Download Colon cancer cells maintain low levels of pyruvate to avoid cell

Biochem. J. (2009) 417, 379–389 (Printed in Great Britain)
379
doi:10.1042/BJ20081132
Colon cancer cells maintain low levels of pyruvate to avoid cell death
caused by inhibition of HDAC1/HDAC3
Muthusamy THANGARAJU, Kristina N. CARSWELL, Puttur D. PRASAD and Vadivel GANAPATHY1
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A.
Human colon cancer cells and primary colon cancer silence the
gene coding for LDH (lactate dehydrogenase)-B and up-regulate
the gene coding for LDH-A, resulting in effective conversion of
pyruvate into lactate. This is associated with markedly reduced
levels of pyruvate in cancer cells compared with non-malignant
cells. The silencing of LDH-B in cancer cells occurs via DNA
methylation, with involvement of the DNMTs (DNA methyltransferases) DNMT1 and DNMT3b. Colon cancer is also associated
with the expression of pyruvate kinase M2, a splice variant with
low catalytic activity. We have shown recently that pyruvate is
an inhibitor of HDACs (histone deacetylases). Here we show that
pyruvate is a specific inhibitor of HDAC1 and HDAC3. Lactate
has no effect on any of the HDACs examined. Colon cancer cells
exhibit increased HDAC activity compared with non-malignant
cells. HDAC1 and HDAC3 are up-regulated in colon cancer cells
and in primary colon cancer, and siRNA (small interfering RNA)mediated silencing of HDAC1 and HDAC3 in colon cancer cells
induces apoptosis. Colon cancer cells silence SLC5A8, the gene
coding for a Na+ -coupled pyruvate transporter. Heterologous
expression of SLC5A8 in the human colon cancer cell line
SW480 leads to inhibition of HDAC activity when cultured in the
presence of pyruvate. This process is associated with an increase
in intracellular levels of pyruvate, increase in the acetylation status
of histone H4, and enhanced cell death. These studies show that
cancer cells effectively maintain low levels of pyruvate to prevent
inhibition of HDAC1/HDAC3 and thereby to evade cell death.
INTRODUCTION
supported by the findings that mutations in the citric acid cycle
enzymes, succinate dehydrogenase and fumarate hydratase, are
associated with cancer and with concomitant increase in HIF-1α
levels [9–11]. Lactate produced by the tumour cells may also have
its own effects related to tumour progression and metastasis [12].
Conversion of pyruvate into lactate is obligatory for aerobic
glycolysis when mitochondrial function is defective. This reaction
is mediated by LDH (lactate dehydrogenase). There are two LDH
subunits coded by two separate genes, LDH-A and LDH-B. These
two subunits associate to form five different isoforms of tetrameric
LDH: LDH1 (LDH-B4), LDH2 (LDH-B3A1), LDH3 (LDHB2A2), LDH4 (LDH-B1A3), and LDH5 (LDH-A4). LDH1 is
effective in the conversion of lactate into pyruvate, whereas
LDH5 is effective in the conversion of pyruvate into lactate.
Since tumour cells robustly convert pyruvate into lactate, one
would expect a differential expression of LDH-A and LDH-B
in these cells. Available evidence indicates that LDH-A is upregulated and LDH-B is silenced in a variety of cancers [13–18].
In addition, tumour-associated fibroblasts and endothelial cells
exhibit a complementary LDH-A/LDH-B phenotype, with high
LDH-B and low LDH-A [19], facilitating metabolic utilization of
tumour-generated lactate to support proliferation and growth
of these cells.
Tumour cells also differ from normal cells in the expression of
pyruvate kinase, the enzyme responsible for the generation
of pyruvate in glycolysis. The splice variant PKM2 (musclespecific pyruvate kinase 2) is expressed specifically in tumour
cells in the dimeric form with low catalytic activity [20]. Most
recent studies have shown that this tumour-specific isoform is
a phosphotyrosine-binding protein [21] and that silencing of
PKM2 and forced expression of PKM1 in cancer cells switch the
metabolic phenotype to one similar to that of normal cells [22].
Enhanced glucose uptake and glycolysis with suppressed oxidative metabolism of pyruvate in mitochondria are a hallmark of
most tumour cells [1–6]. This occurs in the presence of adequate
oxygen supply. The switching to “aerobic glycolysis” from oxidative phosphorylation as the primary source of ATP forms the
central core of the Warburg hypothesis [7]. According to this
hypothesis, the above-mentioned metabolic switching is the
underlying cause of tumourigenesis. Even though the cause/effect
relationship between aerobic glycolysis and tumour formation as
proposed by Warburg [7] is still being debated, it is widely believed that most tumour cells derive a major portion of metabolic
energy from glycolysis rather than from mitochondrial oxidation.
Activation of glycolysis in tumour cells with decreased mitochondrial function obligates the conversion of pyruvate into lactate to
regenerate NAD+ , which is consumed at the level of glyceraldehyde-3-phosphate dehydrogenase. If pyruvate is not converted
into lactate in the cytoplasm, and NADH is not oxidized to NAD+
in mitochondria, glycolysis cannot continue. Therefore, it makes
sense that tumour cells robustly convert pyruvate into lactate.
