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Research paper
Activation of p53 mediated glycolytic inhibition-oxidative stress-
apoptosis pathway in Dalton's lymphoma b...
apoptotic pattern of DNA cleavage which was consistent with the
decreased DL cell viability and increased life span of the...
buffer (20 mM Tris-Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 25 mM Na2 pyrophosphate and 1 mM PMSF)....
analyzed by 1e2% agarose gel electrophoresis, were visualized by
ethidium bromide staining and were found to be; Bcl 2 (35...
mRNA in the DL cells from Ru(II)-CNEB treated DL mice is found to
be unchanged as compared to the untreated counterpart.
3...
Overproduction of lactate is considered as one of the hallmarks
of the tumor growth [44]. It has been reported that tumor ...
the kinase domain of a bi-functional enzyme (PFK2/FBPase2),
synthesizes FBP, the most effective allosteric activator of PF...
H2O2 concentration was found to be remarkably increased (~6Â) in
the DL cells from Ru(II)-CNEB treated DL mice (Fig. 5a). ...
Glutathione content is known to represent actual redox status of
the cells and its enhanced level in the cancer cells is d...
[14] R. Xu, H. Pelicano, Y. Zhou, J.S. Carew, L. Feng, K.N. Bhalla, M.J. Keating,
P. Huang, Inhibition of glycolysis in ca...
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Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide

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There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg
effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is
an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor
progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel
anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered
to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the
DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic
glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to
modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that
Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2:
PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase
(the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the
two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the
enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings
suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor
supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.

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Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide

  1. 1. Research paper Activation of p53 mediated glycolytic inhibition-oxidative stress- apoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide Raj Kumar Koiri a , Surendra Kumar Trigun b, * , Lallan Mishra c a Department of Zoology, Dr. Harisingh Gour Central University, Sagar, Madhya Pradesh 470003, India b Biochemistry Section, Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India c Department of Chemistry, Banaras Hindu University, Varanasi, Uttar Padesh 221005, India a r t i c l e i n f o Article history: Received 25 September 2014 Accepted 30 December 2014 Available online 8 January 2015 Keywords: Ruthenium p53 PFKFB3 LDH Apoptotic factors a b s t r a c t There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was adminis- tered to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2: PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dis- mutase (the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo. © 2015 Elsevier B.V. and Societe française de biochimie et biologie Moleculaire (SFBBM). All rights reserved. 1. Introduction Identification of cellular/molecular targets is of prime concern for formulating novel anticancer agents [1]. Due to effective bio- distribution and multimodal cellular actions, during recent past, ruthenium complexes have drawn much attention as next gener- ation anticancer agents [2]. So far mechanistic aspects of anticancer metal complexes are concerned, DNA, once considered as their main target [3,4], is now evident to be highly unselective [5] and therefore, there is an evolving concept to evaluate whether metal complexes could be able to attenuate certain tumor growth associated biochemical events at cellular level [5e7]. In this respect, Ru-complexes get an upper hand, as metal center of ruthenium has been shown to interact with a variety of ligands [8] and thereby enabling these complexes to affect various cellular activities [9]. We have synthesized and characterized a ruthenium complex; Ru(II)-CNEB, which was found to be highly biocompatible to mice when administered in vivo. Additionally, it could interact with and inhibit M4-LDH non-competitively both in vitro and at tissue level [10]. During pilot experiments, though this compound did not show any DNA cleavage activity in vitro (Supplementary data with this article), when administered to the DL bearing mice, it produced Abbreviations: Bcl-2, B-cell leukemia/lymphoma-2; DL, dalton's lymphoma; FBP, fructose-2,6-bisphosphate; GPx, glutathione peroxidase; GSH, glutathione reduced; iPFK2/PFKFB3, inducible isoform of 6-phosphofructo-2-kinase; LDH, lactate dehy- drogenase; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; Ru(II)-CNEB, [Ru(CNEB-H)2(bpy)2] 2PF6$0.5 NH4PF6 (Ruthenium(II)-complex con- taining 4-carboxy N-ethylbenzamide as ligand); SOD, superoxide dismutase. * Corresponding author. Tel.: þ91 9415811962. E-mail address: sktrigun@gmail.com (S.K. Trigun). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi http://dx.doi.org/10.1016/j.biochi.2014.12.021 0300-9084/© 2015 Elsevier B.V. and Societe française de biochimie et biologie Moleculaire (SFBBM). All rights reserved. Biochimie 110 (2015) 52e61
