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Regression of dalton’s lymphoma in vivo via decline
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    Regression of dalton’s lymphoma in vivo via decline Regression of dalton’s lymphoma in vivo via decline Document Transcript

    • Invest New Drugs (2009) 27:503–516DOI 10.1007/s10637-008-9202-8 PRECLINICAL STUDIESRegression of Dalton’s lymphoma in vivo via declinein lactate dehydrogenase and induction of apoptosisby a ruthenium(II)-complex containing 4-carboxyN-ethylbenzamide as ligandRaj K. Koiri & Surendra K. Trigun & Lallan Mishra &Kiran Pandey & Deobrat Dixit & Santosh K. DubeyReceived: 23 September 2008 / Accepted: 12 November 2008 / Published online: 29 November 2008# Springer Science + Business Media, LLC 2008Summary A novel ruthenium(II)-complex containing 4- inducing mitochondrial dysfunction–apoptosis pathwaycarboxy N-ethylbenzamide (Ru(II)-CNEB) was found to without producing any toxicity to the normal tissues.interact with and inhibit M4-lactate dehydrogenase (M4-LDH), a tumor growth supportive enzyme, at the tissue Keywords Dalton’s lymphoma . Ruthenium .level. The present article describes modulation of M4-LDH Ru(II)-CNEB . Lactate dehydrogenase (LDH) . Apoptosis .by this compound in a T-cell lymphoma (Dalton’s Lym- Anticancer agentphoma: 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.) of IntroductionRu(II)-CNEB was administered to DL bearing mice, it alsoproduced a significant decline in DL cell viability in vivo. Amongst non-platinum anti-cancer metal compounds, Ru-The DL cells from Ru(II)-CNEB treated DL mice showed a thenium complexes are of much current interest due to theirsignificant decline in the level of M4-LDH with a low toxicity, effective bio-distribution, reproducible bio-concomitant release of this protein in the cell free ascitic activities [1, 2] and in some cases their selective anti-fluid. A significant increase of nuclear DNA fragmentation metastatic properties [3]. The DNA, once considered as thein DL cells from Ru(II)-CNEB treated DL mice also main target of metallo-drugs [1, 4, 5], is now evident to becoincided with the release of mitochondrial cytochrome c unselective [6]. Therefore, instead of targeting compoundsin those DL cells. Importantly, neither blood based to interact with DNA, directing them to attenuate certainbiochemical markers of liver damage nor the normal biochemical steps which are over expressed in the tumors ispatterns of LDH isozymes in other tissues were affected an evolving concept [6–8]. Ru-complexes are found to bedue to the treatment of DL mice with the compound. These more versatile in this respect [3], as Ru metal centre canresults were also comparable with the effects of cisplatin interact with and organize different ligands which can(an anticancer drug) observed simultaneously on DL mice. modulate cellular functions differentially [9].The findings suggest that Ru(II)-CNEB is able to regress The glycolytic phenotype of tumor cells, popularlyDalton’s lymphoma in vivo via declining M4-LDH and known as Warburg effect, is now evident to be a near universal trait of all the growing tumors [10]. This ensures adequate production of cellular energy through a non-R. K. Koiri : S. K. Trigun (*) : K. Pandey : D. Dixit mitochondrial route even when O2 supply is not a limitingBiochemistry & Molecular Biology Laboratory, Centreof Advanced Studies in Zoology, Banaras Hindu University, factor and thus, protects tumor cells from oxidative stressVaranasi 221005, India [11–13]. Therefore, to restrict tumor growth, inhibition ofe-mail: sktrigun@sify.com tumor glycolysis could be a logical target for the novelL. Mishra : S. K. Dubey anticancer compounds [11, 12, 14, 15].Department of Chemistry, Banaras Hindu University, Inhibition of glycolysis by 2-deoxy-D-glucose [16] andVaranasi 221005, India inactivation of hexokinase II (HKII), the first committed
    • 504 Invest New Drugs (2009) 27:503–516enzyme of glycolytic pathway, by 3-bromopyruvate [17], tissue level [24]. The present article investigates whetherhave been reported to kill certain tumors in hypoxic this compound is (a) able to decline the level of active M4-condition. Importantly, in colon cancer cells with mito- LDH and to induce apoptosis in the tumor cells in vivo andchondrial defects, it has been demonstrated that inactivation (b) effective in restricting tumor growth without being toxicof tumor glycolysis activates glycolysis–apoptosis pathway to the normal tissues.with a concomitant increase in tumor cell death [18].Nonetheless, effectiveness of 2-deoxy-D-glucose is signifi-cantly masked by the presence of normal glucose in Materials and methodscirculation [17]. In addition, if inactivation of HKII is nottumor specific, it is likely to affect normal cell energy Chemicalsmetabolism also by restricting substrate supply to mito-chondrial oxidative phosphorylation. Therefore, it is im- Ru(II)-CNEB was synthesized and characterized as de-portant to identify certain enzymatic proteins which are scribed in an earlier report [24]. The ligand 4-carboxy N-over expressed selectively in the cancer cells, and thus can ethylbenzamide (CNEB) was characterized by elementalbe targeted by the novel compounds. analysis and single crystal X-ray crystallography. On the In growing tumors, a hypoxia induced factor (HIF1α) is basis of elemental analysis and mass spectroscopy dataknown to activate the genes of glycolytic enzymes under a [24], empirical composition of the final Ru(II)-CNEB com-variety of oncogenic stimulations [12, 19]. HIF1α also plex was assigned as [Ru(CNEB-H)2(bpy)2]2PF6·0.5NH4PF6.restricts entry of pyruvate to TCA cycle by inhibiting The structure of the complex has been presented in Fig. 1,pyruvate dehydrogenase (PDH) complex. As a result, which suggests coordination of ligand with the metal throughmitochondrial function gets attenuated and pyruvate is its amide N due to the presence of electron releasing ethylchanneled to produce lactate by LDH [12]. Thus, enhanced group attached to it. The crystallographic data has beenproduction of lactate becomes a survival factor for deposited to the Cambridge Crystallographic Data Center,malignant tumors [20]. Contrary to this, in normal cells, CCDC No. 618507.due to the less activity of HIF1α, pyruvate pool is β-NADH (β-nicotinamide adeninedinucleotide, reduced),channeled to mitochondria for oxidative phosphorylation NAD, Na-pyruvate, trypan blue, agarose, 4-carboxybenzalde-without implicating LDH. Therefore, selective inactivation hyde, RuCl3·3H2O, ammonium hexafluorophosphate andof LDH is less likely to hamper energy metabolism in anti β-actin were purchased from Sigma-Aldrich Co., USA.normal cells, however, it can inhibit energy yielding HRP-conjugated