This research paper examines how a novel ruthenium(II) complex containing 4-carboxy N-ethylbenzamide (Ru(II)-CNEB) activates the p53-mediated glycolytic inhibition-oxidative stress-apoptosis pathway in Dalton's lymphoma (DL) cells in vivo. The study finds that Ru(II)-CNEB decreases expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2/PFKFB3), a key regulator of glycolysis in DL cells. It also activates superoxide dismutase but decreases catalase and glutathione peroxidase, imposing oxidative stress in DL cells. This is consistent with enhanced p

1. Research paper
Activation of p53 mediated glycolytic inhibition-oxidative stress-
apoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex
containing 4-carboxy N-ethylbenzamide
Raj Kumar Koiri a
, Surendra Kumar Trigun b, *
, Lallan Mishra c
a
Department of Zoology, Dr. Harisingh Gour Central University, Sagar, Madhya Pradesh 470003, India
b
Biochemistry Section, Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India
c
Department of Chemistry, Banaras Hindu University, Varanasi, Uttar Padesh 221005, India
a r t i c l e i n f o
Article history:
Received 25 September 2014
Accepted 30 December 2014
Available online 8 January 2015
Keywords:
Ruthenium
p53
PFKFB3
LDH
Apoptotic factors
a b s t r a c t
There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg
effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is
an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor
progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel
anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was adminis-
tered to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the
DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic
glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to
modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that
Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2:
PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dis-
mutase (the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the
two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the
enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings
suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor
supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.
© 2015 Elsevier B.V. and Societe française de biochimie et biologie Moleculaire (SFBBM). All rights
reserved.
1. Introduction
Identification of cellular/molecular targets is of prime concern
for formulating novel anticancer agents [1]. Due to effective bio-
distribution and multimodal cellular actions, during recent past,
ruthenium complexes have drawn much attention as next gener-
ation anticancer agents [2]. So far mechanistic aspects of
anticancer metal complexes are concerned, DNA, once considered
as their main target [3,4], is now evident to be highly unselective
[5] and therefore, there is an evolving concept to evaluate whether
metal complexes could be able to attenuate certain tumor growth
associated biochemical events at cellular level [5e7]. In this
respect, Ru-complexes get an upper hand, as metal center of
ruthenium has been shown to interact with a variety of ligands [8]
and thereby enabling these complexes to affect various cellular
activities [9].
We have synthesized and characterized a ruthenium complex;
Ru(II)-CNEB, which was found to be highly biocompatible to mice
when administered in vivo. Additionally, it could interact with and
inhibit M4-LDH non-competitively both in vitro and at tissue level
[10]. During pilot experiments, though this compound did not show
any DNA cleavage activity in vitro (Supplementary data with this
article), when administered to the DL bearing mice, it produced
Abbreviations: Bcl-2, B-cell leukemia/lymphoma-2; DL, dalton's lymphoma; FBP,
fructose-2,6-bisphosphate; GPx, glutathione peroxidase; GSH, glutathione reduced;
iPFK2/PFKFB3, inducible isoform of 6-phosphofructo-2-kinase; LDH, lactate dehy-
drogenase; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species;
Ru(II)-CNEB, [Ru(CNEB-H)2(bpy)2] 2PF6$0.5 NH4PF6 (Ruthenium(II)-complex con-
taining 4-carboxy N-ethylbenzamide as ligand); SOD, superoxide dismutase.
* Corresponding author. Tel.: þ91 9415811962.
E-mail address: sktrigun@gmail.com (S.K. Trigun).
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
http://dx.doi.org/10.1016/j.biochi.2014.12.021
0300-9084/© 2015 Elsevier B.V. and Societe française de biochimie et biologie Moleculaire (SFBBM). All rights reserved.
Biochimie 110 (2015) 52e61

2. apoptotic pattern of DNA cleavage which was consistent with the
decreased DL cell viability and increased life span of the tumor
bearing mice [11]. Importantly, since, this complex increased
certain apoptotic markers and also inhibited M4-LDH (LDH-5) in
those DL cells, it was argued that this complex, instead of targeting
DNA directly, is able to activate glycolytic inhibition-apoptosis
pathway in a tumor cell in vivo [11]. This necessitated further
studies on characterizing regulatory factors of this pathway as
target of Ru (II)-CNEB in the DL cells in vivo.
Switching over to aerobic glycolysis, known as ‘Warburg effect’,
is considered to be a common trait of most of the growing tumors
[12] and therefore, inhibition of enhanced tumor glycolysis is now
advocated as one of the therapeutic strategies in cancer therapy
[13,14]. In this regard, targeting regulatory enzymes of glycolytic
pathway assumes special importance for the novel anticancer
agents. Phosphofructokinase1 (PFK1) catalyzes committed step of
glycolysis. PFK2 domain of D-fructose-6-phosphate-2-kinase/fruc-
tose-2,6-bisphosphatase (PFK2/FBPase2) synthesizes fructose-2,6-
bisphosphate (FBP) to activate PFK1 and thereby, it acts as main
glycolytic regulator under a variety of pathological challenges
including tumor progression [15]. Cancer cells express C type PFK1
which is more sensitive to FBP [16] and also over express a cata-
lytically more efficient inducible form of PFK2 (iPFK2: PFKFB3 gene)
[17]. Since, iPFK2 repression has been reported to inhibit tumor cell
growth in vitro [13], this glycolytic activator could be considered as
relevant therpautic target for the novel anticancer agents. Similarly,
to sustain enhanced glycolysis, tumor cells adapt to produce lactate
from pyruvate by activating LDH-5. Reports also suggest that over
expression of LDH-5 gene (LDH-A) is associated with tumor growth
[12,18] and thus, advocating repression of LDH-5 gene as another
mechanism to define therapeutic target for a novel anticancer
agent.