Enhanced glycolysis/suppressed mitochondrial function certainly play a role in tumour progression. Since optimal mitochondrial respiration is dependent on adequate oxygen supply,
suppression of mitochondrial function may lead the tumour cells
to behave as if they are in hypoxia (pseudohypoxia). Heightened
hypoxic response such as stabilization of HIF-1α (hypoxia-inducible factor-1α) with subsequent up-regulation of glucose uptake,
glycolysis and vascular endothelial growth factor will promote
tumour progression through alternative sources of metabolic
energy (i.e. glycolysis instead of oxidative phosphorylation),
as well as neovascularization and nutrient supply [4,8]. This is
Key words: cancer cell death, histone deacetylase (HDAC), lactate
dehydrogenase (LDH), pyruvate, pyruvate kinase, SLC5A8.
Abbreviations used: DKO, double knockout; DNMT, DNA methyltransferase; HDAC, histone deacetylase; HIF-1α, hypoxia-inducible factor-1α; HPRT1,
hypoxanthine phosphoribosyltransferase; LDH, lactate dehydrogenase; PKM, muscle-specific pyruvate kinase; RT–PCR, reverse transcription–PCR;
siRNA, small interfering RNA.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
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M. Thangaraju and others
Glutaminolysis is another metabolic pathway that is highly active
in tumour cells, generating lactate, with pyruvate as the intermediate [23]. Thus pyruvate is at the intersection of the metabolic
switching that is characteristic of tumour cells, but little is known
on the relevance of pyruvate to cancer. We recently reported that
SLC5A8, a Na+ -coupled transporter for pyruvate and other monocarboxylates, triggers tumour cell apoptosis through pyruvatedependent inhibition of HDACs (histone deacetylases) [24]. This
phenomenon is related to the entry of extracellular pyruvate
into tumour cells. The expression of SLC5A8 is silenced in a
variety of cancers [25–27], suggesting that tumour cells prevent
the entry of extracellular pyruvate to avoid pyruvate-induced cell
death. In the present study, we investigated the effects of the
reciprocal regulation of LDH-A and LDH-B on intracellular levels
of pyruvate in colon cancer cells and its significance in the light
of our recent finding that pyruvate is an HDAC inhibitor and a
tumour suppressor [24]. These studies have generated new and
important data which propel pyruvate to the forefront of cancer
biology as a critical metabolite and have clinical and therapeutic
implications.
MATERIALS AND METHODS
Collection of normal and cancer tissue specimens
from human colon
This study received the approval of the Medical College of
Georgia Institutional Human Assurance Committee. The research
was carried out in accordance with the Declaration of Helsinki
(2000) of the World Medical Association. Adult patients with
colorectal adenocarcinoma (n = 18), without a history of prior
chemoradiation, were included in this study after obtaining
their informed consent. A pathologist harvested normal colorectal epithelium and tissue from the luminal surface of the
colorectal cancer from the freshly resected surgical specimens.
Portions of the tissues (0.3 to 0.5 g from each site) were processed
for total RNA extraction using TRIzol® reagent (Invitrogen
Life Technologies). Some of these tissue specimens have been
used previously for the expression analysis of the amino acid
transporter ATB0,+ [28].
Colon cell lines
Two non-malignant colonic epithelial cell lines (NCM460 and
CCD841) and nine malignant colonic epithelial cell lines
(SW480, SW620, KM12C, KM12L4, HT29, HCT116, Colo201,
Colo205 and Ls174T) were used in the present study. The
wild-type HCT116 cell line, which is positive for DNMTs
(DNA methyltransferases) (DNMT+/+ ) and the isogenic cell lines
with homologous deletion of DNMT1 (DNMT1−/− ), DNMT3b
(DNMT3b−/− ), DNMT1 plus DNMT3b (DKO) were provided by
Dr. Bert Vogelstein (Johns Hopkins University, Balitimore, MD,
U.S.A.).
RT–PCR (reverse transcription–PCR)
RNA prepared from colon tissue specimens and cell lines was
used for semi-quantitative RT–PCR. RNA (2 μg) was reverse
transcribed into cDNA using the GeneAmp PCR system (Roche).
HPRT1 (hypoxanthine phosphoribosyltransferase 1) mRNA was
used as the internal control.
were prepared by sonication in 10 mM Tris/HCl buffer (pH 7.6),
supplemented with a cocktail of protease inhibitors (50 mM
NaF, 0.2 mM vanadate, 1 mM PMSF, 5 μg/ml aprotinin, 1 μg/ml
pepstatin and 2 μg/ml leupeptin) and 1 % Triton X-100. Proteins
were size-fractionated on to SDS/PAGE gels and then transferred
on to Protran nitrocellulose membranes (Schliecher & Schuell).
Membranes were blocked with BSA, treated with primary
antibody at 4 ◦C overnight, followed by treatment with appropriate
secondary antibody conjugated to horseradish peroxidase. The
antigen/antibody reaction was detected by Enhanced Chemiluminescence SuperSignal Western System (Amersham). Primary
antibodies were obtained from the following sources: LDH-A
(Santa Cruz), LDH-B (Sigma–Aldrich), HDACs 1–5 (Sigma–
Aldrich or BioVision), and histones H3 and H4, and their
acetylated forms (Santa Cruz or Upstate Biotechnology).
Measurement of pyruvate and lactate levels in cultured cells
The levels of pyruvate and lactate were measured using a fluorescence-based assay (BioVision). For the pyruvate assay, subconfluent cells were collected and washed three times with PBS.
Cells were then lysed in pyruvate assay buffer and the clear lysate
was collected by centrifugation at 50 000 g for 15 min at 4 ◦C.