anti rabbit IgG and cisplatin [cis-diammi-pathway of tumor cells. Thus, LDH could be a target of nedichloroplatinum (II)] were obtained from Genei, andtherapeutic intervention for restricting tumor growth. Cipla respectively. Anti cytochrome c, hydroxylamine Lactate dehydrogenase (LDH; EC: is a tetra- hydrochloride and ECL super signal western pico kit weremeric protein consisting of two types of subunits, the M/A purchased from Santa Cruz, Fluka and Pierce respectively.type (preferentially catalyzes conversion of pyruvate to cis-Ru(bpy)2Cl2·2H2O was prepared by a reported procedurelactate) and the H/B type (pre-dominantly expressed in the [25]. SGOT (serum glutamate oxaloacetate transaminase)aerobic tissues and catalyzes conversion of lactate to and SGPT (serum glutamate pyruvate transaminase) assaypyruvate). Combination of these two sub-units in different kits were purchased from Span Diagnostics Ltd, India. Nitroratio gives rise five LDH isozymes (M4, M3H, M2H2, blue tetrazolium (NBT), phenazine methosulfate (PMS), Li-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 to 2+ Osupport increased production of lactate from pyruvate [21]. N NThe tumorogenic potential of M4-LDH deficient cells wasfound to be diminished drastically, however, it was found to N N Ru R=be 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 chemotherapeutic C2H5 NH HN C2H5 HO Oagents [22, 23]. R R Recently, we have synthesized and characterized a Ru [Ru(CNEB-H)2(bpy)2 ]2PF6.0.5NH4PF6(II)-CNEB complex which was found to be highlybiocompatible to mice in vivo and could interact with and Fig. 1 Structure of Ru(II)-CNEB: [Ru(CNEB-H)2(bpy)2] 2PF6·0.5inhibit M4-LDH non-competitively both in vitro and at the NH4PF6
    • Invest New Drugs (2009) 27:503–516 505lactate and other general chemicals were purchased from both the treated groups were analyzed by Kaplan–MeierSISCO Research Laboratory, Mumbai, India. curve.Induction of Dalton’s lymphoma (DL) in mice Preparation of samples for biochemical studiesInbred AKR strain mice of 16–18 weeks age weighing 24– For biochemical studies, three to four mice from each group26 g were used for the experiments. Mice were maintained were sacrificed, volume of the collected tumor ascite fromunder standard laboratory conditions, as per the guidelines each group was measured and DL cells were pelleted byand approval from the institutional animal ethical commit- centrifugation of ascites at 2,000×g for 10 min at 4°C.tee, with free access to commercially available food pellets Other tissues like liver, kidney and brain were dissectedand water. As described earlier [26, 27], Dalton lymphoma out, washed in ice-cold physiological saline, and stored at(DL) was induced by intraperitoneal (ip) serial trans- −70°C.plantations of 1×107 viable tumor cells (assayed by trypan For blood based studies, blood samples from three toblue method) per mice with 100% success each time. four mice in each group were collected into sterilized tubesDevelopment of DL was confirmed by abnormal belly containing heparin (15–20 IU/ml). For collecting serum, theswelling and increased body weight which were visible on blood was collected in unheparinized tubes, allowed to clot10–12 days of implantation. The untreated DL mice at room temperature (22°C) and centrifuged at 1,000×g forsurvived for 18±2 days. 15 min.Treatment schedule and study on survival time Short term cytotoxicity assayRu(II)-CNEB was first dissolved in the minimum volume Through pilot experiments it was determined that 1×106–of 0.01% methanol followed by its further dilution in 107 DL cells collected from ascitic fluids could beKreb’s ringer buffer (KRB) composed of 9 mM D-glucose, maintained in sterilized KRB medium at 37°C up to0.23 mM MgCl2, 4.5 mM KCl, 20 mM NaCl, 0.7 mM >24 h without any loss of cell viability. Accordingly, forNa2HPO4, 1.5 mM NaH2PO4 and 15 mM NaHCO3 in vitro cytotoxicity assay of Ru(II)-CNEB, 1×106 viable(pH 7.3). Different concentrations of the compound were DL cells were suspended in 0.25 ml KRB and werealso prepared in KRB for ip injections. Through pilot incubated with the increasing concentrations of Ru(II)-experiments, a dose of 10 mg/kg bw of Ru(II)-CNEB, CNEB (0.005–10 mg/ml) at 37°C for 30 min and 20 hgiven intraperitoneally, was found to be a sub-lethal dose to duration separately. After respective time intervals, thenormal mice and could increase the life span of DL bearing number of viable DL cells was determined in each set bymice significantly. Therefore, this dose of Ru(II)-CNEB trypan blue exclusion method. A 10 μl sample of cellwas selected for the present study. The DL mice were suspension was mixed with an equal volume of 0.4%randomly divided into three groups with nine to ten mice in trypan blue and the cells were counted using hemocytom-each. The first group DL mice were treated with Ru(II)- eter. Similar method was adopted to determine the numberCNEB complex (10 mg/kg bw/day, ip), second group with of viable DL cells pelleted from the ascites of differentcisplatin (2 mg/kg bw/day, ip) and the third group, experimental groups. The DL cell viability was recorded asdesignated as DL control, were similarly injected with % DL cell viability=(Total no of cells − trypan blue-stainedequal volume of KRB. As DL becomes visible on day 10– cells)/total no of cells)×100.11 and DL bearing mice survived up to 18–20 days posttransplantation, the treatments of DL mice with the DNA fragmentation assaycompounds were started from day 11 of tumor transplantand continued up to day 17th. The normal control group Quantitative determination of fragmented DNA was carriedmice were also treated simultaneously with KRB. To study out as described earlier [28] with slight modifications.biochemical/molecular parameters, three to four mice from Briefly, DL cells were lysed in 0.5 ml of Tris–EDTA buffereach group were sacrificed on day 18th. The remaining (pH 7.4) containing 0.2% (v/v) triton X-100 and centrifugedmice in each group were allowed to be maintained on at 13,000×g at 4°C for 10 min. The pellets containing totalnormal diets to study their survival time after the treatment. intact DNA (designated P) and the supernatants containing In order to assess the effects of compounds on general smaller fragments of DNA (designated S) were treatedappearance of DL mice, body weight of mice was recorded separately with 0.5 ml of 25% trichloroacetic acid (TCA).at an interval of 3 days starting from the day of Both the sets were left overnight at 4°C and DNAtransplantation up to 21 days. The mortality was noted in precipitated were collected by centrifugation. Each sampleeach group and increases in the survival time of mice of was treated with 80 μl of 5% TCA followed by heat
