The_bioenergetics_of_cancer_Is_glycolysi

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The bioenergetics of cancer: Is
glycolysis the main ATP supplier in
all tumor cells?
Rafael Moreno-Sánchez,* Sara Rodrı́guez-Enrı́quez, Emma Saavedra, Alvaro Marı́n-Hernández, Juan Carlos Gallardo-Pérez
Instituto Nacional de Cardiologı́a, Departamento de Bioquı́mica, Juan Badiano 1, Tlalpan, México DF, Mexico
Abstract.
The molecular mechanisms by which tumor cells achieve an
enhanced glycolytic flux and, presumably, a decreased
oxidative phosphorylation are analyzed. As the O2
concentration in hypoxic regions of tumors seems not
limiting for oxidative phosphorylation, the role of this
mitochondrial pathway in the ATP supply is re-evaluated.
Drugs that inhibit glycoysis and oxidative phosphorylation
are analyzed for their specificity toward tumor cells and
effect on proliferation. The energy metabolism mechanisms
involved in the use of positron emission tomography are
revised and updated. It is proposed that energy metabolism
may be an alternative therapeutic target for both hypoxic
(glycolytic) and oxidative tumors.
VC 2009 International Union of Biochemistry and Molecular Biology, Inc.
Volume 35, Number 2, March/April 2009, Pages 209–225 �
E-mail: rafael.moreno@cardiologia.org.mx or morenosanchez@hotmail.com
Keywords: glycolysis, oxidative phosphorylation, mitochondrial
function, PET, metabolic control analysis
1. Introduction
All tumor cell types show an altered energy metabolism in
comparison to their tissue of origin (Figs. 1 and 2). The best
characterized energy metabolism modification in tumor cells
is an increased glycolytic capacity even in the presence of a
high O2 concentration ([1], reviewed in ref. 2). Several mech-
anisms for the enhanced glycolysis in tumor cells have been
proposed (Table 1). However, each particular tumor cell line
has its own combination of mechanisms to increase glycoly-
sis and, therefore, generalizations should be avoided.
2. Genetic regulation of glycolysis
In tumor cells, there is an enhanced transcription of genes
of several or all glycolytic pathway enzymes and transport-
ers that is accompanied by an enhanced protein synthesis
[2]. It is usually assumed that these transcriptional changes
lead to increased activity and pathway flux. Unfortunately,
the required biochemical (metabolic, kinetic) experimenta-
tion is considered ‘‘old-fashioned’’ and, only in few of the
molecular biology studies, transporter and enzyme activities
and flux rate have been determined.
In comparison with normal rat hepatocytes, the activity
of all glycolytic enzymes are overexpressed in rat AS-30D
hepatoma by 2–4 times (HPI, ALD, TPI, GAPDH, PGK, PGAM,
ENO, and LDH), 8- to 10-fold for PYK, and 17–300 times for
PFK-1 and HK (Fig. 1) [10]. For human cervix HeLa cells, all
enzymes including HK and PFK-1 are overexpressed by 2–7
times, with the exception of PGAM and LDH which are 2–7
times lower than in rat hepatocytes [10]. However, for this
last case a more rigorous comparison should be made with
normal uterine cervix epithelial cells, the original source,
when data become available. In rat Morris hepatomas, the
activities of HK, PFK and PYK are 5- to 500-fold higher than
in liver [26], whereas in human breast cancer, the activities
of HK, ALD, PYK, and LDH are 3.7–7 times higher than in
normal tissue [27]. In several human carcinomas, overexpres-
sion (Table 1) of GLUT [3], GAPDH, ENO-1, PYK, and LDH
[28,29], correlates with a high glycolysis rate. In contrast, in
human cartilage and bone marrow cancer, glycolysis is high
but only sporadic expression of GAPDH, ENO-1, and PYK is
observed [29]. Unfortunately, the expression pattern of the
whole set of glycolytic enzymes is not always analyzed,
which frequently leads to extend these observations to all
types of cancer.
In cancer cells, the hypoxia inducible factor 1a (HIF-1a)
is a transcriptional factor that upregulates the expression of
specific isoforms of GLUT, HK, PFK-1, PFK-2, ALD, GAPDH,
Abbreviations: ALD, aldolase; AcCoA, acetyl CoA; Cit, citrate; DHAP, dihydroxyacetone
phosphate; ENO, enolase; F6P, fructose-6-phosphate; F1,6BP, fructose-1, 6-
bisphosphate; F2,6BP, fructose-2, 6-bisphosphate; Fum, fumarate; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; aGPDH, a-glycerophosphate
dehydrogenase; GLUT, glucose transporter; G6P, glucose-6-phosphate; Gln, glutamine;
Glut, glutamate; HK, hexokinase; HPI, hexose-6-phosphate isomerase; LDH, lactate
dehydrogenase; OxPhos, oxidative phosphorylation; 2-Oxo, 2-oxoglutarate; PDH,
pyruvate dehydrogenase complex; PET, positron energy transfer; PFK-1,
phosphofructokinase type 1; PGK, phosphoglycerate kinase; PGAM, phosphoglycerate
mutase; PYK, pyruvate kinase; PEP, phosphoenolpyruvate; 1,3 BPG, 1,3-
bisphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3 phosphoglycerate; Pyr,
pyruvate;TPI, triosephosphate isomerase.
*Address for correspondence: Rafael Moreno-Sánchez, Ph.D., Instituto Nacional de
Cardiologı́a, Departamento de Bioquı́mica, Juan Badiano No. 1, Col. Seccion XVI,
Tlalpan, México DF 14080, Mexico. Tel: þ5255 5573 2911 (ext. 1422, 1298);
E-mail: rafael.moreno@cardiologia.org.mx or morenosanchez@hotmail.com.
Received 3 December 2008; accepted 18 January 2009
DOI: 10.1002/biof.31
Published online 11 March 2009 in Wiley InterScience
(www.interscience.wiley.com)
209
https://www.researchgate.net/publication/6502225_Energy_metabolism_in_tumor_cells_FEBS_J?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
https://www.researchgate.net/publication/7139756_Determining_and_understanding_the_control_of_glycolysis_in_fast-growth_tumor_cells_Flux_control_by_an_over-expressed_but_strongly_product-inhibited_hexokinase_FEBS_J?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
https://www.researchgate.net/publication/7139756_Determining_and_understanding_the_control_of_glycolysis_in_fast-growth_tumor_cells_Flux_control_by_an_over-expressed_but_strongly_product-inhibited_hexokinase_FEBS_J?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
https://www.researchgate.net/publication/16614624_Enzyme_activities_in_normal_dysplastic_and_cancerous_breast_tissues?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
https://www.researchgate.net/publication/10925652_Understanding_the_Tumor_Metabolic_Phenotype_in_the_Genomic_Era?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
https://www.researchgate.net/publication/10267165_On_the_Origin_of_Cancer_Cell?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
PGK, PGAM, ENO, PYK, and LDH (Table 1). In consequence,
the glycolytic flux and the levels of intermediaries are
increased. Notwithstanding the O2 level, metastatic tumor
cell lines show high levels of HIF-1a, overexpression of gly-
colytic enzymes, and high glycolysis. Nonmetastatic tumor
cells increase HIF-1a, enzyme overexpression and glycolysis
only under hypoxia [30], although VHL-deficient tumors [31]
and tumors with mutations of succinate dehydrogenase or
fumarate hydratase (see later) have constitutive and aerobic
HIF-1a stabilization.
