ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7                                                                   Leukemia ...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 72                                               R.K. Koiri, S.K. Trigun / Le...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7                                                       R.K. Koiri, S.K. Trig...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 74                                                      R.K. Koiri, S.K. Trig...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7                                                        R.K. Koiri, S.K. Tri...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 76                                                      R.K. Koiri, S.K. Trig...
ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7                                                           R.K. Koiri, S.K. ...
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  1. 1. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7 Leukemia Research xxx (2011) xxx–xxx Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukresDimethyl sulfoxide activates tumor necrosis factor -p53 mediated apoptosis anddown regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5in Dalton’s lymphoma in vivoRaj K. Koiri, Surendra K. Trigun ∗Department of Zoology, Biochemistry Section, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, Indiaa r t i c l e i n f o a b s t r a c tArticle history: Dimethyl sulfoxide (DMSO) is evident to induce apoptosis in certain tumor cells in vitro. However, itsReceived 23 September 2010 apoptotic mechanism remains unexplored in in vivo tumors. This article describes that DMSO, beingReceived in revised form non-toxic to the normal lymphocytes, up regulated TNF and p53, declined Bcl-2/Bax ratio, activated20 December 2010 caspase 9 and PARP-1 cleavage and produced apoptotic pattern of DNA ladder in Dalton’s lymphomaAccepted 29 December 2010 (DL) in vivo. This was consistent with the declined expressions of tumor growth supportive glycolyticAvailable online xxx enzymes; inducible d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in the DL cells. The findings suggest induction of TNF -p53-mitochondrial pathway of apoptosis by DMSO in a non-Hodgkin’sKeywords:DMSO lymphoma and support evolving concept of glycolytic inhibition led apoptosis in a tumor cell in vivo.Apoptosis © 2011 Elsevier Ltd. All rights reserved.Inducible d-fructose-6-phosphate-2-kinase(iPFK2)Lactate dehydrogenase-5 (LDH-5)Dalton’s lymphomaTNFp531. Introduction The glycolytic phenotype of tumor cells, popularly known as ‘Warburg effect’, is now evident to be a near universal Dimethyl sulfoxide (DMSO) is primarily used as a solvent for trait of all the growing tumors [10]. Though limited, but somepharmaceutics and as a cryopreservant of cell lines. However, its studies suggest a link between depletion of glycolytic factorsbeneficial effects observed against many ailments [1,2] necessitate and induction of apoptosis in the cancer cells in vitro [11]investigations on therapeutic properties of this compound. DMSO and in vivo [12–14]. PFK2 domain of d-fructose-6-phosphate-dependent differentiation of leukemia cells [3] and induction of 2-kinase/fructose-2,6-bisphosphatase (PFK2/FBPase2) synthesizesapoptosis in a number of cell lines [4,5] suggest that this com- fructose-2,6-bisphosphate (FBP2) which in turn, activates phos-pound could be a potent anticancer agent. Up regulation of a tumor phofructokinase1 (PFK1) and thereby, regulates committed step ofsuppressor protein (PTEN) in HL-60 cells [6] and differentiation of glycolysis. Cancer cells express C type PFK1 which is more sensitiveHuh7 cells by DMSO via increased expressions of drug metabolizing to FBP2 [15] and concordantly, over express a catalytically moreenzymes and many transcription factors [7] suggest gene modu- efficient inducible PFK2 (iPFK2: PFKFB3 gene) [16], whose inacti-lating actions of DMSO in the tumor cells. Induction of apoptosis vation has been reported to regress tumor cell growth in vitro [11].in a murine leukemia cell via DMSO dependent alterations in p53 In growing tumors, hypoxia induced factor1 (HIF1 ) inhibitsconformation [8] and inhibition of telomerase activity in a Burkitt pyruvate dehydrogenase complex and thereby, restricts entry oflymphoma cell [9] further suggest protein modulating activities of pyruvate into tri-carboxylic acid cycle. This drives shunting of pyru-this compound in the tumors. Such multimodal actions of DMSO, vate to produce lactate by lactate dehydrogenase-5 (LDH-5). Overobserved in vitro, invite special attention to explore tumor growth expression of LDH-5 gene (LDH-A) is associated with tumor growthassociated molecular targets for DMSO in in vivo tumors. [10,17]. Decline of LDH-5 by a ruthenium complex was also found to induce apoptosis in the Dalton’s lymphoma (DL) in vivo [14]. Thus, inducing apoptosis in tumor cells by inhibiting tumor glycolysis is of current interest [12,14]. ∗ Corresponding author. Tel.: +91 0542 2575199; fax: +91 0542 368174. Although DMSO has been observed to induce apoptosis in lym- E-mail address: sktrigun@sify.com (S.K. Trigun). phoma cells in vitro [4,5], reports are scanty on DMSO dependent0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2010.12.029 Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
  2. 2. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 72 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxxcell death mechanisms in the tumor cells in vivo. In this paper, transferred to nitrocellulose membrane followed by detection ofwe have investigated whether administration of a pharmaceutical different proteins against 1:1000 times diluted protein specificdose of DMSO to DL bearing mice was able to induce pro-apoptotic polyclonal antibodies. Protein bands were detected by ECL kit. Asmechanisms and to modulate expressions of PFKFB3 and LDH-A in loading control, -actin was probed similarly using monoclonalthe DL cells without affecting normal lymphocytes. anti- -actin-peroxidase antibody (1:10,000). Protein bands were quantified using gel densitometry software AlphaImager 2200.2. Materials and methods 2.6. Analysis of LDH isozymes by non-denaturing polyacrylamide2.1. Induction of Dalton’s lymphoma (DL) in mice gel electrophoresis (PAGE) Inbred AKR strain mice of 16–18 weeks age weighing 24–26 g, Non-denaturing PAGE analysis of LDH employs substrate speci-used for this experiment, were maintained at laboratory condi- ficity based detection of all LDH isozymes distinctly in the same gel,tions and subjected to various treatments as per the guidelines and and it is considered highly relevant for interpreting LDH isozymeapproval from institutional animal ethical committee. based alterations at cellular level [14,18]. DL was induced by transplantation of 1 × 107 viable tumor cells LDH isozymes in various tissue extracts, in DL cell extracts and(assayed by trypan blue method; 14) i.p. per mice. Development in cell free ascitic fluid were analyzed using 10% PAGE as describedof DL was confirmed by belly swelling and increased body weight earlier [18]. The extracts containing 60 g protein were loaded inwhich became visible on 10–12th post transplantation day. The DL each lane and electrophoresed under non-denaturing conditions atbearing mice survived up to 18 ± 2 days. 4 ◦ C. Gels were subjected to activity based detection and identifi- cation of different LDH isozymes in the gel.2.2. Experimental protocol 2.7. Semi-quantitative RT-PCR DL bearing mice were randomly divided into two groups with4–5 mice in each. The experimental group (DL + DMSO) mice were Total RNA was isolated from DL cells using TRI reagent fol-treated with 200 l DMSO (∼7.5 g/kg b.w., i.p.) and those of DL con- lowing manufacturer’s protocol. After DNase I (DNA free-Ambion)trol were similarly injected with equal volume of normal saline on digestion, reverse transcription of 2 g RNA was done using 200 Upost transplantation day 10. For biochemical and molecular studies, of reverse transcriptase and 200 ng random hexamer to make ss-3–4 mice from each group were sacrificed on day 18th. cDNA (Revert Aid First strand cDNA synthesis kit, MBI fermentas). The PCR reaction mixture contained 1× Taq polymerase buffer,2.3. Collection and preparation of cell/tissue extracts 0.2 mM dNTPs, 1 U of Taq polymerase, and 10 pmol of specific primer. DL cells were collected by centrifuging tumor ascites pooled The mouse gene-specific primers used were: PFKFB3from 3 to 4 DL mice from each group at 2000 × g at 4 ◦ C. Normal lym- (forward 5 -GGCAAGATTGGG GGCGACTC-3 ; reverse 5 -phocytes were separated from heparin containing blood, collected GGCTCCAGGCGTTGGACAAG-3 ); LDH A (forward 5 -ATG CACC-from severed neck of mice, using density gradient centrifugation at CGCCTAAGGTTCTT-3 ; reverse 5 -TGCCTACGAGGTGATCAAGCT-400 × g for 30 min with histopaque-1077 reagent (Sigma diagnos- 3 ); Bcl-2 (forward 5 -TACCGTCGTGACTTCGCAGAG-3 ; reversetics protocol). 5 -GGCAGGCTGAGCAGGGT TT-3 ); Bax (forward 5 -CGGCGAAT- The DL cell and normal lymphocyte extracts were prepared TGGAGATGAACTG-3 ; reverse 5 -GCAAAGTAGAAG AGGGCAACC-using lysis buffer (20 mM Tris–Cl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 3 ); TNF˛ (forward 5 -ATGAGCACAGAAAGCATGATCC-3 ; reverse1 mM EGTA, 1% triton X-100, 25 mM Na2 pyrophosphate and 1 mM 5 -GAAGATGATCTGAGTGTG-3 ) and ˇ Actin (forward 5 -ATCG-PMSF). The cell lysates were centrifuged at 10,000 × g for 30 min TGGGCCGCTCTAGGCAC C-3 ; reverse 5 -CTCTTTGATGTCACGAT-and supernatants obtained were used for biochemical and molec- TTC-3 ). PCR were run as: for -actin, 26 cycles; for LDH and Bcl-2,ular studies. Liver and spleen extracts were prepared in a protease 31 cycles of 45 s at 94 ◦ C, 45 s at 55 ◦ C, and 1 min at 72 ◦ C. For Bax,inhibitor containing extraction medium as described previously 31 cycles of 60 s at 56 ◦ C, and 1 min at 72 ◦ C; for PFKFB3, 30 cycles[14,18]. of 60 s at 95 ◦ C, 60 s at 60 ◦ C, and 1 min at 72 ◦ C. Amplification products were analyzed by 1–2% agarose gel electrophoresis and2.4. DNA ladder study visualized by ethidium bromide staining. -Actin amplification served as a control. Total DNA from DL cells was isolated as described previously[14]. Briefly, 5 × 106 DL cells were lysed in 1 ml lysis buffer for 2.8. Statistical analysis1 h, added with 0.4 ml 5 M NaCl and after 5 min, centrifuged at3000 × g for 30 min. The supernatants were treated with RNase Experimental data were expressed as mean ± SD and Student’s(20 g/ml) for 15 min. DNA was precipitated by adding 2× (v/v) t-test was applied for determining the level of significance betweenchilled ethanol. DNA collected after centrifugation was dissolved DL control vs DMSO treated DL groups.in TAE buffer (40 mM Tris–acetate + 1 mM EDTA). For agarose gel electrophoresis, DNA samples were prepared in 3. Resultsa loading solution (0.25% bromophenol blue, 0.25% xylene cyanol FFand 30% glycerol) in the ratio of 1:5 and samples containing 10 g 3.1. DMSO induced apoptosis in the DL cells in vivoDNA were electrophoresed on 1% agarose gel containing 0.5 g/mlethidium bromide in TAE buffer for 2–3 h. The DNA bands in gel In general, TNF mediated apoptosis implicates p53 inducedwere observed under UV transilluminator. mitochondrial mechanism. We have compared expression levels of the key partners of this pathway in the DL cells from DMSO treated2.5. Western blotting DL mice with that from the untreated DL group. Based on RT-PCR analysis, level of TNF mRNA was observed to be ∼2-times higher DL cell extracts containing 60 g protein, were subjected with a significant increment in the level of p53 protein (p < 0.05)to 10% SDS-PAGE. As described previously [14], proteins were in the DL cells from DMSO treated DL mice than that from the Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