    • 506 Invest New Drugs (2009) 27:503–516treatment at 90°C for 15 min. Freshly prepared 1 ml quantified using gel densitometry software AlphaImagerdiphenylamine (DPA) reagent was added in each sample, 2200.tubes were allowed to stand overnight at room temperatureand OD was recorded at 600 nm. Percent DNA fragmen- Analysis of LDH isozymes by non-denaturingtation was calculated as: polyacrylamide gel electrophoresis (PAGE)% DNA fragmentation ¼ ½S=ðS þ Pފ  100 Non-denaturing PAGE is a preferred method to analyze expression pattern of LDH isozymes. It employs substrateAgarose gel electrophoresis of fragmented DNA specificity based detection of all the isozymes of LDH distinctly in the same gel, and thus, it is considered highlyFor electrophoretic analysis of fragmented DNA, the total relevant for correlating a change in the level of a specificnuclear DNA was isolated from the DL cells according to LDH isozyme with that of a metabolic alteration at cellularthe method of Kuo et al. [29]. Briefly, 5×106 cells were level. This method has been successfully applied tolysed in 1 ml of lysis buffer [20 mM Tris–Cl (pH 7.5), understand the implications of critical enzymes like SOD0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X- and LDH in cancer and neuropathology [31–33].100 and 25 mM Na2 pyrophosphate] at 37°C for 1 h. To Tissue extracts were prepared as described earlier fromprecipitate out proteins, 0.4 ml of saturated NaCl was added this lab [30]. LDH isozymes in DL cell lysates and otherin each set of cell lysate, tubes were left on ice for 5 min tissues were analyzed on non-denaturing 10% PAGEand centrifuged at 3,000×g for 30 min. To separate DNA following the method described earlier [32]. Briefly, thefrom the intact chromatin, RNase (20 μg/ml) was added to extracts containing 60 μg protein were loaded in each lanethe supernatants collected and allowed to stand at 37°C for and electrophoresis was performed at 4°C. Gels were15 min. DNA was then precipitated by adding two times subjected to substrate specificity based detection of LDHchilled ethanol (v/v). Samples were frozen at −70°C bands [32] followed by scanning and quantification of theovernight. The DNA precipitated was collected by centri- bands as described earlier. The different isozymes of LDHfugation and dissolved in TAE buffer (40 mM Tris-acetate + in the gel were characterized by comparing their relative1 mM EDTA). migration (from cathodic to anodic) with those of the tissue The DNA samples were prepared in a loading solution specific standard LDH bands [2, 24, 32].(0.25% bromophenol blue, 0.25% xylene cyanol FF and30% glycerol) in the ratio of 1:5. The samples containing Analysis of superoxide dismutase (SOD: Mn-SOD)10 μg DNA were loaded in each well of 1% agarose gel by non-denaturing PAGEcontaining 0.5 μg/ml ethidium bromide. The electrophore-sis was carried out in TAE buffer for 2–3 h. The DNA As described in case of analysis of LDH isozymes, 12%bands in gel were observed under UV transilluminator and non-denaturing PAGE was performed to determine the levelphotographed. of active fraction of SOD2 in different DL cell extracts. The active SOD bands were developed following the methodWestern blotting for cytochrome c release reported recently from our lab [33]. The gel was scannedfrom the mitochondria and SOD bands were quantified as described in the previous text. The Mn-SOD (SOD2) in the gel wasDL cells were lysed in the lysis buffer containing 1 mM identified by its greater cathodic migration than that ofPMSF. The cell lysate was incubated on ice for 15 min, SOD1 (Cu/Zn-SOD) which migrates faster towards anodevortexed and centrifuged at 700×g for 10 min. To obtain [33].mitochondria free cytosolic fraction, supernatant obtainedwas centrifuged at 10,000×g for 30 min. Following the Other biochemical measurementsmethod described earlier [30], the samples containing60 μg protein, prepared in Laemmli buffer, were subjected Protein concentrations in tissue extracts and in the blood/to 15% SDS-PAGE (sodium dodecyl sulphate-polyaryla- serum were measured following the method of Bradfordmide gel electrophoresis). Proteins were transferred to [34]. The activity of LDH in cell extracts was measured asnitrocellulose membrane followed by detection of cyto- described in an earlier report [24] and oxidation of 1 μmolchrome c against a polyclonal anti-cytochrome c (1:1,000). of NADH per min at 25°C was defined as 1 U of theThe ECL super signal west pico kit was used to develop the enzyme and values were presented as unit per milligrambands on X-ray films. Using monoclonal anti-β-actin protein.peroxidase antibody (1:10,000), level of β-actin was The levels of SGOT and SGPT were determinedprobed as a loading control. The bands were analyzed and following the manual of the kits used. Non-denaturing
    • Invest New Drugs (2009) 27:503–516 507 120PAGE was performed to analyze LDH isozymes in the 30 min Viabilty of DL cellsserum also. 100 ** ** 20 h (% of control) 80 ***Statistical analysis 60 40 *** ***Kaplan–Meier survival curves for the treated and untreated 20group of DL mice were compared by using the log-rank *** 0test. Other experimental data were expressed as mean±SD 0.0 0.05 0.5 5.0 10.0and wherever required, Student’s t test was applied for Ru(II)-CNEB (mg/ml)determining the level of significance. p<0.05 was consid- Fig. 2 Effect of increasing concentrations of Ru(II)-CNEB on DLered significant. cells in vitro. DL cells (1×106) for each set were maintained in KRB medium and incubated with the indicated concentrations of the compound for different time intervals. Viability of DL cells afterResults 30 min and 20 h incubation was determined by trypan blue exclusion method. The data represent mean±SD from three to four experimental repeats. **p<0.01; ***p<0.001 (control versus respective experimen-To evaluate anticancer potential of Ru(II)-CNEB in vivo, tal sets)we selected a transplantable T cell lymphoma (Dalton’slymphoma: DL) as a tumor model, because, DL can beinduced in rodents within a short period of time with >95% decrease the DL cell viability in mice, ascitic fluid collectedreproducibility and with clear visible symptoms which can from untreated and treated DL bearing mice were analyzed.be used for monitoring the progression as well as regression As compared to the untreated DL mice, there was aof the DL throughout the period of experimentation. In significant decrease (p<0.01) in the volume of asciteaddition, homogeneous DL cells can be precipitated from collected from the DL mice treated with the both, Ru(II)-the ascitic fluid for studying the biochemical and molecular CNEB and cisplatin (Fig. 3a). Moreover, there was a drasticchanges associated with development/regression of the decline (~80%) in the number of viable DL cells in