  2. 2. apoptotic pattern of DNA cleavage which was consistent with the decreased DL cell viability and increased life span of the tumor bearing mice [11]. Importantly, since, this complex increased certain apoptotic markers and also inhibited M4-LDH (LDH-5) in those DL cells, it was argued that this complex, instead of targeting DNA directly, is able to activate glycolytic inhibition-apoptosis pathway in a tumor cell in vivo [11]. This necessitated further studies on characterizing regulatory factors of this pathway as target of Ru (II)-CNEB in the DL cells in vivo. Switching over to aerobic glycolysis, known as ‘Warburg effect’, is considered to be a common trait of most of the growing tumors [12] and therefore, inhibition of enhanced tumor glycolysis is now advocated as one of the therapeutic strategies in cancer therapy [13,14]. In this regard, targeting regulatory enzymes of glycolytic pathway assumes special importance for the novel anticancer agents. Phosphofructokinase1 (PFK1) catalyzes committed step of glycolysis. PFK2 domain of D-fructose-6-phosphate-2-kinase/fruc- tose-2,6-bisphosphatase (PFK2/FBPase2) synthesizes fructose-2,6- bisphosphate (FBP) to activate PFK1 and thereby, it acts as main glycolytic regulator under a variety of pathological challenges including tumor progression [15]. Cancer cells express C type PFK1 which is more sensitive to FBP [16] and also over express a cata- lytically more efficient inducible form of PFK2 (iPFK2: PFKFB3 gene) [17]. Since, iPFK2 repression has been reported to inhibit tumor cell growth in vitro [13], this glycolytic activator could be considered as relevant therpautic target for the novel anticancer agents. Similarly, to sustain enhanced glycolysis, tumor cells adapt to produce lactate from pyruvate by activating LDH-5. Reports also suggest that over expression of LDH-5 gene (LDH-A) is associated with tumor growth [12,18] and thus, advocating repression of LDH-5 gene as another mechanism to define therapeutic target for a novel anticancer agent. So far up stream regulation of the cell bioenergetics is con- cerned, p53, a tumor suppressor protein, has been found to modulate overall balance between glycolysis and mito-OXPHOS [19]. The loss of normal p53 has been reported to be critically associated with tumor progression, particularly in leukemia and lymphoma [20]. Similarly, p53 over expression has been demon- strated to have prognostic significance in lymphoma [21]. Recently, p53 has been reported to regulate tumor cell energy metabolism [22] via down regulating expression of glucose transporters GLUT1 and GLUT4 [23] and also by activating the TIGAR gene which re- presses iPFK2/PFKFB3 [24]. Thus, when tumor cells switch over to glycolytic phenotype, it is accompanied with declined p53 expression [25]. Similarly, inhibition of glycolysis has been shown to activate p53 [26]. It has been suggested that ROS are down stream mediators of p53 dependent apoptosis [27], wherein, Bcl-2/Bax ratio acts as main determinant of this pathway [28]. Low level of p53 induces expression of antioxidant enzymes responsible to maintain ROS level within a permissive range which otherwise can cause DNA damage and genomic instability in the cells [29]. Higher level of p53 protein, on the other hand, is known to enhance the expression of pro-oxidant and proapoptotic factors [30]. Tumor cells, in addition to acquiring glycolytic phenotype, also modulate their oxygen metabolism in many ways [12,31]. Super- oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) constitute main antioxidant defense mechanism in most of the cells. The oxygen free redicals (O2 .- ), produced during mitochondrial oxidative phosphorylation, are dismutated to hydrogen peroxide (H2O2) by SOD followed by con- version of H2O2 into water by catalase and GPx [32]. As SOD is the committed enzyme of this pathway responsible to produce H2O2, it appears to be the most relevant target for therapeutic intervention in the tumor cells. In the recent past, both SOD isoforms (CueZn- SOD: cytosolic; SOD1 and Mn-SOD: mitochondrial; SOD2) have been found to be implicated in tumerogenesis [33]. Some earlier reports have described that reduced levels of SOD1 SOD2 facilitate tumerogenesis [34,35] via maintaining low level of H2O2 in the cancerous cells and accordingly, increased SOD ac- tivity and in turn, higher level of H2O2 may contribute for tumor suppression [36,37]. However, effectiveness of this SOD mediated mechanism depends on the status of the two down stream en- zymes; catalase and GPx in the cancerous cells. Indeed, SOD and catalase double transfectant (SOCAT3) cells and cells with over activated GPx1 have shown protection from oxidative cell damage [38,39]. Thus, to characterize oxidative mechanism based anti- cancer potential of a novel anticancer agent, it is important to study comparative profile of the three antioxidant enzymes involved in H2O2 metabolism in the cancer cells. In the present article, we have investigated whether Ru(II)-CNEB is able to activate p53 mediated glycolytic inhibition-oxidative stress-apoptosis pathway in the DL cells in vivo. 2. Materials and methods 2.1. Chemicals Ruthenium(II)-complex containing 4-carboxy N-ethyl- benzamide as ligand, Ru(II)-CNEB; [Ru(CNEB-H)2(bpy)2] 2PF6$0.5 NH4PF6, whose structural details have already been described previously [10,11], was used in the present study. Antibodies against p53, caspase 9, Bcl-2, Bax, SOD1, SOD2 PFK 2 were purchased from Santa Cruz and b-actin was purchased from SigmaeAldrich Co., USA. HRP- conjugated anti rabbit/goat/mouse IgG were obtained from Genei. 