HIF-1a upregulates PDH kinase 1, which inhibits by
phosphorylation the PDH complex. As a consequence, it has
been proposed that PDH inactivation ocurrs in cancer cells,
which leads to decreased pyruvate oxidation through the
Krebs cycle and decreased mitochondrial oxygen consump-
tion [32,33]. Indeed, overexpression of PDK1 induces an
increase of three times in ATP content during hypoxia in the
absence of HIF-1a, and knockdown of PDK1 increases respi-
ration by 25% during hypoxia [32,33]. This PDK1-mediatedmechanism would impair the mitochondrial function inducing
a shift to a glycolytic metabolism in cancer cells. However,
this fashionable proposal [34,35] makes several assumptions
which have not been experimentally validated. For instance,
full PDH phosphorylation and inactivation are not achieved.
Neither significant diminution of the respiratory and ATP syn-
thesis rates nor, in contrast, a glycolysis enhancement, has
been demonstrated. Furthermore, tumor mitochondria are
able to oxidize several other substrates such as glutamine,
glutamate, fatty acids, and ketone bodies (reviewed in ref.
2) which do not depend on the PDH complex activity.
Building theories on metabolic changes in cancer cells
based only on transcriptional/proteomic profiling, without
making an actual assessment of the function claimed to be
affected, although informative, does not allow for a complete
understanding of the actual in vivo energy metabolism of
cancer cells. Unfortunately, the field is abundant in this type
of generalizations. Combination of newly developed experi-
mental strategies with classical biochemical and metabolic
Fig. 1. Glycolysis in fast-growing cancer cells. In tumor cells, there is over-expression of GLUT1 and GLUT3, HKII
(which may bind to the outer mitochondrial membrane, thus readily accessing to the newly synthesized ATP by
OxPhos), PFK1, PFK-2FB3, and LDH-A among others. Tumor HK is strongly inhibited by its product G6P, whereas PFK-1
activation by F2,6BP overcomes the citrate and ATP inhibition. In some tumors, aGPDH is down-regulated. An
increased flux towards ribose-5-phosphate (and nucleotides) synthesis is documented for several tumor cells. In
addition to be transformed in L-lactate, pyruvate may be oxidized by mitochondria (MIT), generate alanine, and
synthesizes malate in a reaction catalyzed by an over-expressed cytosolic malic enzyme. Other relevant branches of
the glycolytic pathway are also indicated. Abbreviations: Ala, alanine; ANT, adenine nucleotide translocator; Cys,
cysteine; ERI4P, erythrose 4-phosphate; GLU, glucose; Gly, glycine; LAC, lactate; Mal, malate; MCT, monocarboxylate
transporter; ME, malic enzyme; Ser, serine; TGs, triglycerides; PM, plasma membrane. [Color figure can be viewed in
the online issue, which is available at www.interscience.wiley.com.]
210 BioFactors
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https://www.researchgate.net/publication/5314945_Pyruvate_Dehydrogenase_Complex_Activity_Controls_Metabolic_and_Malignant_Phenotype_in_Cancer_Cells?el=1_x_8&enrichId=rgreq-e4392ea4-f659-4597-9cdf-54af3672b40b&enrichSource=Y292ZXJQYWdlOzI0NDM0MzA4O0FTOjEwNDEzMDM2NjQxMDc2NEAxNDAxODM4MDI1NTA2
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strategies will better help to the understanding of the mo-
lecular mechanisms underlying the metabolic and functional
changes observed in cancer cells.
Another transcription factor, the oncoprotein c-Myc
also regulates the expression of GLUT1, HK, HPI, PFK-1,
GAPDH, PGK, ENO, PYK, and LDH, thus, increasing glycolysis
under aerobiosis [36–38]. This transcription factor also pro-
motes mitochondrial biogenesis under nonhypoxic conditions
[39]. The c-Myc oncogene is overexpressed in several tumors
such as human LoVo colorectal tumor [40], glioblastoma
multiforme [41], Burkitt’s lymphoma, prostatic intraepithelial
neoplasias and prostatic carcinoma, melanoma, multiple my-
eloma, breast carcinoma, and myeloid leukemia (reviewed in
ref. 42). In primary breast cancer cells and Burkitt’s lym-
phoma, a tight correlation between glycolytic rates and lev-
els of c-Myc and HIF-1a is observed, suggesting crosstalk
between the transcriptional factors [37,43].
3. Glycolytic isoform expression
and activity
GLUT, HK, and PFK-1 are the main controlling steps of the
glycolytic flux in erythrocytes, hepatocytes, and cardiac and
skeletal muscle cells [44–47]. Changes in the isoform pattern
of HK and PFK-1 expression occur in several tumor cells (Fig.
1) in comparison with normal cells (reviewed in ref. 2). As
described later, it seems that such modifications in these
and other glycolytic steps are part of the mechanisms
involved in the increased glycolytic flux of tumor cells.