So far up stream regulation of the cell bioenergetics is con-
cerned, p53, a tumor suppressor protein, has been found to
modulate overall balance between glycolysis and mito-OXPHOS
[19]. The loss of normal p53 has been reported to be critically
associated with tumor progression, particularly in leukemia and
lymphoma [20]. Similarly, p53 over expression has been demon-
strated to have prognostic significance in lymphoma [21]. Recently,
p53 has been reported to regulate tumor cell energy metabolism
[22] via down regulating expression of glucose transporters GLUT1
and GLUT4 [23] and also by activating the TIGAR gene which re-
presses iPFK2/PFKFB3 [24]. Thus, when tumor cells switch over to
glycolytic phenotype, it is accompanied with declined p53
expression [25]. Similarly, inhibition of glycolysis has been shown
to activate p53 [26].
It has been suggested that ROS are down stream mediators of
p53 dependent apoptosis [27], wherein, Bcl-2/Bax ratio acts as
main determinant of this pathway [28]. Low level of p53 induces
expression of antioxidant enzymes responsible to maintain ROS
level within a permissive range which otherwise can cause DNA
damage and genomic instability in the cells [29]. Higher level of p53
protein, on the other hand, is known to enhance the expression of
pro-oxidant and proapoptotic factors [30].
Tumor cells, in addition to acquiring glycolytic phenotype, also
modulate their oxygen metabolism in many ways [12,31]. Super-
oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and
glutathione reductase (GR) constitute main antioxidant defense
mechanism in most of the cells. The oxygen free redicals (O2
.-
),
produced during mitochondrial oxidative phosphorylation, are
dismutated to hydrogen peroxide (H2O2) by SOD followed by con-
version of H2O2 into water by catalase and GPx [32]. As SOD is the
committed enzyme of this pathway responsible to produce H2O2, it
appears to be the most relevant target for therapeutic intervention
in the tumor cells. In the recent past, both SOD isoforms (CueZn-
SOD: cytosolic; SOD1 and Mn-SOD: mitochondrial; SOD2) have
been found to be implicated in tumerogenesis [33].
Some earlier reports have described that reduced levels of SOD1
SOD2 facilitate tumerogenesis [34,35] via maintaining low level
of H2O2 in the cancerous cells and accordingly, increased SOD ac-
tivity and in turn, higher level of H2O2 may contribute for tumor
suppression [36,37]. However, effectiveness of this SOD mediated
mechanism depends on the status of the two down stream en-
zymes; catalase and GPx in the cancerous cells. Indeed, SOD and
catalase double transfectant (SOCAT3) cells and cells with over
activated GPx1 have shown protection from oxidative cell damage
[38,39]. Thus, to characterize oxidative mechanism based anti-
cancer potential of a novel anticancer agent, it is important to study
comparative profile of the three antioxidant enzymes involved in
H2O2 metabolism in the cancer cells.
In the present article, we have investigated whether Ru(II)-CNEB
is able to activate p53 mediated glycolytic inhibition-oxidative
stress-apoptosis pathway in the DL cells in vivo.
2. Materials and methods
2.1. Chemicals
Ruthenium(II)-complex containing 4-carboxy N-ethyl-
benzamide as ligand, Ru(II)-CNEB; [Ru(CNEB-H)2(bpy)2] 2PF6$0.5
NH4PF6, whose structural details have already been described
previously [10,11], was used in the present study. Antibodies
against p53, caspase 9, Bcl-2, Bax, SOD1, SOD2 PFK 2 were
purchased from Santa Cruz and b-actin was purchased from
SigmaeAldrich Co., USA. HRP- conjugated anti rabbit/goat/mouse
IgG were obtained from Genei. 20,70-dichlorofluorescin diacetate
and ECL super signal western pico kit were purchased from Fluka
and Pierce respectively. Lactate estimation kit was purchased
from Biorex Diagnostics, Ltd, UK. Other general chemicals and
reagents were obtained from Merck or SRL unless otherwise
specified.
2.2. Induction of Dalton's lymphoma (DL) in mice
Inbred AKR strain mice of 16e18 weeks age weighing 24e26 g
were used for the experiments. Mice were maintained at standard
laboratory conditions with the supply of food and water ad libitum.
This work was approved by the institutional animal ethical com-
mittee (Dan/2006-07/962). Dalton's lymphoma was induced by
transplantation of 1 Â 107
viable tumor cells (assayed by trypan
blue method) i.p. per mice. Development of DL was confirmed by
abnormal belly swelling and increased body weight which became
visible on 10e12th post transplantation day. The DL bearing mice
survived up to 18 ± 2 days.
