Biochem. J. (2009) 417, 717–726 (Printed in Great Britain)
717
doi:10.1042/BJ20080805
Inhibition of energy-producing pathways of HepG2 cells
by 3-bromopyruvate1
Ana Paula PEREIRA DA SILVA*†, Tatiana EL-BACHA†, Nattascha KYAW*, Reinaldo Sousa DOS SANTOS*,
Wagner Seixas DA-SILVA*, Fabio C. L. ALMEIDA‡, Andrea T. DA POIAN† and Antonio GALINA*2
*Laboratório de Bioenergética e Fisiologia Mitocondrial, Programa de Bioquı́mica e Biofı́sica Celular, Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Av.
Carlos Chagas Filho 373 – CCS, Bl. D, ss13, 21941-902, RJ, Brazil, †Laboratório de Bioquı́mica de Vı́rus, Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, RJ,
Brazil, and ‡Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas-Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
3-BrPA (3-bromopyruvate) is an alkylating agent with antitumoral activity on hepatocellular carcinoma. This compound
inhibits cellular ATP production owing to its action on glycolysis
and oxidative phosphorylation; however, the specific metabolic
steps and mechanisms of 3-BrPA action in human hepatocellular
carcinomas, particularly its effects on mitochondrial energetics,
are poorly understood. In the present study it was found that
incubation of HepG2 cells with a low concentration of 3-BrPA
for a short period (150 μM for 30 min) significantly affected both
glycolysis and mitochondrial respiratory functions. The activity
of mitochondrial hexokinase was not inhibited by 150 μM 3BrPA, but this concentration caused more than 70 % inhibition
of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells,
even when glucose was withdrawn from the incubation medium.
Oxygen consumption of HepG2 cells supported by either
pyruvate/malate or succinate was inhibited when cells were pre-
incubated with 3-BrPA in glucose-free medium. On the other
hand, when cells were pre-incubated in glucose-supplemented
medium, oxygen consumption was affected only when succinate
was used as the oxidizable substrate. An increase in oligomycinindependent respiration was observed in HepG2 cells treated with
3-BrPA only when incubated in glucose-supplemented medium,
indicating that 3-BrPA induces mitochondrial proton leakage
as well as blocking the electron transport system. The activity
of succinate dehydrogenase was inhibited by 70 % by 3-BrPA
treatment. These results suggest that the combined action of 3BrPA on succinate dehydrogenase and on glycolysis, inhibiting
steps downstream of the phosphorylation of glucose, play an
important role in HepG2 cell death.
INTRODUCTION
transformed cells [13–15]. As a result, 3-BrPA has been considered a potent agent in the treatment against the proliferation of
HCC [5,13,14]. In addition to 3-BrPA effects on energy homoeostasis, few studies have suggested that this compound induces
cell death either through activation of the mitochondrial pathway
of apoptosis or necrosis [5,12,15,16]. Given that the precise
mechanisms by which 3-BrPA affects mitochondrial physiology
and bioenergetics are still unknown, a detailed characterization
of the pharmacological action of this anti-tumoral agent is crucial
to the development of more efficient treatments.
In the present study it is demonstrated for the first time
that brief exposure of a HCC cell line, HepG2, to 3-BrPA at
a micromolar concentration leads to a severe impairment of
mitochondrial respiratory function by specifically affecting SDH
(succinate dehydrogenase) activity. An interesting observation is
that when cells are forced to rely on mitochondrial oxidative
phosphorylation rather than glycolysis, 3-BrPA effects on respiratory parameters appear to be more pronounced. Moreover, we
show that 3-BrPA, at the concentrations used, inhibits glycolytic
flux in a way not dependent on mt-HK II inhibition, but due to
enzyme inhibition downstream of the phosphorylation of glucose.
Cancer cells depend on large quantities of energy to maintain
elevated rates of proliferation. In this regard, specific adaptations
in energy-yielding pathways are observed, including activation
of glycolysis and down-regulation of oxidative phosphorylation,
also known as the Warburg effect [1,2], a metabolic signature
of many tumour cells. Accordingly, the effects of specific drugs
designed to interfere with the energy-yielding pathways of many
types of cancer have been investigated [3–5]. This is the case of
the analogue of pyruvate/lactate, 3-BrPA (3-bromopyruvate), an
alkylating agent able to react with thiol (SH) and hydroxy (OH)
groups of several enzymes [6–10].
Studies with HCC (hepatocellular carcinoma) cells have
demonstrated that the main blocking site of 3-BrPA is the mt-HK
(hexokinase) type II (mitochondrial HK II), which causes an
impairment of glycolytic flux [11,12]. In addition, 3-BrPA seems
to affect mitochondrial respiration, although the exact mechanism
of mitochondrial collapse was not identified [11]. Subsequent
studies have confirmed 3-BrPA as an agent able to cause ATP
depletion in tumour cells, with no apparent effect on non-
Key words: 3-bromopyruvate, glycolysis, hepatocellular carcinoma, HepG2 cell, mitochondrion, oxygen consumption, tumour
cell.
Abbreviations used: ANT, adenine nucelotide transporter; 3-BrPA, 3-bromopyruvate; DCPIP, 2,6-dichlorophenol-indophenol; DMEM, Dulbecco’s modified
Eagle’s medium; DTT, dithiothreitol; ETS, electron transport system; FBS, foetal bovine serum; FCCP, carbonyl cyanide p -trifluoromethoxyphenylhydrazone;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFM, glucose-free medium; GK, glucokinase; GM, GFM supplemented with 5 mM glucose
or 5 mM D-[U-13 C]glucose; G6P, glucose 6-phosphate; HCC, hepatocellular carcinoma; HK, hexokinase; MEM, minimal essential medium; mt-HK,
mitochondrial HK; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H -tetrazolium bromide; PFK, phosphofructokinase; PGI, phosphoglucose isomerase;
PGK, phosphoglycerate kinase; PK, pyruvate kinase; SDH, succinate dehydrogenase; VDAC, voltage-dependent anion channel.
1
This work is dedicated to Leopoldo de Meis on his 70th birthday
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
718
A. P. Pereira da Silva and others
The metabolic implications of such results to HepG2 cell death are
discussed in an attempt to better understand the action of 3-BrPA
in vivo.
added to the resulting supernatant and centrifuged at 15 000 g
for 10 min. The final pellet was suspended in a small volume of
medium and used for enzymatic assays.