Fig. 2. Mitochondrial metabolism in tumor cells. In glycolytic tumors: (1) Cytosolic pyruvate is transported into
mitochondria through a deficient pyruvate transporter; (2) mitochondrial pyruvate is decarboxylated to acetoin which
inhibits the tumor pyruvate dehydrogenase (PDH) complex; (3) truncated Krebs cycle with low aconitase and isocitrate
dehydrogenase activities diminishes NADH pool; (4) increased citrate (Cit) efflux for cholesterol and fatty acids
synthesis is developed; (5) lower expression of the respiratory chain complexes promotes a low H1 electrochemical
gradient; (6) an increase in the inhibitory protein decreases the ATP synthase hydrolytic activity; (7) the close vicinity
of HKII and ANT favors the direct transfer of mitochondrial ATP to HKII for glucose phosphorylation. In oxidative
tumors: (8) Changes in the lipid composition of the inner mitochondrial membrane brings about a lower passive H1
permeability and a higher H1 gradient across the inner mitochondrial membrane; (9) a complete and fully functional
Krebs cycle; (10) malate is transformed to pyruvate by an increased NADP1-dependent intramitochondrial malic
enzyme; (11) glutamine (Gln) is actively oxidized by an over-expressed phosphate-activated glutaminase to form
glutamate (Glut); (12) acetoacetate and b-hydroxibutyrate are actively oxidized to acetyl-CoA (AcCoA) by means of an
increased succinyl-CoA acetoacetyl transferase (SCAAT); (13) a fraction of mitochondrial ATP is exported tothe cytosol
to be used for cellular work. Abbreviations: Cit, citrate; OAA, oxaloacetate; Fum, fumarate; 2-Oxo, 2-oxoglutarate; RCC,
respiratory chain complexes. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
Energy metabolism of cancer cells 211
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3.1. Glucose transporter
In several tumor cells, the predominant overexpressed iso-
form is GLUT1 (Table 1) [3,4]. However, other isoforms, which
are not usually found in the tissue of origin, may also be
overexpressed in some human leukemia, in kidney, lung,
ovarian, and gastric cancers [4,5]. This de novo synthesis of
GLUT might be part of the mechanisms promoting the
increased glycolysis in tumor cells, because the GLUT activity
also increases and significant control of the pathway resides
in this step [10]; on the contrary, if this step does not exert
control of glycolysis in some tumors, its increased level
seems meaningless.
In most studies on GLUT expression in human tumor
cells (Barret, breast, cervical, colorectal, testicular carcino-
mas, and glioblastoma), an enhanced mRNA or protein con-
tent has been detected [4,48–50], but unfortunately GLUT ac-
tivity was not determined. The GLUT1 Vmax is lower than the
Vmax values of the glycolytic enzymes, suggesting that GLUT
is the step with the higher control in glycolysis of rodent
and human cancer cell lines. This finding emphasizes the
relevance of developing new anti-GLUT target drugs, particu-
larly for highly glycolytic-dependent tumors.
3.2. Hexokinase
In tumor cells, HKII is the overexpressed isoform in the ma-
jority of malignant cancer cells (Table 1), except for brain
tumors in which HKI become the predominant isoform
[51,52]. The HKII activity is 300–500 times higher in some
rodent hepatomas than in rat liver cells, whereas in HeLa
cells it is only seven times higher (reviewed in ref. 2, [10]).
In mammalian normal cells, G6P is a potent inhibitor of
HKI, HKII, and HKIII [51]. Also, the enhanced HK activity in
tumor cells is counterbalanced by a strong G6P inhibition
[10]. HK binding to mitochondria was proposed as a mecha-
nism to circumvent the G6P blockade (reviewed in ref. 2).
However, when assayed under near-physiological conditions
of pH (7.0), temperature (37�C) and concentrations of glu-
cose and G6P (>1 mM), the mitochondrial HK exhibits a sim-
ilar sensitivity to G6P as its cytosolic counterpart in
Table 1
Molecular mechanism involved in the enhanced glycolysis of human fast-growing tumors
Human tumor type Ref.
1. Overexpression of selected HIF-1a glycolytic target enzymes and glucose transporters
GLUT1 Bone (HOS), brain (Hs683, H4, A-172), breast (MDA-MB-231, MDA-MB-435, MCF-7,
T47D), cervical (HeLa), choriocarcinoma (BeWo, JEG-3, JAr), colorectal (Caco-2,
WiDr), epidermoid (A431), leukemia (K562, U937, HL60, Jurkat, H9), liver (HepG2),
ovarian (A2780), pancreatic, renal (786-0), skin (HTB 63), squamous (UM-SCC22A,
Scc-25) carcinomas
[3–5]
GLUT3 Breast (MDA-MB-231, T47D, ZR-75), choriocarcinoma (BeWo, JEG-3, JAr), Ischikawa en-
dometrial cells, thyroid (papillary, follicular); lung, ovarian, and gastric carcinomas
(biopsies)
[4,6–8]
HKI Colorectal (SW480, SW620) carcinoma [9]
HKII Ependymoma, astrocitoma, glioma; pancreatic and cholangiocellular carcinoma
(biopsies); cervical (HeLa), hepatocellular (HepG2) and alveolar (A549)
carcinomas; stomach (AGS), and epidermoid (A431) cancers
[10,11–13]
PFK-L HL-60, KG-1, K-562 myeloid leukemia, MOLT-4 leukemia, lymphoma, HeLa and KB
carcinoma, glioma
[11,14]
ALD-A and C, PGK-1,
PGAM-B, ENO-a, LDHA
Colorectal (SW480, SW620) carcinoma, hepatocellular carcinoma, squamous cell lung
and pancreatic carcinomas and lung adenocarcinomas (biopsies)
[15–18]
2. Low number of mitochondria or mitochondrial DNA content per cell
Ovarian cancer, breast cancer, renal cell carcinoma [19,20]
3. Low expression and activity of OxPhos enzymes and transporters
Krebs cycle enzymes Transformed mesenchymal cells, phaeochromocytomas, paragangliomas, liomyomas,
leiomyosarcomas, renal cell, gastric and colon carcinomas, and papillary thyroid
cancer
[2,18,21–24]
Respiratory complexes
and ATP synthase
Brain, mammary gland neoplasias; renal, squamous esophageal, lung, breast and
gastric carcinomas. HeLa carcinoma; mammary tumors (Cf7, C3H); meningioma;
ependymoma; pituitary adenoma; human kidney carcinoma
4. Increased mitochondrial DNA-sensitivity to reactive oxygen species
Breast, colon, stomach, liver, kidney, bladder, head/neck and lung carcinomas;
leukemia, and lymphoma.
25
212 BioFactors
hepatocarcinoma AS-30D [10]. The presence of this G6P reg-
ulatory mechanism of tumor HK supports the proposal of an
essential role for this enzyme in the control of tumor glycol-
ysis, despite its elevated overexpression [10]. This HK regu-
lation mechanism has not been explored in other cancer
cells, although it is expected to be present in those cells
overexpressing HKI or HKII.
Apparent specific inhibition of HK by 3-bromopyruvate
(Fig. 3) was reported [53]. However, its cytotoxic activity
against cancer cells was of low potency (IC90 ¼ 50–100 lM)
[54]; and other glycolytic (GAPDH, PGK) and mitochondrial
(PDH, SDH, glutamate dehydrogenase, pyruvate transporter)
enzymes [53,55] are also sensitive to this compound, indicat-
ing poor specificity. Clotrimazole (Fig. 3) induces HK detach-
ment from mitochondria in B16 melanoma cells, but also
detachment of PFK-1 and ALD from cytoskeleton in mouse
LL/2 Lewis lung cancer and CT-26 colon adenocarcinoma,
leading to diminished G6P, F1,6BP, and ATP levels, and gly-
colytic flux (reviewed in ref. 56). Clotrimazole induces dimi-
nution of cellular proliferation and viability in CT-26 colon
carcinoma, Lewis lung carcinoma and breast cancer MCF-7
(IC50 ¼ 50–90 lM); and diminution of the size and develop-
ment of intracranial gliomas (C6 and 9L), prolonging survival
inrodents [56]. Lonidamine (Fig. 3) also blocks mitochondrial
HK activity (65%) at lower doses (5–75 lM) than those used
to induce tumor mortality in human fibrosarcoma (IC50 ¼
150–500 lM) [56]. Although there are not specific HK inhibi-
tors, some of the known HK inhibitors have been success-
fully used in the cancer treatment by inducing increased
sensitization to other anticancer drugs.