2.3. Treatment schedule
The DL mice were randomly divided into 2 groups with 10 mice
in each. The first group DL mice were treated with Ru(II)-CNEB
complex (10 mg/kg bw/day, ip), and the second group, designated
as DL control, were similarly injected with equal volume of Krebs
Ringer Buffer (KRB). As DL becomes visible on day 10e11 and DL
bearing mice survived up to 18e20 days post DL transplantation,
the treatments with the compounds were started from day 11 of
tumor transplantation and continued up to day 17th. The normal
control group mice were also treated simultaneously with KRB. To
study biochemical/molecular parameters, 3-4 mice from each
group were sacrificed on day 18th and tumor ascites pooled from 3
to 4 DL mice from each group were centrifuged at 2000 Â g at 4 C
to collect DL cells. The DL cell extract was prepared using lysis
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 53

3. buffer (20 mM Tris-Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 25 mM Na2 pyrophosphate and 1 mM PMSF). The
cell lysates were centrifuged at 20,000 Â g for 30 min and super-
natant obtained were used for biochemical and molecular studies.
Protein concentrations in the extracts were measured following the
method of Lowry et al. [40].
2.4. Western blotting
For western blot analysis of different proteins, DL cell extracts
containing 60 mg proteins, were subjected to 10% SDS-PAGE. As
described previously [11], proteins were transferred to nitrocellu-
lose membrane followed by detection of p53, caspase 9, Bcl-2, Bax,
iPFK2, SOD1 and SOD2 against specific polyclonal antibodies
(1:1000). Proteins on membrane were detected by ECL west pico
kit. As loading control, b-actin was probed similarly using mono-
clonal anti-b-actin-peroxidase antibody (1:10,000). Protein bands
were quantified using gel densitometry software AlphaImager
2200.
2.5. Assay of the antioxidant enzymes
Catalase activity was measured following an earlier method [41]
with some modifications. Briefly, 1 ml reaction mixture consisted of
0.05 M phosphate buffer (pH 7.0) and 0.003% H2O2. The reaction
was started by the addition of suitably diluted cell extract and
decrease in absorbance at 240 nm was recorded for 10 min. Unit of
the enzyme was defined as mmol of H2O2 depleted/min. The activity
was expressed as units/mg protein.
Glutathione peroxidase (GPx) activity was determined as
described earlier [42] with slight modifications. Briefly, cell ex-
tracts were added to the assay buffer consisting of 100 mM Tris-
Cl (pH 7.2), 3 mM EDTA, 1 mM sodium azide, 0.25 mM H2O2,
0.5 mM NADPH, 0.17 mM GSH, and 1 unit GR. The change in
absorbance per minute at 340 nm was recorded for 10 min and
enzyme activity was expressed as mmole NADPH oxidized/min/
mg protein.
2.6. Analysis of SOD and Gpx by non-denaturing PAGE
The active levels of superoxide dismutase (SOD) and glutathione
peroxidase (GPx) was determined using non-denaturing PAGE of
the cell extracts following the method recently reported from our
lab [42,43]. The cell extract containing 60 mg protein were loaded in
each lane of 10% non-denaturing PAGE. After electrophoresis at
4 ± 2 C, the gels were subjected to substrate specific staining of
different antioxidant enzymes.
The staining mixture for SOD consisted of 2.5 mM NBT, 28 mM
riboflavin, and 28 mM TEMED. Gels were incubated for 20 min in
the dark, followed by exposing them to a fluorescent light till
achromatic SOD bands developed against dark blue background.
GPx specific staining mixture was composed of 50 mM TriseCl
buffer (pH 7.9), 3 mM GSH, 0.004% H2O2, 1.2 mM NBT and 1.6 mM
PMS. Achromatic bands corresponding to GPx activity appeared
against a violeteblue background.
The intensities of all the bands were quantified by gel densi-
tometry using AlphaImager 2200 gel documentation software.
Specificity of the PAGE bands of different enzymes were confirmed
by obtaining clear negative results when similarly run gels were
treated in the absence of the enzyme specific substrates. In each
case, PAGE was performed at least 3 times and mean ± SD of
densitometry values of the bands, as percentage of the control lane,
have been presented in the result with one representative gel
photograph.
2.7. Biochemical estimations
2.7.1. Lactate
The concentration of lactate was determined by measuring
lactate oxidation by lactate oxidase as per the manufacturer's in-
structions given in the lactate assay kit obtained from Biorex Di-
agnostics Ltd, UK.
2.7.2. H2O2
Following the method described earlier [42]; intracellular H2O2
was determined by measuring H2O2 dependent oxidation of DCFH-
DA into DCF. Briefly, 100 ml of cell extracts were incubated with
10 mM DCFH-DA at 37 C for 30 min in dark. Using excitation at
504 nm and emission at 529 nm, intensity of DCF fluorescence was
measured. H2O2 concentration in the extract was determined by
using a calibration plot made against different H2O2 concentrations
(5e100 mM).
2.7.3. Total glutathione (GSH þ GSSG)
Following the method described earlier [42], DL cell extracts
were precipitated with 5% sulfosalicylic acid in the ratio of 1:2 and
centrifuged. The supernatant collected was neutralized. In a 96 well
micro plate, 50 ml neutralized supernatant was incubated with
100 ml of the reagent containing 0.30 mM NADPH, 0.22 mM DTNB
and 1.6 units/ml GR prepared in 100 mM phosphate buffer (pH 7.4)
containing 1 mM EDTA. The absorbance was recorded at 412 nm for
10 min using Micro Scan MS5608A (ECIL) micro plate reader. Total
glutathione content was expressed in terms of nmol/mg protein.