Lysate (50 μl) was aliquoted into each well in a 96-well plate in
triplicate. The reaction assay mixture was prepared according to
the manufacturer’s instructions, and 50 μl of this mixture was then
added to each well. In this assay, pyruvate oxidation generates
a fluorescent signal which is measured in a fluorimeter at an
excitation wavelength of 535 nm and an emission wavelength of
590 nm. The procedure was exactly the same for measurement
of lactate levels, except that the assay mixture contained the
enzyme for lactate oxidation, generating a fluorescent signal.
Measurement of HDAC activity
The measurement of HDAC activity in lysates from colonic cell
lines or immunoprecipitates was carried out using a commercially
available assay kit (Cayman Chemical Company). The activity of
recombinant human HDAC isoforms was also measured using
the same assay kit. The recombinant HDAC isoforms were
purchased from Cayman Chemical Company. Cell lysate protein
(50 μg) or recombinant HDAC (10 ng) was incubated with or
without butyrate, pyruvate or lactate, and the reaction was initiated
by the addition of HDAC substrate. The enzyme activity was
monitored using a fluorimetric assay. The immunoprecipitates
were prepared from the malignant human colon cancer cell line
SW480 as follows. Sub-confluent cells were washed twice and
collected in PBS. Cells were solubilized with 25 mM Tris/HCl
buffer (pH 7.5) containing 100 mM NaCl, 1 μl/ml of 2-mercaptoethanol and protease inhibitor cocktail (Roche). Cell lysate
was incubated on ice for 20 min followed by sonication, and
then centrifuged at 14 000 g for 15 min. Soluble proteins (1 mg)
were incubated with anti-HDAC1, anti-HDAC2 and anti-HDAC3
antibodies (Sigma–Aldrich) individually for 3 h at 4 ◦C. The
resultant immunocomplexes were collected using Protein A/G
plus-agarose beads (Santa Cruz Biotechnology) at 4 ◦C overnight.
Beads were then washed three times with RIPA buffer (Sigma–
Aldrich) and resuspended in 700 μl of HDAC assay buffer. These
immunoprecipitates (50 μl) were used for measurement of HDAC
activity.
Flow cytometric analysis of apoptosis
Western blot
This was done for LDH-A, LDH-B, various isoforms of HDAC,
histones H3 and H4, and their acetylated forms. Cell lysates
c The Authors Journal compilation c 2009 Biochemical Society
Cells were transiently transfected with either pcDNA3.1 or
SLC5A8 cDNA, and cultured in the presence or absence of pyruvate (1 mM) for 48 h. Cells were then fixed in 50 % ethanol,
Cancer cell death via pyruvate-induced inhibition of HDAC1/HDAC3
treated with 0.1 % sodium citrate, 1 mg/ml RNase A and 50 μg/ml
propidium iodide, and then subjected to FACS (FACS Caliber,
Becton Dickinson) analysis. For siRNA (small interfering RNA)
studies, CCD841 (a non-malignant human colon cell line) and
SW480 (a malignant human colon cell line) cells were seeded in
6-well plates and cultured in RPMI 1640 medium. After 24 h, cells
were transfected with HDAC1 siRNA, HDAC2 siRNA or HDAC3
siRNA [Santa Cruz Biotechnology; catalogue numbers: sc-29343
for HDAC1 siRNA (h); sc-29345 for HDAC2 siRNA (h); sc35538 for HDAC3 siRNA (h)] according to the manufacturer’s
instructions. Scrambled siRNA (Santa Cruz Biotechnology;
catalogue number: sc-37007) was used as a negative control. After
48 h, cells were collected and processed for the FACS analysis.
RESULTS
Differential expression of LDH-A and LDH-B in colon cancer and its
relevance to intracellular levels of pyruvate and lactate
Pyruvate is generated in tumour cells principally by glycolysis
and glutaminolysis. The levels of pyruvate in these cells are
controlled not only by these two metabolic pathways but also
by the cells’ ability to convert pyruvate into lactate. Since
LDH1, a tetramer of LDH-B, preferentially functions in the
conversion of lactate into pyruvate, whereas LDH5, a tetramer
of LDH-A, preferentially functions in the conversion of pyruvate
into lactate, we anticipated that the differential expression of
LDH-A and LDH-B in normal cells compared with cancer
cells would be a key determinant of the intracellular levels of
pyruvate. Therefore, we examined the expression of these two
genes in primary colon cancer and in colon cancer cell lines.
With paired specimens of normal and cancer colon tissues, we
found the expression of LDH-B to be markedly silenced in
cancer, along with up-regulation of LDH-A (Figure 1A). Of
the 18 paired tissue samples evaluated, the silencing of LDH-B
was seen in 16 cases and the up-regulation of LDH-A was seen
in all cases. The decrease in the expression of LDH-B mRNA in
cancer tissues compared with normal tissues was ∼ 5-fold and the
increase in the expression of LDH-A mRNA was ∼ 6-fold. We
also compared the expression levels of these two genes between
non-malignant and malignant human colonic cell lines. The nonmalignant cell lines NCM460 and CCD841 expressed high levels
of LDH-B and low levels of LDH-A. This was evident both
at mRNA level and protein level (Figure 1B). In contrast, the
expression of LDH-B was lower and that of LDH-A was higher
in nine different malignant cell lines. We then measured the intracellular levels of pyruvate and lactate in these cells. The levels
of pyruvate were markedly reduced in cancer cell lines compared
with non-malignant cell lines, the concentration in the former being only about 20 % of the concentration in the latter (Figure 1C).