EXPERIMENTAL
Measurements of enzymatic activities
Cell culture
Recovery of glycolytic enzyme activities after treatment with 3-BrPA
HepG2, a human HCC cell line, was obtained from American
Type Culture Collection and grown in MEM (minimal essential
medium) with 5 mM glucose, supplemented with 10 % (v/v) FBS
(foetal bovine serum), 0.22 % sodium bicarbonate and 0.2 %
Hepes (pH 7.4) at 37 ◦C in a humidified incubation chamber with
5 % CO2 . For viability assays, cells were grown on 24-well plates.
For all other assays, cells were grown on plastic Petri dishes. In
each assay, cells were seeded at a density of 105 cells/ml. Cells
were sub-cultured every 2– 4 days and were used for experiments
when they were nearly 95 % confluent.
The activity of the glycolytic enzymes HK, PGI, PFK, GAPDH,
PGK and PK were measured in cellular extracts after cell pretreatment with 3-BrPA. Culture medium was replaced with
fresh medium (MEM) containing 150 μM 3-BrPA and cells
were incubated for 30 min at 37 ◦C. Enzymatic activities were
measured using the following reaction media: (a) for HK, 10 mM
Tris/HCl (pH 7.4), 5 mM MgCl2 , 0.5 mM β-NADP+ , 1 unit/ml
GAPDH (Leuconostoc mesenteroides), 0.1 % Triton X-100,
1 μg/ml antimycin A, 1 μM rotenone, 2 mM sodium azide, 2 mM
ATP and 50 –100 μg/ml protein [20]; (b) for PGI, 50 mM Tris/HCl
(pH 7.4), 0.5 mM β-NAD+ , 1 unit/ml GAPDH (Leuconostoc
mesenteroides), 1 mM fructose 6-phosphate and 50 –100 μg/ml
protein [21]; (c) for PFK, 50 mM Tris/HCl (pH 7.4),
5 mM MgCl2 , 5 mM ammonium sulfate, 1.5 units/ml aldolase,
4 units/ml triose phosphate isomerase, 4 units/ml α-glycerophosphate dehydrogenase, 0.25 mM β-NADH, 0.1 mM ATP
and 50 –100 μg/ml protein [22]; (d) for GAPDH, 50 mM Tris/
HCl (pH 7.4), 2 mM MgCl2 , 1 mM ATP, 1 mM EDTA, 13 units/ml
PGK, 0.25 mM β-NADH and 50 –100 μg/ml protein [23]; (e)
for PGK, 50 mM Tris/HCl (pH 7.4), 2 mM MgCl2 , 1 mM ATP,
0.2 mM β-NADH, 5 units/ml GAPDH and 50 –100 μg/ml protein
[23]; and (f) for PK, 50 mM imidazole buffer (pH 7.4), 2 mM
MgCl2 , 100 mM KCl, 0.5 mM β-NADH, 1 mM ADP, 5 units/ml
lactate dehydrogenase and 50 –100 μg/ml protein [24]. The
reactions were started by the addition of 1 mM glucose (for
HK), 1 mM fructose 6-phosphate (for PGI and PFK), 5 mM
3-phosphoglycerate (for GAPDH), 5 mM 3-phosphoglycerate
(for PGK) or 1 mM phosphoenolpyruvate (for PK). For all of
the reactions, the baseline was followed for 3 min.
Cell viability
Cells were incubated with different concentrations of 3-BrPA at
37 ◦C for 30, 60, 90 and 180 min. After each treatment, cells were
washed twice with PBS and cell viability was assessed using the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide] assay as described previously [17] and cross-checked
by the Trypan Blue dye-exclusion assay.
Animal care and use
The experimental protocols using animals were approved by the
Committee for Ethics in Animal Research of the Universidade
Federal do Rio de Janeiro in compliance with the Brazilian
College for Animal Experimentation.
Preparation of cellular extracts
HepG2 cells were disrupted by liquid nitrogen addition and mixed
in a regular lysis buffer containing 10 mM Tris/HCl (pH 7.0),
20 mM NaF, 1 mM DTT (dithiothrietol), 250 mM sucrose, 5 mM
EDTA, 1 mM PMSF, 10 μM leupeptin and 1 μM pepstatin A.
The suspension was centrifuged at 100 g for 5 min at 4 ◦C,
and the resulting supernatant was used for measurement of the
recovered activities of HK, PGI (phosphoglucose isomerase), PFK
(phosphofructokinase), GAPDH (glyceraldehyde-3-phosphate
dehydrogenase), PGK (phosphoglycerate kinase) and PK
(pyruvate kinase) after 3-BrPA treatment [18].
Preparation of HK isoforms
Mitochondrial fractions for measurements of type II mt-HK
(HepG2 cells) activities were obtained by centrifugation of the
cellular extracts at 10 000 g for 15 min at 4 ◦C. The resulting
pellets were resuspended in lysis buffer and used for enzymatic
assays [18]. Mitochondrial fractions for measurements of type I
mt-HK (mouse brain) activities were obtained as previously
described [19]. GK (glucokinase) activity was measured in soluble
fractions of mouse liver. Briefly, liver was excised and homogenized with a Potter–Elvehjem-type homogenizer in a medium
containing 20 mM triethanolamine (pH 7.5), 5 mM EGTA,
100 mM glucose, 100 mM KCl, 1 mM DTT, 5 % glycerol, 5 mM
EDTA, 0.25 % sodium azide and 0.5 mM PMSF. The homogenate
was centrifuged at 100 000 g for 40 min. Ammonium sulfate
(45 % saturation) was added to the supernatant and centrifuged
at 15 000 g for 10 min. Ammonium sulfate (65 % saturation) was
c The Authors Journal compilation c 2009 Biochemical Society
Direct effect of 3-BrPA on enzyme preparations
The direct effect of 3-BrPA on three isoforms of HK and on
SDH was evaluated by incubating the enzyme preparations with
different concentrations of 3-BrPA. The activities of mt-HK I, mtHK II and GK were measured using an adaptation of a radiometric
assay described previously [25]. The assay was performed at
37 ◦C in 0.4 ml of reaction medium containing 20 mM Tris/HCl
(pH 7.5), 5 mM MgCl2 , 1 mM [γ -32 P]ATP (3000 c.p.m./nmol of
ATP), 10 mM glucose (mt-HK types I and II) or 100 mM glucose
(GK), 1 mM sodium orthovanadate, 5 mM sodium azide and
different concentrations of 3-BrPA. Duplicates were performed
for all assays and blanks were obtained in the absence of glucose.