2.8. Semi-quantitative RT-PCR
Total RNA was isolated from DL cells using TRI reagent
following the manufacturer's protocol. DNA-free™ (Ambion) was
used to remove any contaminating DNA from the RNA preparation
following the manufacturer's protocol. Briefly, reaction mixture
consisting of DNase I, 10Â DNase I buffer and RNA sample was
incubated at 37 C for 20 min. Thereafter, DNase I inactivation
reagent slurry was added and incubated for 2 more min at room
temperature and centrifuged. The upper phase was collected as
DNA free RNA solution. From this RNA isolate, 2 mg RNA was
subjected for reverse transcription using 200 U of reverse tran-
scriptase and 200 ng random hexamer to make ss-cDNA (Revert
Aid First strand cDNA synthesis kit, MBI fermentas). The PCR re-
action mixture contained 1Â Taq polymerase buffer, 0.2 mM each
of the four dNTPs, 1.0 U of Taq polymerase, and 10 pmol of the
specific primer. The mouse gene-specific primers used were: Bcl-2
(forward 50-TAC CGT CGT GAC TTC GCA GAG-30; reverse 50-GGC
AGG CTG AGC AGG GTC TT-30); Bax (forward 50-CGG CGA ATT GGA
GAT GAA CTG-30; reverse 50-GCA AAG TAG AAG AGG GCA ACC-30);
PFKFB3 (forward 50-GGC AAG ATT GGG GGC GAC TC-30; reverse 50-
GGC TCC AGG CGT TGG ACA AG-30); LDH A (forward 50-ATG CAC
CCG CCT AAG GTT CTT-30; reverse 50-TGC CTA CGA GGT GAT CAA
GCT-30); SOD2 (forward 50-GCA CAT TAA CGC GCA GAT CA-30;
reverse 50-AGC CTC CAG CAA CTC TCC TT-30) and b actin (forward
50-ATC GTG GGC CGC TCT AGG CAC C-30; reverse 50-CTC TTT GAT
GTC ACG ATT TC-30). Linearity of PCR amplifications was checked
for each gene using various cycles (20, 24, 28, 32, 36 and 40 cycles)
vs densitometric value of the PCR product of the corresponding
gene. Accordingly, the optimal number of cycles from the linear
phase and other conditions were chosen for amplification for each
gene as; b actin, 30 cycles of 30 s at 94 C, 30 s at 52 C, and 30 s at
72 C; Bcl-2 LDH A, 34 cycles of 45 s at 94 C, 45 s at 55 C, and
1 min at 72 C; Bax, 30 cycles of 30 s at 94 C; 60 s at 56 C, and
1 min at 72 C; PFKFB3, 31 cycles of 60 s at 95 C, 60 s at 50 C, and
1 min at 72 C; SOD2, 27 cycles. The amplification products,
R.K. Koiri et al. / Biochimie 110 (2015) 52e6154

4. analyzed by 1e2% agarose gel electrophoresis, were visualized by
ethidium bromide staining and were found to be; Bcl 2 (350 bp),
Bax (160 bp), PFKFB3 (329 bp), LDH A (103 bp), SOD2 (241 bp) and
b actin (543 bp). Amplification of b actin served as a control. Three
RT-PCR repeats from 3 RNA isolates for each gene were performed.
The expression levels were measured by densitometry using
AlphaImager 2200.
2.9. Statistical analysis
Experimental data were expressed as mean ± SD and Student's t
test was applied for determining the level of significance between
control and experimental groups and value of p 0.05 was
considered significant.
3. Results
3.1. Ru(II)-CNEB mediated decline of lactate level in the DL cells
in vivo
Lactate serves as an alternate metabolic fuel for the high energy
demanding tumor cells. Therefore, overproduction of lactate is
considered one of the hallmarks of the tumor growth. According to
Fig. 1b, as compared to the DL cells from untreated group, there is a
significant decline (p 0.001) in lactate level in case of the DL cells
from Ru(II)-CNEB treated DL mice. To ascertain, whether decline in
lactate level is due to decline in the expression of M4-LDH, the level
of LDH-A mRNA was measured in the DL cells from the untreated
the treated group DL mice. According to Fig. 1a, the level of LDH-A
Fig. 1. Effect of Ru(II)-CNEB (Rc) on the expression of LDH-A and iPFK2 lactate production in the DL cells in vivo. (a) shows representative RT-PCR photograph for LDH-A with the
ratio of LDH-A/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b) shows lactate level represent as mean ± SD where n ¼ 4. (c) shows representative RT-PCR
photograph for PFKFB3 with the ratio of PFKFB3/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. In (d), a representative western blot photograph, with 60 mg
protein in each lane, presented with the relative densitometric values of iPFK2/b actin as mean ± SD from three western blot repeats. *p 0.05; ***p 0.001 (untreated vs. treated
DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 55

5. mRNA in the DL cells from Ru(II)-CNEB treated DL mice is found to
be unchanged as compared to the untreated counterpart.