20,70-dichlorofluorescin diacetate and ECL super signal western pico kit were purchased from Fluka and Pierce respectively. Lactate estimation kit was purchased from Biorex Diagnostics, Ltd, UK. Other general chemicals and reagents were obtained from Merck or SRL unless otherwise specified. 2.2. Induction of Dalton's lymphoma (DL) in mice Inbred AKR strain mice of 16e18 weeks age weighing 24e26 g were used for the experiments. Mice were maintained at standard laboratory conditions with the supply of food and water ad libitum. This work was approved by the institutional animal ethical com- mittee (Dan/2006-07/962). Dalton's lymphoma was induced by transplantation of 1 Â 107 viable tumor cells (assayed by trypan blue method) i.p. per mice. Development of DL was confirmed by abnormal belly swelling and increased body weight which became visible on 10e12th post transplantation day. The DL bearing mice survived up to 18 ± 2 days. 2.3. Treatment schedule The DL mice were randomly divided into 2 groups with 10 mice in each. The first group DL mice were treated with Ru(II)-CNEB complex (10 mg/kg bw/day, ip), and the second group, designated as DL control, were similarly injected with equal volume of Krebs Ringer Buffer (KRB). As DL becomes visible on day 10e11 and DL bearing mice survived up to 18e20 days post DL transplantation, the treatments with the compounds were started from day 11 of tumor transplantation and continued up to day 17th. The normal control group mice were also treated simultaneously with KRB. To study biochemical/molecular parameters, 3-4 mice from each group were sacrificed on day 18th and tumor ascites pooled from 3 to 4 DL mice from each group were centrifuged at 2000 Â g at 4 C to collect DL cells. The DL cell extract was prepared using lysis R.K. Koiri et al. / Biochimie 110 (2015) 52e61 53
  3. 3. buffer (20 mM Tris-Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM Na2 pyrophosphate and 1 mM PMSF). The cell lysates were centrifuged at 20,000 Â g for 30 min and super- natant obtained were used for biochemical and molecular studies. Protein concentrations in the extracts were measured following the method of Lowry et al. [40]. 2.4. Western blotting For western blot analysis of different proteins, DL cell extracts containing 60 mg proteins, were subjected to 10% SDS-PAGE. As described previously [11], proteins were transferred to nitrocellu- lose membrane followed by detection of p53, caspase 9, Bcl-2, Bax, iPFK2, SOD1 and SOD2 against specific polyclonal antibodies (1:1000). Proteins on membrane were detected by ECL west pico kit. As loading control, b-actin was probed similarly using mono- clonal anti-b-actin-peroxidase antibody (1:10,000). Protein bands were quantified using gel densitometry software AlphaImager 2200. 2.5. Assay of the antioxidant enzymes Catalase activity was measured following an earlier method [41] with some modifications. Briefly, 1 ml reaction mixture consisted of 0.05 M phosphate buffer (pH 7.0) and 0.003% H2O2. The reaction was started by the addition of suitably diluted cell extract and decrease in absorbance at 240 nm was recorded for 10 min. Unit of the enzyme was defined as mmol of H2O2 depleted/min. The activity was expressed as units/mg protein. Glutathione peroxidase (GPx) activity was determined as described earlier [42] with slight modifications. Briefly, cell ex- tracts were added to the assay buffer consisting of 100 mM Tris- Cl (pH 7.2), 3 mM EDTA, 1 mM sodium azide, 0.25 mM H2O2, 0.5 mM NADPH, 0.17 mM GSH, and 1 unit GR. The change in absorbance per minute at 340 nm was recorded for 10 min and enzyme activity was expressed as mmole NADPH oxidized/min/ mg protein. 2.6. Analysis of SOD and Gpx by non-denaturing PAGE The active levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) was determined using non-denaturing PAGE of the cell extracts following the method recently reported from our lab [42,43]. The cell extract containing 60 mg protein were loaded in each lane of 10% non-denaturing PAGE. After electrophoresis at 4 ± 2 C, the gels were subjected to substrate specific staining of different antioxidant enzymes. The staining mixture for SOD consisted of 2.5 mM NBT, 28 mM riboflavin, and 28 mM TEMED. Gels were incubated for 20 min in the dark, followed by exposing them to a fluorescent light till achromatic SOD bands developed against dark blue background. GPx specific staining mixture was composed of 50 mM TriseCl buffer (pH 7.9), 3 mM GSH, 0.004% H2O2, 1.2 mM NBT and 1.6 mM PMS. Achromatic bands corresponding to GPx activity appeared against a violeteblue background. The intensities of all the bands were quantified by gel densi- tometry using AlphaImager 2200 gel documentation software. Specificity of the PAGE bands of different enzymes were confirmed by obtaining clear negative results when similarly run gels were treated in the absence of the enzyme specific substrates. In each case, PAGE was performed at least 3 times and mean ± SD of densitometry values of the bands, as percentage of the control lane, have been presented in the result with one representative gel photograph. 