The reaction was started by the addition of protein (0.1 mg/ml) and
quenched by the addition of 1 ml of activated charcoal in 0.1 M
HCl. After centrifugation (1000 g for 15 min), 0.4 ml aliquots of
supernatant were added to liquid-scintillation vials and counted.
The resulting radioactivity corresponds to [32 P]G6P (glucose 6phosphate) formed from the HK-catalysed reaction. The activity
of SDH was determined spectrophotometrically using DCPIP
(2,6-dichlorophenol-indophenol) as an artificial electron acceptor
and succinate as the substrate [26]. The assay was performed at
room temperature (25 ◦C) in 1.0 ml of reaction medium containing
20 mM phosphate buffer (pH 7.2), 0.1 % Triton X-100, 4 mM sodium azide, 5 mM succinate, 50 μM DCPIP and 2.5–300 μM
3-BrPA. Blanks were obtained in the absence of succinate. The
reaction was started by adding 0.1 mg of HepG2 mitochondrial
Inhibition of HepG2 energy pathways by 3-bromopyruvate
fraction (prepared as described for the mt-HK activity using the
same buffer, but without DTT) and the reduction of DCPIP was
monitored for 3 min at 600 nm. The SDH activity was calculated using the molar absorption coefficient of reduced DCPIP
(21.0 mM−1 · cm−1 ). SDH activity in the presence of 3-BrPA is
presented as a percentage of the control activity.
Metabolomic analyses of HepG2 cells by NMR
Culture medium was replaced by fresh glucose-free DMEM
(Dulbecco’s modified Eagle’s medium; Invitrogen) supplemented
with 5 mM D-[U-13 C]glucose and 0.2 % Hepes. Following this,
cells were incubated for 30 min at 37 ◦C in the absence or in the
presence of 150 μM 3-BrPA. Following the incubation period,
medium was removed and ∼ 8 × 107 cells were suspended in
glucose-free DMEM containing 10 % (v/v) deuterium oxide to a
final volume of 600 μl. One-dimensional 13 C spectra of the HepG2
cellular metabolites was obtained at 5 ◦C using 45o pulses with
a repetition time of 0.6 s, 16 000 complex points, ∼ 20 000 scans
and a spectral width of 200 p.p.m. The free-induction decays
were zero-filled to 16384 points and apodized with exponential
multiplication using line broadening of 20 Hz. One-dimensional
31
P spectra were obtained using 45o pulses with a repetition time
of 0.6 s, 16 000 complex points, ∼ 2000 scans and a spectral
width of 120 p.p.m. The free-induction decays were zero-filled
to 16384 points, and apodized with exponential multiplication
using line broadening of 10 Hz. Spectra of HepG2 cell metabolites
were acquired with a Bruker DRX 400 MHz using a broadband
inverse detection probe (BBI). Spectral processing and analysis
was performed using Topspin 2.0. Analysis and assignment of
the metabolites were obtained using the Human Metabolome
Database v1.0 using the 13 C one-dimensional spectra search mode
with a tolerance of 0.25 p.p.m. [27].
719
Oxygen consumption of intact and digitonin-permeabilized
HepG2 cells
Oxygen consumption rates were measured polarographically in
an oximeter using a Clark-type oxygen electrode (Oxytherm,
Hansatech Instruments) or using high-resolution respirometry
(Oroboros Oxygraph-O2K). The electrode was calibrated between 0 and 100 % saturation with atmospheric oxygen at 37 ◦C.
To measure the direct effect of 3-BrPA concentrations on mitochondrial respiration, HepG2 cells were removed from culture
dishes through trypsinization and approx. 2.5–5 × 106 cells were
added to a respiration medium containing 0.25 M mannitol,
0.1 % fatty-acid-free BSA, 10 mM MgCl2 and 10 mM KH2 PO4
(pH 7.2) as previously described [29], followed by the sequential
addition of 0.005 % digitonin, 10 mM succinate (complex II
substrate), 100 μM ADP (ATP synthesis substrate), 0.05–5 mM
3-BrPA and 1 μM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone).
For measurements of mitochondrial respiration following treatment with 3-BrPA, HepG2 cells were pre-incubated with 150 μM
3-BrPA for 30 min at 37 ◦C, with GM or GFM. After the incubation period, media were removed and cells were either suspended
in the same culture medium used for the pre-incubation (for measurements of oxygen consumption of intact cells), or in the
respiration medium described above (for evaluation of respiratory
complexes of permeabilized cells). Basal consumption, oligomycin-independent respiration (proton leak) and FCCP-stimulated respiration (maximum respiration) were assessed in intact
HepG2 cells as described previously [30]. 3-BrPA effects
on respiratory complexes of 0.005 % digitonin-permeabilized
HepG2 cells were performed following the addition of different substrates/modulators as 10 mM pyruvate + 10 mM malate
(complex I-linked substrates), 10 mM succinate, 1 μM rotenone,
50 μM ADP, 1 μg/ml oligomycin and 1 μM FCCP, or titration
from 250 –750 nM FCCP.
Detection of lactate in the culture medium
Culture medium was replaced with fresh medium containing
150 μM 3-BrPA and cells were incubated at 37 ◦C. Two different
culture media were used: GFM (glucose-free medium; DMEM),
which contains only glutamine as an oxidizable substrate, and
GM {GFM supplemented with 5 mM glucose or 5 mM D-[U13
C]glucose (for NMR assays)}. Both media were supplemented
with 0.2 % Hepes. At the times indicated, samples of 50 or
100 μl from culture medium were collected to evaluate lactate
accumulation as described below.
Enzymatic assay
Lactate measurement was performed in a hydrazine/glycine buffer
(pH 9.2), containing 5 mg/ml β-NAD+ and 15 units/ml lactate
dehydrogenase to a final volume of 200 μl. The absorbance due
to formation of NADH was monitored in a microplate reader
(SpectraMax M5, Molecular Devices) for 30 min at 340 nm and
was correlated with the presence of lactate on samples from a
standard curve [28].
Detection by NMR
One-dimensional 13 C spectra for the kinetics of lactate cell export
were obtained at 5 ◦C using 45o pulses with a repetition time of
0.6 s, 16 000 complex points, 2028 scans and a spectral width
of 200 p.p.m. The free-induction decays were zero-filled to 16384
points and apodized with exponential multiplication using line
broadening of 10 Hz. Spectra were acquired and analysed as
described for metabolomic analysis as described above.
