PRECLINICAL STUDIES

Regression of Dalton’s lymphoma in vivo via declinein lactate dehydrogenase and induction of apoptosisby a ruthenium(II)-complex containing 4-carboxyN-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-carboxy N-ethylbenzamide (Ru(II)-CNEB) was found tointeract with and inhibit M4-lactate dehydrogenase (M4-LDH), a tumor growth supportive enzyme, at the tissuelevel. The present article describes modulation of M4-LDHby this compound in a T-cell lymphoma (Dalton’s Lym-phoma: DL) vis a vis regression of the tumor in vivo. Thecompound showed a dose dependent cytotoxicity to DLcells in vitro. When a non toxic dose (10 mg/kg bw i.p.) ofRu(II)-CNEB was administered to DL bearing mice, it alsoproduced a significant decline in DL cell viability in vivo.The DL cells from Ru(II)-CNEB treated DL mice showed asignificant decline in the level of M4-LDH with aconcomitant release of this protein in the cell free asciticfluid. A significant increase of nuclear DNA fragmentationin DL cells from Ru(II)-CNEB treated DL mice alsocoincided with the release of mitochondrial cytochrome cin those DL cells. Importantly, neither blood basedbiochemical markers of liver damage nor the normalpatterns of LDH isozymes in other tissues were affecteddue to the treatment of DL mice with the compound. Theseresults were also comparable with the effects of cisplatin(an anticancer drug) observed simultaneously on DL mice.The findings suggest that Ru(II)-CNEB is able to regressDalton’s lymphoma in vivo via declining M4-LDH and

inducing mitochondrial dysfunction–apoptosis pathwaywithout producing any toxicity to the normal tissues.

Keywords Dalton’s lymphoma . Ruthenium .

Ru(II)-CNEB . Lactate dehydrogenase (LDH) . Apoptosis .

Anticancer agent

Introduction

Amongst non-platinum anti-cancer metal compounds, Ru-thenium complexes are of much current interest due to theirlow toxicity, effective bio-distribution, reproducible bio-activities [1, 2] and in some cases their selective anti-metastatic properties [3]. The DNA, once considered as themain target of metallo-drugs [1, 4, 5], is now evident to beunselective [6]. Therefore, instead of targeting compoundsto interact with DNA, directing them to attenuate certainbiochemical steps which are over expressed in the tumors isan evolving concept [6–8]. Ru-complexes are found to bemore versatile in this respect [3], as Ru metal centre caninteract with and organize different ligands which canmodulate cellular functions differentially [9].

The glycolytic phenotype of tumor cells, popularlyknown as Warburg effect, is now evident to be a nearuniversal trait of all the growing tumors [10]. This ensuresadequate production of cellular energy through a non-mitochondrial route even when O2 supply is not a limitingfactor and thus, protects tumor cells from oxidative stress[11–13]. Therefore, to restrict tumor growth, inhibition oftumor glycolysis could be a logical target for the novelanticancer compounds [11, 12, 14, 15].

Inhibition of glycolysis by 2-deoxy-D-glucose [16] andinactivation of hexokinase II (HKII), the first committed

Invest New Drugs (2009) 27:503–516DOI 10.1007/s10637-008-9202-8

R. K. Koiri : S. K. Trigun (*) :K. Pandey :D. DixitBiochemistry & Molecular Biology Laboratory, Centreof Advanced Studies in Zoology, Banaras Hindu University,Varanasi 221005, Indiae-mail: sktrigun@sify.com

L. Mishra : S. K. DubeyDepartment of Chemistry, Banaras Hindu University,Varanasi 221005, India

enzyme of glycolytic pathway, by 3-bromopyruvate [17],have been reported to kill certain tumors in hypoxiccondition. Importantly, in colon cancer cells with mito-chondrial defects, it has been demonstrated that inactivationof tumor glycolysis activates glycolysis–apoptosis pathwaywith 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 incirculation [17]. In addition, if inactivation of HKII is nottumor specific, it is likely to affect normal cell energymetabolism also by restricting substrate supply to mito-chondrial oxidative phosphorylation. Therefore, it is im-portant to identify certain enzymatic proteins which areover expressed selectively in the cancer cells, and thus canbe targeted by the novel compounds.

In growing tumors, a hypoxia induced factor (HIF1α) isknown to activate the genes of glycolytic enzymes under avariety of oncogenic stimulations [12, 19]. HIF1α alsorestricts entry of pyruvate to TCA cycle by inhibitingpyruvate dehydrogenase (PDH) complex. As a result,mitochondrial function gets attenuated and pyruvate ischanneled to produce lactate by LDH [12]. Thus, enhancedproduction of lactate becomes a survival factor formalignant tumors [20]. Contrary to this, in normal cells,due to the less activity of HIF1α, pyruvate pool ischanneled to mitochondria for oxidative phosphorylationwithout implicating LDH. Therefore, selective inactivationof LDH is less likely to hamper energy metabolism innormal cells, however, it can inhibit energy yieldingpathway of tumor cells. Thus, LDH could be a target oftherapeutic intervention for restricting tumor growth.

Lactate dehydrogenase (LDH; EC: 1.1.1.27) is a tetra-meric protein consisting of two types of subunits, the M/Atype (preferentially catalyzes conversion of pyruvate tolactate) and the H/B type (pre-dominantly expressed in theaerobic tissues and catalyzes conversion of lactate topyruvate). Combination of these two sub-units in differentratio gives rise five LDH isozymes (M4, M3H, M2H2,MH3 and H4), which are expressed in a tissue specificmanner in most of the animals. M4-LDH (LDH-5, LDH-A)has been found to be over expressed in tumor cells tosupport increased production of lactate from pyruvate [21].The tumorogenic potential of M4-LDH deficient cells wasfound to be diminished drastically, however, it was found tobe recovered by complementation with the human orthologof M4-LDH [11]. Also, there are some reports on decreasein the growth of certain tumor cells in vitro due toinhibition of LDH activity by certain chemotherapeuticagents [22, 23].

Recently, we have synthesized and characterized a Ru(II)-CNEB complex which was found to be highlybiocompatible to mice in vivo and could interact with andinhibit M4-LDH non-competitively both in vitro and at the

tissue level [24]. The present article investigates whetherthis compound is (a) able to decline the level of active M4-LDH and to induce apoptosis in the tumor cells in vivo and(b) effective in restricting tumor growth without being toxicto the normal tissues.