  3. 3. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx 3Fig. 1. DMSO caused increased expressions of TNF (A) and p53 (B) in the DL cells in vivo. (A) A representative RT-PCR photograph with the normalized values of TNF / actinmRNA levels as mean ± SD from three RT-PCR repeats. (B) A representative western blot photograph with the normalized values of p53/ actin protein levels as mean ± SDfrom three western blot repeats. * p < 0.05; *** p < 0.001.untreated group (Fig. 1A and B). Furthermore, levels of Bcl-2 mRNA 9 and PARP-1 with proportionate decline of their respective pro-and its protein were found to be declined markedly with the con- caspases were observed in the DL cells from DMSO treated DL micecomitant increments in Bax expression resulting into a significant than that from the untreated group (Fig. 3A). Additionally, as com-decline in Bcl-2/Bax ratio (Fig. 2A and B) in the DL cells from DMSO pared to a single intact genomic DNA band seen in case of DL cellstreated DL mice than that of the untreated counterpart. from untreated DL group, DNA from DL cells of DMSO treated group Caspase 9 activation is associated with induction of mitochon- showed many DNA fragments in the range of ∼800–200 bp (Fig. 3B).drial pathway of apoptosis and that of PARP-1 cleavage with DNAfragmentation in the apoptotic cells. As prescribed in supplier’s 3.2. Down regulation of PFKFB3 and LDH-A by DMSO in the DLmanual, anti-caspase 9 and PARP-1 antibodies could detect pro- cellscaspases as well as their cleaved products; 46- and 35 kDa and116 and 24 kDa for caspase 9 and PARP-1 respectively (Fig. 3A). Over expression of iPFK2 (PFKFB3) and LDH-A are associatedAccordingly, ∼2-times increases in the levels of cleaved caspase with tumor growth. As illustrated in Fig. 4A and B, the levels ofFig. 2. DMSO caused declined expression of Bcl-2 with concomitantly increased expression of Bax in the DL cells in vivo. (A) Representative western blot photographs withBcl-2/Bax ratio as mean ± SD from three western blot repeats. (B) Representative RT-PCR photographs with the ratio of Bcl-2/Bax mRNA level as mean ± SD from three RT-PCRrepeats. ** p < 0.01. Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
  4. 4. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 74 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxxFig. 3. DMSO caused activations of caspase 9 and PARP 1 (A) and DNA fragmentation (B) in the DL cells in vivo. (A) Representative western blot photographs from threewestern blot repeats for each protein; caspase 9 and PARP 1 and that of -actin as loading control. (B) Represents a representative (out of three repeats) ethidium bromidestained DNA gel photograph.PFKFB3 mRNA and its protein product were declined significantly apoptotic proteins, is the most plausible mechanism [20–23]. We(p < 0.01 and 0.05 respectively) in the DL cells from DMSO treated observed ∼2-times increment in the level of TNF mRNA with sig-DL mice than that from the untreated counterpart. nificantly increased level of p53 protein in the DL cells from DMSO For native PAGE analysis based identification of LDH isozymes, treated DL mice (Fig. 1). The TNF -p53 related apoptotic mecha-the LDH band obtained from DL extracts was compared with a nism is suggested to involve mitochondrial pathway of apoptosisstandard pattern showing all the five LDH isozymes in the kidney [21,22] wherein, p53 induced alterations in Bcl-2/Bax ratio acts as aextracts of normal mice (Fig. 4C(a)). Accordingly, an over activated determining factor [23]. Thus, significantly declined levels of Bcl-2LDH-5 band was observed in the DL cell extracts of the untreated DL mRNA and its protein with concomitant increments in Bax mRNAmice (Fig. 4C(b)), however, with a significant decline (p < 0.001) in and its protein (Fig. 2), in effect, represented a