On the contrary, the reverse was true with lactate. Non-malignant cells had 4-fold lower levels of lactate compared with
malignant cells (Figure 1D).
Role of DNA methylation in cancer-associated silencing of LDH-B
Even though the silencing of LDH-B has been demonstrated in a
variety of tumours, the underlying molecular mechanism has not
been investigated in detail. In a recent report, Leiblich et al. [18]
showed that LDH-B is silenced by promoter hypermethylation in
prostate cancer. Here we examined the role of DNA methylation
in the silencing of LDH-B in colon cancer cell lines. The expression of LDH-B in non-malignant cell lines, which constitutively
express this gene at high levels, was not affected by treatment with
5-azadeoxycytidine, an inhibitor of DNA methylation (Fig-
381
ure 2A). But in malignant cell lines, which express very low
levels of LDH-B, treatment with 5-azadeoxycytidine induced the
expression of LDH-B, detectable at the level of both mRNA
and protein. Procainamide, a specific inhibitor of DNMT1 [29],
was unable to induce the expression of LDH-B, suggesting that
DNMT1 alone is insufficient to silence LDH-B. Studies with wildtype HCT116 cell line and isogenic cell lines with deletion of
DNMT1, DNMT3b or both showed that LDH-B expression was
induced only in the cell line which lacked both DNMT1 and
DNMT3b (Figure 2B). These data demonstrate for the first time
the involvement of specific DNMTs in the silencing of LDH-B in
cancer cells. We then correlated the intracellular levels of pyruvate
in HCT116 cells with the expression of LDH-B (Figure 2C). In
the double knockout cell line (DKO), in which both DNMT1 and
DNMT3b have been deleted with consequent expression of LDHB, intracellular pyruvate levels were several-fold higher than in
the wild-type cell line. Interestingly, the DNMT1−/− cell line had
significantly higher levels of pyruvate compared with the wildtype cell line, even though there was no expression of LDH-B. This
was principally due to the induction of the expression of SLC5A8,
a Na+ -coupled transporter for pyruvate. We have demonstrated that DNMT1 alone is sufficient to cause the silencing
of SLC5A8 [30]. Since the cells were cultured in the presence of
pyruvate, the induction of SLC5A8 in these cells leads to an
increase in intracellular pyruvate levels by facilitating the entry
of pyruvate from the medium into the cells. This was not the case
with DNMT3b−/− cells which do not express SLC5A8. The
intracellular levels of pyruvate correlated inversely with HDAC
activity in these cell lines (Figure 2D). Wild-type cells and the
DNMT3b−/− cells, which had low levels of pyruvate, had high
HDAC activity. In contrast, DNMT1−/− and DKO cells, which
had high levels of pyruvate, had low HDAC activity. This agrees
with our recent findings that pyruvate is an HDAC inhibitor [24].
Differential expression of the pyruvate kinase isoforms PKM1 and
PKM2 in colon cancer
The concentration of pyruvate in cancer cells is controlled not
only by the expression pattern of LDH-A and LDH-B isoforms
but also by pyruvate kinase. Pyruvate is generated in cancer cells
by glycolysis as well as by glutaminolysis. There is evidence that
cancer cells use the carbon atoms present in glucose for the synthesis of nucleotides, fatty acids, lipids, and non-essential amino
acids, and that glutamine is metabolized preferentially into lactate
via pyruvate [23]. For the utilization of glucose carbon in biosynthetic processes, the conversion of phosphoenolpyruvate into
pyruvate must be decreased. Recent studies have shown that
cancer cells express pyruvate kinase primarily in the form of
PKM2 dimer which possesses low catalytic activity [20–22]. We
confirmed the cancer-associated expression of the PKM2 variant
with primary colon cancer and colon cancer cell lines (Figure 3).
PKM1 was expressed at high levels in normal colon tissues and in
non-malignant colon cell lines, whereas PKM2 was expressed at
high levels in colon cancer and in colon cancer cell lines. In paired
samples of primary tumour tissues and normal colon tissues, the
cancer-associated decrease in PKM1 expression was 7-fold and
the cancer-associated increase in PKM2 expression was 3-fold.
These findings suggest that the differential expression of PKM
isoforms also contributes to the lower levels of pyruvate in colon
cancer cells.
Isoform selectivity for the inhibition of HDACs by pyruvate
We have demonstrated previously that pyruvate is an inhibitor of
HDAC [24]; however, the isoform specificity of this inhibition was
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M. Thangaraju and others
Figure 1 Correlation between the differential expression of LDH-A/LDH-B and intracellular levels of lactate/pyruvate in primary human colon cancer and in
colon cancer cell lines
(A) Semi-quantitative RT–PCR analysis of the steady-state levels of LDH-A mRNA and LDH-B mRNA in paired tissue specimens of primary colon cancer (T) and adjacent normal tissue (N). HPRT1
mRNA was used as the internal control. (B) Comparison of LDH-A and LDH-B mRNA and protein levels between non-malignant and malignant colon cell lines. mRNA levels were monitored by
semi-quantitative RT–PCR and protein levels by Western blot. (C) and (D) Steady-state levels of intracellular pyruvate and lactate in non-malignant and malignant colon cell lines. Results are
means +
− S.E.M. (n = 3). The levels of pyruvate and lactate were significantly different between colon tumour cells and normal colon cells (P < 0.05).