2.7. Biochemical estimations 2.7.1. Lactate The concentration of lactate was determined by measuring lactate oxidation by lactate oxidase as per the manufacturer's in- structions given in the lactate assay kit obtained from Biorex Di- agnostics Ltd, UK. 2.7.2. H2O2 Following the method described earlier [42]; intracellular H2O2 was determined by measuring H2O2 dependent oxidation of DCFH- DA into DCF. Briefly, 100 ml of cell extracts were incubated with 10 mM DCFH-DA at 37 C for 30 min in dark. Using excitation at 504 nm and emission at 529 nm, intensity of DCF fluorescence was measured. H2O2 concentration in the extract was determined by using a calibration plot made against different H2O2 concentrations (5e100 mM). 2.7.3. Total glutathione (GSH þ GSSG) Following the method described earlier [42], DL cell extracts were precipitated with 5% sulfosalicylic acid in the ratio of 1:2 and centrifuged. The supernatant collected was neutralized. In a 96 well micro plate, 50 ml neutralized supernatant was incubated with 100 ml of the reagent containing 0.30 mM NADPH, 0.22 mM DTNB and 1.6 units/ml GR prepared in 100 mM phosphate buffer (pH 7.4) containing 1 mM EDTA. The absorbance was recorded at 412 nm for 10 min using Micro Scan MS5608A (ECIL) micro plate reader. Total glutathione content was expressed in terms of nmol/mg protein. 2.8. Semi-quantitative RT-PCR Total RNA was isolated from DL cells using TRI reagent following the manufacturer's protocol. DNA-free™ (Ambion) was used to remove any contaminating DNA from the RNA preparation following the manufacturer's protocol. Briefly, reaction mixture consisting of DNase I, 10Â DNase I buffer and RNA sample was incubated at 37 C for 20 min. Thereafter, DNase I inactivation reagent slurry was added and incubated for 2 more min at room temperature and centrifuged. The upper phase was collected as DNA free RNA solution. From this RNA isolate, 2 mg RNA was subjected for reverse transcription using 200 U of reverse tran- scriptase and 200 ng random hexamer to make ss-cDNA (Revert Aid First strand cDNA synthesis kit, MBI fermentas). The PCR re- action mixture contained 1Â Taq polymerase buffer, 0.2 mM each of the four dNTPs, 1.0 U of Taq polymerase, and 10 pmol of the specific primer. The mouse gene-specific primers used were: Bcl-2 (forward 50-TAC CGT CGT GAC TTC GCA GAG-30; reverse 50-GGC AGG CTG AGC AGG GTC TT-30); Bax (forward 50-CGG CGA ATT GGA GAT GAA CTG-30; reverse 50-GCA AAG TAG AAG AGG GCA ACC-30); PFKFB3 (forward 50-GGC AAG ATT GGG GGC GAC TC-30; reverse 50- GGC TCC AGG CGT TGG ACA AG-30); LDH A (forward 50-ATG CAC CCG CCT AAG GTT CTT-30; reverse 50-TGC CTA CGA GGT GAT CAA GCT-30); SOD2 (forward 50-GCA CAT TAA CGC GCA GAT CA-30; reverse 50-AGC CTC CAG CAA CTC TCC TT-30) and b actin (forward 50-ATC GTG GGC CGC TCT AGG CAC C-30; reverse 50-CTC TTT GAT GTC ACG ATT TC-30). Linearity of PCR amplifications was checked for each gene using various cycles (20, 24, 28, 32, 36 and 40 cycles) vs densitometric value of the PCR product of the corresponding gene. Accordingly, the optimal number of cycles from the linear phase and other conditions were chosen for amplification for each gene as; b actin, 30 cycles of 30 s at 94 C, 30 s at 52 C, and 30 s at 72 C; Bcl-2 LDH A, 34 cycles of 45 s at 94 C, 45 s at 55 C, and 1 min at 72 C; Bax, 30 cycles of 30 s at 94 C; 60 s at 56 C, and 1 min at 72 C; PFKFB3, 31 cycles of 60 s at 95 C, 60 s at 50 C, and 1 min at 72 C; SOD2, 27 cycles. The amplification products, R.K. Koiri et al. / Biochimie 110 (2015) 52e6154
  4. 4. analyzed by 1e2% agarose gel electrophoresis, were visualized by ethidium bromide staining and were found to be; Bcl 2 (350 bp), Bax (160 bp), PFKFB3 (329 bp), LDH A (103 bp), SOD2 (241 bp) and b actin (543 bp). Amplification of b actin served as a control. Three RT-PCR repeats from 3 RNA isolates for each gene were performed. The expression levels were measured by densitometry using AlphaImager 2200. 2.9. Statistical analysis Experimental data were expressed as mean ± SD and Student's t test was applied for determining the level of significance between control and experimental groups and value of p 0.05 was considered significant. 3. Results 3.1. Ru(II)-CNEB mediated decline of lactate level in the DL cells in vivo Lactate serves as an alternate metabolic fuel for the high energy demanding tumor cells. Therefore, overproduction of lactate is considered one of the hallmarks of the tumor growth. According to Fig. 1b, as compared to the DL cells from untreated group, there is a significant decline (p 0.001) in lactate level in case of the DL cells from Ru(II)-CNEB treated DL mice. To ascertain, whether decline in lactate level is due to decline in the expression of M4-LDH, the level of LDH-A mRNA was measured in the DL cells from the untreated the treated group DL mice. According to Fig. 1a, the level of LDH-A Fig. 1. Effect of Ru(II)-CNEB (Rc) on the expression of LDH-A and iPFK2 lactate production in the DL cells in vivo. (a) shows representative RT-PCR photograph for LDH-A with the ratio of LDH-A/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b) shows lactate level represent as mean ± SD where n ¼ 4. (c) shows representative RT-PCR photograph for PFKFB3 with the ratio of PFKFB3/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. In (d), a representative western blot photograph, with 60 mg protein in each lane, presented with the relative densitometric values of iPFK2/b actin as mean ± SD from three western blot repeats. *p 0.05; ***p 0.001 (untreated vs. treated DL groups). R.K. Koiri et al. / Biochimie 110 (2015) 52e61 55