Materials and methods

Chemicals

Ru(II)-CNEB was synthesized and characterized as de-scribed in an earlier report [24]. The ligand 4-carboxy N-ethylbenzamide (CNEB) was characterized by elementalanalysis and single crystal X-ray crystallography. On thebasis of elemental analysis and mass spectroscopy data[24], empirical composition of the final Ru(II)-CNEB com-plex was assigned as [Ru(CNEB-H)2(bpy)2]2PF6·0.5NH4PF6.The structure of the complex has been presented in Fig. 1,which suggests coordination of ligand with the metal throughits amide N due to the presence of electron releasing ethylgroup attached to it. The crystallographic data has beendeposited to the Cambridge Crystallographic Data Center,CCDC No. 618507.

β-NADH (β-nicotinamide adeninedinucleotide, reduced),NAD, Na-pyruvate, trypan blue, agarose, 4-carboxybenzalde-hyde, RuCl3·3H2O, ammonium hexafluorophosphate andanti β-actin were purchased from Sigma-Aldrich Co., USA.HRP-conjugated anti rabbit IgG and cisplatin [cis-diammi-nedichloroplatinum (II)] were obtained from Genei, andCipla respectively. Anti cytochrome c, hydroxylaminehydrochloride and ECL super signal western pico kit werepurchased from Santa Cruz, Fluka and Pierce respectively.cis-Ru(bpy)2Cl2·2H2O was prepared by a reported procedure[25]. SGOT (serum glutamate oxaloacetate transaminase)and SGPT (serum glutamate pyruvate transaminase) assaykits were purchased from Span Diagnostics Ltd, India. Nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), Li-

N

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2+

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Fig. 1 Structure of Ru(II)-CNEB: [Ru(CNEB-H)2(bpy)2] 2PF6·0.5NH4PF6

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lactate and other general chemicals were purchased fromSISCO Research Laboratory, Mumbai, India.

Induction of Dalton’s lymphoma (DL) in mice

Inbred AKR strain mice of 16–18 weeks age weighing 24–26 g were used for the experiments. Mice were maintainedunder standard laboratory conditions, as per the guidelinesand approval from the institutional animal ethical commit-tee, with free access to commercially available food pelletsand water. As described earlier [26, 27], Dalton lymphoma(DL) was induced by intraperitoneal (ip) serial trans-plantations of 1×107 viable tumor cells (assayed by trypanblue method) per mice with 100% success each time.Development of DL was confirmed by abnormal bellyswelling and increased body weight which were visible on10–12 days of implantation. The untreated DL micesurvived for 18±2 days.

Treatment schedule and study on survival time

Ru(II)-CNEB was first dissolved in the minimum volumeof 0.01% methanol followed by its further dilution inKreb’s ringer buffer (KRB) composed of 9 mM D-glucose,0.23 mM MgCl2, 4.5 mM KCl, 20 mM NaCl, 0.7 mMNa2HPO4, 1.5 mM NaH2PO4 and 15 mM NaHCO3

(pH 7.3). Different concentrations of the compound werealso prepared in KRB for ip injections. Through pilotexperiments, a dose of 10 mg/kg bw of Ru(II)-CNEB,given intraperitoneally, was found to be a sub-lethal dose tonormal mice and could increase the life span of DL bearingmice significantly. Therefore, this dose of Ru(II)-CNEBwas selected for the present study. The DL mice wererandomly divided into three groups with nine to ten mice ineach. The first group DL mice were treated with Ru(II)-CNEB complex (10 mg/kg bw/day, ip), second group withcisplatin (2 mg/kg bw/day, ip) and the third group,designated as DL control, were similarly injected withequal volume of KRB. As DL becomes visible on day 10–11 and DL bearing mice survived up to 18–20 days posttransplantation, the treatments of DL mice with thecompounds were started from day 11 of tumor transplantand continued up to day 17th. The normal control groupmice were also treated simultaneously with KRB. To studybiochemical/molecular parameters, three to four mice fromeach group were sacrificed on day 18th. The remainingmice in each group were allowed to be maintained onnormal diets to study their survival time after the treatment.

In order to assess the effects of compounds on generalappearance of DL mice, body weight of mice was recordedat an interval of 3 days starting from the day oftransplantation up to 21 days. The mortality was noted ineach group and increases in the survival time of mice of

both the treated groups were analyzed by Kaplan–Meiercurve.

Preparation of samples for biochemical studies

For biochemical studies, three to four mice from each groupwere sacrificed, volume of the collected tumor ascite fromeach group was measured and DL cells were pelleted bycentrifugation of ascites at 2,000×g for 10 min at 4°C.Other tissues like liver, kidney and brain were dissectedout, washed in ice-cold physiological saline, and stored at−70°C.

For blood based studies, blood samples from three tofour mice in each group were collected into sterilized tubescontaining heparin (15–20 IU/ml). For collecting serum, theblood was collected in unheparinized tubes, allowed to clotat room temperature (22°C) and centrifuged at 1,000×g for15 min.

Short term cytotoxicity assay

Through pilot experiments it was determined that 1×106–107 DL cells collected from ascitic fluids could bemaintained in sterilized KRB medium at 37°C up to>24 h without any loss of cell viability. Accordingly, forin vitro cytotoxicity assay of Ru(II)-CNEB, 1×106 viableDL cells were suspended in 0.25 ml KRB and wereincubated with the increasing concentrations of Ru(II)-CNEB (0.005–10 mg/ml) at 37°C for 30 min and 20 hduration separately. After respective time intervals, thenumber of viable DL cells was determined in each set bytrypan blue exclusion method. A 10 μl sample of cellsuspension was mixed with an equal volume of 0.4%trypan blue and the cells were counted using hemocytom-eter. Similar method was adopted to determine the numberof viable DL cells pelleted from the ascites of differentexperimental groups. The DL cell viability was recorded as% DL cell viability=(Total no of cells − trypan blue-stainedcells)/total no of cells)×100.

