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cell death mechanisms in the tumor cells in vivo. In this paper, transferred to nitrocellulose membrane followed by detection of
we have investigated whether administration of a pharmaceutical different proteins against 1:1000 times diluted protein specific
dose of DMSO to DL bearing mice was able to induce pro-apoptotic polyclonal antibodies. Protein bands were detected by ECL kit. As
mechanisms and to modulate expressions of PFKFB3 and LDH-A in loading control, -actin was probed similarly using monoclonal
the 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 polyacrylamide
2.1. 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 isozyme
approval 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 described
of DL was confirmed by belly swelling and increased body weight earlier [18]. The extracts containing 60 g protein were loaded in
which became visible on 10–12th post transplantation day. The DL each lane and electrophoresed under non-denaturing conditions at
bearing 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 with
4–5 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 U
post 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: PFKFB3
from 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 ; reverse
tics 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 ; reverse
1 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 and
2.4. 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 analysis
1 h, added with 0.4 ml 5 M NaCl and after 5 min, centrifuged at
3000 × 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 between
chilled 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. Results
a loading solution (0.25% bromophenol blue, 0.25% xylene cyanol FF
and 30% glycerol) in the ratio of 1:5 and samples containing 10 g 3.1. DMSO induced apoptosis in the DL cells in vivo
DNA were electrophoresed on 1% agarose gel containing 0.5 g/ml
ethidium bromide in TAE buffer for 2–3 h. The DNA bands in gel In general, TNF mediated apoptosis implicates p53 induced
were observed under UV transilluminator. mitochondrial mechanism. We have compared expression levels of
the key partners of this pathway in the DL cells from DMSO treated
2.5. 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
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sis and down regulates d-fructose-6-phosphate-2-kinase and lactate dehydrogenase-5 in Dalton’s lymphoma in vivo. Leuk Res (2011),
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Fig. 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 / actin
mRNA 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 ± SD
from 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 mice
comitant 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 cells
treated 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 DNA
fragmentation in the apoptotic cells. As prescribed in supplier’s 3.2. Down regulation of PFKFB3 and LDH-A by DMSO in the DL
manual, anti-caspase 9 and PARP-1 antibodies could detect pro- cells
caspases as well as their cleaved products; 46- and 35 kDa and
116 and 24 kDa for caspase 9 and PARP-1 respectively (Fig. 3A). Over expression of iPFK2 (PFKFB3) and LDH-A are associated
Accordingly, ∼2-times increases in the levels of cleaved caspase with tumor growth. As illustrated in Fig. 4A and B, the levels of
Fig. 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 with
Bcl-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-PCR
repeats. ** p < 0.01.
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Fig. 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 three
western 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 bromide
stained 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 apoptosis
standard pattern showing all the five LDH isozymes in the kidney [21,22] wherein, p53 induced alterations in Bcl-2/Bax ratio acts as a
extracts of normal mice (Fig. 4C(a)). Accordingly, an over activated determining factor [23]. Thus, significantly declined levels of Bcl-2
LDH-5 band was observed in the DL cell extracts of the untreated DL mRNA and its protein with concomitant increments in Bax mRNA
mice (Fig. 4C(b)), however, with a significant decline (p < 0.001) in and its protein (Fig. 2), in effect, represented a significantly reduced
the 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 present
DMSO 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 membrane
DL 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 of
ascitic fluid (Fig. 5A and B). This was consistent with a declining apoptosis [23] and that of PARP-1 cleavage is associated with DNA
trend 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 in
a susceptible parameter, was compared in normal lymphocytes, the DL cells from DMSO treated DL mice (Fig. 3A). Thus, together
spleen and liver from normal, DL bearing and DMSO treated DL with the production of DMSO dependent apoptotic pattern of DNA
mice. 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 of
in 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 steps
non-toxic to the animals. In one such evaluation, a single dose in tumor cells is of current focus as a novel anticancer strategy
of 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 cells
administered to the DL bearing mice, it significantly declined ascitic express a C-type PFK1 with increased sensitivity for FBP2 activation
volume and caused LDH-5 release in the cell free ascitic fluid (Fig. 5A [15] and concordantly, a catalytically more efficient iPFK2: PFKFB3
and B). As reported earlier [14], these findings suggested DL cell gene [11,16]. We observed DMSO mediated decline in the levels of
death/regression in vivo by DMSO. Additionally, apoptotic pattern PFKFB3 mRNA and its protein (iPFK2) in the DL cells (Fig. 4A and
of 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 certain
apoptosis 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 apoptosis
primarily 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 vivo
TNF 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
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Fig. 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 western
blot 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 the
ratio 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 for
tissue 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 of
repression of LDH-5 can affect tumor cell bioenergetics severely. cellular energy substrates and induction of apoptosis in the HeLa
Like 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 also
with 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 vivo
these 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 and
led 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 in
to DMSO mediated decreased expression of LDH-A in the tumor many tumor models. In this respect, this is a first report to describe
cells. 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, TNF
colysis for their energy requirements [17]. Depletion of energy induced apoptosis in another non-Hodgkin’s lymphoma has been
substrates 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
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Fig. 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 normal
lymphocytes (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 as
mean ± 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 of
growth and tissue toxicity [14,17,27]. We observed unaltered pat- Zoology, BHU, are also acknowledged.
tern of LDH-5 in normal lymphocytes, in spleen (lymphocyte
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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