  5. 5. mRNA in the DL cells from Ru(II)-CNEB treated DL mice is found to be unchanged as compared to the untreated counterpart. 3.2. Downregulation of iPFK2/PFKFB3 in the DL cells due to the treatment with Ru(II)-CNEB Over expression of iPFK2/PFKFB3 is associated with tumor growth. Recently, p53 has been reported to regulate tumor cell energy metabolism indirectly by activation of the TIGAR gene which declines iPFK2/PFKFB3 induced fructose-2,6-bisphosphate production in the tumor cells. As illustrated in Fig. 1c and d, the levels of PFKFB3 mRNA and its protein product declined signifi- cantly (p 0.05e0.001) in the DL cells from Ru(II)-CNEB treated DL mice than that from the untreated counterpart. 3.3. Ru(II)-CNEB increases p53 level in the DL cells in vivo Restoration of p53 level is an important strategy of anticancer chemotherapy. Based on immunoblot analysis, level of p53 was observed to be ~5 times higher in the DL cells (p 0.001) from the Ru(II)-CNEB treated DL mice than that from the control group mice (Fig. 2). 3.4. Enhanced expressions of SOD1 SOD2 in the DL cells from Ru(II)-CNEB treated DL mice Oxidative stress and mitochondrial dysfunction are known to initiate final steps of apoptosis. Infact ROS has been suggested to act as a down stream mediator of p53 dependent apoptosis. SOD is the first commited enzyme that neutralizes superoxide anion free radical (O2 À ) based oxidative stress in the cells. According to Fig. 3a, the level of active SOD2 SOD1, both shows significant increments in the DL cells from Ru(II)-CNEB treated DL mice. To confirm whether activity increment is contributed due to the similar in- crease in the expression of these antioxidant enzymes, RT-PCR and western blot analysis was performed. Fig. 3bed illustrate that Ru(II)-CNEB is able to increase the expression of both, SOD1 and SOD2 significantly (p 0.05e0.001) in the DL cells in vivo. 3.5. Ru(II)-CNEB mediated in vivo modulation of pro-oxiative factors in the DL cells The two antioxidant enzymes, catalase GPx, down stream to SOD, show significant decline in their activity (p 0.05) in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 4aec). However, treatment with Ru(II)-CNEB caused a significant decrease in the activity of GPx1, with a concomitant increase in the activity of GPx2 (Fig. 4c). Such a reciprocal change between SODs and catalase GPx is likely to allow accumulation of H2O2 at cellular level. Fig. 5a shows that indeed there is a marked increase (~6 times; p 0.001) in H2O2 level in the DL cells from Ru(II)-CNEB treated DL mice than those from the untreated groups. Level of glutathione is another marker of oxidative stress in the cells. According to Fig. 5b, as compared to the untreated DL group, the DL cells from Ru(II)-CNEB treated DL mice show a significant decline (p 0.05) in total glutathione (GSH þ GSSG) level. 3.6. Ru(II)-CNEB mediated in vivo modulation of pro-apoptotic factors in the DL cells Bcl2/Bax ratio serves as one of the critical determinants of apoptotic induction at cellular level. As compared to the untreated DL mice, treatment with Ru(II)-CNEB caused a significant decline (p 0.001) in the level of Bcl2 protein in the DL cells with a concomitant increase in the Bax protein (Fig 6b). Such reciprocal changes in Bcl2 vs Bax resulted into a significantly declined Bcl2/ Bax ratio (~3.5 times; p 0.01) in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 6b). The RT-PCR results (Fig 6a) also demon- strate a significant decline in Bcl2/Bax mRNA level, however, mainly due to a significant decrease in Bcl2 mRNA level in the DL cells from the treated rats. Caspase 9 cleavage is an important event of the mitochondrial pathway of apoptosis and that of PARP-1 cleavage associates with DNA fragmentation in the apoptotic cells. As shown in Fig. 6c, treatment with Ru(II)-CNEB is able to produce cleaved products of caspase 9. Full length PARP-1 is a 116 kDa protein, which, upon activation, is cleaved into a 89 kDa C-terminal a 24 kDa N-ter- minal protein fragments. Fig 6d illustrates ~1.5 times increase in the levels of the cleaved PARP-1 fragments with a proportionate decline in its full length PARP-1. This was consistent with the DNA fragmentation pattern of the DL cells reported previously [11]. 