DNA fragmentation assay

Quantitative determination of fragmented DNA was carriedout as described earlier [28] with slight modifications.Briefly, DL cells were lysed in 0.5 ml of Tris–EDTA buffer(pH 7.4) containing 0.2% (v/v) triton X-100 and centrifugedat 13,000×g at 4°C for 10 min. The pellets containing totalintact DNA (designated P) and the supernatants containingsmaller fragments of DNA (designated S) were treatedseparately with 0.5 ml of 25% trichloroacetic acid (TCA).Both the sets were left overnight at 4°C and DNAprecipitated were collected by centrifugation. Each samplewas treated with 80 μl of 5% TCA followed by heat

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treatment at 90°C for 15 min. Freshly prepared 1 mldiphenylamine (DPA) reagent was added in each sample,tubes were allowed to stand overnight at room temperatureand OD was recorded at 600 nm. Percent DNA fragmen-tation was calculated as:

% DNA fragmentation ¼ S= S þ Pð Þ½ � � 100

Agarose gel electrophoresis of fragmented DNA

For electrophoretic analysis of fragmented DNA, the totalnuclear DNA was isolated from the DL cells according tothe method of Kuo et al. [29]. Briefly, 5×106 cells werelysed in 1 ml of lysis buffer [20 mM Tris–Cl (pH 7.5),0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and 25 mM Na2 pyrophosphate] at 37°C for 1 h. Toprecipitate out proteins, 0.4 ml of saturated NaCl was addedin each set of cell lysate, tubes were left on ice for 5 minand centrifuged at 3,000×g for 30 min. To separate DNAfrom the intact chromatin, RNase (20 μg/ml) was added tothe supernatants collected and allowed to stand at 37°C for15 min. DNA was then precipitated by adding two timeschilled ethanol (v/v). Samples were frozen at −70°Covernight. The DNA precipitated was collected by centri-fugation and dissolved in TAE buffer (40 mM Tris-acetate +1 mM EDTA).

The DNA samples were prepared in a loading solution(0.25% bromophenol blue, 0.25% xylene cyanol FF and30% glycerol) in the ratio of 1:5. The samples containing10 μg DNA were loaded in each well of 1% agarose gelcontaining 0.5 μg/ml ethidium bromide. The electrophore-sis was carried out in TAE buffer for 2–3 h. The DNAbands in gel were observed under UV transilluminator andphotographed.

Western blotting for cytochrome c releasefrom the mitochondria

DL cells were lysed in the lysis buffer containing 1 mMPMSF. The cell lysate was incubated on ice for 15 min,vortexed and centrifuged at 700×g for 10 min. To obtainmitochondria free cytosolic fraction, supernatant obtainedwas centrifuged at 10,000×g for 30 min. Following themethod described earlier [30], the samples containing60 μg protein, prepared in Laemmli buffer, were subjectedto 15% SDS-PAGE (sodium dodecyl sulphate-polyaryla-mide gel electrophoresis). Proteins were transferred tonitrocellulose membrane followed by detection of cyto-chrome c against a polyclonal anti-cytochrome c (1:1,000).The ECL super signal west pico kit was used to develop thebands on X-ray films. Using monoclonal anti-β-actinperoxidase antibody (1:10,000), level of β-actin wasprobed as a loading control. The bands were analyzed and

quantified using gel densitometry software AlphaImager2200.

Analysis of LDH isozymes by non-denaturingpolyacrylamide gel electrophoresis (PAGE)

Non-denaturing PAGE is a preferred method to analyzeexpression pattern of LDH isozymes. It employs substratespecificity based detection of all the isozymes of LDHdistinctly in the same gel, and thus, it is considered highlyrelevant for correlating a change in the level of a specificLDH isozyme with that of a metabolic alteration at cellularlevel. This method has been successfully applied tounderstand the implications of critical enzymes like SODand LDH in cancer and neuropathology [31–33].

Tissue extracts were prepared as described earlier fromthis lab [30]. LDH isozymes in DL cell lysates and othertissues were analyzed on non-denaturing 10% PAGEfollowing the method described earlier [32]. Briefly, theextracts containing 60 μg protein were loaded in each laneand electrophoresis was performed at 4°C. Gels weresubjected to substrate specificity based detection of LDHbands [32] followed by scanning and quantification of thebands as described earlier. The different isozymes of LDHin the gel were characterized by comparing their relativemigration (from cathodic to anodic) with those of the tissuespecific standard LDH bands [2, 24, 32].

Analysis of superoxide dismutase (SOD: Mn-SOD)by non-denaturing PAGE

As described in case of analysis of LDH isozymes, 12%non-denaturing PAGE was performed to determine the levelof active fraction of SOD2 in different DL cell extracts. Theactive SOD bands were developed following the methodreported recently from our lab [33]. The gel was scannedand SOD bands were quantified as described in theprevious text. The Mn-SOD (SOD2) in the gel wasidentified by its greater cathodic migration than that ofSOD1 (Cu/Zn-SOD) which migrates faster towards anode[33].

Other biochemical measurements

Protein concentrations in tissue extracts and in the blood/serum were measured following the method of Bradford[34]. The activity of LDH in cell extracts was measured asdescribed in an earlier report [24] and oxidation of 1 μmolof NADH per min at 25°C was defined as 1 U of theenzyme and values were presented as unit per milligramprotein.

The levels of SGOT and SGPT were determinedfollowing the manual of the kits used. Non-denaturing

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PAGE was performed to analyze LDH isozymes in theserum also.

Statistical analysis

Kaplan–Meier survival curves for the treated and untreatedgroup of DL mice were compared by using the log-ranktest. Other experimental data were expressed as mean±SDand wherever required, Student’s t test was applied fordetermining the level of significance. p<0.05 was consid-ered significant.

Results

To evaluate anticancer potential of Ru(II)-CNEB in vivo,we selected a transplantable T cell lymphoma (Dalton’slymphoma: DL) as a tumor model, because, DL can beinduced in rodents within a short period of time with >95%reproducibility and with clear visible symptoms which canbe used for monitoring the progression as well as regressionof the DL throughout the period of experimentation. Inaddition, homogeneous DL cells can be precipitated fromthe ascitic fluid for studying the biochemical and molecularchanges associated with development/regression of thetumor [26, 35]. Importantly, some mechanistic aspects ofanticancer activity of cisplatin have been worked out usingthis model [26, 36, 37]. We could also induce DL in micewith 100% success each time and used DL bearing mice forin vivo evaluation of Ru(II)-CNEB as an anticancer agent.

