Inhibition of Glycolysis in Cancer Cells: A Novel Strategy to
Overcome Drug Resistance Associated with Mitochondrial
Respiratory Defect and Hypoxia
Michael J. Keating,
1Jennifer S. Carew,
1Kapil N. Bhalla,
Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, China; and
H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida
1Molecular Pathology and
2Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas;
4Department of Interdisciplinary Oncology,
Cancer cells generally exhibit increased glycolysis for ATP
generation (the Warburg effect) due in part to mitochondrial
respiration injury and hypoxia, which are frequently associ-
ated with resistance to therapeutic agents. Here, we report
that inhibition of glycolysis severely depletes ATP in cancer
cells, especially in clones of cancer cells with mitochondrial
respiration defects, and leads to rapid dephosphorylation of
the glycolysis-apoptosis integrating molecule BAD at Ser112,
relocalization of BAX to mitochondria, and massive cell
death. Importantly, inhibition of glycolysis effectively kills
colon cancer cells and lymphoma cells in a hypoxic environ-
ment in which the cancer cells exhibit high glycolytic activ-
ity and decreased sensitivity to common anticancer agents.
Depletion of ATP by glycolytic inhibition also potently in-
duced apoptosis in multidrug-resistant cells, suggesting that
deprivation of cellular energy supply may be an effective
way to overcome multidrug resistance. Our study shows a
promising therapeutic strategy to effectively kill cancer cells
and overcome drug resistance. Because the Warburg effect
and hypoxia are frequently seen in human cancers, these
findings may have broad clinical implications. (Cancer Res
2005; 65(2): 613-21)
Over 70 years ago, Warburg (1) observed that cancer cells
frequently exhibit increased glycolysis and depend largely on this
injury’’ and considered this as the most fundamental metabolic
alteration in malignanttransformation or ‘‘the origin of cancer cells’’
(2). During the past several decades, the Warburg effect has been
consistently observed in a wide spectrum of human cancers,
although the underlying biochemical and molecular mechanisms
are extremely complex and remain to be defined. Among the
tumor microenvironment are considered two major factors
contributing to the Warburg effect. However, whether the increase
of glycolytic activity in cancer cells is mainly due to inherent
metabolic alterations or due to anaerobic environment in the tumor
tissues remains controversial (3).
Under physiologic conditions, generation of ATP through
oxidative phosphorylation in the mitochondria is an efficient and
preferred metabolic process, which produces far more ATP
molecules from a given amount of glucose compared with
glycolysis. However, when the ability of cells to generate ATP
through mitochondrial oxidative phosphorylation is compro-
mised, cells are able to adapt alternative metabolic pathways,
such as increasing glycolytic activity, to maintain their energy
supply. Mitochondrial respiratory function can be negatively
affected by multiple factors, including mutations in mitochon-
drial DNA (mtDNA), malfunction of the electron transport chain,
aberrant expression of enzymes involved in energy metabolism,
and insufficient oxygen available in the cellular microenviron-
ment. It is known that mtDNA contains a displacement loop,
and the coding gene sequence for 13 important protein
components of the mitochondrial respiratory complexes without
introns. Mutations in mtDNA are likely to cause alterations of
the encoded protein and compromise the respiratory chain
function. Thus, the frequent mtDNA mutations observed in a
variety of human cancers are thought to contribute to
respiratory malfunction in cancer cells (4–6). The constant
generation of reactive oxygen species within the mitochondria
and the increased free radical stress in cancer cells may cause
further damage to both mtDNA and the electron transport
chain, thus amplifying respiratory malfunctions and dependency
on glycolysis (7).
Hypoxia is another important factor that contributes to the
Warburg effect. The fast growth of cancer cells and rapid expansion
of the tumor mass usually outpace new vascular generation,
resulting in an insufficient blood supply to certain area of the
tumor tissues. Such a hypoxic environment within the tumor mass
limits the availability of oxygen for use in mitochondrial respiration
and synthesis of ATP and forces the cancer cells to up-regulate the
glycolytic pathway as the main route of energy production (8).
The ability of oxygen to regulate glucose metabolism is know as the
Pasteur effect and is mediated through several pathways involving
various kinases (9, 10). In this case, the increased glycolytic activity
in cancer cells is not necessarily due to intrinsic mitochondrial
defects but is induced by the tumor microenvironment through a
series of metabolic adaptation processes, including preferentially
increased expression of enzymes required for glycolysis (11).