Protein determination
The protein concentration in the samples was determined as
described by Lowry et al. [31].
Statistical analysis
Statistical analyses were performed using Origin® 7.5
(OriginLab). All results are expressed as means +
− S.E.M. for n
independent experiments. Statistical significance was determined
using a Student’s t test. Differences were considered statistically
significant for P < 0.05 or P < 0.01.
RESULTS
3-BrPA affects HepG2 viability in a dose- and time-dependent
manner
Figure 1(A) shows that cell incubation with 10 –100 μM 3-BrPA
for up to 60 min caused a small 10 % decrease in viability. On
the other hand, 50 μM 3-BrPA incubation for 180 min caused
a significant 50 % decrease in HepG2 cell viability (P < 0.05).
Higher concentrations of 3-BrPA (1–5 mM) were effective to
promote a more than 30 % reduction in viability at all times
tested (P < 0.05). These results were confirmed by a Trypan Blue
exclusion assay (Figure 1B) and suggest that, depending on the
concentration and period of exposure to 3-BrPA, this compound
may act on different intracellular targets with distinct affinities
and reactivity.
c The Authors Journal compilation c 2009 Biochemical Society
720
Figure 1
A. P. Pereira da Silva and others
Time course of the effects of 3-BrPA on HepG2 cell viability
HepG2 cells in culture were incubated with different concentrations of 3-BrPA (50 –5000 μM) for
30 (䊉), 60 (䊊), 90 (䊏) and 180 () min. Following the incubation procedure, cell viability was
accessed by MTT assay (A) or Trypan Blue exclusion assay (B) as described in the Experimental
section. Values represent means +
− S.E.M. (n = 5).
Figure 3 Effects of 3-BrPA on mt-HK II activity and on the recovery of
glycolytic enzyme activities in HepG2 cells
Figure 2
Lactate production by HepG2 cells in the presence of 3-BrPA
(A) HepG2 cells in culture were incubated in the absence (open symbols) or in the presence
(closed symbols) of 150 μM 3-BrPA in glucose-supplemented medium (GM-cells) (circles) or
in glucose-free medium (GFM-cells) (triangles) for up to 60 min as described in the Experimental
section. At 30 min of incubation (indicated by arrows) 2 μg/ml antimycin A (Ant A) was added
to the culture. Medium samples were removed at the times indicated and used to measure
lactate production. (B) Results obtained in the absence of glucose presented in (A), showing a
magnification of the antimycin A effect on lactate production. Values represent means +
− S.E.M.
(n = 4). Broken lines show lactate production if antimycin A had not been added.
3-BrPA affects lactate production by HepG2 cells
Glucose availability is an important factor controlling energy
production, which is important for cancer cell survival. It has
been proposed that 3-BrPA inhibits glycolytic flux and that this
inhibition is associated with a decrease in cell viability [11,14]. To
check whether this is the case for HepG2 cells, lactate production
was followed in cells incubated with 150 μM 3-BrPA for up to
60 min (Figure 2), a condition that caused a 25 % decrease in cell
viability (Figure 1). In order to also evaluate the contribution of the
utilization of endogenous substrates, such as glycogen (bypassing
HK reaction), on lactate production, cells were incubated with
glucose-supplemented (GM-cells) or glucose-free (GFM-cells)
medium (Figure 2). As expected, GFM-cells presented a 16-fold
decrease (P < 0.05) in the rate of lactate formation compared with
GM-cells (Figures 2A and 2B). Treatment of GM-cells with 3BrPA led to a 3-fold decrease (P < 0.05) in lactate production,
whereas the absence of glucose in the medium resulted in a 1.5fold decrease in lactate production. The addition of antimycin A,
an inhibitor of oxidative phosphorylation, forces cells to rely only
c The Authors Journal compilation c 2009 Biochemical Society
(A) Activities of mt-HK type I from mouse brain (䉱), type II from HepG2 cells (䊊) and GK
from mouse liver (䊉) were measured in the presence of different concentrations of 3-BrPA
(0.01–5 mM) as described in the Experimental section. (B) Time course of mt-HK II activity in
the absence (䊊), 150 μM (䉱) or 5 mM (䊉) 3-BrPA. (C) Cells in culture were pre-incubated
with 150 μM 3-BrPA for 30 min and the activities of HK, PGI, PFK, GAPDH, PGK and PK
were measured as described in the Experimental section. Open bars, control cells; closed bars,
cells pre-incubated with 3-BrPA. The relative activity of each enzyme is related to the control
+
condition. The maximal activities (m-units) of controls were: HK = 33 +
− 4; PGI = 1040 − 27;
+
+
+
PFK = 35 +
− 3; GAPDH = 515 − 41; PGK = 637 − 15; and PK = 654 − 39. Values represent
means +
− S.E.M. (n = 4).
on glycolysis for ATP production. In this situation, it is expected
that lactate formation is stimulated, mimicking the Pasteur effect
[32]. Indeed, addition of antimycin A to GM-cells caused a 3-fold
increase (P < 0.05) in lactate production (Figure 2A), indicating
that cells were already near maximum glycolytic capacity. An
intriguing finding was that no net increase in lactate production
was observed when antimycin A was added to GM-cells treated
with 3-BrPA (Figure 2A). Figure 2(B) shows a magnification of
results presented in Figure 2(A) of the effects of 3-BrPA and
antimycin A on lactate production by GFM-cells. In contrast with
GM-cells, antimycin A induced an 8.5-fold increase (P < 0.05)
in the rate of lactate production when glucose was withdrawn from
the medium (Figure 2B). An interesting finding was that, even
in the presence of antimycin A, no increase in lactate production
was observed after treatment with 3-BrPA (Figure 2B).
3-BrPA targets HepG2 cell glycolysis: inhibition of GAPDH
and 3-PGK, but not of HK
Since it has been demonstrated that 3-BrPA has an important
inhibitory effect on mt-HK type II of tumour cells [11], we
evaluated whether mt-HK inhibition of HepG2 cells would be
responsible for the observed effects on lactate production. The
direct effect of 3-BrPA on mt-HK activity is shown in Figure 3(A).