Cytotoxicity of Ru(II)-CNEB on DL cells in vitro

Cytotoxicity assay was done to ascertain whether Ru(II)-CNEB is able to kill DL cells in vitro. For this, DL cellswere maintained in a physiological buffer medium and theirviability was assayed after incubating them with increasingconcentrations of the compound for 30 min and 20 h. Asshown in Fig. 2, in 30 min set, though DL cell viability wasnot affected up to 0.5 mg/ml Ru(II)-CNEB, a significantdecline in the number of viable DL cells was observed athigher concentrations of the compound (at 5 mg/ml; p<0.01and at 10 mg/ml; p<0.001). Moreover, when incubationperiod was increased to 20 h, a linear decline in the numberof viable DL cells was observed starting from 0.05 mg/ml(p<0.01) to 10 mg/ml (p<0.001) of the compound. Thus, itwas evident that Ru(II)-CNEB is able to kill DL cells in vitroin a dose and incubation time dependent manner.

Effect of Ru(II)-CNEB on regression of DL cells in vivo

In order to confirm whether administered dose of Ru(II)-CNEB is able to restrict the development of DL and/or to

decrease the DL cell viability in mice, ascitic fluid collectedfrom untreated and treated DL bearing mice were analyzed.As compared to the untreated DL mice, there was asignificant decrease (p<0.01) in the volume of ascitecollected from the DL mice treated with the both, Ru(II)-CNEB and cisplatin (Fig. 3a). Moreover, there was a drasticdecline (~80%) in the number of viable DL cells in thesamples of ascites collected from both, the Ru(II)-CNEBand cisplatin treated DL mice (Fig. 3b).

Release of LDH in cell free medium indicates celldamage in vitro as well as in vivo. In comparison to thelevel of M4-LDH in the cell free ascitic fluid fromuntreated DL mice, there was a significant increase in thelevel of M4-LDH in those from Ru(II)-CNEB and cisplatintreated DL mice (Fig. 3c). These findings clearly suggestthat both the compounds tested are able to restrict DLdevelopment and also to induce death of DL cells in vivo.

Effect of Ru(II)-CNEB on the level of M4-LDH in DL cells

Increased LDH activity is associated with tumor develop-ment. To ascertain whether Ru(II)-CNEB and cisplatin areable to decrease the activity of this enzyme in DL cells,LDH activity was compared in DL cells from untreated andtreated DL mice. According to Fig. 4a, as compared to theLDH activity observed in DL cells from the untreatedgroup, there was a significant decrease (p<0.01) in theactivity of this enzyme in DL cells from Ru(II)-CNEBtreated DL mice. However, activity of LDH was found tobe increased significantly in DL cells from the cisplatintreated DL mice.

In order to ascertain whether Ru(II)-CNEB affects thelevel of active M4-LDH and/or the level of other LDHisozymes in DL cells, non-denaturing PAGE analysis waspreferred over the immunostaining methods. Though this

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Fig. 2 Effect of increasing concentrations of Ru(II)-CNEB on DLcells in vitro. DL cells (1×106) for each set were maintained in KRBmedium and incubated with the indicated concentrations of thecompound for different time intervals. Viability of DL cells after30 min and 20 h incubation was determined by trypan blue exclusionmethod. The data represent mean±SD from three to four experimentalrepeats. **p<0.01; ***p<0.001 (control versus respective experimen-tal sets)

Invest New Drugs (2009) 27:503–516 507

method is relatively less sensitive than that of immunode-tection, however, it is more relevant for interpretingmetabolic changes associated with the enzymatic altera-tions. Because, this method utilizes substrate specificitybased detection of only active fraction of the enzymeexcluding inactive/denatured proteins, which otherwise cannot be excluded by immunostaining method.

Figure 4b shows that DL cells from the untreated DLmice expressed high amount of only M4-LDH which wasfound to be decreased significantly (p<0.01) in the DL cellsfrom Ru(II)-CNEB treated DL mice. However, there wasno change in the level of M4-LDH in the DL cells fromcisplatin treated DL mice. This suggest that among the twocompounds tested, only Ru(II)-CNEB caused a decline inthe level of M4-LDH in DL cells in vivo.

Apoptosis of DL cells in vivo by Ru(II)-CNEB

The DNA fragmentation assay is a reliable tool to ascertainapoptotic cell death. In the present context, using a standardmethod, the percentage of fragmented DNA in DL cellextracts was measured followed by its analysis on agarosegel electrophoresis. There was a significant increase (p<0.01) in the level of fragmented DNA in DL cells fromboth, the Ru(II)-CNEB and cisplatin treated DL mice(Fig. 5a). This was further confirmed by the results ofagarose gel electrophoresis (Fig. 5b) wherein, DNAsamples from the DL cells collected from both the treated

group mice showed the smears of fragmented DNA. Whereas,the DNA isolated from DL cells of untreated mice showed asingle DNA band at higher molecular weight (MW) range.These results clearly suggest that both the compounds testedwere able to induce apoptosis in DL cells in vivo.

Oxidative stress and mitochondrial dysfunction areknown to initiate final steps of apoptosis. Superoxidedismutase (SOD) is the first committed enzyme thatneutralizes oxygen free radical (O2)

− based oxidative stressin the cells. Therefore, a decrease in the level of Mn-SOD(SOD2: mitochondrial isoform) may be considered as anindicator of oxidative stress in mitochondria. As shown inFig. 6a, there was a significant increase (p<0.05) in thelevel of active SOD2 in DL cells from Ru(II)-CNEB treatedDL mice than that from the untreated DL group. However,the DL cells from cisplatin treated DL mice showed asignificant decrease (p<0.01) in the level of active SOD2.

Release of mitochondrial cytochrome c in the cytosolindicates for induction of mitochondrial dysfunction–apoptotic pathway in the cells. Therefore, the level ofcytochrome c in the cytosol of DL cells from untreated andtreated DL groups was compared. As compared to the DLcells from untreated DL group, cytosolic fractions of DLcells from Ru(II)-CNEB and cisplatin treated DL miceshowed a significant increase (p<0.05) in the level ofcytochrome c (Fig. 6b). The results suggest induction ofmitochondrial dysfunction–apoptotic pathway in the DLcells in vivo by both the compounds tested.