3.3. Phosphofructokinase-1
In different malignant tumors, PFK1-C, -L, or both, prevail
over the M isoform (Table 1). On the other hand, overexpres-
sion of both L and M isoforms are detected in human glio-
mas, whereas in MOLT-4 leukemia, murine ascites and cervix
carcinomas (HeLa, KB) the C isoform predominates [14,57].
PFK-1 from different sources shows variable kinetic
properties depending on the isoform expressed and the tetra-
meric complexes formed [58]. For example, PFK1-C from mu-
rine ascites has a lower sensitivity to PEP, one of the physio-
logical allosteric inhibitors of PFK-1, favoring an increase in
the glycolytic flux [59]. Tumor PFK-1-M is less sensitive to in-
hibition by ATP and citrate, and more sensitive to F2,6BP acti-
vation, than normal PFK-1-C [60,61]. AMP activation of tumor
PFK-1 is also enhanced, but this has not been further eval-
uated probably because AMP seems ineffective to relieving
the citrate and ATP inhibition (reviewed in refs. 2,10).
Fig. 3. Anticancer drugs targeting glycolysis and OxPhos. Abbreviations: 3-BrPyr, 3-bromopyruvate; CTZ, clotrimazole;
Goss, gossypol; IAA, iodoacetic acid; Rote, rotenone; Anti, antimycin; Cas IIgly, casiopeina-II glycine; cyanide-3-
chlorophenylhydrazone, CCCP; Oligo, oligomycin; LCD, lipophilic cationic drugs; a-TOS, a-tocopherylsuccinate; DW,
mitochondrial electrochemical gradient. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
Energy metabolism of cancer cells 213
In several malignant rodent and human cells and
established human tumor cell lines (Table 1), the PFK-1 activ-
ity is 22–56 times higher than in non-tumorigenic cells
[10,14]. In contrast, in human and rodent carcinomas (KB,
thyroid) and brain tumors (gliomas, meningiomas, schwan-
nomas, meduloblastoma), the PFK-1 activity is similar or
even 1.3–2.5 times lower than its non-tumorigenic counter-
part [14,61]. As the glycolytic flux is also enhanced in these
other tumor cells, the decreased PFK-1 activity (i.e., content
of active enzyme) suggests negligible control exerted by this
step (6% in AS-30D glycolysis; [10]). These observations
emphasize that not only changes on mRNA, protein content,
or even enzyme activity, suffice to draw conclusions about
how glycolysis changes in tumor cells. It is also necessary to
analyze how the activity of key (e.g., controlling) pathway
enzymes is regulated through changes in pathway intermedi-
ary metabolites and modulator concentrations.
3.4. Lactate dehydrogenase
A high LDH-A expression level correlates with aggressive
forms of several different tumor types [62]. LDH-A knock-
down in human breast cancer cells and LDH-A dowregulation
in lymphoblastoid cells compromise their ability to prolifer-
ate under hypoxia [63,64]. In addition, the tumorigenicity of
the LDH-A-deficient cells is severely diminished and their
respiration rate is increased by 50–60%; this phenotype is
reversed by complementation with the human ortholog
LDH-A protein [63]. Although it remains to be determined
whether similar knockdown or downregulation of other gly-
colytic steps also induces the same described phenotype,
these studies show that the cancer energy metabolism can
be a suitable therapeutic target.
It was recently shown that lactate can be actively oxi-
dized by aerobic tumor cells [65]. The authors proposed
that, in solid tumors, lactate released from hypoxic cells
may fuel the oxidative metabolism of the aerobic cellular
subpopulation. For this lactate cycling, it is kinetically
required that hypoxic cells express LDH-A and monocarboxy-
late transporter 4 (MCT4), whereas aerobic cells should
express LDH-B and MCT1; however, these aspects remain to
be experimentally tested.
4. Evaluation of mitochondrial
oxidative metabolism in cancer cells
Warburg originally proposed that the driving force of the
enhanced glycolysis in tumor cells was the energy deficiency
caused by an irreversible damage of the mitochondrial func-
tion [1]. There indeed seems to be a diminished oxidative
metabolism in many tumor cell types. On this regard, some
explanations have been advocated (Fig. 2), which, however,
require to be critically reevaluated because several unsub-
stantiated assumptions have been made.
4.1. Lower content of mitochondria per cell
and deficient respiratory chain
In 1978, Pedersen [66] proposed that the respiratory activity
of isolated tumor mitochondria was as efficient as that of
normal mitochondria, but that the diminished oxidative
phosphorylation (OxPhos) observed in tumor cells was the
result of a 20–50% lower content of mitochondria (Fig. 2; Ta-
ble 1). This conclusion extended the original 1956 argument
by Warburg [1] that the high glycolytic rate in tumor cells
was due to a damaged respiratory chain (Fig. 2). Thus, a
lower number of mitochondria per cell suggest that in tumor
cells there are more active degradation mechanisms of mito-
chondria (i.e., mitophagy; [67]) and/or a diminished de novo
mitochondrial synthesis. These events have not been studied
in tumors with a diminished number of mitochondria (Table
1). However, in von-Hippel-Lindau protein-deficient renal car-
cinoma, HIF-1a expression diminishes mitochondrial biogene-
sis and in consequence the cellular oxygen consumption
[68]. In several carcinomas, HIF-1a is overexpressed suggest-
ing that de novo mitochondrial synthesis may also be
affected. Also, mitochondrial DNA diminishes during human
ovarian and breast cancer progression and in highly aggres-
sive tumors (renal cell carcinoma) [19,20,69], implying
mitophagy activation.
However, these results are in contradiction with other
studies showing that the mitochondrial content of Morris 16
and 7800 hepatomas is similar to that of liver cells [66]; and
that bronchial carcinoid and hepatoma mitochondria are
abundant and exhibit normal morphology [66,70], although
the mitochondrial functions were not determined. In Morris
7777 hepatoma similar COX, but lower succinase oxidase
activity to those attained in rat liver mitochondria were
described [71].
