here sounds further better and thereby, indicated the possibility of liver toxicity by
than only a 12% increase observed in the life span of cisplatin. This also corroborates an earlier report on the
Ehrlich ascite bearing mice due to the treatment with a Ru effect of cisplatin on liver LDH of DL mice [26].
(II)-complex, [cis-Ru (II) DMSO Cl2] [49]. Thus, our In summary, the present study demonstrates that a
findings suggest potent anticancer activity of Ru(II)-CNEB nontoxic dose of Ru(II)-CNEB is able to decrease the
against Dalton’s lymphoma in mice and thereby, provide a viability of DL cells in vivo with a concomitant increase in

13. Invest New Drugs (2009) 27:503–516 515
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Acknowledgment This work was financially supported by a project
Recently elucidated energy catabolism pathways provide oppor-
from Department of Biotechnology (DBT), Govt. of India, (BT/
tunities for novel treatments in hepatocellular carcinoma. Expert
PR5910/BRB/10/406/2005) sanctioned jointly to LM and SKT. The
Rev Anticancer Ther 4:449–457. doi:10.1586/14737140.4.3.449
authors are thankful to UGC Centre of Advanced Studies programme
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to Department of Zoology, BHU, for providing infrastructural
(2005) Inhibition of glycolysis in cancer cells: a novel strategy to
facilities. The help extended by Mr. S. Bhattacharyya, Ms. S.
overcome drug resistance associated with mitochondrial respira-
Srivastav, and Ms. B. Mishra is also acknowledged.
tory defect and hypoxia. Cancer Res 65:613–621. doi:10.1158/
0008-5472.CAN-04-4313
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