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Fig. 3 Effects of Ru(II)-CNEB and cisplatin treatment on ascitic fluidvolume (a), viability of DL cells (b) and on the release of M4-LDH inthe cell free ascitic fluid (c) of DL bearing mice. In case of (a) and (b),the data represents mean±SD where n=4. In (c), pooled cell freeascitic fluid from three to four mice containing 60 μg protein wasloaded in each lane, 10% non-denaturing PAGE was performed and

substrate specific LDH bands were developed in the gel. The gelphotograph is a representative of the three PAGE repeats. In lowerpanel of (c), relative densitometric values of LDH bands fromexperimental group, as percent of the control DL lane, have beenpresented as mean±SD from three PAGE repeats. *p<0.05; **p<0.01; ***p<0.001 (DL control versus treated DL groups)

508 Invest New Drugs (2009) 27:503–516

Improvements in the survival parameters of Ru(II)-CNEBtreated 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 assesswhether Ru(II)-CNEB was able to bring recovery in theDL associated symptoms in mice. As compared to thecontrol mice, DL implanted mice showed a significantincrease 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 cisplatinfrom day 11 to 17, a significant decrease (p<0.01) in thebody weight of DL mice was observed. In addition, ascompared to the mean survival time of untreated DL mice(18 days), the DL mice treated with Ru(II)-CNEB andcisplatin could survive up to 24 and 26 days respectively.Comparison of the survival time data on Kaplan–Meiersurvival curves (Fig. 8) using log-rank statistics suggests asignificant increase (p<0.001) in the survival time of DL

mice treated with Ru(II)-CNEB, which was comparablewith the effect of cisplatin treatment also. Thus, it wasevident that like cisplatin, Ru(II)-CNEB is also able tocause an increase in the survival period of DL bearing micewith remarkable improvements in DL associated symptoms.

Effect of Ru(II)-CNEB treatment on normal tissues of DLmice

Liver metabolizes most of the drugs and kidney filters outall the unwanted exogenous substances. Therefore, thesetwo organs are likely to be affected up to a greater extent bythe drug treatment. Also, it is important to ensure that ananticancer compound does not cross the blood brain barrierand central nervous system remains protected during thetreatment. Therefore, these three tissues were selected toassess toxicity of Ru(II)-CNEB on the normal tissue.

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Fig. 5 Effects of Ru(II)-CNEB and cisplatin treatment on DNAfragmentation in DL cells of DL bearing mice. The values in (a)represent mean±SD of three experimental repeats from the pooled DLcell extracts collected from four DL mice. In case of (b), 10 μg DNAextracted from the pooled DL cells from three to four DL mice wasloaded in each lane and subjected to 1% agarose gel electrophoresisfollowed by detection of ethidium bromide stained DNA bands underUV transilluminator. The photograph is a representative of threerepeats. *p<0.05 (untreated versus treated groups)

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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.The values in (a) represent mean±SD where n=4 and each experimentdone in duplicate. In case of (b), pooled DL cell extracts from fourmice containing 60 μg protein in each lane was electrophoresed on10% non-denaturing PAGE followed by substrate specific develop-ment of LDH bands. The gel photograph is a representative of thethree PAGE repeats. In lower panel of (b), relative densitometricvalues of LDH bands from experimental group, as percent of thecontrol DL lane, have been presented as mean±SD from three PAGErepeats. **p<0.01 (DL control versus treated DL groups)

Invest New Drugs (2009) 27:503–516 509

Increased level of serum LDH indicates for vital tissuedamage and the levels of SGOT and SGPT are used asblood based markers of liver damage. The serum of controlas well as all the DL group mice showed the presence ofmainly M4-LDH with a minor fraction of M3H isozyme(Fig. 9). As compared to their levels in the serum of normalmice, both these isozymes were significantly increased (p<0.05) in the serum of untreated DL mice. However, both ofthem remained unchanged, as compared to the normalmice, in the serum of Ru(II)-CNEB and cisplatin treated DLgroup mice. Also, the levels of SGOT and SGPT werefound to be unaltered among the normal, untreated DL and

Ru(II)-CNEB as well as cisplatin treated DL group mice(Fig. 10a,b). Though there was a significant increase in thelevel of M4-LDH (p<0.01) in the liver of control DL micethan that of the normal mice, no change in the level of thisenzyme was observed in the liver of DL mice treated withRu(II)-CNEB (Fig. 10c). However, as compared to theuntreated DL group, a significant decline in the level ofM4-LDH could be seen in the liver of cisplatin treated DLmice.

40

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Fig. 8 Kaplan–Meier survival curve for untreated DL mice and theDL mice treated with Ru(II)-CNEB (DL + Rc) and cisplatin (DL +Cpt). The log-rank analysis was performed to examine the level ofsignificance and a p value of <0.001 was obtained in case of both thetreated group of DL mice versus untreated DL mice

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Fig. 6 Effects of Ru(II)-CNEB and cisplatin treatment on the level ofactive SOD2 (a) and on cytochrome c release in the cytosol of DLcells (b) of DL bearing mice. In (a), pooled DL cell extracts from fourmice containing 60 μg protein in each lane were electrophoresed on12% non-denaturing PAGE followed by substrate specific develop-ment of SOD2 bands in the gel. The gel photograph is a representativeof the three PAGE repeats. In lower panel of (a), relativedensitometric values of SOD2 bands from the treated group, aspercent of the control lane (untreated DL group), have been presentedas mean±SD from three PAGE repeats. b Immunoblotting results

wherein, pooled DL cell extracts from three to four DL micecontaining 60 μg protein in each lane was subjected to 15% SDS-PAGE followed by western transfer on nitrocellulose membrane anddetection of cytochrome c bands using a polyclonal anti cytochrome c.The level of β actin was probed as the loading control. Thephotograph is a representative of the three western blot repeats. Inlower panel of (b), normalized values of cytochrome c/β actin havebeen presented as mean±SD from three western blot repeats.*p<0.05; **p<0.01; (untreated versus treated groups)

510 Invest New Drugs (2009) 27:503–516

Results in Fig. 11a and b re-confirm that mice kidneyand brain express all the five LDH isozymes. However, ascompared to the LDH pattern observed in the kidney ofnormal mice, there was a significant decrease (p<0.05) inthe level of all the five isozymes in that of untreated DLmice, but with no change in case of Ru(II)-CNEB and

cisplatin treated DL group mice (Fig. 11a). Unchangedpatterns of all the five LDH isozymes were also observed inthe brain of control and all the DL (untreated and treated)group mice (Fig. 11b). These results clearly suggest that theadministered dose of Ru(II)-CNEB was nontoxic to thevital tissues of the DL bearing mice.