4. Discussion The two main findings reported in our previous paper [11]; one, Ru(II)-CNEB could inhibit M4-LDH and second, it induced release of mitochondrial cytochrome c in the DL cells in vivo, hinted towards induction of metabolic derangement led apoptosis in the DL cells due to the treatment with this compound. This tempted us to explore the regulatory aspects of such cellular effects produced by Ru(II)-CNEB. Since, decline in M4-LDH activity in the DL cells, due to the treatment with an anti-DL agent, could be attributed to the reduced expression of this enzyme [28], first we checked the mRNA level of LDH-A (M4-LDH) in the DL cells from Ru(II)-CNEB treated DL mice. The RT-PCR result (Fig. 1a) shows no change in the level of LDH-A mRNA in the DL cells due to the treatment with Ru(II)-CNEB. Thus, suggesting that Ru(II)-CNEB does not affect expression of M4- LDH. This implies that decline in M4-LDH activity, observed pre- viously due to the treatment with Ru(II)-CNEB [11], is a conse- quence of inhibiting catalytic efficiency of this LDH isozyme by the compound at protein level. Indeed, this complex has already been demonstrated to interact with and inhibit M4-LDH non- competitively [10]. β actin DL DL+Rc p53 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 DL DL+Rc Densitometricvalue (p53/βactin) *** Fig. 2. Treatment with Ru(II)-CNEB (Rc) increased the level of p53 in the DL cells in vivo. A representative western blot photograph is presented with the relative densitometric values of p53/b actin as mean ± SD from three western blot repeats. ***p 0.001 (untreated vs. treated DL groups). R.K. Koiri et al. / Biochimie 110 (2015) 52e6156
  6. 6. Overproduction of lactate is considered as one of the hallmarks of the tumor growth [44]. It has been reported that tumor cells survival is greatly supported by excess of lactate produced by the tumor cells [45]. Also, blockage of tumor M4-LDH, responsible to synthesize lactate, has been found to suppress this additional route of metabolic supplementation and thereby renders tumor cells susceptible to death [45]. Therefore, keeping aside the mechanism by which Ru(II)-CNEB decreases M4-LDH activity, the resultant decline in lactate production could be of high therapeutic relevance for this compound. Indeed, Ru(II)-CNEB, earlier found to inhibit M4-LDH [11], is also able to significantly decline lactate level in the DL cells (Fig. 1b), and thus, advocating M4-LDH as a therapeutic target for this compound at least in case of the DL cells in vivo. Such a mechanism gets support from inhibition of tumor cell prolifera- tion in vitro due to the treatment with certain N-hydroxyindole- based inhibitors of M4-LDH [46]. Moreover, M4-LDH catalyzed rapid production of lactate is mainly dependent on the adequate supply of pyruvate as a conse- quence of enhanced glycolysis in the tumor cells [18,47]. The committed step of glycolysis is catalyzed by PFK1 which is evident to be the target of multimodal regulation under a variety of path- ophysiological conditions including tumor development [16]. PFK2, Fig. 3. Effect of Ru(II)-CNEB (Rc) on (a) active levels of SOD2 SOD1, (b) on the expression of SOD1 and (c d) on the expression of SOD 2 in the DL cells from untreated and treated DL mice. (a) gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the relative densitometric values for the respective SOD bands from 3 PAGE repeats. (c) shows a representative RT-PCR photograph with the densitometric values of SOD2/b actin mRNA presented as mean ± SD from 3 RT-PCR repeats. (b and d) shows representative western blot photographs, with 60 mg protein in each lane, presented with the densitometric values of SOD1/b actin and SOD2/b actin respectively as mean ± SD from 3 western blot repeats. *p 0.05; **p 0.01; ***p 0.001 (untreated vs. treated DL groups). R.K. Koiri et al. / Biochimie 110 (2015) 52e61 57
  7. 7. the kinase domain of a bi-functional enzyme (PFK2/FBPase2), synthesizes FBP, the most effective allosteric activator of PFK1, and thereby, considered as a master regulator of the glycolytic pathway. In general, tumor cells are evident to express a C-type PFK1 that shows greater sensitivity for FBP activation [16] and also express a catalytically more efficient isoform of PFK2 (iPFK2: PFKFB3) [13,17]. Importantly, iPFK2 is found to be over expressed as a universal trait of most of the growing tumor cells including DL [28] and many tumors of human origin as well [17]. It was interesting to observe that this compound could significantly decline the expression of iPFK2, both at mRNA and at protein levels (Fig. 1c and d). This clearly suggested that Ru(II)-CNEB is able to downregulate the synthesis of master regulator of the glycolytic pathway in the DL cells in vivo. The argument is well supported by a report describing correlation between PFKFB3 gene silencing by siRNA and apoptosis in the Hela cells in culture [13]. Though information is scanty on modulation of iPFK2 by synthetic compounds in tumor cells, a 3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one has been found to inhibit iPFK2 resulting into decreased glucose uptake by the tumor cells leading into tumor growth suppression in vivo [48]. In this context, the findings of Fig. 1c and d are first of its kind to demonstrate that Ru(II)-CNEB is able to repress iPFK2 in a tumor cell in vivo and thereby speculated to render less production of adequate glycolytic intermediates to sustain high glycolytic effi- ciency of the DL cells in vivo. So far tumor growth associated regulator of cell bioenergetics is concerned, p53, a tumor suppressor protein, has been given much emphasis as it has been reported to be involved in imposing War- burg effect during tumor development [19,22]. It is now evident that switching over to glycolytic phenotype by the tumor cells is accompanied with the declined p53 level [25]. Similarly, the enhanced level of p53 has been found to repress glucose trans- porters GLUT1 and GLUT4 [23]. Also, increased