Inhibition of HepG2 energy pathways by 3-bromopyruvate
Figure 4
721
NMR spectroscopy analysis of HepG2 cell metabolites treated with 3-BrPA
HepG2 cells in culture were pre-incubated with 150 μM 3-BrPA for 30 min. (A) Spectra highlight the enrichment of informative metabolic intermediates. Top spectrum, control cells; bottom
spectrum, cells pre-incubated with 3-BrPA. (B) Culture medium samples were removed at the times indicated and subjected to [13 C]lactate determination. The numbers indicate specific
13
C-containing chemicals of the following intermediates: (1) fructose 1,6-bisphosphate, (2) ribose 5-phosphate, (3) G6P, (4) glyceraldehyde 3-phosphate, (5) dihidroxyacetone phosphate, (6)
fructose 6-phosphate, (7) glucose 1-phosphate, (8) 6-phosphoglucoronate, (9) 2-phosphoglycerate, (10) L-aspartate, (11) L-asparagine, (12) L-alanine, (13) L-glutamate, (14) L-lactate, (15)
2-acetolactate.
Unexpectedly, only a 20 % decrease in mt-HK activity was
observed when cells were incubated for 20 min with 3-BrPA at
concentrations as high as 5 mM (P < 0.05) (Figure 3A). To check
whether the inhibition of mt-HK activity was dependent on the
time of incubation with 3-BrPA, G6P production was followed
for up to 60 min (Figure 3B). The results showed that 150 μM
3-BrPA did not affect the time course of the reaction and 5 mM
of the compound promoted a 30 % inhibition (3.01 +
− 0.3 to 2.1 +
−
0.1 μmol of G6P/mg of protein) of mt-HK activity (P < 0.05).
Since 3-BrPA reacts with SH or/and OH groups of many enzymes
[6–10], differences in the reactivity of these groups in HK from
distinct tissues could explain the differences between the results
of the present study and those shown in the literature [11]. For
that reason, the inhibition pattern of different HK isoforms by 3BrPA (mt-HK I and GK) was evaluated. It becomes apparent
that GK presented higher sensitivity for 3-BrPA than mt-HK
I or II (Figure 3A). The half-maximum inhibition of GK was
obtained with 300 – 400 μM 3-BrPA, whereas for the other two
HK isoforms, 5 mM 3-BrPA only slightly affected their activity
(20 % inhibition) (P < 0.05). These results indicate that mt-HK II
from HepG2 cells is unlikely to be an important target of 3-BrPA
at the conditions that promoted inhibition of the glycolytic flux
(Figure 2).
In order to search for the 3-BrPA target in HepG2 cell glycolysis, the activities of different enzymes from the ATP-consuming
and ATP-producing phases of this pathway were measured. After
incubation of HepG2 cells with 150 μM 3-BrPA for 30 min, the
recovered activities of HK, PGI and PFK were not altered by
the treatment. On the other hand, activities of the enzymes
involved in the glycolytic ATP synthesis, GAPDH, PGK and PK,
were significantly modified (Figure 3C). The activities of GAPDH
and PGK were strongly suppressed by 3-BrPA treatment (90 and
70 % of control activities respectively, P < 0.01). Conversely, the
recovery of PK activity was almost twice that of control (P < 0.01)
(Figure 3C).
3-BrPA treatment affects the glycolytic metabolite profile
According to the results showing the inhibition of specific glycolytic enzymes (Figure 3C), it can be assumed that 3-BrPA treatment may compromise glycolysis downstream of the glyceraldehyde 3-phosphate formation step. To further characterize
this metabolic failure, the intermediates of glycolysis in HepG2
cells treated with 3-BrPA were analysed non-invasively by NMR
spectroscopy. For this purpose, 5 mM D-[U-13 C]glucose was
added to GFM in the presence or in the absence of 150 μM 3-BrPA
for 30 min. The 13 C-NMR spectra of the cells revealed that
the assigned glycolytic metabolites such as sugar phosphates
(fructose 1,6-bisphosphate, ribose 5-phosphate, G6P, fructose
6-phosphate, glucose 1-phosphate and 6-phosphoglucoronate)
and triose phosphates (glyceraldehyde 3-phosphate and dihidroxyacetone phosphate) (see 13 C chemical shift from 66 to
105 p.p.m. in Figure 4A) are clearly presented in large excess
after treatment with 3-BrPA. Additionally, it is evident that [13 C]2phosphoglycerate derived from D-[U-13 C]glucose is decreased in
3-BrPA-treated HepG2 cells (Figure 4A). These spectral profiles
are in agreement with the inhibition of the glycolytic steps
involved in ATP synthesis, i.e. GAPDH and PGK (Figure 3C),
indicating that these enzymes are the main targets of 3BrPA in HepG2 cells. This hypothesis was reinforced by the
decreased levels of 2-acetolactate and amino acids (L-aspartate,
L-asparagine, L-alanine and L-glutamate) (see 13 C chemical shift
from 30 to 55 p.p.m. in Figure 4A). Unexpectedly, higher amounts
of intracellular L-lactate was observed in HepG2 cells treated with
3-BrPA (Figure 4A). The inhibition of lactate production (and
export) by 3-BrPA in HepG2 cells was further confirmed using
NMR spectroscopy analysis (Figure 4B). The most abundant
13
C-labelled metabolite detected in the culture medium was
[13 C]lactate, which steadily accumulated.
Rapid (∼ 20 min) 31 P spectra of HepG2 cells were acquired
immediately before the 13 C spectra acquisition. The spectra
c The Authors Journal compilation c 2009 Biochemical Society
722
A. P. Pereira da Silva and others
Figure 6 Effect of pre-incubation with 3-BrPA on oxygen consumption of
intact HepG2 cells
Figure 5 Titration of mitochondrial oxygen consumption of HepG2
permeabilized cells by 3-BrPA
(A) Representative record of mitochondrial oxygen consumption of HepG2-permeabilized cells
under different regimes. Cells were added to a standard respiration buffer and permeabilized by
the addition of 0.005 % digitonin as described in the Experimental section. Arrows indicate the
addition of substrates/modulators as follows: 10 mM succinate, 200 μM ADP and 3-BrPA to
obtain the final concentration as indicated, and 1 μM FCCP. (B) Rates of oxygen consumption
in the presence of different concentrations of 3-BrPA. Values represent means +
− S.E.M. (n = 4).
showed accumulation of phosphomonoesters in the cells treated
with 3-BrPA (results not shown). 31 P spectra are consistent with
the accumulation of the phosphorylated hexoses detected by 13 C
NMR.
3-BrPA strongly affects mitochondrial oxygen consumption
in HepG2 cells
Although it has been suggested that mitochondrial respiration is
affected by 3-BrPA [11], there are no available data in the literature
with regard to the mechanisms involved in this process. Direct
effects of 3-BrPA on mitochondrial respiration of HepG2 cells
were evaluated after cell permeabilization and 3-BrPA titration.