Discussion

Low toxicity and efficient bio-distribution of Ru-complexesare of great advantage over other metal complexes forevaluating their anticancer potential in vivo [2, 3]. This wasfound to be true in case of Ru(II)-CNEB also. When anontoxic dose of the compound, determined for normalmice, was administered to DL bearing mice, it resulted in asignificant decrease in the number of viable DL cell in vivo(Fig. 3b) without producing any toxic effect on the othernormal tissues (Figs. 9, 10, and 11).

The in vitro studies provide primary level information oncytotoxic potentials of a novel compound. We could alsoobserve a dose and time dependent decrease in the numberof viable DL cells by Ru(II)-CNEB in vitro (Fig. 2).However, a more pronounced decrease observed in theviability of DL cells from Ru(II)-CNEB treated DL mice(Fig. 3b) clearly indicated a greater anticancer activity ofthis compound in vivo than ex vivo. Some other Ru-complexes have also been shown to be less toxic in vitrobut could cause potent anti tumor activity in vivo [38].

It has been suggested that different Ru-complexes showtheir anticancer activities via distinctly different mecha-nisms such as by interacting with DNA and some serum

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Fig. 9 Effects of Ru(II)-CNEB and cisplatin treatment on the releaseof LDH in the serum of DL mice. Pooled serum from three to fourmice containing 60 μg protein in each lane was electrophoresed on10% non-denaturing PAGE followed by substrate specific develop-ment of LDH bands. The gel photograph is a representative of thethree PAGE repeats. In lower panel, relative densitometric values ofLDH bands from experimental group, as percent of the control lane,have been presented as mean±SD from three PAGE repeat experi-ments. #p<0.05 (normal control versus untreated DL group)

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Fig. 10 Effects of Ru(II)-CNEB and cisplatin treatment on the levelsof SGOT (a), SGPT (b) and M4-LDH in the liver (c) of DL bearingmice. The values in (a) and (b) represent mean±SD where n=4 andeach assay done in triplicate. In (c), pooled liver extracts from fourmice containing 60 μg protein in each lane was electrophoresed on10% non-denaturing PAGE followed by substrate specific develop-

ment of LDH bands. The gel photograph is a representative of thethree PAGE repeats. In lower panel of (c), relative densitometricvalues of LDH bands from experimental group, as percent of thenormal control lane, have been presented as mean±SD from threePAGE repeats. **p<0.01 (untreated DL versus treated DL groups);##p<0.01 (normal control versus untreated DL group)

Invest New Drugs (2009) 27:503–516 511

proteins and also by inhibiting certain enzymes likecytochrome c, protein kinase C, topoisomerase II etc [3].Being highly unselective, DNA is considered an unsuitabletarget for anticancer agents [7]. Alternatively, selecting aprotein as pharmacological target sounds better, however, itis important to first ensure that inactivation of a cellularprotein is cancer cell specific and does not hamper normalcell metabolism. In this respect, inhibiting glycolyticefficiency of tumor cells seems to be the most relevanttarget, as all tumor cells switch over to enhanced aerobicglycolysis [10] for their additional energy needs [11, 12].Also, the two key glycolytic enzymes, PKM2 (a fetalisoform of pyruvate kinase) and M4-LDH, have been foundto be over expressed selectively in most of the tumors andtherefore, both of these enzymes are argued to be the potentialtargets for novel anticancer compounds [8, 11, 21, 24].

Based upon our recent findings on inhibition of M4-LDH by Ru(II)-CNEB [24] and modulation of this enzymeby other metal complexes [32], we selected M4-LDH as atarget protein for evaluating anticancer activity of Ru(II)-CNEB. It has been reported that like most of the tumors,DL cells also over express M4-LDH [26, 27]. A highlyintense band of M4-LDH in the DL cell extracts fromuntreated DL mice (Fig. 4b; lane 1) also corroborate theseearlier findings and accordingly, a significant decline in thelevel of M4-LDH in DL cells from Ru(II)-CNEB treatedmice (Fig. 4b; lane 2) suggests that this compound was ableto decline the active level of this enzyme in DL cells invivo. Though immunostaining of M4-LDH would be of

future interest to determine the actual levels of this proteinin DL cells from treated and untreated mice, in the presentcontext, a significant decline in the level of active M4-LDHin DL cells of Ru(II)-CNEB treated mice suggests decreasein energy metabolism of the cancerous cell due to thetreatment with this compound. The tumor cells rely muchon the energy pathway lead by M4-LDH dependentproduction of lactate from pyruvate [11, 20, 21]. Thus,inactivation of this isozyme can severely affect only tumorcell energy metabolism. Contrary to this, as normal cellsutilize pyruvate for mitochondrial oxidative phosphoryla-tion rather than to produce lactate by M4-LDH, decline ofthis isozyme in normal tissues is less likely to affect theirenergy metabolism. This argument also justifies a greaterdecrease in the number of viable DL cells in vivo than invitro due to the treatment with Ru(II)-CNEB (Figs. 2 and3b). Isolated tumor cells maintained in vitro are devoid oftrue hypoxia and they can exploit aerobic pathway forenergy production even if LDH activity is declinedsignificantly and thus, can survive better. Contrary to this,due to greater hypoxia faced by the tumor cells in vivo,they rely much on anaerobic glycolysis [12] and thus, as aconsequence of diminished M4-LDH activity, they can bedeprived of adequate energy production resulting into poorsurvival. In addition, it has been reported [20] that tumorstroma associated fibroblasts help in the survival of tumorcells via recycling of lactate produced in excess by thetumor cells. However, the blockage of tumor LDH-5 (M4-LDH) suppresses this additional route of metabolic supple-

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Fig. 11 Effects of Ru(II)-CNEB and cisplatin treatment on the levelof LDH isozymes in kidney (a) and brain (b) of DL mice. In upperpanels of (a) and (b), the pooled tissue extracts from four micecontaining 60 μg protein in each lane was electrophoresed on 10%non-denaturing PAGE followed by substrate specific development ofLDH bands. The gel photographs are the representative of the three

PAGE repeats for each tissue. In lower panel of (a) and (b), relativedensitometric values of LDH bands from experimental group, aspercent of the control DL lane, have been presented as mean±SDfrom three PAGE repeats. ##p<0.01 (normal control versus untreatedDL group)

512 Invest New Drugs (2009) 27:503–516

mentation and thus, can render tumor cells susceptible todeath [20].