thetumor [26, 35]. Importantly, some mechanistic aspects of samples of ascites collected from both, the Ru(II)-CNEBanticancer activity of cisplatin have been worked out using and cisplatin treated DL mice (Fig. 3b).this model [26, 36, 37]. We could also induce DL in mice Release of LDH in cell free medium indicates cellwith 100% success each time and used DL bearing mice for damage in vitro as well as in vivo. In comparison to thein vivo evaluation of Ru(II)-CNEB as an anticancer agent. level of M4-LDH in the cell free ascitic fluid from untreated DL mice, there was a significant increase in theCytotoxicity of Ru(II)-CNEB on DL cells in vitro level of M4-LDH in those from Ru(II)-CNEB and cisplatin treated DL mice (Fig. 3c). These findings clearly suggestCytotoxicity assay was done to ascertain whether Ru(II)- that both the compounds tested are able to restrict DLCNEB is able to kill DL cells in vitro. For this, DL cells development and also to induce death of DL cells in vivo.were maintained in a physiological buffer medium and theirviability was assayed after incubating them with increasing Effect of Ru(II)-CNEB on the level of M4-LDH in DL cellsconcentrations of the compound for 30 min and 20 h. Asshown in Fig. 2, in 30 min set, though DL cell viability was Increased LDH activity is associated with tumor develop-not affected up to 0.5 mg/ml Ru(II)-CNEB, a significant ment. To ascertain whether Ru(II)-CNEB and cisplatin aredecline in the number of viable DL cells was observed at able to decrease the activity of this enzyme in DL cells,higher concentrations of the compound (at 5 mg/ml; p<0.01 LDH activity was compared in DL cells from untreated andand at 10 mg/ml; p<0.001). Moreover, when incubation treated DL mice. According to Fig. 4a, as compared to theperiod was increased to 20 h, a linear decline in the number LDH activity observed in DL cells from the untreatedof viable DL cells was observed starting from 0.05 mg/ml group, there was a significant decrease (p<0.01) in the(p<0.01) to 10 mg/ml (p<0.001) of the compound. Thus, it activity of this enzyme in DL cells from Ru(II)-CNEBwas evident that Ru(II)-CNEB is able to kill DL cells in vitro treated DL mice. However, activity of LDH was found toin a dose and incubation time dependent manner. be increased significantly in DL cells from the cisplatin treated DL mice.Effect of Ru(II)-CNEB on regression of DL cells in vivo In order to ascertain whether Ru(II)-CNEB affects the level of active M4-LDH and/or the level of other LDHIn order to confirm whether administered dose of Ru(II)- isozymes in DL cells, non-denaturing PAGE analysis wasCNEB is able to restrict the development of DL and/or to preferred over the immunostaining methods. Though this
    • 508 Invest New Drugs (2009) 27:503–516 a Vol. of ascitic fluid (ml) 16 14 12 c DL DL+Rc DL+Cpt 10 8 6 ** ** 4 M4-LDH 2 0 DL DL+Rc DL+Cpt b Densitometry of LDH bands 120 350 Viability of DL cells *** (% of DL control) (% of DL control) 100 300 80 250 200 60 *** 150 40 *** 100 20 *** 50 0 0 DL DL+Rc DL+Cpt DL DL+RC DL+CPTFig. 3 Effects of Ru(II)-CNEB and cisplatin treatment on ascitic fluid substrate specific LDH bands were developed in the gel. The gelvolume (a), viability of DL cells (b) and on the release of M4-LDH in photograph is a representative of the three PAGE repeats. In lowerthe cell free ascitic fluid (c) of DL bearing mice. In case of (a) and (b), panel of (c), relative densitometric values of LDH bands fromthe data represents mean±SD where n=4. In (c), pooled cell free experimental group, as percent of the control DL lane, have beenascitic fluid from three to four mice containing 60 μg protein was presented as mean±SD from three PAGE repeats. *p<0.05; **p<loaded in each lane, 10% non-denaturing PAGE was performed and 0.01; ***p<0.001 (DL control versus treated DL groups)method is relatively less sensitive than that of immunode- group mice showed the smears of fragmented DNA. Whereas,tection, however, it is more relevant for interpreting the DNA isolated from DL cells of untreated mice showed ametabolic changes associated with the enzymatic altera- single DNA band at higher molecular weight (MW) range.tions. Because, this method utilizes substrate specificity These results clearly suggest that both the compounds testedbased detection of only active fraction of the enzyme were able to induce apoptosis in DL cells in vivo.excluding inactive/denatured proteins, which otherwise can Oxidative stress and mitochondrial dysfunction arenot be excluded by immunostaining method. known to initiate final steps of apoptosis. Superoxide Figure 4b shows that DL cells from the untreated DL dismutase (SOD) is the first committed enzyme thatmice expressed high amount of only M4-LDH which was neutralizes oxygen free radical (O2)− based oxidative stressfound to be decreased significantly (p<0.01) in the DL cells in the cells. Therefore, a decrease in the level of Mn-SODfrom Ru(II)-CNEB treated DL mice. However, there was (SOD2: mitochondrial isoform) may be considered as anno change in the level of M4-LDH in the DL cells from indicator of oxidative stress in mitochondria. As shown incisplatin treated DL mice. This suggest that among the two Fig. 6a, there was a significant increase (p<0.05) in thecompounds tested, only Ru(II)-CNEB caused a decline in level of active SOD2 in DL cells from Ru(II)-CNEB treatedthe level of M4-LDH in DL cells in vivo. DL mice than that from the untreated DL group. However, the DL cells from cisplatin treated DL mice showed aApoptosis of DL cells in vivo by Ru(II)-CNEB significant decrease (p<0.01) in the level of active SOD2. Release of mitochondrial cytochrome c in the cytosolThe DNA fragmentation assay is a reliable tool to ascertain indicates for induction of mitochondrial dysfunction–apoptotic cell death. In the present context, using a standard apoptotic pathway in the cells. Therefore, the level ofmethod, the percentage of fragmented DNA in DL cell cytochrome c in the cytosol of DL cells from untreated andextracts was measured followed by its analysis on agarose treated DL groups was compared. As compared to the DLgel electrophoresis. There was a significant increase (p< cells from untreated DL group, cytosolic fractions of DL0.01) in the level of fragmented DNA in DL cells from cells from Ru(II)-CNEB and cisplatin treated DL miceboth, the Ru(II)-CNEB and cisplatin treated DL mice showed a significant increase (p<0.05) in the level of(Fig. 5a). This was further confirmed by the results of cytochrome c (Fig. 6b). The results suggest induction ofagarose gel electrophoresis (Fig. 5b) wherein, DNA mitochondrial dysfunction–apoptotic pathway in the DLsamples from the DL cells collected from both the treated cells in vivo by both the compounds tested.