Figures 5(A) and 5(B) show that when respiration was supported
by succinate, oxygen consumption of digitonin-permeabilized
HepG2 cells was strongly inhibited by 3-BrPA addition. When
400 μM of the compound was added, state 3 respiration (ADPstimulated respiration coupled to ATP synthesis) was completely
inhibited (Figures 5A and 5B). The IC50 value for 3-BrPA inhibition was 150 μM (Figure 5B). The inhibition of oxygen consumption by 3-BrPA is probably due to a blockage of electron transport
in the ETS (electron transport system), since addition of a proton
ionophore (FCCP), that would fully stimulate respiration if the
inhibition was at the level of the ADP ↔ ATP recycling system
[ANT (adenine nucelotide transporter)/Fo F1 -ATP synthase], was
not able to increase oxygen consumption (Figure 5A). No effect
of the same concentration of 3-BrPA was observed in the azidesensitive ATPase activity (results not shown).
Pre-incubation with 3-BrPA affects mitochondrial respiratory
parameters of intact and permeabilized HepG2 cells
In addition to the direct effects of 3-BrPA on mitochondria, the
respiratory parameters of HepG2 cells were also assessed after
pre-incubation with this compound (Figures 6 –8). As for lactate
production, cells were incubated with glucose-supplemented
(GM-cells) or glucose-free (GFM-cells) medium to analyse
possible differences in the effects of 3-BrPA when glycolysis
and oxidative phosphorylation were differentially stimulated.
Figure 6(A) shows that GFM-cells pre-incubated with 150 μM
c The Authors Journal compilation c 2009 Biochemical Society
HepG2 cells in culture were pre-incubated with 150 μM 3-BrPA for 30 min in the absence
(GFM-cells) (A) or presence (GM-cells) (B) of glucose as described in the Experimental
section. Oxygen consumption was measured in the same culture medium used in the incubation
step without FBS. The histogram represents the rates of oxygen consumption under different
regimes (basal respiration, BASAL; FCCP-induced maximum respiration, 1 μM FCCP; and
oligomycin-independent respiration, 1 μg/ml OLIGO). Open bars, control cells; closed bars,
cells pre-incubated with 3-BrPA. Values represent means +
− S.E.M. (n = 5). **P < 0.01 and
*P < 0.05, significantly different from control cells.
3-BrPA for 30 min presented a 50 % decrease in basal respiration
−1
−6
(3.1 +
− 0.19 to 1.6 +
− 0.13 nmol of O2 · min · 10 cells) and
FCCP-stimulated maximum respiration (3.5 +
0.22
to 1.9 +
−
−
0.3 nmol of O2 · min−1 · 10−6 cells) (P < 0.01). Additionally, no
difference was observed in the respiratory rate not coupled to ATP
synthesis, the proton leak (oligomycin-independent respiration)
(Figure 6A). A different situation was observed in GM-cells (Figure 6B), where 3-BrPA caused only a 20 % inhibition of basal
−1
−6
respiration (2.1 +
− 0.12 to 1.6 +
− 0.1 nmol of O2 · min · 10 cells)
(P < 0.05) and no difference was detected on FCCP-stimulated
maximum respiration. Curiously, despite the apparent small
effect of 3-BrPA treatment on the basal respiration of GMcells, oligomycin-independent respiration was almost 2-fold
higher than that measured on control cells (0.6 +
− 0.1 to 1.1 +
−
0.1 nmol of O2 · min−1 · 10−6 cells) (P < 0.05) (Figure 6B). These
results indicate that, depending on the carbon source utilized,
3-BrPA has distinct effects on the energy metabolism of HepG2
cells.
To analyse in more detail the effects of 3-BrPA on mitochondrial
function and ETS complex activities, oxygen consumption was
measured on digitonin-permeabilized cells using high-resolution
respirometry (Figures 7 and 8). Following incubation with
150 μM 3-BrPA for 30 min, GFM-cells presented a significant
−1
decrease in complex I (7.8 +
− 0.6 pmol of O2 · s ·
− 1.4 to 3.8 +
−6
10 cells) and succinate-driven respiration (54 +
− 9.3 to 29.6 +
−
3.5 pmol of O2 · s−1 · 10−6 cells) (P < 0.05) (Figures 7C and
8A). Total electron transport capacity after FCCP titrations
was significantly decreased (84.6 +
− 8.3 to 44.4 +
− 9 pmol of
O2 · s−1 · 10−6 cells) (P < 0.05) (Figures 7C and 8A), whereas
oxygen flow related to proton leak was not significantly altered
(Figures 7C and 8A), as observed with intact cells (Figure 6A).
Different effects of 3-BrPA treatment on respiratory parameters
were observed in GM-cells when compared with GFM-cells
(Figures 7D and 8B). 3-BrPA was able to significantly decrease
only the oxygen flow induced by succinate (35.7 +
− 5.7 to 15 +
−
4.4 pmol of O2 · s−1 · 10−6 cells) (P < 0.05). In a similar manner
to the respiratory flow observed for intact GM-cells (Figure 6B),
3-BrPA did not affect maximum respiration flow induced by
FCCP, and promoted a 3-fold increase in proton leak respiration
−1
of permeabilized cells (4.4 +
− 0.95 to 14.2 +
− 4.2 pmol of O2 · s ·
−6
10 cells) (P < 0.05) (Figures 7D and 8B). Moreover, the respiratory control induced by ADP was delayed after 3-BrPA treatment in
Inhibition of HepG2 energy pathways by 3-bromopyruvate
Figure 7
723
Representative records of oxygen concentration (A and B) or oxygen flow (C and D) of permeabilized HepG2 cells pre-incubated with 3-BrPA
HepG2 cells in culture were pre-incubated with 150 μM 3-BrPA for 30 min in the absence (GFM-cells) (A and C) or presence (GM-cells) (B and D) of glucose as described in the Experimental
section. Oxygen consumption was measured using high-resolution respirometry on standard respiration buffer after the addition of cells and 0.005 % digitonin. Arrows indicate the addition of
substrates/modulators as follows: 10 mM pyruvate + malate (Pyr/Mal), 50 μM ADP, 1 μM rotenone (Rot), 10 mM succinate (Succ), 1 μg/ml oligomycin (Oligo) and FCCP as indicated. Continuous
traces, control cells; broken traces, cells pre-incubated with 3-BrPA.