3.2. Downregulation of iPFK2/PFKFB3 in the DL cells due to the
treatment with Ru(II)-CNEB
Over expression of iPFK2/PFKFB3 is associated with tumor
growth. Recently, p53 has been reported to regulate tumor cell
energy metabolism indirectly by activation of the TIGAR gene
which declines iPFK2/PFKFB3 induced fructose-2,6-bisphosphate
production in the tumor cells. As illustrated in Fig. 1c and d, the
levels of PFKFB3 mRNA and its protein product declined signifi-
cantly (p 0.05e0.001) in the DL cells from Ru(II)-CNEB treated DL
mice than that from the untreated counterpart.
3.3. Ru(II)-CNEB increases p53 level in the DL cells in vivo
Restoration of p53 level is an important strategy of anticancer
chemotherapy. Based on immunoblot analysis, level of p53 was
observed to be ~5 times higher in the DL cells (p 0.001) from the
Ru(II)-CNEB treated DL mice than that from the control group mice
(Fig. 2).
3.4. Enhanced expressions of SOD1 SOD2 in the DL cells from
Ru(II)-CNEB treated DL mice
Oxidative stress and mitochondrial dysfunction are known to
initiate final steps of apoptosis. Infact ROS has been suggested to act
as a down stream mediator of p53 dependent apoptosis. SOD is the
first commited enzyme that neutralizes superoxide anion free
radical (O2
À
) based oxidative stress in the cells. According to Fig. 3a,
the level of active SOD2 SOD1, both shows significant increments
in the DL cells from Ru(II)-CNEB treated DL mice. To confirm
whether activity increment is contributed due to the similar in-
crease in the expression of these antioxidant enzymes, RT-PCR and
western blot analysis was performed. Fig. 3bed illustrate that
Ru(II)-CNEB is able to increase the expression of both, SOD1 and
SOD2 significantly (p 0.05e0.001) in the DL cells in vivo.
3.5. Ru(II)-CNEB mediated in vivo modulation of pro-oxiative
factors in the DL cells
The two antioxidant enzymes, catalase GPx, down stream to
SOD, show significant decline in their activity (p 0.05) in the DL
cells from Ru(II)-CNEB treated DL mice (Fig. 4aec). However,
treatment with Ru(II)-CNEB caused a significant decrease in the
activity of GPx1, with a concomitant increase in the activity of GPx2
(Fig. 4c). Such a reciprocal change between SODs and catalase
GPx is likely to allow accumulation of H2O2 at cellular level. Fig. 5a
shows that indeed there is a marked increase (~6 times; p 0.001)
in H2O2 level in the DL cells from Ru(II)-CNEB treated DL mice than
those from the untreated groups. Level of glutathione is another
marker of oxidative stress in the cells. According to Fig. 5b, as
compared to the untreated DL group, the DL cells from Ru(II)-CNEB
treated DL mice show a significant decline (p 0.05) in total
glutathione (GSH þ GSSG) level.
3.6. Ru(II)-CNEB mediated in vivo modulation of pro-apoptotic
factors in the DL cells
Bcl2/Bax ratio serves as one of the critical determinants of
apoptotic induction at cellular level. As compared to the untreated
DL mice, treatment with Ru(II)-CNEB caused a significant decline
(p 0.001) in the level of Bcl2 protein in the DL cells with a
concomitant increase in the Bax protein (Fig 6b). Such reciprocal
changes in Bcl2 vs Bax resulted into a significantly declined Bcl2/
Bax ratio (~3.5 times; p 0.01) in the DL cells from Ru(II)-CNEB
treated DL mice (Fig. 6b). The RT-PCR results (Fig 6a) also demon-
strate a significant decline in Bcl2/Bax mRNA level, however, mainly
due to a significant decrease in Bcl2 mRNA level in the DL cells from
the treated rats.
Caspase 9 cleavage is an important event of the mitochondrial
pathway of apoptosis and that of PARP-1 cleavage associates with
DNA fragmentation in the apoptotic cells. As shown in Fig. 6c,
treatment with Ru(II)-CNEB is able to produce cleaved products of
caspase 9. Full length PARP-1 is a 116 kDa protein, which, upon
activation, is cleaved into a 89 kDa C-terminal a 24 kDa N-ter-
minal protein fragments. Fig 6d illustrates ~1.5 times increase in the
levels of the cleaved PARP-1 fragments with a proportionate
decline in its full length PARP-1. This was consistent with the DNA
fragmentation pattern of the DL cells reported previously [11].
4. Discussion
The two main findings reported in our previous paper [11]; one,
Ru(II)-CNEB could inhibit M4-LDH and second, it induced release of
mitochondrial cytochrome c in the DL cells in vivo, hinted towards
induction of metabolic derangement led apoptosis in the DL cells
due to the treatment with this compound. This tempted us to
explore the regulatory aspects of such cellular effects produced by
Ru(II)-CNEB. Since, decline in M4-LDH activity in the DL cells, due to
the treatment with an anti-DL agent, could be attributed to the
reduced expression of this enzyme [28], first we checked the mRNA
level of LDH-A (M4-LDH) in the DL cells from Ru(II)-CNEB treated
DL mice. The RT-PCR result (Fig. 1a) shows no change in the level of
LDH-A mRNA in the DL cells due to the treatment with Ru(II)-CNEB.