    • Invest New Drugs (2009) 27:503–516 509 a 6 ** mice treated with Ru(II)-CNEB, which was comparable 5 with the effect of cisplatin treatment also. Thus, it was (U/mg protein) LDH Activity 4 evident that like cisplatin, Ru(II)-CNEB is also able to 3 cause an increase in the survival period of DL bearing mice ** with remarkable improvements in DL associated symptoms. 2 1 0 Effect of Ru(II)-CNEB treatment on normal tissues of DL DL DL+Rc DL+Cpt mice b DL DL+Rc DL+Cpt Liver metabolizes most of the drugs and kidney filters out M4-LDH all the unwanted exogenous substances. Therefore, these two organs are likely to be affected up to a greater extent by Densitometry of LDH bands 140 the drug treatment. Also, it is important to ensure that an anticancer compound does not cross the blood brain barrier (% of DL control) 120 and central nervous system remains protected during the 100 ** treatment. Therefore, these three tissues were selected to 80 assess toxicity of Ru(II)-CNEB on the normal tissue. 60 40 20 a 0 160 140 * * Fragmented DNA (% of DL control) DL DL+Rc DL+Cpt 120 100Fig. 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. 80The values in (a) represent mean±SD where n=4 and each experiment 60done in duplicate. In case of (b), pooled DL cell extracts from four 40mice containing 60 μg protein in each lane was electrophoresed on 2010% non-denaturing PAGE followed by substrate specific develop- 0ment of LDH bands. The gel photograph is a representative of the DL DL+Rc DL+Cptthree 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 PAGE brepeats. **p<0.01 (DL control versus treated DL groups)Improvements in the survival parameters of Ru(II)-CNEBtreated DL miceDevelopment 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 cisplatin DL DL+Rc DL+Cptfrom day 11 to 17, a significant decrease (p<0.01) in the Fig. 5 Effects of Ru(II)-CNEB and cisplatin treatment on DNAbody weight of DL mice was observed. In addition, as fragmentation in DL cells of DL bearing mice. The values in (a)compared to the mean survival time of untreated DL mice represent mean±SD of three experimental repeats from the pooled DL(18 days), the DL mice treated with Ru(II)-CNEB and cell extracts collected from four DL mice. In case of (b), 10 μg DNAcisplatin could survive up to 24 and 26 days respectively. extracted from the pooled DL cells from three to four DL mice was loaded in each lane and subjected to 1% agarose gel electrophoresisComparison of the survival time data on Kaplan–Meier followed by detection of ethidium bromide stained DNA bands undersurvival curves (Fig. 8) using log-rank statistics suggests a UV transilluminator. The photograph is a representative of threesignificant increase (p<0.001) in the survival time of DL repeats. *p<0.05 (untreated versus treated groups)
    • 510 Invest New Drugs (2009) 27:503–516 a b DL DL+Rc DL+Cpt DL DL+Rc DL+Cpt Cyto C SOD 2 β Actin Densitometry of SOD bands 140 * Densitometric value 120 2.0 (% of DL control) (Cyt C/β actin) 100 1.5 * 80 ** * 60 1.0 40 0.5 20 0 0.0 DL DL+Rc DL+Cpt DL DL+Rc DL+CptFig. 6 Effects of Ru(II)-CNEB and cisplatin treatment on the level of wherein, pooled DL cell extracts from three to four DL miceactive SOD2 (a) and on cytochrome c release in the cytosol of DL containing 60 μg protein in each lane was subjected to 15% SDS-cells (b) of DL bearing mice. In (a), pooled DL cell extracts from four PAGE followed by western transfer on nitrocellulose membrane andmice containing 60 μg protein in each lane were electrophoresed on detection of cytochrome c bands using a polyclonal anti cytochrome c.12% non-denaturing PAGE followed by substrate specific develop- The level of β actin was probed as the loading control. Thement of SOD2 bands in the gel. The gel photograph is a representative photograph is a representative of the three western blot repeats. Inof the three PAGE repeats. In lower panel of (a), relative lower panel of (b), normalized values of cytochrome c/β actin havedensitometric values of SOD2 bands from the treated group, as been presented as mean ± SD from three western blot repeats.percent of the control lane (untreated DL group), have been presented *p<0.05; **p<0.01; (untreated versus treated groups)as mean±SD from three PAGE repeats. b Immunoblotting resultsIncreased level of serum LDH indicates for vital tissue Ru(II)-CNEB as well as cisplatin treated DL group micedamage and the levels of SGOT and SGPT are used as (Fig. 10a,b). Though there was a significant increase in theblood based markers of liver damage. The serum of control level of M4-LDH (p<0.01) in the liver of control DL miceas well as all the DL group mice showed the presence of than that of the normal mice, no change in the level of thismainly M4-LDH with a minor fraction of M3H isozyme enzyme was observed in the liver of DL mice treated with(Fig. 9). As compared to their levels in the serum of normal Ru(II)-CNEB (Fig. 10c). However, as compared to themice, both these isozymes were significantly increased (p< untreated DL group, a significant decline in the level of0.05) in the serum of untreated DL mice. However, both of M4-LDH could be seen in the liver of cisplatin treated DLthem remained unchanged, as compared to the normal mice.mice, in the serum of Ru(II)-CNEB and cisplatin treated DLgroup mice. Also, the levels of SGOT and SGPT were 100found to be unaltered among the normal, untreated DL and Logrank p=0.0007 80 Survival rate (%) N DL DL+Rc DL+Cpt 60 40 Start of treatmentBody weight (g) ## ## DL 40 35 DL+Rc # * 20 ** DL+Cpt 30 0 25 0 10 20 30 40 1 7 14 21 Days after DL transplantation Period (days) Fig. 8 Kaplan–Meier survival curve for untreated DL mice and theFig. 7 Effects of Ru(II)-CNEB and cisplatin treatment on the body DL mice treated with Ru(II)-CNEB (DL + Rc) and cisplatin (DL +weight of DL mice. The data represents mean±SD where n=5–6. # p< Cpt). The log-rank analysis was performed to examine the level of0.05; ## p<0.01 (normal control versus untreated DL group). *p<0.05; significance and a p value of <0.001 was obtained in case of both the**p<0.01 (DL control versus treated DL groups) treated group of DL mice versus untreated DL mice