Figure 8
Rates of oxygen consumption of permeabilized HepG2 cells pre-incubated with 3-BrPA
HepG2 cells in culture were pre-incubated with 150 μM 3-BrPA for 30 min in the absence (GFM-cells) (A) or presence (GM-cells) (B) of glucose as described in the Experimental section.
Oxygen consumption was measured using high-resolution respirometry on standard respiration buffer after the addition of cells and 0.005 % digitonin. The following substrate and/or modulator
concentrations were added: 10 mM pyruvate + malate (Pyr/Mal), 50 μM ADP, 1 μM rotenone (Rot), 10 mM succinate (Succ), 1 μg/ml oligomycin (Oligo) and 1 μM FCCP. St4(CI) and St4(CII) mean
state 4 obtained when complex I (Pyr/Mal) or complex II (Succ) were stimulated respectively. Open bars, control cells; closed bars, cells pre-incubated with 3-BrPA. Values represent means +
− S.E.M.
(n = 5). *P < 0.05, significantly different from control cells.
GFM-cells either with pyruvate/malate or succinate as respiratory
substrates (Figure 7C); however, in GM-cells, 3-BrPA treatment
did not change the response to ADP when pyruvate/malate was
used to induce respiration, but the respiratory control induced by
ADP was slowed when succinate was the respiratory substrate
(Figure 7D).
c The Authors Journal compilation c 2009 Biochemical Society
724
A. P. Pereira da Silva and others
Figure 9 Titration and threshold curves of SDH activity in HepG2
mitochondria
(A) 3-BrPA titration curve. SDH activity was measured by a spectrophotometric method using
DCPIP as an artificial electron acceptor as described in the Experimental section. The reaction
was started with the addition of HepG2 mitochondrial fraction (0.01 mg/ml) in the absence or
2.5–300 μM of 3-BrPA. Values are expressed as a percentage of control activity. Control activity:
−1
−1
9.1 +
− 1.1 nmol of DCPIP · min · mg of protein . (B) Threshold curves. The percentage of
respiratory rate as a function of the percentage of SDH inhibition by 3-BrPA. Each point comes
from the experimental titration curves and represents the percentage of the respiratory rate as a
function of the percentage of the SDH activity for the same 3-BrPA concentration.
SDH as a target of 3-BrPA in the mitochondria of HepG2 cells
The results presented in Figures 7 and 8 point to a preferential
inhibition of mitochondrial respiratory complex II (SDH) activity
after 3-BrPA treatment in both GM- and GFM-cells. To check
this hypothesis, the direct effect of 3-BrPA on SDH activity in
the HepG2 mitochondrial fraction was measured (Figure 9A).
Accordingly, the results confirmed that SDH from HepG2 cells
was inhibited by 3-BrPA, presenting an IC50 value between 10
and 20 μM.
The SDH inhibition profile may not be strictly correlated
with oxidative phosphorylation inhibition and, in fact, may vary
among different cell types [33]. This phenomenon of biochemical
threshold can be demonstrated by threshold curves, which may
indicate either a deficiency or an ‘excess of enzyme activity’
or a ‘buffering effect by the metabolic network’ of a given
complex of oxidative phosphorylation responses. Figure 9(B)
shows a threshold curve of the rates of oxygen consumption and
the degree of SDH inhibition attained by 3-BrPA. It becomes
apparent that at the level of 50 % inhibition of SDH activity,
no significant decrease in HepG2 mitochondrial respiration was
observed; however, above this degree of SDH inhibition, a large
fall in the respiratory rate was detected. More importantly, at
150 μM 3-BrPA, which corresponds to 70 % inhibition of SDH
activity, respiration was inhibited in 60 % (Figure 9B).
DISCUSSION
HCC is an important health problem, being one of the most
common fatal cancers worldwide [34]. The search for an efficient
therapy led to the proposal of a new approach which consists of the
direct intra-arterial administration of 3-BrPA to the tumour.
The treatment with 3-BrPA eradicates HCC without affecting
healthy surrounding tissues of the host [13,14]. The beneficial properties of this agent rely on its capacity to inhibit cellular energy
machinery systems, i.e. glycolysis and oxidative phosphorylation
[11]; however, the understanding of the specific metabolic steps
and mechanisms of 3-BrPA action in human HCC cell lines,
particularly its effects on mitochondria bioenergetics, are very
limited. In the present study, we could establish the time course of
c The Authors Journal compilation c 2009 Biochemical Society
3-BrPA effects on the viability of HepG2 cells, which indicated
that, depending on the concentration, period of incubation and
the substrates oxidized by the cells, 3-BrPA affects different
metabolic pathways.
Cancer cells are believed to present an accelerated glycolysis,
even in the presence of sufficient amounts of oxygen [1,2]. HepG2
cells follow this pattern, since only 3-fold stimulation on lactate
production was observed when oxidative phosphorylation was
inhibited by antimycin A addition (Figure 2A). In non-transformed cell lines, the stimulatory effect on lactate production by
antimycin A may be as high as 10-fold [32]. In the presence of 3BrPA, the amount of lactate produced was significantly decreased,
indicating an inhibitory effect on glycolysis (Figure 2A). The
70 % inhibition of glycolysis could not be explained by mtHK inhibition (Figures 2A, 3A and 3B). Other candidate target
enzymes for 3-BrPA action could be GAPDH and PGK, which, in
a previous study, were immediately inhibited by 3-BrPA in a competitive manner [35]. Indeed, in the present work we demonstrated
that treatment of HepG2 cells with 150 μM 3-BrPA by short
periods of incubation is sufficient to promote an irreversible
inhibition of both GAPDH and PGK activities by more than
70 % (Figure 3C), despite the fact that cell viability is
almost 100 % (Figure 1). The inhibition of GAPDH and PGK
activities dramatically altered the steady-state levels of glycolytic
intermediates in HepG2 cells (Figure 4A). It is remarkable that the
ATP-consuming reactions of glycolysis (HK and PFK) were not
affected by 3-BrPA, but the downstream ATP-producing steps of
this pathway (GAPDH and PGK), together with oxidative phosphorylation, were severely inhibited (Figures 3, 4 and 8). The
glycolytic ATP-consuming steps (i.e. HK and PFK) would
facilitate the bioenergetic collapse of tumour cells by accelerating
the intracellular ATP depletion. It should be mentioned that,
besides the bioenergetic alterations, 3-BrPA-treated cells showed
increased levels of intracellular lactate (Figure 4A) which would
affect the internal pH of the cell. The reasons for this increase
in intracellular lactate are not yet understood, but it might be
speculated that lactate transport across cellular membranes may
be affected by 3-BrPA treatment, since this compound shares the
same transporter as lactate.