In a recent microarray analysis of the genes of the
Krebs cycle enzymes it was found that citrate synthase, aco-
nitase, isocitrate and succinate dehydrogenases, and fuma-
rase, but not 2-oxoglutarate and malate dehydrogenases
were downregulated in tumors while the opposite pattern
was attained in transformed cells (Table 1) [21]. Furthermore,
marked deficiencies have been identified in some mitochon-
drial respiratory chain components from several human and
rodent carcinomas (Table 1; Fig. 2) (reviewed in ref. 2). In
contrast, an increase (2–5 times) in the activity of NADH
cytochrome c reductase has also been determined in the
same brain tumors and no differences in oxidative enzyme
activities with normal cells have been detected for Morris
and Novikoff hepatomas (reviewed in ref. 2) (Fig. 2). In mito-
chondria isolated from some rat hepatomas, the ATP/ADP
translocase activity was lower (5.4 times) than in normal
liver mitochondria. In several human carcinomas, the ATP
synthase was lower than in healthy human liver (Table 1)
(reviewed in ref. 2, [22]).
The tumor suppressor p53 activates TIGAR, a PFK-2
isoform that generates low F2,6BP levels, and hence unfa-
vors glycolysis [72]. Moreover, p53 upregulates the COX sub-
unit II protein expression [73], and down-regulates mRNA
and protein expression of PGAM [74]. In cancer cells, p53 is
214 BioFactors
often mutated, reduced, or deleted [75]. Some authors have
proposed that this p53 inactivation provides a metabolic
shift from respiratory to glycolytic metabolism although, in
accordwith the TIGAR observations, it would rather favor mi-
tochondrial oxidative metabolism [76]. Contradictory effects
by the p53-modulated Erk and PI3K signaling pathways on
the energy metabolism pathways awaits further experimen-
tation to clearly establish the p53 role.
It is pertinent to emphasize that diminution of one
enzyme or transporter does not ensure a diminution in the
pathway flux or metabolite concentration; the altered steps
have to exert significant metabolic control, otherwise the
alteration is silent. Unfortunately, the determination of the
enzyme activities in tumor cells has not been always accom-
panied by measurements of flux rate and steady-state
metabolite concentrations. Likewise, detection of protein lev-
els by western blot analysis or gene transcription by north-
ern blotting or microarray analysis provides information with
little functional meaning unless these measurements are
accompanied by determination of activity and pathway flux.
4.2. Increased mitochondrial DNA (mtDNA)
mutations and sensitivity to oxidative stress
Thirteen protein components of the respiratory chain and
ATP synthase are encoded by mtDNA. As mtDNA lacks histo-
nes and there is lower capacity for DNA repair in normal mi-
tochondria in comparison with the nuclear DNA (reviewed in
ref. 2), it is thought that the enhanced oxidative stress in tu-
mor cells induced by certain drugs may modify the normal
transcription of mitochondrial genes (Table 1). Likewise, the
higher frequency of somatic mtDNA mutations found in
breast (61–74% of patients), bladder (51–100%), head and
neck (51%), gastric (48%), colorectal (44–70%), brain (40%),
hepatocellular (39–68%), thyroid (37–52%), lung (33–51%),
ovarian (20%), prostate (11–19%), and other human cancers,
might also presumably contribute to mitochondrial dysfunc-
tion, although only a few mutations are known to have path-
ological significance (reviewed in refs. 2,22). Surprisingly,
72% of the somatic mtDNA mutations found in cancer cells
are also detected in the general healthy population [77] sug-
gesting that most of these mutations are, simply, normal
DNA duplication errors with no functional consequences.
Germ-line mutations or downregulation of succinate
dehydrogenase (SDH) and fumarate hydratase (FH) are asso-
ciated with development of a variety of rather benign forms
of phaeochromocytomas, paragangliomas, liomyomas, leio-
myosarcomas, renal cell, gastric and colon carcinomas, and
papillary thyroid cancer. These mutations inhibit SDH and FH
activities, inducing succinate and fumarate accumulation
and, hence, prolyl-hydroxylases inhibition, which in turn
prompts an increased HIF-1a level (Table 1) (reviewed in ref.
78). These observations certainly suggest a diminished mito-
chondrial function, but they do not demonstrate a complete
blockade of the Krebs cycle flux or negligible respiratory or
OxPhos rates. All these functions must be experimentally
determined.
4.3. Lower Pyr oxidation; truncated Krebs cycle
and lower reducing equivalents transfer
These proposals were previously reviewed and analyzed [2].
No new information on this regard has been published, but
recently it was demonstrated that prostate PC3 cancer accu-
mulates high Zn2þ levels in mitochondria and cytosol. Zinc
may inhibit aconitase, ‘‘truncating’’ the Krebs cycle flux (Fig.
2) and diminishing the reducing equivalent pool and cellular
oxygen consumption [79]; however, by adding glutamine to
the PC3 culture, the intracellular ATP level is restored, over-
coming the aconitase inhibition by Zn2þ [80]. Therefore, the
available data on the proposed deficiency of cancer cells in
transfer equivalents from cytosol to mitochondria, acetoin
inhibition and truncated Krebs cycle do not unequivocally
establish a lower mitochondrial function in tumor cells.
Furthermore, significantly marked differences should
be found in the mechanism proposed for decreasing OxPhos
in several tumors, not only in a selected one or two tumor
types. Because of the genetic heterogeneity among the dif-
ferent tumor cell types, it should not be expected a similar
degree of modification in the mechanism proposed, but at
least it should be observed in several tumor cell lines.
In conclusion, there are tumor cell lines that certainly
exhibit a decreased mitochondrial function, but that observa-
tion does not seem to apply to all tumor cell types. There-
fore, due to the genetic heterogeneity of tumor cells,
OxPhos capacity should be experimentally evaluated for
each particular tumor, to assess whether the enhanced gly-
colysis is indeed accompanied by a significant depressed
mitochondrial function. This last statement widely spread in
the field (reviewed in refs. 2,76,81), has been taking as an
established fact for tumor cell metabolism for many years,
but because of the absence of hard experimental data it has
rather become the metabolic central dogma of cancer.
5. Is oxidative phosphorylation
damaged in most cancer cells?
According to the Warburg hypothesis, the main oxidizable
substrates in cancer cells should be hexoses such as glu-
cose, fructose, and mannose; in turn, substrates oxidized by
mitochondria should not be metabolized by cancer cells. In
consequence, the main and only pathway providing ATP in
cancer cells should be glycolysis; thus, negligible respiratory
rates and insensitivity to mitochondrial inhibitors should
also be expected. These assumptions are critically analyzed
in the following sections.
5.1. Substrate utilization
It is well-established that glucose is one of the carbon sour-
ces preferentially consumed by fast-growth tumor cells (Fig.