Thus, suggesting that Ru(II)-CNEB does not affect expression of M4-
LDH. This implies that decline in M4-LDH activity, observed pre-
viously due to the treatment with Ru(II)-CNEB [11], is a conse-
quence of inhibiting catalytic efficiency of this LDH isozyme by the
compound at protein level. Indeed, this complex has already been
demonstrated to interact with and inhibit M4-LDH non-
competitively [10].
β actin
DL DL+Rc
p53
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
DL DL+Rc
Densitometricvalue
(p53/βactin)
***
Fig. 2. Treatment with Ru(II)-CNEB (Rc) increased the level of p53 in the DL cells
in vivo. A representative western blot photograph is presented with the relative
densitometric values of p53/b actin as mean ± SD from three western blot repeats.
***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e6156

6. Overproduction of lactate is considered as one of the hallmarks
of the tumor growth [44]. It has been reported that tumor cells
survival is greatly supported by excess of lactate produced by the
tumor cells [45]. Also, blockage of tumor M4-LDH, responsible to
synthesize lactate, has been found to suppress this additional route
of metabolic supplementation and thereby renders tumor cells
susceptible to death [45]. Therefore, keeping aside the mechanism
by which Ru(II)-CNEB decreases M4-LDH activity, the resultant
decline in lactate production could be of high therapeutic relevance
for this compound. Indeed, Ru(II)-CNEB, earlier found to inhibit
M4-LDH [11], is also able to significantly decline lactate level in the
DL cells (Fig. 1b), and thus, advocating M4-LDH as a therapeutic
target for this compound at least in case of the DL cells in vivo. Such
a mechanism gets support from inhibition of tumor cell prolifera-
tion in vitro due to the treatment with certain N-hydroxyindole-
based inhibitors of M4-LDH [46].
Moreover, M4-LDH catalyzed rapid production of lactate is
mainly dependent on the adequate supply of pyruvate as a conse-
quence of enhanced glycolysis in the tumor cells [18,47]. The
committed step of glycolysis is catalyzed by PFK1 which is evident
to be the target of multimodal regulation under a variety of path-
ophysiological conditions including tumor development [16]. PFK2,
Fig. 3. Effect of Ru(II)-CNEB (Rc) on (a) active levels of SOD2 SOD1, (b) on the expression of SOD1 and (c d) on the expression of SOD 2 in the DL cells from untreated and treated
DL mice. (a) gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the relative densitometric values for the respective SOD bands from
3 PAGE repeats. (c) shows a representative RT-PCR photograph with the densitometric values of SOD2/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b and d) shows
representative western blot photographs, with 60 mg protein in each lane, presented with the densitometric values of SOD1/b actin and SOD2/b actin respectively as mean ± SD from
3 western blot repeats. *p 0.05; **p 0.01; ***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 57

7. the kinase domain of a bi-functional enzyme (PFK2/FBPase2),
synthesizes FBP, the most effective allosteric activator of PFK1, and
thereby, considered as a master regulator of the glycolytic pathway.
In general, tumor cells are evident to express a C-type PFK1 that
shows greater sensitivity for FBP activation [16] and also express a
catalytically more efficient isoform of PFK2 (iPFK2: PFKFB3) [13,17].
Importantly, iPFK2 is found to be over expressed as a universal trait
of most of the growing tumor cells including DL [28] and many
tumors of human origin as well [17]. It was interesting to observe
that this compound could significantly decline the expression of
iPFK2, both at mRNA and at protein levels (Fig. 1c and d). This
clearly suggested that Ru(II)-CNEB is able to downregulate the
synthesis of master regulator of the glycolytic pathway in the DL
cells in vivo. The argument is well supported by a report describing
correlation between PFKFB3 gene silencing by siRNA and apoptosis
in the Hela cells in culture [13]. Though information is scanty on
modulation of iPFK2 by synthetic compounds in tumor cells, a 3-(3-
pyridinyl)-1-(4-pyridinyl)-2-propen-1-one has been found to
inhibit iPFK2 resulting into decreased glucose uptake by the tumor
cells leading into tumor growth suppression in vivo [48]. In this
context, the findings of Fig. 1c and d are first of its kind to
demonstrate that Ru(II)-CNEB is able to repress iPFK2 in a tumor
cell in vivo and thereby speculated to render less production of
adequate glycolytic intermediates to sustain high glycolytic effi-
ciency of the DL cells in vivo.
So far tumor growth associated regulator of cell bioenergetics is
concerned, p53, a tumor suppressor protein, has been given much
emphasis as it has been reported to be involved in imposing War-
burg effect during tumor development [19,22]. It is now evident
that switching over to glycolytic phenotype by the tumor cells is
accompanied with the declined p53 level [25]. Similarly, the
enhanced level of p53 has been found to repress glucose trans-
porters GLUT1 and GLUT4 [23]. Also, increased p53 has been
demonstrated to decline the expression of iPFK2 via activating the
TIGAR gene [24] and consequently, it inhibits glycolysis [26]. We
have observed significant increase in p53 level (Fig. 2) vis a vis a
significantly declined iPFK2 expression (Fig.1c and d) in the DL cells
due to the treatment with Ru(II)-CNEB and thus suggesting an as-
sociation between Ru(II)-CNEB mediated enhanced p53 level and
declined activity of the committed step of glycolytic pathway in the
DL cells in vivo.