not known. Therefore, we examined the effects of pyruvate on the
activity of different isoforms of human recombinant HDACs. We
used butyrate as a positive control. Butyrate inhibited HDAC1
and HDAC3, but had no effect on HDAC2, HDAC5, HDAC6,
HDAC8 and HDAC9 (Figure 4A). Pyruvate showed similar
isoform specificity. It also inhibited only HDAC1 and HDAC3
(Figure 4B). On the other hand, lactate had no effect on any
of the HDAC isoforms. Butyrate and pyruvate had comparable
inhibitor potency (Figures 4C and 4D). HDAC1 was inhibited
more potently than HDAC3 by these compounds. The IC50 values
c The Authors Journal compilation c 2009 Biochemical Society
for butyrate and pyruvate for the inhibition of HDAC1 were
20 +
− 4 and 24 +
− 5 μM respectively. The corresponding values
for the inhibition of HDAC3 were 75 +
− 10 and 80 +
− 15 μM. We
confirmed this isoform selectivity using endogenous HDAC1 and
HDAC3 expressed in the colon cancer cell line SW480. These
isoforms were immunoprecipitated from SW480 cell lysates with
specific antibodies, and the immunocomplexes were then used
as the source of enzyme activity. HDAC1 and HDAC3 were
inhibited by butyrate and pyruvate, but not by lactate (Figure 4E).
In contrast, HDAC2 was not affected by any of these compounds.
Cancer cell death via pyruvate-induced inhibition of HDAC1/HDAC3
Figure 2
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Involvement of DNMTs in the silencing of LDH-B in colon cancer cell lines
(A) Influence of 5 -azadeoxycytidine, a pan-inhibitor of DNMTs, and procainamide, a specific inhibitor of DNMT1, on the expression of LDH-B mRNA (semi-quantitative RT–PCR) and protein
(Western blot). HPRT1 was used as the internal control for mRNA analysis, whereas β-actin was used as the internal control for protein analysis. (B) Expression of LDH-B mRNA and protein in
the HCT116 cell line (DNMT+/+ ), which expresses DNMT1 and DNMT3b, and in isogenic cell lines which lack either DNMT1 (DNMT1−/− ), DNMT3b (DNMT3b−/− ) or both (DKO ). (C) and (D)
Steady-state levels of pyruvate and HDAC activity in the HCT116 cell line (DNMT+/+ ), which expresses DNMT1 and DNMT3b, and in isogenic cell lines which lack either DNMT1 (DNMT1−/− ),
∗
+/+
cells.
DNMT3b (DNMT3b−/− ) or both (DKO ). Results are means +
− S.E.M. (n = 3). P = 0.05 compared with values in DNMT
Up-regulation of HDAC1 and HDAC3 in colon cancer
Data from wild-type and isogenic HCT116 cell lines indicated an
inverse relationship between cellular pyruvate levels and HDAC
activity (Figures 2C and 2D). To confirm these findings, we
measured HDAC activity in non-malignant and malignant colon
cell lines for which data on cellular levels of pyruvate are available
in the present study. We already showed that the non-malignant
cell lines had much higher levels of pyruvate compared with
malignant cell lines (Figure 1C). As expected from the ability of
pyruvate to inhibit HDAC, the HDAC activity was much lower in
non-malignant cell lines than in malignant cell lines (Figure 5A).
However, since pyruvate is a specific inhibitor of HDAC1 and
HDAC3, the postulated inverse relationship would be expected
only if the higher HDAC activity in cancer cell lines than in nonmalignant cell lines is predominantly due to increased expression
of these two HDAC isoforms in cancer cells. Therefore, we
monitored the expression pattern of HDAC isoforms in nonmalignant and malignant colon cell lines (Figures 5B and 5C).
While the expression levels of HDAC2, HDAC4 and HDAC5
are similar in non-malignant and malignant cell lines, there was
a clear difference in the expression of HDAC1 and HDAC3
between these two groups of cell lines. The expression of these
two HDAC isoforms was higher in malignant cell lines compared
with non-malignant cell lines. These data suggest that the increase
in HDAC activity in cancer cells is primarily due to an increase in
the expression of the pyruvate-sensitive isoforms HDAC1 and
HDAC3. To confirm that this is also true in primary colon cancer,
we compared the expression of HDAC1 and HDAC3 between
normal colon tissue specimens and paired colon cancer specimens
(Figure 5D). The expression of these two isoforms was higher in
cancer tissues compared with adjacent normal tissues (∼ 3-fold
for HDAC1 and ∼ 2-fold for HDAC3), corroborating the findings
in colon cell lines. The expression of HDAC5 was also increased
slightly but significantly in cancer tissues compared with normal
tissues (∼ 70 % increase), whereas there was no difference in the
expression levels of HDAC2 and HDAC4.
Cancer cell-specific apoptosis by inhibition of HDAC1 and HDAC3
SLC5A8 is a tumour suppressor gene that is silenced in colon
cancer and in colon cancer cell lines [31]. We and others have
shown that SLC5A8 is a Na+ -coupled transporter for monocarboxylates, including short-chain fatty acids, pyruvate and lactate [32–34]. In normal intestinal tract, the transporter is expressed
in the lumen-facing apical membrane of epithelial cells [35,36].