1), together with glutamine, an exclusive mitochondrial oxi-
dizable substrate (Fig. 2) (reviewed in ref. 2). In glycolytic
tumors, an increased oxidation of glutamine is also
observed. For the fast growing human cervix (HeLa), breast
(MCF-7), prostate (PC3), colorectal (HT29) carcinomas, and
Energy metabolism of cancer cells 215
slow-growing adenoma-derived cell line AA/C1, the ATP
demand is supported by the aerobic oxidation of both glu-
cose and glutamine [80,82–84], which indicates that glycoly-
sis and OxPhos are both essential for ATP supply in these
human tumor cells.
A fully functional Krebs cycle able to supply NADH for
OxPhos operates in AS-30D hepatoma (reviewed in ref. 2),
and in PC3 carcinoma, in which aconitase activity is restored
to the same level than that found in nontumorigenic tissues,
when Zn2þ is removed [85]. In Caco-2 and Hep-G2 carcino-
mas, several di and tricarboxilic transporters are upregulated
to supply, continuosly, mitochondrial substrates for ATP pro-
duction (Fig. 2) [86,87]. Mitochondrial tumor NADPþ-depend-
ent malic enzyme is 10–20 times more active in tumor cells
than in its origin tissue counterpart, thus, supplying NADH
for the respiratory chain and pyruvate for LDH (Fig. 2). More-
over, other oxidative pathways such as those for glutamine,
glutamate, and ketone bodies are also highly active in tumor
cells (Fig. 2) (reviewed in ref. 2). Cytosolic glutamine is
transported faster (4–10 times) into tumor mitochondria and
further transformed to glutamate by a strongly Pi-activated
glutaminase with also higher activity (10–20 times versus
normal liver mitochondria) (reviewed in ref. 2) (Fig. 2). Gluta-
mine oxidation requires several Kreb cycle enzymes starting
at 2-OGDH. Glutamine oxidation is the major anaplerotic
pathway in gliomas and it is stimulated by estrogen in
breast cancer; in addition, some c-Myc-transformed cells
have an absolute requirement for glutamine in order to
maintain viability through the continuous replenishment of
Krebs cycle intermediates [88]. These observations suggest
that glutamine oxidation is essential for tumor cell
metabolism.
The succinyl-CoA acetoacetyl transferase (SCAAT),
enzyme that initiates ketone bodies oxidation, is 40-fold
more active in AS-30D cells than in hepatocytes (reviewed in
ref. 2) (Fig. 2). On the contrary, many brain tumors have
lower SCAAT activity than normal neuronsand glia and,
hence, are unable to metabolize ketone bodies. Then, as
fatty acids do not pass the blood brain barrier, brain tumors
seem to depend only on glucose, glycolysis and perhaps
OxPhos for ATP supply (reviewed in ref. 2). In addition, dur-
ing tumor cell proliferation, growth-factor signaling inhibits
fatty acid b-oxidation, and stimulates lipid synthesis [88].
These accumulating evidences suggest an active mitochon-
drial-dependent energy metabolism and a fully functional
Krebs cycle in some tumor cell lines.
5.2. Oxygen concentration in tumors
OxPhos is apparently limited by the low O2 availability inside
the tumor [89]. Indeed, the prevalence of hypoxic areas (pO2
< 10 mm Hg; 14 lM; >50% of total tissue heterogeneously
distributed) is characteristic of a wide variety of locally
advanced human solid tumors [90]. In these hypoxic regions
of human tumors as well as in the center of glioma and car-
cinoma multicellular spheroids (a model that simulates the
avascular stages of solid tumors), the determined O2 con-
centration ranges from 1 to 61 lM [89,91,92], which resem-
bles the range of values usually found in several normal tis-
sues with normal blood irrigation (femoral muscle, mammary
gland tissue) [93] (Table 2).
However, OxPhos can only be compromised at O2 con-
centration values lower than 1 lM because KmO2 of COX is
0.1–0.8 lM in different biological systems including ascites
tumor cells [102–105]. In consequence, a saturating O2 con-
centration for COX, and OxPhos, would be >4–8 lM (i.e., a
substrate concentration of 10 times its Km value). Therefore,
tumor mitochondrial metabolism would not be affected, in
principle, by the hypoxia level found in tumors.
On the other hand, prolonged exposure (weeks or
months) to such hypoxic microenvironment indeed alters the
expression of normal and tumor mitochondrial enzymes.
Although the molecular mechanisms are still not clear, it has
been proposed that HIF-1a or p53 are involved [73]. In fact,
it has been described that a lengthy exposure (16–72 h) to
hypoxia (1–20 lM O2) in HepG2 carcinoma and PC12 pheo-
chromocytoma diminishes the mRNA level of mitochondrial
enzymes (NADH dehydrogenase, COX, ATPase, glutaminase)
Table 2
Oxygen concentration in human tumors
Tumor type Organism O2 (lM) Ref.
Head and neck squamous carcinoma Human 1 [94]
Mammary adenocarcinoma Rat 3.5–42 [95,96]
Cervix carcinoma Rat 7–29 [97]
Mammary squamous carcinoma, lung and medulla Human <7 [98]
Prostatic carcinoma Rat 2.0–9.1a [92,99]
11–112b
Dunning Prostatic carcinoma Rat 14–61 [100,101]
Fibrosarcoma Rat 15 [101]
a Oxygen concentration determined in the center of solid tumor.
b Oxygen concentration determined in the periphery of solid tumor.
For other tumors, the oxygen concentration reported did not specify region.
216 BioFactors
[106,107]. Unfortunately in these studies, the enzyme activ-
ities and changes in hypoxia-associated transcription factors
(HIF-1a and p53) were not evaluated.
5.3. Contribution of glycolysis and OxPhos to
ATP supply
It is intriguing that despite the accelerated glycolysis in sev-
eral tumor cells, its total contribution to the cellular ATP
supply only reaches 10% (reviewed in ref. 2) (Table 3). In
marked contrast, in other tumor cell lines (Table 3), glycoly-
sis indeed supports 50–70% of the ATP demand, a contribu-
tion value also estimated by Warburg [1]. Moreover, the con-
tribution of glycolysis and OxPhos to the ATP demand during
the proliferative phase was similar in tumor cells with defi-
cient oxidative system (Ehrlich hepatocarcinoma) (Table 3)
[108].