It is known that activation of aerobic glycolysis by the tumor cells
is a metabolic strategy to prevent production of ROS, an inevitable
outcome of the oxidative energy metabolism, and thereby to protect
tumor cells from ROS induced apoptosis [49]. In addition, tumor
cells are known to modulate main antioxidant enzymes to prevent
oxidative stress [34,35]. Therefore, deranging such enzymatic
mechanisms by a therapeutic agent is argued to be a relevant option
for driving tumor cells to undergo apoptosis [28,43].
SOD is the first and commited enzyme of antioxidant pathway
that neutralizes O2
À
into H2O2. It has been reported that reduced
levels of SOD1 and SOD2 maintain low level of H2O2 in the
cancerous cells to facilitate tumerogenesis [34,35]. Similarly,
increased SOD activity and in turn, higher level of H2O2 is specu-
lated to inhibit tumor progression [36,37]. According to Fig. 3a,
there is a significant increase in the activity of both the SOD iso-
forms; SOD2 SOD1, in the DL cells from Ru(II)-CNEB treated DL
mice. Since, the pattern of active levels of both of them coincided
with the similar increments in expression of both these enzymes
(Fig. 3bed), it is evident that Ru(II)-CNEB is able to enhance
expression of SOD2 and SOD1 to make overall increment in SOD
activity in the DL cells in vivo. Since reports are limited on metal
complex induced expression of antioxidant enzymes, it is a first
report wherein, a Ru(II)-complex is demonstrated to enhance ac-
tivity of SOD by overexpressing SOD2 and SOD1 proteins. Moreover,
keeping aside these explanations, the enhanced level of SODs is
likely to ultimately produce higher level of H2O2 in the DL cells due
to the treatment with Ru(II)-CNEB.
H2O2 produced by SODs is metabolized by GPx and Catalase and
activity of both these enzymes were observed to be declined
significantly in the DL cells from Ru(II)-CNEB treated DL mice
(Fig. 4aec). Such a reciprocal pattern between SOD vs catalase
GPx has been reported accountable for unusually increased level of
H2O2 in the DL cells due to the treatment with emodin [50]. Indeed,
0
1
2
3
4
5
6
DL DL+Rc
Catalaseactivity
(U/mgprotein)
*
a
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
DL DL+Rc
GPxactivity
(U/mgprotein)
*
b
0
10
20
30
40
50
60
DL DL+RcsdnabxPGfoyrtemotisneD
(Relativeintensity)
GPx1 GPx2
**
*
GPx1
DL DL+Rc
GPx2
GPx3
c
Fig. 4. Effect of Ru(II)-CNEB (Rc) on the activity of catalase (a) and GPx (b and c) in the DL cells in vivo. The values in (a and b) are mean ± SD, where n ¼ 4. (c) represents active level
of GPx in the DL cells from untreated and complex treated DL mice. The gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the
relative densitometric values for the respective GPx bands from 3 PAGE repeats. *p 0.05; **p 0.01 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e6158

8. H2O2 concentration was found to be remarkably increased (~6Â) in
the DL cells from Ru(II)-CNEB treated DL mice (Fig. 5a). Thus, it is
argued that Ru(II)-CNEB is able to modulate all the three enzymes
of antioxidant pathway; SOD, catalase and GPx, to maintain higher
level of H2O2 in the DL cells. Such a condition might drive the DL
cells to undergo apoptosis, as increase in intracellular H2O2 is
known to cause a significant drop in cytosolic pH [51] which is
considered accountable for translocation of Bax, a pro-apoptotic
factor, to mitochondria [49].
Though the mechanism by which Ru(II)-CNEB regulates
reciprocal changes in SODs vs catalase and GPx could be a matter
of further investigation, in the present context, however,
enhanced level of p53 protein (Fig. 2) could be considered as one
of the integrators of such enzymatic changes. This is because,
not only a close association between p53 dependent modulation
of ROS metabolism and cell apoptosis is on record [26,27] but
also, higher level of p53 protein has been found to enhance the
expression of pro-oxidant and proapoptotic factors [28,30].
Fig. 5. Effect of Ru(II)-CNEB (Rc) on the level of H2O2 (a) and total glutathione (b) in the DL cells in vivo. (a) represents level of H2O2 in the DL cells. Values are mean ± SD and n ¼ 4.
(b) represents level of total glutathione in the DL cells in vivo. Values are mean ± SD, where n ¼ 3. *p 0.05; ***p 0.001 (untreated vs. treated DL groups).
Fig. 6. Treatment with Ru(II)-CNEB (Rc) caused a significant decline in the expression of Bcl2 with concomitantly increased expression of Bax (a and b), activation of caspase 9 (c)
and PARP1 cleavage (d) in the DL cells in vivo. a shows representative RT-PCR photographs with the b-actin normalized densitometric ratio of Bcl2/Bax mRNA where values
represent mean ± SD from 3 RT-PCR repeats. b shows representative western blot photographs with the b-actin normalized densitometric ratio of Bcl2/Bax where values represent
mean ± SD from three western blot repeats. ***p 0.001 (untreated vs. treated DL groups).