However, when transformed into cancer, these epithelial cells
lose their polarity and the transporter would have access to metabolites present in the circulation. Pyruvate is present in blood
at significant levels (∼ 100 μM) [37]. It is likely that colon
cancer cells silence SLC5A8 to prevent the transporter-mediated
entry of circulating pyruvate into the cells. Thus, the silencing
of SLC5A8 would complement the differential expression of
LDH-A and LDH-B in cancer cells to maintain low intracellular
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Figure 3
M. Thangaraju and others
Semi-quantitative RT–PCR analysis of the steady-state levels of pyruvate kinase PKM1 and PKM2
Semi-quantative RT–PCR of the mRNAs specific for the two splice variants of pyruvate kinase PKM1 and PKM2 in primary colon cancer tissue specimens (T) and in adjacent normal colon tissue
specimens (N) (A) and in non-malignant and malignant colon cell lines (B).
levels of pyruvate as a means to avoid inhibition of HDAC1 and
HDAC3. Then ectopic expression of SLC5A8 in colon cancer
cell lines should increase the cellular levels of pyruvate and
reduce HDAC activity when cultured in a medium containing
pyruvate. To examine this, we transfected the non-malignant cell
line CCD841 and the malignant cell line SW480 with either vector
alone or SLC5A8 cDNA and cultured the cells in a pyruvatecontaining medium for 48 h. CCD841 cells express SLC5A8
constitutively, whereas the gene is silenced in SW480 cells. We
found that the cellular levels of pyruvate in CCD841 cells were
high and not altered in response to overexpression of the transporter (Figure 6A). In contrast, pyruvate levels in SW480 cells
were relatively lower, but the levels increased markedly with the
expression of the transporter (Figure 6A). The changes in pyruvate
levels mirrored the changes in HDAC activity reciprocally. The
HDAC activity was low in CCD841 cells, and the activity was
not influenced by ectopic expression of SLC5A8 (Figure 6B).
This was expected because pyruvate levels were not altered under
these conditions. In contrast, the HDAC activity in SW480 cells
was higher than in CCD841 cells, and the activity was reduced
significantly with ectopic expression of SLC5A8 (Figure 6B).
This corroborated with the increase in pyruvate levels in SW480
cells. We then examined the effects of the changes in pyruvate
levels and HDAC activity in these cell lines on apoptosis. With
CCD841 cells, there was very little apoptosis when transfected
with either vector alone or SLC5A8 cDNA irrespective of
whether or not the cells were cultured in the presence of pyruvate (Figure 6C). With SW480 cells also, there was very little
apoptosis when transfected with either vector alone or SLC5A8
cDNA, but only when cultured in the absence of pyruvate. When
cultured in the presence of pyruvate, vector-transfected cells did
c The Authors Journal compilation c 2009 Biochemical Society
not undergo apoptosis, whereas massive cell death occurred in
SLC5A8-expressing cells (Figure 6C). We confirmed the changes
in HDAC activity in CCD841 and SW480 cells under these
conditions by analysing the acetylation status of histones H3 and
H4 (Figure 6D). The acetylation status of histones H3 and H4
was high in CCD841 cells irrespective of whether the cells were
transfected with vector alone or SLC5A8 cDNA and whether the
cells were cultured in the presence or absence of pyruvate. In
contrast, the acetylation status of histone H4, in particular the
acetylation of H4-Lys12 and H4-Lys16 , was much lower in vectortransfected and SLC5A8 cDNA-transfected SW480 cells than that
in CCD841 cells when the cells were cultured in the absence of
pyruvate. But the acetylation status of histone H4 increased in
SLC5A8-expressing SW480 cells when cultured in the presence
of pyruvate. There was no change in the acetylation status of
histone H3. These results show that elevation of pyruvate levels
in colon cancer cells through SLC5A8-mediated transport leads to
HDAC inhibition, enhances the acetylation status of histone H4,
and consequently induces apoptosis. In contrast, non-malignant
cells do not undergo changes in the acetylation status of histone
H4 under these conditions and hence there is no apoptosis in these
cells.
We confirmed the relevance of HDAC1/HDAC3 inhibition to
cancer cell-specific apoptosis using siRNA-mediated silencing of
these isoforms in SW480 cells (Figure 6E). Transfection of the
non-malignant colon cell line CCD841 with siRNAs specific for
the three isoforms of HDAC (HDAC1, 2 and 3) did not have any
effect. In contrast, there was a differential effect of these siRNAs
on the malignant colon cell line SW480. Transfection of these cells
with HDAC1 siRNA or HDAC3 siRNA induced apoptosis,
whereas transfection with HDAC2 siRNA had no effect.
Cancer cell death via pyruvate-induced inhibition of HDAC1/HDAC3
Figure 4
385
Isoform specificity of HDAC inhibition by pyruvate
(A) and (B) Effects of butyrate (1 mM), lactate (1 mM) and pyruvate (1 mM) on various isoforms of human recombinant HDAC. (C) and (D) Dose/response relationship for the inhibition of human
recombinant HDAC1 and HDAC3 by butyrate and pyruvate. (E) Effects of butyrate (But, 1 mM), pyruvate (Pyr, 1 mM) and lactate (Lac, 1 mM) on the activities of HDAC1, HDAC2 and HDAC3
immunocomplexes prepared from SW480 cell lysates using specific antibodies. Results are means +
− S.E.M. (n = 3). Butyrate and pyruvate significantly inhibited HDAC1 and HDAC3 (P < 0.05).
Scrambled siRNA was used as a negative control in these experiments. These studies show that silencing of HDAC1 and HDAC3
leads to apoptosis in a cancer cell-specific manner.