Although the underlying mechanisms responsible for the
Warburg effect are rather complex and can be attributed to a
variety of factors, such as mitochondrial defects and hypoxia, the
metabolic consequences seem similar. The compromised ability of
cancer cells to generate ATP through oxidative phosphorylation
Requests for reprints: Peng Huang, Department of Molecular Pathology,
University of Texas M.D. Anderson Cancer Center, Box 89, 1515 Holcombe
Boulevard, Houston, TX 77030. Phone: 713-792-7742; Fax: 713-794-4672; E-mail:
I2005 American Association for Cancer Research.
Cancer Res 2005; 65: (2). January 15, 2005
forces the cells to increase glycolysis to maintain their energy
supply and thus renders cancer cells highly dependent on this
metabolic pathway for survival. As such, it is conceivable that the
metabolic alterations in malignant cells may be exploited to serve
as a biochemical basis to develop therapeutic strategies to target
this metabolic abnormality. One possibility is to inhibit glycolysis
and preferentially kill the cancer cells that are dependent on
glycolytic pathway for ATP generation.
Several agents, including 2-deoxyglucose and arsenate com-
pounds, have long been known to abolish ATP generation
through the glycolytic pathway. For instance, 2-deoxyglucose is
an analogue of glucose and is able to bind and suppress
hexokinase II, an enzyme that catalyzes the initial metabolic step
in the conversion of glucose to glucose-6-phosphate during
glycolysis (12). Inhibition of this rate-limiting step by 2-
deoxyglucose causes a depletion of cellular ATP, leading to
blockage of cell cycle progression and cell death in vitro, and
exhibits antitumor activity in vivo (13, 14). However, the
effectiveness of 2-deoxyglucose is significantly affected by the
presence of its natural counterpart glucose and seems to only
partially reduce the availability of glucose for glycolysis (12). In
contrast, the pentavalent arsenic compounds do not directly
inhibit glycolysis but abolish net ATP generation by causing
arsenolysis in the glyceraldehyde-3-phosphate dehydrogenase
reaction, preventing the generation of 1,3-bisphosphoglycerate.
However, the pentavalent arsenic compounds seem to have
limited specificity due to other toxic effects. More recent studies
showed that 3-bromopyruvate (3-BrPA) is a potent inhibitor of
hexokinase II and effectively inhibits glycolysis (12, 15). This
compound is effective in killing liver cancer cells in the rabbit
VX2 tumor implantation animal tumor model when given by
local infusion (16).
In the present study, we used defined experimental systems to
examine the potential therapeutic implications of the Warburg
effect by testing the sensitivity of cancer cells to glycolytic
inhibition under hypoxic and normoxic conditions and of cancer
cells with genetic mitochondrial defects. We showed that cancer
cells with such respiratory abnormalities were less sensitive to
chemotherapeutic agents commonly used in the clinical
treatment of cancer but could be effectively killed by inhibition
of glycolysis using 3-BrPA. We also observed that inhibition of
glycolysis caused a rapid dephosphorylation of BAD protein at
Ser112, leading to BAX localization to mitochondria and massive
cell death. Importantly, cells that express a multidrug-resistant
(MDR) phenotype still remain sensitive to inhibition of glycolysis.
Because alterations in energy metabolism and increased
dependency on glycolysis are commonly seen in cancer cells,
our findings suggest potential broad therapeutic implications of
using glycolytic inhibitors for cancer treatment.
Materials and Methods
Chemicals and Reagents. 3-BrPA was purchased from Sigma (St. Louis,
MO). The compound was dissolved into water and neutralized with NaOH
immediately before use in cell culture. [3H]2-deoxyglucose and [3H]2-leucine
were purchased from Amersham (Piscataway, NJ). Mouse monoclonal anti-
pBAD(Ser112), rabbit anti-caspase-3, rabbit polyclonal anti-pAkt(Ser473), and
rabbit polyclonal anti-BAD antibodies were purchased from Cell Signaling
Technology, Inc. (Beverly, MA). Rabbit polyclonal anti–cytochrome c was
from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-
BAX was purchased from BD Biosciences (San Diego, CA).
Cells and Cell Culture. Human leukemia cell line HL-60 and human
lymphoma cell line Raji were cultured in RPMI 1640 supplemented with
10% fetal bovine serum at 37jC in a humidified atmosphere with 5% CO2.