To emphasize that OxPhos is a more efficient pathway
than glycolysis to generate ATP, it is usually described that
‘‘OxPhos generates 36 or 38 ATP molecules per glucose,
whereas glycolysis only generates 2 ATP molecules per glu-
cose.’’ However, not all glucose, metabolized through the
glycolytic pathway, actually generates lactate molecules,
because a fraction of glucose may be drained to the branch-
ing reactions. Thus, it is more accurate to determine the rate
of lactate (þpyruvate) production as the more adequate pa-
rameter of glycolytic ATP synthesis (1 lactate ¼ 1 ATP), and
correcting for lactate formed from other sources (i.e., gluta-
minolysis) with glycolytic inhibitors such as 2-deoxyglucose
(2DOG). The rate of O2 consumption, sensitive to oligomycin,
a specific ATP synthase inhibitor (Fig. 3), is the most
adequate measurement of OxPhos, which is converted to
rate of ATP synthesis by using the experimentally deter-
mined P/O ratio of 2.5 for NADH and 1.5 for FADH2 in hepa-
toma isolated mitochondria [110]. Thus, 30–32 ATP molecules
generated per glucose fully oxidized is a more accurate
value for OxPhos.
There are few reports where metabolic control analysis
(MCA) of the tumor energy metabolism has been experimen-
tally carried out. MCA allows the quantification of the degree
of control that an enzyme, pathway segment or entire path-
way has on a particular pathway flux, metabolite concentra-
tion, or cellular process [111,112]. By applying elasticity anal-
ysis (an experimental approach of MCA) to glycolysis in AS-
30D tumor cells, it was found that the main flux-control
(71%) resided in the first two pathway steps, that is, GLUT
and HK [10]. The rest of the control (29%) was localized in
the ALD-LDH segment, with a negligible contribution by PFK-
1 (<6 %). It was shown that, despite a remarkable overex-
pression, tumor HK was strongly inhibited by its product
G6P, thus, still being limiting for the pathway flux. On the
other hand, PFK-1 was moderately overexpressed, but the
tumor isoform was highly activated by F2,6BP, thus, surpass-
ing the citrate and ATP inhibition. These findings provided a
mechanistic explanation for the respective high and low flux
control exerted by HK and PFK-1 (see Fig. 1). This study also
showed that a massive over-expression of glycolytic enzymes
does not lead to uncontrolled flux, but rather strict regula-
tory mechanisms are preserved in tumor cells.
Kinetic modeling of glycolysis in AS-30D and HeLa tu-
mor cells has revealed that indeed GLUT and HK are the
main flux-controlling steps in both tumors; and that HPI,
inhibited by erythrose 4-phosphate and F1,6BP, modulates
the supply of G6P towards pentose phosphate and glycogen
synthesis pathways (Fig. 1) (A. Marı́n-Hernández, R. Moreno-
Sánchez, and E. Saavedra, manuscript in preparation).
Flux control analysis of the ATP synthesis in AS-30D
hepatoma cells showed that OxPhos controlled by 66%
whereas the ATP-consuming processes (protein and nucleic
acid synthesis; ion ATPases) controlled by 34%. The main
controlling step within OxPhos was the respiratory chain site
Table 3
Energy metabolism in malignant tumor cells
Tumor Organism Prevailing energy pathway % ATP contribution
Glioma C6 Rat OxPhos 80
Oligodendroglioma, meningioma, medulloblastoma Human Glycolysis 60
Glioblastoma multiforme, Astrocytoma C6 Human, Rat Glycolysis and OxPhos 50
Transformed brain Hamster OxPhos 71
Colon sarcoma Human OxPhos 70
Novikoff hepatoma Rat Glycolysis 75
Ehrlich Lettré, Ehrlich, Walker-256, Morris 3683 and Dunings LC18
hepatomas; ascites mouse cancer; sarcoma 27
Rat, mouse Glycolysis and OxPhos 50
Reuber H-35, Morris (7793,7795, 7800, 5123), and AS-30D hepatomas Rat OxPhos 97
Lung carcinoma Human OxPhos 95
Breast cancer Human OxPhos 95
MCF7 breast carcinoma Human OxPhos 80
Melanoma Human OxPhos 97
HeLa cervix, ovarian and uterus carcinomas Human OxPhos 90
Modified from [2].
Energy metabolism of cancer cells 217
1 [113]. Control analysis of the tumor energy metabolism, ei-
ther by establishing the main sites of control in glycolysis
and OxPhos and/or by assessing the predominant energy
pathway, may provide a more rational and quantitive
approach for the identification and design of more specific
therapeutic strategies whether the tumor cells are either of
the glycolytic or the oxidative type. Therefore, it would be
highly informative to carry out this kind of analysis with
manyother different cancer models.
5.4. Mitochondrial inhibitors and respiration rates
in tumor cells
Some human and rodent gliomas exhibit high or moderate
susceptibility to highly specific respiratory inhibitors; in
addition, gliomas with glycolytic phenotype actively oxidize
pyruvate and glutamine under low glucose [114]. These
observations indicate the presence of fully functional mito-
chondria and dependency on OxPhos. Oligomycin (Fig. 3) at
low doses (0.06–0.7 lM), which do not affect normal cells,
arrests the cell cycle progression from G1 to S phase in
human promyelocytic leukemia (HL-60), and Jurkat T cells,
due to a severe diminution of mitochondrial ATP production
(reviewed in ref. 56). At 3–6 lM oligomycin, more than 50%
of HL-60 cells are arrested in G2/M phase; however, this
drug concentration may also affect normal cells. The respira-
tory chain site 1 inhibitor rotenone (0.1–1 lM) (Fig. 3) arrests
the cell cycle at G2/M promoting a strong inhibition (50–
90%) of cell proliferation in human lymphoma WP and 134 B
osteosarcoma (reviewed in ref. 56). Rotenone inhibits respi-
ratory chain site 1 (Fig. 3) in normal cells, but this drug
might still be advantageously used for tumor cells as long
as site 1 exerts a significantly higher flux control of OxPhos
in tumor cells than in normal cells. Antimycin (IC50 ¼ 50 lM)
(Fig. 3) diminishes proliferation by increasing ROS and hence
apoptosis, loss of mitochondrial membrane potential, Bcl-2
down-regulation and Bax up-regulation in HeLa cells, by
increasing ROS [115]. Classical uncouplers of OxPhos (Fig. 3)
such as CCCP inhibit the growth of several malignant mam-
mary carcinomas (EMT-6, M6H-U1). The action of this iono-
phore consists in the acidification of the mitochondrial ma-
trix, and collapse of the mitochondrial electrochemical Hþ
gradient, which results in significant decrease of cellular ATP
levels [116].
As originally claimed by Weinhouse [117], a wide vari-
ety of tumor cell lines exhibit elevated rates of respiration
(reviewed in refs. 2,56,109); whether this activity is fully
associated with OxPhos remains to be determined (i.e., sen-
sitivity to oligomycin). In AS-30D and HeLa monolayer cell
cultures [83] and HeLa microspheroids [118], the rate of res-
piration is 85–90% sensitive to oligomycin, indicating that
the remaining 10–15% of the cellular O2 consumption was
not associated to OxPhos. In tumor cells, the non-mitochon-
drial oxygen-consuming enzymes, heme oxygenase and cyto-
chrome P450 may also be overexpressed [119]; thus, the oxy-
gen consumption values determined in the absence of
oligomycin overestimate the rate of OxPhos.