DISCUSSION
The most important findings in the present study are: (i) intracellular levels of pyruvate in colon cancer cells are much lower than
those in non-malignant cells; (ii) the lower levels of pyruvate
in colon cancer cells are the result of not only the differential
expression of enzymes involved in the generation and metabolism
of pyruvate, namely increased expression of LDH-A and PKM2,
and decreased expression of LDH-B and PKM1, but also the decreased expression of SLC5A8, a Na+ -coupled active transporter
for pyruvate; (iii) the silencing of LDH-B in colon cancer cells
involves DNMT1 and DNMT3b; (iv) pyruvate is a specific inhib-
itor of the HDAC isoforms HDAC1 and HDAC3; (v) colon cancer
cells have much higher HDAC activity than non-malignant cells,
primarily due to increased expression of HDAC1 and HDAC3;
(vi) elevation of intracellulal levels of pyruvate leads to apoptosis
specifically in colon cancer cells without having any effect in nonmalignant cells; and (vii) siRNA-mediated silencing of HDAC1
and HDAC3 causes apoptosis specifically in cancer cells. These
findings are novel and have immense clinical and therapeutic
significance. It is well known that cancer cells are principally
lactate producers. But this was presumed to be mostly due to
mass action, where increased generation of pyruvate in glycolysis
results in increased production of lactate. Cancer cells have
enhanced glycolysis, the end product of which is pyruvate. Since
mitochondrial function is down-regulated in cancer, what do the
cancer cells do with this glycolytic end product? These cells
induce LDH-A and silence LDH-B so as to convert pyruvate into
lactate. The underlying notion here is that increased generation
c The Authors Journal compilation c 2009 Biochemical Society
386
M. Thangaraju and others
Figure 5 HDAC activity and expression pattern of HDAC isoforms in non-malignant and malignant colon cell lines and in primary human colon cancer and
adjacent normal colon tissue
(A) HDAC activity, measured in a cell-free system, in two non-malignant and nine malignant colon cell lines. Results are means +
− S.E.M. (n = 3). HDAC activity was significantly lower in
non-malignant cells than in malignant cells (P < 0.05). (B) and (C) Steady-state levels of mRNA and protein for the various HDAC isoforms (HDAC 1–5) in two non-malignant and nine malignant
colon cell lines. (D) Steady-state levels of mRNA for vaious HDAC isoforms in paired tissue specimens of colon cancer (T) and adjacent uninvolved normal tissue (N).
of lactate in cancer cells is the direct result of increased pyruvate
levels. But the present study suggests that this is not true. Our
results show that the increased lactate production in cancer cells
is not simply the result of ‘more pyruvate means more lactate’.
Cancer cells generate lactate purposely to reduce the intracellular
levels of pyruvate. The conversion of pyruvate into lactate is not
the only mechanism by which cancer cells manage to keep the
pyurvate levels low. These cells also differentially express
the pyruvate kinase splice variants PKM1 and PKM2 such that the
activity of pyruvate kinase, which converts phosphoenolpyruvate
into pyruvate, is low. In addition, the expression of SLC5A8,
the gene coding for the Na+ -coupled pyruvate transporter, is
silenced in cancer cells, which decreases the entry of bloodborne pyruvate into cancer cells. Collectively, these processes
c The Authors Journal compilation c 2009 Biochemical Society
maintain the intracellular levels of pyruvate low in cancer cells.
To our knowledge, this is the first report describing this unique
phenomenon of significantly reduced pyruvate levels in cancer
cells.
Even though the silencing of LDH-B in cancer has been demonstrated in several studies [13–18], the underlying mechanism is
just beginning to be understood. A recent study has demonstrated
that the silencing of LDH-B in prostate cancer involves promoter
hypermethylation [18]. Here we show a similar mechanism
in colon cancer cells. Treatment of colon cancer cells with
5 -azadeoxycytidine, a pan-inhibitor of DNMTs, induces the
expression of LDH-B. However, procainamide, a specific inhibitor
of DNMT1, has no effect, suggesting that inhibition of DNMT1
alone is not sufficient to induce the re-expression of the gene.
Cancer cell death via pyruvate-induced inhibition of HDAC1/HDAC3
387
Figure 6 Relevance of intracellular pyruvate levels to HDAC activity, histone acetylation, and apoptosis in the non-malignant colon cell line CCD841 and in
the colon cancer cell line SW480
(A) CCD841 and SW480 cells were transfected with either pcDNA or SLC5A8 cDNA, and then cultured in a pyruvate-containing medium. Intracellular levels of pyruvate were then measured.
(B) CCD841 and SW480 cells were transfected with either pcDNA or SLC5A8 cDNA, and then cultured in a pyruvate-containing medium. HDAC activity was then measured using a cell-free system.
(C) CCD841 and SW480 cells were transfected with either pcDNA or SLC5A8 cDNA, and then cultured in the presence or absence of pyruvate for 48 h. Cells were then subjected to FACS analysis to
determine the fraction of the cells undergoing apoptosis. (D) CCD841 and SW480 cells were transfected with either pcDNA or SLC5A8 cDNA, and then cultured in the presence or absence of pyruvate
for 48 h. Cell lysates were then used for Western blot analysis of histone H3, histone H4, and their acetylated forms. (E) CCD841 and SW480 cells were transfected with either scrambled siRNA
∗
(negative control), HDAC1 siRNA, HDAC2 siRNA, or HDAC3 siRNA, cultured for 48 h, and then subjected to FACS analysis to determine apoptosis. Results are means +
− S.E.M. (n = 4). P = 0.05
compared with vector control. For (D), cell death was significantly higher in SW480 cells with HDAC1 siRNA and HDAC3 siRNA than with scrambled siRNA (P < 0.05).