    • Invest New Drugs (2009) 27:503–516 511 NC DL DL+Rc DL+Cpt cisplatin treated DL group mice (Fig. 11a). Unchanged patterns of all the five LDH isozymes were also observed in the brain of control and all the DL (untreated and treated) LDH group mice (Fig. 11b). These results clearly suggest that the M4 administered dose of Ru(II)-CNEB was nontoxic to the M3H vital tissues of the DL bearing mice. Densitometry of LDH bands 140 Discussion 120 (% of DL control) 100 # Low toxicity and efficient bio-distribution of Ru-complexes 80 are of great advantage over other metal complexes for 60 evaluating their anticancer potential in vivo [2, 3]. This was 40 found to be true in case of Ru(II)-CNEB also. When a 20 nontoxic dose of the compound, determined for normal 0 NC DL DL+Rc DL+Cpt mice, was administered to DL bearing mice, it resulted in a significant decrease in the number of viable DL cell in vivoFig. 9 Effects of Ru(II)-CNEB and cisplatin treatment on the release (Fig. 3b) without producing any toxic effect on the otherof LDH in the serum of DL mice. Pooled serum from three to four normal tissues (Figs. 9, 10, and 11).mice containing 60 μg protein in each lane was electrophoresed on10% non-denaturing PAGE followed by substrate specific develop- The in vitro studies provide primary level information onment of LDH bands. The gel photograph is a representative of the cytotoxic potentials of a novel compound. We could alsothree PAGE repeats. In lower panel, relative densitometric values of observe a dose and time dependent decrease in the numberLDH bands from experimental group, as percent of the control lane, of viable DL cells by Ru(II)-CNEB in vitro (Fig. 2).have been presented as mean±SD from three PAGE repeat experi-ments. #p<0.05 (normal control versus untreated DL group) However, a more pronounced decrease observed in the viability of DL cells from Ru(II)-CNEB treated DL mice (Fig. 3b) clearly indicated a greater anticancer activity of Results in Fig. 11a and b re-confirm that mice kidney this compound in vivo than ex vivo. Some other Ru-and brain express all the five LDH isozymes. However, as complexes have also been shown to be less toxic in vitrocompared to the LDH pattern observed in the kidney of but could cause potent anti tumor activity in vivo [38].normal mice, there was a significant decrease (p<0.05) in It has been suggested that different Ru-complexes showthe level of all the five isozymes in that of untreated DL their anticancer activities via distinctly different mecha-mice, but with no change in case of Ru(II)-CNEB and nisms such as by interacting with DNA and some serum a c 200 SGOT (IU/L) 150 NC DL DL+Rc DL+Cpt 100 M4-LDH 50 0 NC DL DL+Rc DL+Cpt Densitometry of LDH bands 120 100 ## ** (% of DL control) b 80 8 SGPT (IU/L) 6 60 4 40 2 20 0 0 NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+CptFig. 10 Effects of Ru(II)-CNEB and cisplatin treatment on the levels ment of LDH bands. The gel photograph is a representative of theof SGOT (a), SGPT (b) and M4-LDH in the liver (c) of DL bearing three PAGE repeats. In lower panel of (c), relative densitometricmice. The values in (a) and (b) represent mean±SD where n=4 and values of LDH bands from experimental group, as percent of theeach assay done in triplicate. In (c), pooled liver extracts from four normal control lane, have been presented as mean±SD from threemice containing 60 μg protein in each lane was electrophoresed on PAGE repeats. **p<0.01 (untreated DL versus treated DL groups);10% non-denaturing PAGE followed by substrate specific develop- ##p<0.01 (normal control versus untreated DL group)
    • 512 Invest New Drugs (2009) 27:503–516 a b NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+Cpt LDH LDH M4 M4 M3H M3H M2H2 M2H2 MH3 MH3 H4 H4 Densitometry of LDH bands Densitometry of LDH bands 200 120 100 (% of DL control) # (% of DL control) 150 80 100 60 40 50 20 0 0 NC DL DL+Rc DL+Cpt NC DL DL+Rc DL+CptFig. 11 Effects of Ru(II)-CNEB and cisplatin treatment on the level PAGE repeats for each tissue. In lower panel of (a) and (b), relativeof LDH isozymes in kidney (a) and brain (b) of DL mice. In upper densitometric values of LDH bands from experimental group, aspanels of (a) and (b), the pooled tissue extracts from four mice percent of the control DL lane, have been presented as mean±SDcontaining 60 μg protein in each lane was electrophoresed on 10% from three PAGE repeats. ##p<0.01 (normal control versus untreatednon-denaturing PAGE followed by substrate specific development of DL group)LDH bands. The gel photographs are the representative of the threeproteins and also by inhibiting certain enzymes like future interest to determine the actual levels of this proteincytochrome c, protein kinase C, topoisomerase II etc [3]. in DL cells from treated and untreated mice, in the presentBeing highly unselective, DNA is considered an unsuitable context, a significant decline in the level of active M4-LDHtarget for anticancer agents [7]. Alternatively, selecting a in DL cells of Ru(II)-CNEB treated mice suggests decreaseprotein as pharmacological target sounds better, however, it in energy metabolism of the cancerous cell due to theis important to first ensure that inactivation of a cellular treatment with this compound. The tumor cells rely muchprotein is cancer cell specific and does not hamper normal on the energy pathway lead by M4-LDH dependentcell metabolism. In this respect, inhibiting glycolytic production of lactate from pyruvate [11, 20, 21]. Thus,efficiency of tumor cells seems to be the most relevant inactivation of this isozyme can severely affect only tumortarget, as all tumor cells switch over to enhanced aerobic cell energy metabolism. Contrary to this, as normal cellsglycolysis [10] for their additional energy needs [11, 12]. utilize pyruvate for mitochondrial oxidative phosphoryla-Also, the two key glycolytic enzymes, PKM2 (a fetal tion rather than to produce lactate by M4-LDH, decline ofisoform of pyruvate kinase) and M4-LDH, have been found this isozyme in normal tissues is less likely to affect theirto be over expressed selectively in most of the tumors and energy metabolism. This argument also justifies a greatertherefore, both of these enzymes are argued to be the potential decrease in the number of viable DL cells in vivo than intargets for novel anticancer compounds [8, 11, 21, 24]. vitro due to the treatment with Ru(II)-CNEB (Figs. 2 and Based upon our recent findings on inhibition of M4- 3b). Isolated tumor cells maintained in vitro are devoid ofLDH by Ru(II)-CNEB [24] and modulation of this enzyme true hypoxia and they can exploit aerobic pathway forby other metal complexes [32], we selected M4-LDH as a energy production even if LDH activity is declinedtarget protein for evaluating anticancer activity of Ru(II)- significantly and thus, can survive better. Contrary to this,CNEB. It has been reported that like most of the tumors, due to greater hypoxia faced by the tumor cells in vivo,DL cells also over express M4-LDH [26, 27]. A highly they rely much on anaerobic glycolysis [12] and thus, as aintense band of M4-LDH in the DL cell extracts from consequence of diminished M4-LDH activity, they can beuntreated DL mice (Fig. 4b; lane 1) also corroborate these deprived of adequate energy production resulting into poorearlier findings and accordingly, a significant decline in the survival. In addition, it has been reported [20] that tumorlevel of M4-LDH in DL cells from Ru(II)-CNEB treated stroma associated fibroblasts help in the survival of tumormice (Fig. 4b; lane 2) suggests that this compound was able cells via recycling of lactate produced in excess by theto decline the active level of this enzyme in DL cells in tumor cells. However, the blockage of tumor LDH-5 (M4-vivo. Though immunostaining of M4-LDH would be of LDH) suppresses this additional route of metabolic supple-