R.K. Koiri et al. / Biochimie 110 (2015) 52e61 59

9. Glutathione content is known to represent actual redox status of
the cells and its enhanced level in the cancer cells is directly
correlated with the tumor progression. Higher glutathione level, in
addition to providing multidrug and radiation resistance [52], has
been found to prevent apoptosis in the tumor cells [53]. Similarly,
depletion of glutathione level has been reported to sensitize tumor
cells to undergo apoptosis via release of cytochrome c [54]. The
anti-apoptotic role of Bcl-2 has also been linked with the gluta-
thione content in the tumor cells [55]. We have shown previously
that Ru(II)-CNEB causes release of mitochondrial cytochrome c [11]
and according to Fig. 6a and b, it is evident to decline Bcl2/Bax ratio
also. Importantly these cellular alterations are consistent with a
significant decline in total glutathione (GSH þ GSSG) level in the DL
cells (Fig. 5b) and thereby providing another biochemical mecha-
nism by which Ru(II)-CNEB could be able to induce apoptosis in the
DL cells.
One of the important aspects of p53 biochemistry is that it is
considered to be involved in regulating apoptosis in the tumor cells
[19,22,27,28]. Normally, p53 remains sequestered with Mdm2 in
the cytosol and thereby prevents cells to undergo apoptosis [56].
This implies that release of p53 from Mdm2 could be one of the
mechanisms to induce apoptosis in the cells. This may happen due
to many cellular changes including increased DNA damage caused
by the exogenous agents [57]. However, we observed that the
increasing concentration of Ru(II)-CNEB, when incubated in vitro at
37 C for 24 h with pBR322 plasmid DNA, it did not convert
supercoiled plasmid into the nicked circular DNA (Supplementary
data; Fig. S1) and thereby excluding direct nuclease activity of
this compound. Therefore, in the present context, as reported
earlier [28,56], abrrent ellular signaling could be argued account-
able for a significantly enhanced level of p53 in the DL cells from
Ru(II)-CNEB treated DL mice (Fig. 2). Moreover, keeping aside the
mechanism by which p53 level gets enhanced in the DL cells, it is
considered to act as a strong apoptotic inducer in multimodal ways.
The enhanced cytoplasmic p53 level has been reported to activate
Bax and its oligomerization [58] which in turn, induces cytochrome
c release from the mitochondria [59]. Bcl2 is an anti-apoptotic
factor and therefore, declined Bcl2/Bax ratio is considered
accountable for initiating intrinsic pathway of apoptosis [28]. In the
present context, Ru(II)-CNEB significantly declined the level of Bcl2
protein with a concomitant increase in Bax level (Fig. 6b). This
suggested that Ru(II)-CNEB is able to reciprocally modulate level of
both these factors resulting into a significant decline in Bcl2/Bax
ratio in the DL cells in vivo.
Caspase 9 activation is a hallmark of mitochondrial pathway of
apoptosis [60] and that of PARP-1 cleavage is associated with DNA
fragmentation in the cells undergoing apoptosis [61]. We observed
significant increase in the levels of cleaved caspase 9 and PARP-1 in
the DL cells from Ru(II)-CNEB treated DL mice (Fig. 6c and d).
Consistent with these observations, Ru(II)-CNEB mediated release of
mitochondrial cytochrome c, the main initiator of the intrinsic
pathway of apoptosis, in those DL cells, is already on record [11].
Thus, taking together, these findings strongly advocate for p53
mediated activation of Bcl2/Bax-cytochrome c release-caspase 9 led
intrinsic pathway of apoptosis in the DL cells due to treatment with
Ru(II)-CNEB in vivo. Though not much information is available on
induction of intrinsic pathway of apoptosis by metal complexes, a
Ru(II)-arene compound [Ru(g6-p-cymene)Cl2(pta)] has been
described to implicate this pathway for inducing apoptosis in the
Ehrlich ascite carcinoma [62]. Recently, another Ru(II)-complex
(Ru(II) b-carboline complex) has also been demonstrated to induce
apoptosis in the cancer cells by involving p53 [63]. In this context,
the findings of this paper are of special merit with regard to eluci-
dation of the biochemical mechanism by which a Ru(II)-complex can
induce p53 dependent apoptosis in the tumor cells in vivo.
In conclusion, there is an evolving concept of restricting tumor
growth by depriving tumor cells from adequate energy production
and by rendering them susceptible to oxidative stress. The present
article demonstrates that a Ru(II)-CNEB, characterized previously as
an anti tumor compound, is evident to activate p53 mediated
glycolytic inhibition-oxidative stress-apoptosis pathway in the DL
cells when administered in vivo. As such these findings provide a
biochemical mechanism which can be utilized for defining phar-
macological targets for the novel anticancer agents suitable for
in vivo applications.
Conflict of interest
The authors declare no conflict of interest with respect to this
article.
Acknowledgements
This work was financially supported by a project from Depart-
ment of Biotechnology (DBT), Govt. of India, (BT/PR5910/BRB/10/
406/2005) sanctioned jointly to LM and SKT at BHU. The contri-
bution of Dr. SK Dubey in synthesizing Ru(II)-CNEB in the lab of LM
is also acknowledged. RKK thanks CSIR, Govt. of India for awarding
Senior Research Fellowship during the tenure of this work at BHU.
The authors are thankful to UGC Centre of Advanced Study pro-
gramme to Department of Zoology and DBT-ISLS, BHU, for
providing facilities and assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biochi.2014.12.021.
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