p53 has been demonstrated to decline the expression of iPFK2 via activating the TIGAR gene [24] and consequently, it inhibits glycolysis [26]. We have observed significant increase in p53 level (Fig. 2) vis a vis a significantly declined iPFK2 expression (Fig.1c and d) in the DL cells due to the treatment with Ru(II)-CNEB and thus suggesting an as- sociation between Ru(II)-CNEB mediated enhanced p53 level and declined activity of the committed step of glycolytic pathway in the DL cells in vivo. It is known that activation of aerobic glycolysis by the tumor cells is a metabolic strategy to prevent production of ROS, an inevitable outcome of the oxidative energy metabolism, and thereby to protect tumor cells from ROS induced apoptosis [49]. In addition, tumor cells are known to modulate main antioxidant enzymes to prevent oxidative stress [34,35]. Therefore, deranging such enzymatic mechanisms by a therapeutic agent is argued to be a relevant option for driving tumor cells to undergo apoptosis [28,43]. SOD is the first and commited enzyme of antioxidant pathway that neutralizes O2 À into H2O2. It has been reported that reduced levels of SOD1 and SOD2 maintain low level of H2O2 in the cancerous cells to facilitate tumerogenesis [34,35]. Similarly, increased SOD activity and in turn, higher level of H2O2 is specu- lated to inhibit tumor progression [36,37]. According to Fig. 3a, there is a significant increase in the activity of both the SOD iso- forms; SOD2 SOD1, in the DL cells from Ru(II)-CNEB treated DL mice. Since, the pattern of active levels of both of them coincided with the similar increments in expression of both these enzymes (Fig. 3bed), it is evident that Ru(II)-CNEB is able to enhance expression of SOD2 and SOD1 to make overall increment in SOD activity in the DL cells in vivo. Since reports are limited on metal complex induced expression of antioxidant enzymes, it is a first report wherein, a Ru(II)-complex is demonstrated to enhance ac- tivity of SOD by overexpressing SOD2 and SOD1 proteins. Moreover, keeping aside these explanations, the enhanced level of SODs is likely to ultimately produce higher level of H2O2 in the DL cells due to the treatment with Ru(II)-CNEB. H2O2 produced by SODs is metabolized by GPx and Catalase and activity of both these enzymes were observed to be declined significantly in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 4aec). Such a reciprocal pattern between SOD vs catalase GPx has been reported accountable for unusually increased level of H2O2 in the DL cells due to the treatment with emodin [50]. Indeed, 0 1 2 3 4 5 6 DL DL+Rc Catalaseactivity (U/mgprotein) * a 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 DL DL+Rc GPxactivity (U/mgprotein) * b 0 10 20 30 40 50 60 DL DL+RcsdnabxPGfoyrtemotisneD (Relativeintensity) GPx1 GPx2 ** * GPx1 DL DL+Rc GPx2 GPx3 c Fig. 4. Effect of Ru(II)-CNEB (Rc) on the activity of catalase (a) and GPx (b and c) in the DL cells in vivo. The values in (a and b) are mean ± SD, where n ¼ 4. (c) represents active level of GPx in the DL cells from untreated and complex treated DL mice. The gel photograph is a representative of 10% native PAGE of 60 mg protein in each lane, presented with the relative densitometric values for the respective GPx bands from 3 PAGE repeats. *p 0.05; **p 0.01 (untreated vs. treated DL groups). R.K. Koiri et al. / Biochimie 110 (2015) 52e6158
  8. 8. H2O2 concentration was found to be remarkably increased (~6Â) in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 5a). Thus, it is argued that Ru(II)-CNEB is able to modulate all the three enzymes of antioxidant pathway; SOD, catalase and GPx, to maintain higher level of H2O2 in the DL cells. Such a condition might drive the DL cells to undergo apoptosis, as increase in intracellular H2O2 is known to cause a significant drop in cytosolic pH [51] which is considered accountable for translocation of Bax, a pro-apoptotic factor, to mitochondria [49]. Though the mechanism by which Ru(II)-CNEB regulates reciprocal changes in SODs vs catalase and GPx could be a matter of further investigation, in the present context, however, enhanced level of p53 protein (Fig. 2) could be considered as one of the integrators of such enzymatic changes. This is because, not only a close association between p53 dependent modulation of ROS metabolism and cell apoptosis is on record [26,27] but also, higher level of p53 protein has been found to enhance the expression of pro-oxidant and proapoptotic factors [28,30]. Fig. 5. Effect of Ru(II)-CNEB (Rc) on the level of H2O2 (a) and total glutathione (b) in the DL cells in vivo. (a) represents level of H2O2 in the DL cells. Values are mean ± SD and n ¼ 4. (b) represents level of total glutathione in the DL cells in vivo. Values are mean ± SD, where n ¼ 3. *p 0.05; ***p 0.001 (untreated vs. treated DL groups). Fig. 6. Treatment with Ru(II)-CNEB (Rc) caused a significant decline in the expression of Bcl2 with concomitantly increased expression of Bax (a and b), activation of caspase 9 (c) and PARP1 cleavage (d) in the DL cells in vivo. a shows representative RT-PCR photographs with the b-actin normalized densitometric ratio of Bcl2/Bax mRNA where values represent mean ± SD from 3 RT-PCR repeats. b shows representative western blot photographs with the b-actin normalized densitometric ratio of Bcl2/Bax where values represent mean ± SD from three western blot repeats. ***p 0.001 (untreated vs. treated DL groups). R.K. Koiri et al. / Biochimie 110 (2015) 52e61 59