    • Invest New Drugs (2009) 27:503–516 513mentation and thus, can render tumor cells susceptible to oxidative stress [47]. We have observed a direct relation-death [20]. ship between the release of cytochrome c and increased Tissue damage causes leakage of LDH in body fluids level of DNA fragmentation in the DL cells from Ru(II)-[39, 40]. Thus, a significant increase in the level of M4- CNEB treated DL mice (Figs. 5 and 6b). A similar patternLDH in the cell free ascitic fluid from Ru(II)-CNEB treated of DNA fragmentation and increased level of cytochrome cDL mice, than that from the untreated DL mice (Fig. 3c; release in the DL cells from cisplatin treated DL mice werelane 1 vs lane 2), indicates for DL cell death caused by this also observed and thus, suggesting that both, Ru(II)-CNEBcompound in vivo. A more pronounced increase in the level and cisplatin, have been able to induce apoptosis in DLof M4-LDH in cell free ascitic fluid from cisplatin treated cells in vivo via release of cytochrome c.DL mice (Fig. 3c; lane 3) further strengthened this There could be more than one mechanism for inducingargument, as cisplatin induced regression of DL cells has apoptosis by chemotherapeutic agents. Anticancer drugbeen shown to accompany the release of LDH in ascitic causing induction of apoptosis via inhibition of glycolysisfluid [26]. Moreover, since cisplatin did not alter the level in tumor cells is a relatively new concept [18, 48]. Thoughof M4-LDH in DL cells, which was observed to be declined the link between inhibition of glycolysis and tumor cellsignificantly by Ru(II)-CNEB, it may be speculated that the apoptosis is yet to be defined, it may be speculated thatmechanism of cell death caused by both the compounds are depletion of energy and growth promoting substrates due todifferent from each other. decline in glycolytic efficiency could act as an inducer of Development of Dalton’s lymphoma is characterized by apoptosis in the tumor cells. Tumor cells show aberrantthe increments in the body weight and volume of the ascitic NADH/NAD shuttle of mitochondria resulting into in-fluid and thus, measurement of both these parameters are creased level of NADH in the cytosol [49]. This may alterused to determine the development of DL and its regression redox state of the cells and can induce final apoptoticin vivo [41, 42]. In comparison to the untreated DL mice, pathway in those cells [50]. Decline in the level of M4-~50% decrease observed in the ascitic volume in case of Ru LDH, which utilizes NADH as substrate, may further(II)-CNEB treated DL mice (Fig. 3a) suggest that this contribute for the accumulation of NADH in cytosol. Thiscompound was able to restrict DL development in mice. argument gets support from ~2 times increase in NADH/The range of reduction observed in ascitic volume is NAD ratio observed in M4-LDH deficient tumor cells [11].comparable with a ∼2 times reduction caused by the extract Thus, it may be argued that the resultant increase in NADH/of a macrofungus [42] and ∼50% reduction in tumor weight NAD ratio, due to a significant decrease in the level of M4-by the extract of Withania somnifera [43]. Reports are LDH, might be implicated as a biochemical mediator toscanty on Ru-complex induced regression of lymphoma in induce apoptosis in DL cells in Ru(II)-CNEB treated DLvivo. Therefore, ∼80% decline in the number of viable DL mice. Mitochondrial dysfunction in DL cells of treatedcells (Fig. 3b) in Ru(II)-CNEB treated DL mice is of great group mice has also been suggested by a significantrelevance. The reductions in ascitic volume and DL cell increase in the release of mitochondrial cytochrome cviability by Ru(II)-CNEB treatment were also comparable (Fig. 6b).with the data obtained with cisplatin treatment and thereby, Alternatively, DNA damage also induces apoptosis,suggesting further for a potent anticancer activity of Ru(II)- however, such a possibility in this case was ruled out byCNEB on DL in vivo. observing a poor DNA-Ru(II)-CNEB interaction in vitro One of the major mechanisms in cancer therapy is to (unpublished data). Also, (O2)− based oxidative stress isinduce apoptosis in transformed cells by chemotherapeutic known to cause cytochrome c release and in turn inductionagents [44, 45]. Some Ru(II)-complexes derived organo- of apoptosis in the affected cells, however, under depletedmetallic compounds have been reported to mediate their antioxidant condition. SOD is the committed enzyme ofcytotoxicity on lymphoma cell lines in vitro via induction antioxidant pathway and Mn-SOD (SOD2) in particularof apoptosis [46]. However, reports are scanty on the plays a critical role in protecting mitochondria from (O2)−induction of apoptosis in tumor cells in vivo by Ru(II)- insult. We observed a significant increase in the level ofcomplexes. Increased fragmentation of DNA is an impor- SOD2 in the DL cells from Ru(II)-CNEB treated than thosetant parameter to suggest apoptotic death of a cell. Thus, a from the untreated DL mice (Fig. 6a). This suggests thatsignificant increase in the level of fragmented DNA in the antioxidant potential of DL cells was not depleted due toDL cells from Ru(II)-CNEB treated DL mice (Fig. 5a, b) the treatment with Ru(II)-CNEB and hence, rules outclearly suggests that Ru(II)-CNEB is able to induce possibility of a role of (O2)− based oxidative stress in Ruapoptosis in DL cells in vivo. Release of cytochrome c (II)-CNEB induced apoptosis in the DL cell.from mitochondria is an indicator of mitochondrial dys- Thus, the results presented here suggest that Ru(II)-function and has been correlated with the induction of CNEB might be implicating the decline of M4-LDH andapoptosis under a variety of metabolic derangements and mitochondrial dysfunction to induce apoptosis in DL cells