Tissue damage causes leakage of LDH in body fluids[39, 40]. Thus, a significant increase in the level of M4-LDH in the cell free ascitic fluid from Ru(II)-CNEB treatedDL mice, than that from the untreated DL mice (Fig. 3c;lane 1 vs lane 2), indicates for DL cell death caused by thiscompound in vivo. A more pronounced increase in the levelof M4-LDH in cell free ascitic fluid from cisplatin treatedDL mice (Fig. 3c; lane 3) further strengthened thisargument, as cisplatin induced regression of DL cells hasbeen shown to accompany the release of LDH in asciticfluid [26]. Moreover, since cisplatin did not alter the levelof M4-LDH in DL cells, which was observed to be declinedsignificantly by Ru(II)-CNEB, it may be speculated that themechanism of cell death caused by both the compounds aredifferent from each other.

Development of Dalton’s lymphoma is characterized bythe increments in the body weight and volume of the asciticfluid and thus, measurement of both these parameters areused to determine the development of DL and its regressionin vivo [41, 42]. In comparison to the untreated DL mice,~50% decrease observed in the ascitic volume in case of Ru(II)-CNEB treated DL mice (Fig. 3a) suggest that thiscompound was able to restrict DL development in mice.The range of reduction observed in ascitic volume iscomparable with a ∼2 times reduction caused by the extractof a macrofungus [42] and ∼50% reduction in tumor weightby the extract of Withania somnifera [43]. Reports arescanty on Ru-complex induced regression of lymphoma invivo. Therefore, ∼80% decline in the number of viable DLcells (Fig. 3b) in Ru(II)-CNEB treated DL mice is of greatrelevance. The reductions in ascitic volume and DL cellviability by Ru(II)-CNEB treatment were also comparablewith the data obtained with cisplatin treatment and thereby,suggesting further for a potent anticancer activity of Ru(II)-CNEB on DL in vivo.

One of the major mechanisms in cancer therapy is toinduce apoptosis in transformed cells by chemotherapeuticagents [44, 45]. Some Ru(II)-complexes derived organo-metallic compounds have been reported to mediate theircytotoxicity on lymphoma cell lines in vitro via inductionof apoptosis [46]. However, reports are scanty on theinduction of apoptosis in tumor cells in vivo by Ru(II)-complexes. Increased fragmentation of DNA is an impor-tant parameter to suggest apoptotic death of a cell. Thus, asignificant increase in the level of fragmented DNA in theDL cells from Ru(II)-CNEB treated DL mice (Fig. 5a, b)clearly suggests that Ru(II)-CNEB is able to induceapoptosis in DL cells in vivo. Release of cytochrome cfrom mitochondria is an indicator of mitochondrial dys-function and has been correlated with the induction ofapoptosis under a variety of metabolic derangements and

oxidative stress [47]. We have observed a direct relation-ship between the release of cytochrome c and increasedlevel of DNA fragmentation in the DL cells from Ru(II)-CNEB treated DL mice (Figs. 5 and 6b). A similar patternof DNA fragmentation and increased level of cytochrome crelease in the DL cells from cisplatin treated DL mice werealso observed and thus, suggesting that both, Ru(II)-CNEBand cisplatin, have been able to induce apoptosis in DLcells in vivo via release of cytochrome c.

There could be more than one mechanism for inducingapoptosis by chemotherapeutic agents. Anticancer drugcausing induction of apoptosis via inhibition of glycolysisin tumor cells is a relatively new concept [18, 48]. Thoughthe link between inhibition of glycolysis and tumor cellapoptosis is yet to be defined, it may be speculated thatdepletion of energy and growth promoting substrates due todecline in glycolytic efficiency could act as an inducer ofapoptosis in the tumor cells. Tumor cells show aberrantNADH/NAD shuttle of mitochondria resulting into in-creased level of NADH in the cytosol [49]. This may alterredox state of the cells and can induce final apoptoticpathway in those cells [50]. Decline in the level of M4-LDH, which utilizes NADH as substrate, may furthercontribute for the accumulation of NADH in cytosol. Thisargument gets support from ~2 times increase in NADH/NAD ratio observed in M4-LDH deficient tumor cells [11].Thus, it may be argued that the resultant increase in NADH/NAD ratio, due to a significant decrease in the level of M4-LDH, might be implicated as a biochemical mediator toinduce apoptosis in DL cells in Ru(II)-CNEB treated DLmice. Mitochondrial dysfunction in DL cells of treatedgroup mice has also been suggested by a significantincrease in the release of mitochondrial cytochrome c(Fig. 6b).

Alternatively, DNA damage also induces apoptosis,however, such a possibility in this case was ruled out byobserving a poor DNA-Ru(II)-CNEB interaction in vitro(unpublished data). Also, (O2)

− based oxidative stress isknown to cause cytochrome c release and in turn inductionof apoptosis in the affected cells, however, under depletedantioxidant condition. SOD is the committed enzyme ofantioxidant pathway and Mn-SOD (SOD2) in particularplays a critical role in protecting mitochondria from (O2)

−

insult. We observed a significant increase in the level ofSOD2 in the DL cells from Ru(II)-CNEB treated than thosefrom the untreated DL mice (Fig. 6a). This suggests thatantioxidant potential of DL cells was not depleted due tothe treatment with Ru(II)-CNEB and hence, rules outpossibility of a role of (O2)

− based oxidative stress in Ru(II)-CNEB induced apoptosis in the DL cell.

Thus, the results presented here suggest that Ru(II)-CNEB might be implicating the decline of M4-LDH andmitochondrial dysfunction to induce apoptosis in DL cells

Invest New Drugs (2009) 27:503–516 513

in vivo. The induction of glycolysis–apoptotic pathway intumor cells due to chemotherapeutic intervention is arelatively less explored area. Therefore, these findings areof much current interest with respect to identify novel Ru-complexes which can inhibit a critical step of glycolyticpathway resulting into induction of apoptosis in the tumorcells in vivo.