significantly reducedthe DL cell extracts from DMSO treated DL group. The RT-PCR result Bcl-2/Bax ratio in the DL cells from DMSO treated group. Keeping(Fig. 4D) of the corresponding gene (LDH-A) further suggested a aside some fragmentary reports from in vitro studies on involve-similar decline in LDH-A mRNA level (p < 0.05) in the DL cells from ment of Bcl-2/Bax in DMSO induced apoptosis [5], the presentDMSO treated group. finding is the first report to demonstrate up regulations of TNF and p53 with concomitant decline in Bcl-2/Bax ratio and thereby, sug-3.3. Regression of DL by DMSO and effect on normal tissues gesting induction of mitochondrial pathway of apoptosis by DMSO in a non-Hodgkin’s lymphoma in vivo. In case of myeloid leukemia Decline in ascitic volume and release of DL specific LDH-5 in U937 cells also, DMSO has been reported to induce death recep-cell free ascitic fluid are considered good parameters to ascertain tor mediated apoptosis via depolarizing mitochondrial membraneDL regression in vivo. DMSO caused significant decline in ascitic [19].volume (p < 0.05) with concomitant release of LDH-5 in the cell free Caspase 9 activation is a hall mark of mitochondrial pathway ofascitic fluid (Fig. 5A and B). This was consistent with a declining apoptosis [23] and that of PARP-1 cleavage is associated with DNAtrend in the body weight of DMSO treated DL mice also. Further, fragmentation in the cells undergoing apoptosis. We observed ∼2-to ascertain DL cell specific effect of DMSO, the level of LDH-5, as times increments in the levels of cleaved caspase 9 and PARP-1 ina susceptible parameter, was compared in normal lymphocytes, the DL cells from DMSO treated DL mice (Fig. 3A). Thus, togetherspleen and liver from normal, DL bearing and DMSO treated DL with the production of DMSO dependent apoptotic pattern of DNAmice. Fig. 5C–E suggests that DMSO did not alter the level of LDH-5 ladder in the DL cells (Fig. 3B), these results suggested induction ofin these tissues. mitochondrial pathway of apoptosis, consistent with up regulation of TNF and p53, in the DL cells due to treatment with DMSO in vivo.4. Discussion There could be many biochemical aberrations accountable for apoptosis in the tumor cells. As tumor cells in vivo depend more DMSO, as a vehicle for hydrophobic compounds, is considered on anaerobic energy production, inhibiting key glycolytic stepsnon-toxic to the animals. In one such evaluation, a single dose in tumor cells is of current focus as a novel anticancer strategyof 200 l (∼7.5 g/kg b.w.) DMSO was also found to be non-toxic [11,13,14,17]. PFK1 catalyzes committed step of glycolysis. FBP2 ,to the normal mice (data of pilot experiments). However, when a metabolic activator of PFK1, is synthesized by PFK2. Tumor cellsadministered to the DL bearing mice, it significantly declined ascitic express a C-type PFK1 with increased sensitivity for FBP2 activationvolume and caused LDH-5 release in the cell free ascitic fluid (Fig. 5A [15] and concordantly, a catalytically more efficient iPFK2: PFKFB3and B). As reported earlier [14], these findings suggested DL cell gene [11,16]. We observed DMSO mediated decline in the levels ofdeath/regression in vivo by DMSO. Additionally, apoptotic pattern PFKFB3 mRNA and its protein (iPFK2) in the DL cells (Fig. 4A andof DNA ladder observed in the DL cells from DMSO treated DL mice B) suggesting down regulation of iPFK2 by DMSO in these cells. As(Fig. 3B) led us to investigate mechanistic aspects of DMSO induced decline of iPFK2 has been correlated with the regression of certainapoptosis in the DL cells. tumors in vitro [11], it is argued that DMSO dependent down reg- Although limited, but some in vitro studies suggest that DMSO ulation of this enzyme could be associated with DL cell apoptosisprimarily potentiates death receptor mediated apoptosis by involv- in vivo.ing different mechanisms in different tumor cells [19,20]. However, Over activation of LDH-5 is associated with tumor growth in vivoTNF mediated apoptosis, involving p53 and p53 related pro- because, it preferentially converts pyruvate into lactate to facilitate Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