The mitochondrial defective clones (U?cells) HL-60/C6F and Raji/C8 were
derived as described previously (17, 18) and maintained in RPMI 1640
supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate,
50 mmol/L uridine, and additional 2.7% glucose. Human colon cancer
HCT116 cells were maintained in McCoy’s 5A medium supplemented with
10% fetal bovine serum. A hypoxic culture condition was created by
incubating cells in a sealed modular incubator chamber (Billups-
Rothenberg, Del Mar, CA) flushed with 5% CO2 and 95% N2. Because
the culture flasks contained ambient oxygen at the beginning of the
experiments, the final oxygen content in the hypoxia chamber wasf0.5%
to 1.0% after achieving air equilibrium.
Cytotoxicity Assays. Cell growth inhibition was determined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide assay using
MTT reagent in 96-well plates. After 72 hours of drug incubation, 50 AL
MTT reagent was added to each well and incubated for an additional
4 hours. The plates were then centrifuged (1,500 ? g, 5 minutes) and the
supernatant was removed. The cell pellets were dissolved in 200 AL DMSO.
Absorbance was determined using a MultiSkan plate reader (LabSystems,
Helsinki, Finland) at a wavelength of 570 nm. Apoptotic or necrotic cell
death was determined by flow cytometric analysis of cells double stained
with Annexin V-FITC and propidium iodide (PI) using an assay kit from
BD PharMingen (San Diego, CA). Briefly, after drug incubation, cells were
collected, washed with cold PBS, and suspended in Annexin V binding
buffer. The cells were stained with Annexin V-FITC for 15 minutes at room
temperature in the dark, washed, and stained with PI. The samples were
analyzed with a FACSCalibur flow cytometer with CellQuest Pro software.
Measurement of Cellular ATP Pool. Cells were incubated with various
concentrations of 3-BrPA for indicated time intervals. Intracellular
nucleotides were extracted from the cells with 0.4 N perchloric acid,
neutralized with concentrated KOH, and quantitated as described
previously (19). Briefly, nucleotides in the cell extracts were analyzed by
HPLC using an anion exchange Partisil-10 SAX column with an elution
flow rate of 1.5 mL/min over a 50-minute buffer gradient [curve 5, buffer A:
0.005 mol/L NH4H2PO4(pH 2.8); buffer B: 0.5 mol/L NH4H2PO4+ 0.25 mol/L
KCl (pH 2.8)]. The eluting flow was continuously monitored by UV
absorption at 262 nm, and the peaks of nucleoside triphosphates were
quantitated by electronic integration with reference to external standards.
The intracellular ATP contents were calculated and normalized by equal
cell number and expressed as percentage of the control cells.
Determination of Glycolytic Activity. The cellular glycolytic activity
under various experimental conditions was evaluated by measuring the
glycolytic index, which was calculated by the formula: glycolytic index =
(L ? G) / O, where L is the cellular lactate generation rate, G is glucose
uptake rate, and O is the oxygen consumption rate. To determine the
cellular lactate production, cells in exponential growth phase were
washed and incubated with fresh medium for the indicated times.
Aliquots of the culture medium were removed for the analysis of lactate
content using an Accutrend lactate analyzer with a linear range of
standard lactate concentrations according to the procedures recommen-
ded by the manufacturer (Roche, Mannheim, Germany). Cellular glucose
uptake was measured by incubating cells in glucose-free RPMI 1640
with 0.2 Ci/mL [3H]2-deoxyglucose (specific activity, 40 Ci/mmol) for
60 minutes. After the cells were washed with ice-cold PBS, the radio-
activity in the cell pellets was quantified by liquid scintillation counting.
To determine cellular oxygen consumption, cells were resuspended in
1 mL fresh warm medium pre-equilibrated with 21% oxygen and placed
in the sealed respiration chamber equipped with a thermostat control,
a microstirring device, and a Clark-type oxygen electrode disc (Oxytherm,
Hansatech Instrument, Cambridge, United Kingdom). The oxygen content
in the cell suspension medium was constantly monitored and oxygen
consumption rate was determined as described previously (17).
Western Blot Analysis. Proteins in whole cell lysates, mitochondrial
fractions, or cytosolic fractions were resolved by electrophoresis on SDS-
PAGE and transferred to nitrocellulose membranes. The membranes were
Cancer Res 2005; 65: (2). January 15, 2005
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Overcome Drug Resistance by Inhibition of Glycolysis
Cancer Res 2005; 65: (2). January 15, 2005