Since no stimulation of glycolysis was observed following the
addition of antimycin A, the rate of lactate production by HepG2
cells appeared to be on its maximum flux, which indicates that
3-BrPA also affects mitochondrial function. This is more clearly
observed when lactate production was measured in GFM-cells
(Figure 2). In this situation, the carbon sources for lactate synthesis
are glycogen and mainly glutamine oxidation, as demonstrated
previously [36]. Since replacement of glucose by glutamine
induces a maximum stimulation of oxidative phosphorylation, the
observed decrease in lactate formation due to 3-BrPA treatment
indicates a decrease in glutaminolysis and, ultimately, a decreased
mitochondrial oxidative capacity. In addition, one can hypothesize
that inhibition of glutamate dehydrogenase [37], SDH [6] and
malic enzyme [9], all of them involved in lactate production
from glutamine by 3-BrPA, could also contribute to the inhibitory
effects shown in Figure 2(B).
The results presented in Figures 5–8 show in more detail the
effects of 3-BrPA on mitochondrial respiratory parameters. Preincubation with 3-BrPA caused a significant decrease in the rate of
oxygen consumption either in intact (Figure 6) or permeabilized
(Figures 7 and 8) HepG2 cells, indicating that 3-BrPA may
be reacting irreversibly on SH groups of the ETS under cellculture conditions, in accordance with previous in vitro assays
shown for both SDH and PDH (pyruvate dehydrogenase) [6,10].
Unexpectedly, the degree of inhibition of respiration by 3-BrPA
depends on the carbon source supplied to cells (Figures 2 and
Inhibition of HepG2 energy pathways by 3-bromopyruvate
6–8), glucose or glutamine, both of which are rapidly consumed
during proliferation of tumours. Accordingly, in results with
GFM-cells, we observed significant alterations in mitochondrial
bioenergetics. The inhibition of ETS by 3-BrPA may be related
to an imbalance of cellular redox state and to the absence of
substrates, turning the cellular enzymes more reactive to 3-BrPA
[7,10]. On the other hand, in the presence of glucose (GM-cells),
the inhibition of complex-I linked substrates (pyruvate/malate)
was not apparent (Figures 7D and 8B), indicating the protective
effect of this substrate. Despite the distinct effects on complex-I
respiratory activity, it is important to emphasize that, under both
conditions, complex II-driven respiration, i.e. SDH activity, was
significantly reduced (Figures 7C, 7D and 8).
An interesting finding was that the proton leak was increased in
the GM-cells (Figures 6B and 8B). This proton leak may reflect
the permeability transition pore formed in the mitochondrial
membrane, as observed in apoptosis or necrosis [38– 41]. One
possibility is that the inhibition of glycolysis at the levels of the
GAPDH and PGK reactions (Figure 3C) increased the intracellular levels of G6P (Figure 4A), which leads to mt-HK dissociation
from the mitochondrial membrane and favours the binding of proapoptotic proteins to VDACs (voltage-dependent anion channels)
[42]. In fact, the results from 13 C-NMR spectra show that the
level of G6P is higher in HepG2-treated cells than in control cells
(Figure 4A). Actually, it was demonstrated that 3-BrPA promotes
the dissociation of mt-HK from VDACs derived from HCC HuhBAT and MH134 cells [16]. On the other hand, one can speculate
that, in GFM-cells, mt-HK dissociation does not occur since G6P
levels are low (Figure 4A) and proton leak is decreased (Figures 6–
8). Additionally, we cannot exclude the possibility that a reaction
of 3-BrPA with crucial cysteine residues (e.g. Cys56 ) located at
the ANT may lead to the formation of a permeability transition
pore [43], contributing to the increase in proton leak in GM-cells.
Analysing the effects of 3-BrPA on other HK isoforms, the
results of the present study showed that GK, the major isoform
in normal liver, was much more sensitive to 3-BrPA inhibition
(Figure 3A) when compared with the mitochondrial isoforms
found in tumour cells. GK is characterized by a high susceptibility
to SH group oxidation [44] and a strong sensitivity towards SH
group reactive reagents [45,46]. The SH group of the cysteine
residues has been shown to modulate enzyme activity [47]. In
contrast with GK, low-K m HKs show an approx. 100-fold lower
sensitivity towards inhibition of enzyme activity by SH group
reagents, such as alloxan [46]. The results in Figure 3(A) show a
weaker inhibition by 3-BrPA treatment of HepG2 mt-HK II when
compared with mice liver GK. Thus it may be concluded that
3-BrPA acts like a SH group reagent following a very similar
pattern to those SH group reagents described previously for
GK and low-K m HKs [47]. Previous results have shown that 3BrPA almost completely inhibits mt-HK II from HCC [11]. The
apparent contrasting results shown in Figure 3 and those described
previously [11] may be explained, in part, by the protocol used to
assay mt-HK activities in the present study, in which the reactions
were started by the addition of enzymes in a medium containing
ATP, glucose and 3-BrPA. In the previous study, 5 mM 3-BrPA
was added to mt-HK preparations before the addition of glucose
[11]. It had been shown that cysteine residues of the catalytic site
of GK and mt-HK II are much less sensitive to SH group reagents
while in contact with their substrates [47].
In conclusion, it seems that 3-BrPA may act on several cellular
enzymes and, based on the threshold inhibition curve and lactate
production (Figures 2, 4B and 9B) we propose that GAPDH, PGK
and SDH are important targets of this compound. Therefore the
mutual effects of 3-BrPA on glycolysis and mitochondria may be
responsible for the decrease in viability and death of HCC cells.
725
We believe that this information will help to design new analogues
and compounds that might contribute to more effective treatment
of HCC.
ACKNOWLEDGEMENTS
We are grateful to Dr Erich Gnaiger for kind technical advice with regard to the use of the
high-resolution respirometry in O2K Oroboros. We also would like to thank the unknown
reviewers for their valuable comments in improving the paper.
FUNDING
This work was supported by the Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico [grant number 480378/2004-5] and the Fundação Carlos Chagas Filho de
Amparo à Pesquisa do Estado do Rio de Janeiro [grant number E-26/ 111.911/2008].
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