  9. 9. Glutathione content is known to represent actual redox status of the cells and its enhanced level in the cancer cells is directly correlated with the tumor progression. Higher glutathione level, in addition to providing multidrug and radiation resistance [52], has been found to prevent apoptosis in the tumor cells [53]. Similarly, depletion of glutathione level has been reported to sensitize tumor cells to undergo apoptosis via release of cytochrome c [54]. The anti-apoptotic role of Bcl-2 has also been linked with the gluta- thione content in the tumor cells [55]. We have shown previously that Ru(II)-CNEB causes release of mitochondrial cytochrome c [11] and according to Fig. 6a and b, it is evident to decline Bcl2/Bax ratio also. Importantly these cellular alterations are consistent with a significant decline in total glutathione (GSH þ GSSG) level in the DL cells (Fig. 5b) and thereby providing another biochemical mecha- nism by which Ru(II)-CNEB could be able to induce apoptosis in the DL cells. One of the important aspects of p53 biochemistry is that it is considered to be involved in regulating apoptosis in the tumor cells [19,22,27,28]. Normally, p53 remains sequestered with Mdm2 in the cytosol and thereby prevents cells to undergo apoptosis [56]. This implies that release of p53 from Mdm2 could be one of the mechanisms to induce apoptosis in the cells. This may happen due to many cellular changes including increased DNA damage caused by the exogenous agents [57]. However, we observed that the increasing concentration of Ru(II)-CNEB, when incubated in vitro at 37 C for 24 h with pBR322 plasmid DNA, it did not convert supercoiled plasmid into the nicked circular DNA (Supplementary data; Fig. S1) and thereby excluding direct nuclease activity of this compound. Therefore, in the present context, as reported earlier [28,56], abrrent ellular signaling could be argued account- able for a significantly enhanced level of p53 in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 2). Moreover, keeping aside the mechanism by which p53 level gets enhanced in the DL cells, it is considered to act as a strong apoptotic inducer in multimodal ways. The enhanced cytoplasmic p53 level has been reported to activate Bax and its oligomerization [58] which in turn, induces cytochrome c release from the mitochondria [59]. Bcl2 is an anti-apoptotic factor and therefore, declined Bcl2/Bax ratio is considered accountable for initiating intrinsic pathway of apoptosis [28]. In the present context, Ru(II)-CNEB significantly declined the level of Bcl2 protein with a concomitant increase in Bax level (Fig. 6b). This suggested that Ru(II)-CNEB is able to reciprocally modulate level of both these factors resulting into a significant decline in Bcl2/Bax ratio in the DL cells in vivo. Caspase 9 activation is a hallmark of mitochondrial pathway of apoptosis [60] and that of PARP-1 cleavage is associated with DNA fragmentation in the cells undergoing apoptosis [61]. We observed significant increase in the levels of cleaved caspase 9 and PARP-1 in the DL cells from Ru(II)-CNEB treated DL mice (Fig. 6c and d). Consistent with these observations, Ru(II)-CNEB mediated release of mitochondrial cytochrome c, the main initiator of the intrinsic pathway of apoptosis, in those DL cells, is already on record [11]. Thus, taking together, these findings strongly advocate for p53 mediated activation of Bcl2/Bax-cytochrome c release-caspase 9 led intrinsic pathway of apoptosis in the DL cells due to treatment with Ru(II)-CNEB in vivo. Though not much information is available on induction of intrinsic pathway of apoptosis by metal complexes, a Ru(II)-arene compound [Ru(g6-p-cymene)Cl2(pta)] has been described to implicate this pathway for inducing apoptosis in the Ehrlich ascite carcinoma [62]. Recently, another Ru(II)-complex (Ru(II) b-carboline complex) has also been demonstrated to induce apoptosis in the cancer cells by involving p53 [63]. In this context, the findings of this paper are of special merit with regard to eluci- dation of the biochemical mechanism by which a Ru(II)-complex can induce p53 dependent apoptosis in the tumor cells in vivo. In conclusion, there is an evolving concept of restricting tumor growth by depriving tumor cells from adequate energy production and by rendering them susceptible to oxidative stress. The present article demonstrates that a Ru(II)-CNEB, characterized previously as an anti tumor compound, is evident to activate p53 mediated glycolytic inhibition-oxidative stress-apoptosis pathway in the DL cells when administered in vivo. As such these findings provide a biochemical mechanism which can be utilized for defining phar- macological targets for the novel anticancer agents suitable for in vivo applications. Conflict of interest The authors declare no conflict of interest with respect to this article. Acknowledgements This work was financially supported by a project from Depart- ment of Biotechnology (DBT), Govt. of India, (BT/PR5910/BRB/10/ 406/2005) sanctioned jointly to LM and SKT at BHU. The contri- bution of Dr. SK Dubey in synthesizing Ru(II)-CNEB in the lab of LM is also acknowledged. 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