Cisplatin is a well studied compound on a variety oftumors [51]. It was interesting to note that cisplatin alsoinduced apoptosis in DL cells in vivo via release ofcytochrome c, however, without affecting the level of M4-LDH. Thus, it is likely that cisplatin might be adoptingLDH independent mechanism to induce apoptosis in DLcells. DNA has been shown to be the major target ofcisplatin induced cytotoxicity, wherein, cisplatin-DNAadduct formation is known to induce oxidative stress andfinally to initiate tumor cell death [52]. There was asignificant decline in the level of SOD2 with a concomitantincrease in the level of cytochrome c in the DL cells fromcisplatin treated DL mice (Fig. 6a, b). Thus, it may beargued that, as against the role of Ru(II)-CNEB in DL cellapoptosis, cisplatin adopts (O2)

− dependent mitochondrialdysfunction pathway to induce apoptosis in these cells.Caspase 9, an important component of oxidative stressinduced apoptosis, has also been reported to be implicatedin apoptotic death of certain tumor cells by cisplatin [45].

Increase in the life span and improvement in overallappearance of cancerous animal after the treatment are theultimate criteria to ascertain anticancer potential of achemotherapeutic agent. A significant decline (∼50% ) inbody weight (Fig. 7) of the Ru(II)-CNEB treated DL miceand a significant increase in their survival period (Fig. 8)suggest that the molecular alterations induced by thiscompound has resulted into an overall improvement in thelife of the cancerous mice. The ranges of decrease in thebody weight and increase in the survival period reportedhere are well correlated with the similar findings on DLbearing animal treated with the extracts of a macrofungus[42] and that on TLX5 lymphoma bearing mice treated withthe different antimetastatic agents [38]. Also, the findingson Ru(II)-CNEB treated DL mice were comparable andvery close to the data obtained from the DL mice treatedwith cisplatin (Figs. 7 and 8). NAMI-A is the most widelystudied Ru-complex as an anticancer agent which was alsoshown to reduce the increased body weight of thecancerous animal maximum up to 50% that too when givenin combination with cisplatin [53]. The data on theincreased survival time reported here sounds further betterthan only a 12% increase observed in the life span ofEhrlich ascite bearing mice due to the treatment with a Ru(II)-complex, [cis-Ru (II) DMSO Cl2] [49]. Thus, ourfindings suggest potent anticancer activity of Ru(II)-CNEBagainst Dalton’s lymphoma in mice and thereby, provide a

basis for standardizing the dose and the treatment scheduleof this compound against a variety of tumors in vivo.

The major limitation of cancer therapy is the injury ofnormal tissues leading to multiple organ toxicity [54].Detection of increased level of LDH in serum is a widelyused parameter for blood based diagnosis of tissue damageas well as to characterize the rapid turnover of cancerouscells in vivo [39]. Therefore, unaltered patterns of serumLDH observed in case of Ru(II)-CNEB and cisplatin treatedDL mice (Fig. 9; lanes 3 and 4 versus lane 1) clearlysuggest that no damage has occurred to the normal tissuesdue to the treatment with both these compounds. Accord-ingly, a significant increase in the level of M4-LDH inserum of untreated DL mice (Fig. 9; lane 2) may becorrelated with the rapid turnover of DL cells in theuntreated DL mice.

Most of the drugs given through systemic routes undergotheir final metabolism in liver, and therefore, liver is likelyto be affected adversely during chemotherapeutic treat-ments [54]. Increased levels of SGOT and SGPT are themost widely used blood based markers to ascertain liverdysfunction. Therefore, unaltered patterns of SGOT andSGPT in the treated and untreated DL mice (Fig. 10a, b)indicate that doses of both the compounds tested are nontoxic to the liver. In addition, corroborating an earlierfinding [27], though M4-LDH was slightly increased in theliver of untreated DL mice, it remained unaltered in theliver of Ru(II)-CNEB treated DL mice (Fig. 10c) and thus,suggesting further that liver of DL mice was unaffected dueto the treatment with this compound. Kidney is involved inthe filtration of blood born factors continuously. Thoughthe levels of all LDH isozymes were found to be decreasedin the kidney of DL bearing mice, they remained unalteredin the DL mice treated with Ru(II)-CNEB (Fig. 11a). Bloodbrain barrier protects brain from most of the exogenousfactors. It was evident in the present context also. All thefive LDH isozymes were found to remain unaltered in thebrain of both, the treated and the untreated group of DLmice (Fig. 11b) and thus, suggesting that neither thedevelopment of DL nor Ru(II)-CNEB treatment causedany alteration in the expression pattern of any of the LDHisozymes in the mouse brain. Thus, it is evident that thedose of Ru(II)-CNEB used in this experiment did notproduce any damage to the normal tissues in vivo. The doseof cisplatin used also did not produce much change in thelevel of LDH isozymes in the normal tissues. However, itcaused a significant decline in the level of M4-LDH in liverand thereby, indicated the possibility of liver toxicity bycisplatin. This also corroborates an earlier report on theeffect of cisplatin on liver LDH of DL mice [26].

In summary, the present study demonstrates that anontoxic dose of Ru(II)-CNEB is able to decrease theviability of DL cells in vivo with a concomitant increase in

514 Invest New Drugs (2009) 27:503–516

the life span of the tumor bearing mice without producingany toxicity to the normal tissues. The findings on Ru(II)-CNEB induced decline in M4-LDH and increments in thelevels of DNA fragmentation & release of cytochrome c inthe DL cells suggest that decreased tumor glycolysis andinduction of mitochondrial dysfunction–apoptosis pathwaycould be implicated in the anticancer activity of thiscompound. The precise mechanism by which the declineof M4-LDH by Ru(II)-CNEB causes mitochondrial dys-function and induces apoptosis in DL cells needs to bedefined further. Nonetheless, the findings reported here areof great significance with respect to identification of aprotein based pharmacological target in vivo for the novelchemotherapeutic agents.

Acknowledgment This work was financially supported by a projectfrom Department of Biotechnology (DBT), Govt. of India, (BT/PR5910/BRB/10/406/2005) sanctioned jointly to LM and SKT. Theauthors are thankful to UGC Centre of Advanced Studies programmeto Department of Zoology, BHU, for providing infrastructuralfacilities. The help extended by Mr. S. Bhattacharyya, Ms. S.Srivastav, and Ms. B. Mishra is also acknowledged.

Conflict of interest The authors declare that there are no conflicts ofinterest.

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