Additional studies with isogenic HCT116 cell lines show that both
DNMT1 and DNMT3b are involved in the silencing of the gene.
This is in contrast with the mechanism associated with the
silencing of SLC5A8 in these cells, where inhibition of DNMT1
alone is enough to induce the re-expression of the transporter [30].
The metabolic rationale underlying the conversion of pyruvate
into lactate in cancer cells is understandable in terms of regeneration of NAD+ and maintenance of enhanced glycolytic rate under
conditions of decreased mitochondrial function. But what is the
need for cancer cells to maintain reduced intracellular levels of
pyruvate? The findings of the present study provide an answer to
this question. While pyruvate is an energy-rich nutrient necessary
for growth in non-malignant cells, this metabolite is a tumour suppressor and an inducer of apoptosis in cancer cells. The tumoursuppressive function of pyruvate is related to its ability to inhibit
HDAC1 and HDAC3. We have already shown in a previous study
c The Authors Journal compilation c 2009 Biochemical Society
388
M. Thangaraju and others
that pyruvate is an HDAC inhibitor and a tumour suppressor [24].
Here we describe for the first time the isoform specificity of
pyruvate inhibition of HDAC. It is interesting and important to
note that HDAC1 and HDAC3, which are inhibitable by pyruvate,
are the two isoforms which are up-regulated in cancer cells. The
elevation of HDAC activity is presumably necessary for the cancer
cells to maintain their malignant phenotype. Therefore, cancer cells must maintain the low intracellular levels of pyruvate, lest
HDAC1 and HDAC3 will be inhibited and cell growth prevented
by enhanced apoptosis. This is supported by the findings of the
present study, where we show marked induction of apoptosis
in cancer cells when the intracellular levels of pyruvate were
forced to increase through ectopic expression of the Na+ -coupled
pyruvate transporter SLC5A8. This is confirmed further by the
induction of apoptosis specifically in colon cancer cells by siRNAmediated silencing of HDAC1 and HDAC3.
We stumbled upon the tumour-suppressive function of pyruvate
when we were investigating the significance of the silencing of
SLC5A8 in cancer. The silencing of this gene was first reported in
colon cancer [31]. This led us to postulate that SLC5A8 may be
a transporter for the short-chain fatty acid butyrate, which is an
HDAC inhibitor, a tumour suppressor, and a bacterial fermentation
product generated in the colonic lumen. Subsequent studies from
our laboratory and from others have shown that SLC5A8 is indeed
a Na+ -coupled transporter for butyrate and other short-chain fatty
acids such as acetate and propionate [32–34]. Interestingly, the
transporter is expressed not only in the colon, where its role
in butyrate transport may provide the reason for its expression,
but also in the kidney, where there is no possible relevance of
butyrate transport. This led us to hypothesize that SLC5A8 in the
kidney may be responsible for the reabsorption of lactate, which
is a monocarboxylate similar to butyrate. Subsequent studies
from our laboratory have shown that this is indeed true [39,40].
Detailed investigations of the substrate specificity of SLC5A8
followed, which showed that the transporter is able to transport a
variety of other monocarboxylates, including pyruvate [32,39],
nicotinate [41], β-hydroxybutyrate [42] and monocarboxylate
drugs [36]. During this time, studies from other laboratories
have demonstrated that the silencing of SLC5A8 is not unique
to colon cancer. The gene is silenced in thyroid cancer [43–45],
stomach cancer [46] and brain cancer [47]. This was puzzling
because these non-colonic tissues are not exposed to butyrate.
The butyrate connection to the tumour-suppressive function of
SLC5A8 is not relevant to non-colonic tissues, such as the thyroid,
stomach and brain. This suggested that some other endogenous
metabolite commonly found in circulation must be a substrate for
SLC5A8 and function as a tumour suppressor similar to butyrate.
This rationale led us to the discovery that pyruvate, a high affinity substrate for SLC5A8, is an HDAC inhibitor and a tumour
suppressor [24]. Since then, the silencing of SLC5A8 has been
shown to occur in other non-colonic tissues such as the mammary
gland [24], pancreas [48] and prostate [49], extending the relevance of the pyruvate/SLC5A8 connection to cancer in a broader
range of tissues.
The studies reported here demonstrate that the silencing of
SLC5A8 is not the only means used by cancer cells to avoid
pyruvate-induced HDAC inhibition and cell death. These cells
have an elaborate mechanism to maintain low intracellular levels
of pyruvate. It includes differential expression of LDH isoforms
and pyruvate kinase splice variants. These studies suggest that
elevation of intracellular levels of pyruvate may provide a
potential therapeutic strategy in the treatment of cancer. Possible
means to achieve this goal include induction of SLC5A8 and
LDH-B expression with the use of DNA methylase inhibitors,
and inhibition of LDH-A. These therapeutic strategies may be
c The Authors Journal compilation c 2009 Biochemical Society
applicable to cancer treatment in a wide variety of tissues because
the differential expression of LDH-A and LDH-B and the silencing
of SLC5A8 appear to be a common phenomenon in cancer.
FUNDING
This research received no specific grant from any funding agency in the public, commercial
or not-for-profit sectors.
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Received 4 June 2008/5 September 2008; accepted 12 September 2008
Published as BJ Immediate Publication 12 September 2008, doi:10.1042/BJ20081132
c The Authors Journal compilation c 2009 Biochemical Society