    • 514 Invest New Drugs (2009) 27:503–516in vivo. The induction of glycolysis–apoptotic pathway in basis for standardizing the dose and the treatment scheduletumor cells due to chemotherapeutic intervention is a of this compound against a variety of tumors in vivo.relatively less explored area. Therefore, these findings are The major limitation of cancer therapy is the injury ofof much current interest with respect to identify novel Ru- normal tissues leading to multiple organ toxicity [54].complexes which can inhibit a critical step of glycolytic Detection of increased level of LDH in serum is a widelypathway resulting into induction of apoptosis in the tumor used parameter for blood based diagnosis of tissue damagecells in vivo. as well as to characterize the rapid turnover of cancerous Cisplatin is a well studied compound on a variety of cells in vivo [39]. Therefore, unaltered patterns of serumtumors [51]. It was interesting to note that cisplatin also LDH observed in case of Ru(II)-CNEB and cisplatin treatedinduced apoptosis in DL cells in vivo via release of DL mice (Fig. 9; lanes 3 and 4 versus lane 1) clearlycytochrome c, however, without affecting the level of M4- suggest that no damage has occurred to the normal tissuesLDH. Thus, it is likely that cisplatin might be adopting due to the treatment with both these compounds. Accord-LDH independent mechanism to induce apoptosis in DL ingly, a significant increase in the level of M4-LDH incells. DNA has been shown to be the major target of serum of untreated DL mice (Fig. 9; lane 2) may becisplatin induced cytotoxicity, wherein, cisplatin-DNA correlated with the rapid turnover of DL cells in theadduct formation is known to induce oxidative stress and untreated DL mice.finally to initiate tumor cell death [52]. There was a Most of the drugs given through systemic routes undergosignificant decline in the level of SOD2 with a concomitant their final metabolism in liver, and therefore, liver is likelyincrease in the level of cytochrome c in the DL cells from to be affected adversely during chemotherapeutic treat-cisplatin treated DL mice (Fig. 6a, b). Thus, it may be ments [54]. Increased levels of SGOT and SGPT are theargued that, as against the role of Ru(II)-CNEB in DL cell most widely used blood based markers to ascertain liverapoptosis, cisplatin adopts (O2)− dependent mitochondrial dysfunction. Therefore, unaltered patterns of SGOT anddysfunction pathway to induce apoptosis in these cells. SGPT in the treated and untreated DL mice (Fig. 10a, b)Caspase 9, an important component of oxidative stress indicate that doses of both the compounds tested are noninduced apoptosis, has also been reported to be implicated toxic to the liver. In addition, corroborating an earlierin apoptotic death of certain tumor cells by cisplatin [45]. finding [27], though M4-LDH was slightly increased in the Increase in the life span and improvement in overall liver of untreated DL mice, it remained unaltered in theappearance of cancerous animal after the treatment are the liver of Ru(II)-CNEB treated DL mice (Fig. 10c) and thus,ultimate criteria to ascertain anticancer potential of a suggesting further that liver of DL mice was unaffected duechemotherapeutic agent. A significant decline (∼50% ) in to the treatment with this compound. Kidney is involved inbody weight (Fig. 7) of the Ru(II)-CNEB treated DL mice the filtration of blood born factors continuously. Thoughand a significant increase in their survival period (Fig. 8) the levels of all LDH isozymes were found to be decreasedsuggest that the molecular alterations induced by this in the kidney of DL bearing mice, they remained unalteredcompound has resulted into an overall improvement in the in the DL mice treated with Ru(II)-CNEB (Fig. 11a). Bloodlife of the cancerous mice. The ranges of decrease in the brain barrier protects brain from most of the exogenousbody weight and increase in the survival period reported factors. It was evident in the present context also. All thehere are well correlated with the similar findings on DL five LDH isozymes were found to remain unaltered in thebearing animal treated with the extracts of a macrofungus brain of both, the treated and the untreated group of DL[42] and that on TLX5 lymphoma bearing mice treated with mice (Fig. 11b) and thus, suggesting that neither thethe different antimetastatic agents [38]. Also, the findings development of DL nor Ru(II)-CNEB treatment causedon Ru(II)-CNEB treated DL mice were comparable and any alteration in the expression pattern of any of the LDHvery close to the data obtained from the DL mice treated isozymes in the mouse brain. Thus, it is evident that thewith cisplatin (Figs. 7 and 8). NAMI-A is the most widely dose of Ru(II)-CNEB used in this experiment did notstudied Ru-complex as an anticancer agent which was also produce any damage to the normal tissues in vivo. The doseshown to reduce the increased body weight of the of cisplatin used also did not produce much change in thecancerous animal maximum up to 50% that too when given level of LDH isozymes in the normal tissues. However, itin combination with cisplatin [53]. The data on the caused a significant decline in the level of M4-LDH in liverincreased survival time reported here sounds further better and thereby, indicated the possibility of liver toxicity bythan only a 12% increase observed in the life span of cisplatin. This also corroborates an earlier report on theEhrlich ascite bearing mice due to the treatment with a Ru effect of cisplatin on liver LDH of DL mice [26].(II)-complex, [cis-Ru (II) DMSO Cl2] [49]. Thus, our In summary, the present study demonstrates that afindings suggest potent anticancer activity of Ru(II)-CNEB nontoxic dose of Ru(II)-CNEB is able to decrease theagainst Dalton’s lymphoma in mice and thereby, provide a viability of DL cells in vivo with a concomitant increase in
    • Invest New Drugs (2009) 27:503–516 515the life span of the tumor bearing mice without producing physiology and tumour maintenance. Cancer Cell 9:425–434.any toxicity to the normal tissues. The findings on Ru(II)- doi:10.1016/j.ccr.2006.04.023 12. Kim JW, Dang CV (2006) Cancers molecular sweet tooth and theCNEB induced decline in M4-LDH and increments in the Warburg effect. Cancer Res 66:8927–8930. doi:10.1158/0008-levels of DNA fragmentation & release of cytochrome c in 5472.CAN-06-1501the DL cells suggest that decreased tumor glycolysis and 13. Kondoh H, Lleonart ME, Gil J, Beach D, Peters G (2005)induction of mitochondrial dysfunction–apoptosis pathway Glycolysis and cellular immortalization. Drug Discov Today 2:263–267. doi:10.1016/j.ddmec.2005.05.001could be implicated in the anticancer activity of this 14. Gaber K (2006) Energy deregulation: licensing tumors to grow.compound. 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Geschwind JF, Georgiades CS, Ko YH, Pedersen PL (2004)Acknowledgment This work was financially supported by a project Recently elucidated energy catabolism pathways provide oppor-from Department of Biotechnology (DBT), Govt. of India, (BT/ tunities for novel treatments in hepatocellular carcinoma. ExpertPR5910/BRB/10/406/2005) sanctioned jointly to LM and SKT. The Rev Anticancer Ther 4:449–457. doi:10.1586/14737140.4.3.449authors are thankful to UGC Centre of Advanced Studies programme 18. Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN et alto Department of Zoology, BHU, for providing infrastructural (2005) Inhibition of glycolysis in cancer cells: a novel strategy tofacilities. The help extended by Mr. S. Bhattacharyya, Ms. S. overcome drug resistance associated with mitochondrial respira-Srivastav, and Ms. B. Mishra is also acknowledged. tory defect and hypoxia. 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