  5. 5. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 7 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxx 5Fig. 4. DMSO declined the levels of iPFK2 (A) and its mRNA (B) and those of active LDH-5 (C) and LDH-A mRNA (D) in the DL cells in vivo. (A) A representative westernblot photograph and values of iPFK2/ actin as mean ± SD from three western blot repeats. (B and D) Representative RT-PCR photographs for PFKFB3 and LDH-A with theratio of PFKFB3/ actin and LDH-A/ actin mRNA respectively as mean ± SD from 3 RT-PCR repeats. (C) Representative photographs from three PAGE repeats obtained fortissue specific standard LDH isozymes in the kidney extract (C a) and for the LDH-5 bands in the DL extracts (C b) with relative densitometry values as mean ± SD. * p < 0.05;** p < 0.01, *** p < 0.001.anaerobic energy production in the tumor cells [10,17,24]. Thus, gene silencing by siRNA has been correlated with the depletion ofrepression of LDH-5 can affect tumor cell bioenergetics severely. cellular energy substrates and induction of apoptosis in the HeLaLike most of the tumors, DL cells also over activate LDH-5, whose cells [11]. Blockage of tumor LDH-A has also been reported to ren-inactivation by a novel anticancer compound has been correlated der tumor cells susceptible to death [24]. Recently, we have alsowith apoptosis of DL in vivo [14]. An intense band of active LDH-5 demonstrated a correlation between decline of LDH-5 and induc-in DL cell extracts from untreated DL mice (Fig. 4C(b)) corroborated tion of apoptosis by a ruthenium complex in the DL cells in vivothese earlier findings and accordingly, significantly declined level of [14]. In the present context, therefore, it is argued that as a con-active LDH-5 in DL cells from DMSO treated mice suggested DMSO sequence of DMSO mediated decreased expressions of PFKFB3 andled decline in the activity of this enzyme in the DL cells in vivo. As LDH-A (Fig. 4), DL cells might be deprived of adequate energy pro-RT-PCR band for the corresponding gene (LDH-A) was also observed duction and concomitantly forced to undergo apoptosis. Moreover,to be declined similarly in the DL cells from DMSO treated group induction of apoptosis due to inhibition of tumor glycolysis is a rel-(Fig. 4D), it is argued that decline in LDH-5 activity resulted due atively newer concept [12,14] and therefore, need to be defined into DMSO mediated decreased expression of LDH-A in the tumor many tumor models. In this respect, this is a first report to describecells. declined expressions of tumor growth supportive glycolytic factors As compared to in vitro conditions, tumor cells in vivo face consistent with the induction of TNF -p53-mitochondrial path-greater hypoxia and therefore, they rely much on anaerobic gly- way of apoptosis by DMSO in a tumor cell in vivo. Indeed, TNFcolysis for their energy requirements [17]. Depletion of energy induced apoptosis in another non-Hodgkin’s lymphoma has beensubstrates is considered as strong apoptotic signal [25]. PFKFB3 found associated with changes in the LDH isozymes [26]. Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
  6. 6. ARTICLE IN PRESSG ModelLR-4131; No. of Pages 76 R.K. Koiri, S.K. Trigun / Leukemia Research xxx (2011) xxx–xxxFig. 5. DMSO declined ascitic volume (A) and enhanced LDH-5 release in the cell free ascitic fluid (B) without any adverse effects on active level of LDH-5 in the normallymphocytes (C), spleen (D) and liver (E) of DL mice. (A) Data as mean ± SD where n = 4. (B) A representative photograph with relative densitometry of LDH-5 bands asmean ± SD from three PAGE repeats. (C–E) Representative photographs from three PAGE repeats obtained for normal lymphocytes, spleen and liver respectively. *p < 0.05. Tumor specificity is the main limitation of anticancer agents. Fellowship to R.K.K. for working on this topic. The facilities pro-LDH is a highly sensitive enzyme to undergo change during tumor vided due to UGC-CAS and DST-FIST programmes to Department ofgrowth and tissue toxicity [14,17,27]. We observed unaltered pat- Zoology, BHU, are also acknowledged.tern of LDH-5 in normal lymphocytes, in spleen (lymphocytematuration organ) and in liver (affected during drug treatment) of Referencesnormal and DMSO treated DL mice (Fig. 5C–E). This suggested thatDMSO did not affect normal lymphocyte biochemistry and other [1] Santos NC, Figueira-Coelho J, Martins-Silva J, Saldanha C. Multidisciplinarynormal tissues as well. An earlier report has also described that utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem Pharmacol 2003;65:1035–41.DMSO was more toxic to a lymphocytic leukemia than to lympho- [2] Parisi A, Alfieri A, Mazzella M, Mazzella A, Scognamiglio M, Scognamiglio G,cytes from normal patients and also to lymphomas of AKR mice et al. Protective effect of dimethyl sulfoxide on acute myocardial infarction inthan to the normal lymphocytes [28]. rats. J Cardiovasc Pharmacol 2010;55:106–9. [3] Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Terminal differentiation of In conclusion, for the first time we have demonstrated that a human promyelocytic leukemia HL-60 cells induced by dimethyl sulfoxide andpharmaceutical dose of DMSO was able to regress a non-Hodgkin’s other polar compounds. Proc Natl Acad Sci USA 1978;75:2458–62.lymphoma: DL in vivo via inducing TNF -p53-mitochondrial path- [4] Trubiani O, Ciancarelli M, Rapino M, Primio RD. Dimethyl sulfoxide induces programmed cell death and reversible G1 arrest in the cell cycle of humanway of apoptosis. As such, these findings would be of immense lymphoid pre-T cell line. Immunol Lett 1996;50:51–7.applications to examine DMSO, a neutral and freely absorbable [5] Liu J, Yoshikawa H, Nakajima Y, Tasaka K. Involvement of mitochondrial per-compound, for its anticancer potential against several in vivo meability transition and caspase-9 activation in dimethyl sulfoxide-induced apoptosis of EL-4 lymphoma cells. Int Immunopharmacol 2001;1:63–74.tumors. Additionally, though DMSO was found to modulate brain [6] Lee YR, Shim HJ, Yu HN, Song EK, Park J, Kwon KB, et al. Dimethylsulfoxidemetabolism [29], there is no previous report on modulating tumor induces upregulation of tumor suppressor protein PTEN through nuclear factor-cell biochemistry by DMSO. Thus, DMSO led down regulation of B activation in HL-60 cells. Leuk Res 2005;29:401–5.PFKFB3 and LDH-A in the DL cells is a first report to suggest mod- [7] Choi S, Sainz B, Corcoran P, Uprichard S, Jeong H. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hep-ulations of the key glycolytic steps by DMSO in a cancerous cell atoma cells. Xenobiotica 2009;39:205–17.in vivo. Taken together, these findings support the evolving concept [8] Ryan JJ, Clarke MF. 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Inhibition of glycoly-Acknowledgements sis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res 2005;65:613–21. This work had a genesis from a Department of Biotechnology [13] Gaber K. Energy deregulation: licensing tumors to grow. Science 2006;312:1158–9.(DBT), Govt. of India project (No. BT/PR5910/BRB/10/406/2005). [14] Koiri RK, Trigun SK, Mishra L, Pandey K, Dixit D, Dubey SK. Regression of Dalton’sThe authors thank CSIR, Govt. India, for awarding Senior Research lymphoma in vivo via decline in lactate dehydrogenase and induction of apop- Please cite this article in press as: Koiri RK, Trigun SK. Dimethyl sulfoxide activates tumor necrosis factor -p53 mediated apopto- sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011), doi:10.1016/j.leukres.2010.12.029
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