Therefore, the generalized statement that glycolysis
predominates over OxPhos for ATP supply in tumor cells
(reviewed in refs. 2,76,81) should be re-evaluated and exper-
imentally determined for each particular type of tumor cell.
An energy deficiency caused by a deteriorated OxPhos might
indeed be the driving force behind the enhanced glycolysis
in hypoxic tumors (in a process mediated perhaps by HIF-1a
or p53), but a mitochondrial impairment does not seem to
apply in nonhypoxic, oxidative tumors. Thus, the main ther-
modynamic reason for increasing glycolysis in tumor cells
(associated with either a damaged or an unaltered OxPhos)
might rather be an energy deficiency induced by highly ATP-
demanding processes such as an accelerated cellular prolif-
eration and exacerbated nucleic acid, protein, and choles-
terol synthesis.
6. Energy metabolism in tumor cells
as therapeutic target
A proportional relationship between the rate of cellular pro-
liferation and the rate of ATP supply has been established
for human (erythroleukemia K 562) and rodent (Ehrlich
mouse carcinoma) fast-growth tumor cells [120,121]. How-
ever, there is some discrepancy regarding the correlation
between the degree of malignancy and the rate of ATP syn-
thesis from glycolysis or OxPhos. Some authors have pro-
posed that the glycolytic activity correlates with the degree
of tumor malignancy, so that glycolysis is faster and OxPhos
is slower in highly de-differentiated and fast-growing tumors
than in slow-growing tumors or normal cells [66,122]. In fact,
a high level of lactate has been proposed as a predictor of
malignancy [123]. High serum LDH levels predict a poor out-
come in pancreatic, lung, renal and testicular carcinomas,
osteosarcoma, and melanoma. For human lung, colorectal,
ovarian, larynx, breast, and hepatocellular carcinomas, high
levels of GLUT1 or GLUT3, ALD-B, and HKII have been used
as indicators of unfavorable prognosis [124,125]. There is a
direct correlation between the tumor progression and the HK
[10,52] and PFK-1 [10,59] activities. A high intracellular LDH-A
(LDH-5) level correlates with a high serum level and with
aggressive forms of several tumor types [62]. However, these
correlations rely largely on nuclear LDH-5 expression. As
localization of this glycolytic enzyme in the nucleus is
atypical, the functional relevance of this expression pattern
requires further experimentation [90].
Accordingly, it has been postulated that tumor cells
that exhibit deficiencies in their oxidative capacity are more
malignant than those that have an active OxPhos [126]. Fur-
thermore, it seems that mtDNA mutations confer resistance
to anticancer drugs. For instance, mutant mtDNA cybrids are
more resistance to apoptosis and cisplatin than wild-type
cells, and rho cells are extremely resistant to radiation, as
well as to adriamycin, cisplatin, and etoposide [22,127].
Prostate cancer mutant (T8993G) cybrids (with an ATPase
mutation) generated seven times larger tumors in nude mice
than wild-type cybrids; this mutation causes impaired mito-
chondrial ATPase synthesis [22].
218 BioFactors
6.1. Energy metabolism anticancer drugs
Certainly, drug efficacy, delivery, and side effects are prob-
lems to be solved in developing new chemotherapies. In
solid tumors, delivery to a hypoxic region may be difficult
whether the drug does not easily permeate through the
different cellular layers or whether drug expelling systems
(i.e., P-glycoprotein, an organic anion ATPase that efficiently
expels xenobiotics) are overexpressed [128]. To overcome
these uncertainties, further searching and design of new
specific drugs (i.e., molecules with inhibition constants in, at
least, the submicromolar range and with superior membrane
permeability) is required.
Another problem associated with chemotherapy is that
in many tumor types there is either inherent or acquired re-
sistance to antineoplastic drugs. A significant advance has
been the elucidation of the metabolic changes developed by
the tumor cells for drug resistance. The drug-resistant cells
decrease the mitochondrial Hþ electrochemical gradient by
overexpressing the uncoupler protein 2 (UCP-2), which acts
as a mitochondrial Hþ channel, collapsing the Hþ gradient
generated by the respiratory chain. These cells also increase
the utilization of alternative oxidizable substrates (fatty
acids) [129]. Furthermore, drug-resistant tumor cells may
also overexpress the P-glycoprotein to expel drugs such as
doxorubicin, paclitaxel, vinblastine, and epirubicin [35].
Therefore, more selective targeting is required for the treat-
ment of cancer.
The observations of the previous section suggest that
the blocking of glycolysis should arrest tumor progression.
Indeed, inhibition of tumor glycolysis with 2DOG, 3-bromo-
pyruvate, or lonidamide increases the sensitivity towards
anticancer drugs commonly used in chemo-therapy such as
cisplatin, 4-hydroperoxycyclophosphamide, melphalan, car-
mustine, 1-b-D-arabinofuranosylcytocine, vincristine, taxol,
adriamycin, etoposide, and camptothecin (reviewed in ref.
56). However, glycolytic inhibitors display low efficacy in
arresting tumor growth, when used alone, which is also usu-
ally accompanied by severe side effects in the host
(reviewed in ref. 56).
Muller et al. [108] originally proposed that a biochemi-
cal strategy to efficiently suppress the acceleratedEhrlich
mouse hepatoma proliferation was the simultaneous block-
ade of both ATP-generating pathways. It appears difficult to
target the energy metabolism of tumors as also the host
cells depend on the same essential pathways for ATP supply.
However, by identifying the most significant differences in
the energy metabolism between tumor cells versus host
cells, it might be possible to detect suitable antineoplastic
targets (Figs. 1–3). It is worth noting that drugs used for in-
hibiting OxPhos in cancer cells are not the classical specific
and potent inhibitors such as rotenone, antimycin, oligomy-
cin, and carboxyatractyloside, because they block the path-
way in both malignant and healthy cells. Instead, these
drugs are designed to be mitochondrially targeted only for
cancer cells in virtue of their delocalized positive charge.
Interestingly, the lipophilic cationic drugs rhodamines 6G
and 123, and casiopeina-IIgly at low micromolar concentra-
tions drastically abolish OxPhos and cell proliferation of
Walker and AS-30D hepatomas, whereas glycolytic drugs
such as gossypol, arsenite, and iodoacetate are ineffective
(reviewed in ref. 56).
Lipophilic cationic drugs (LCD) (Fig. 3) are accumulated
in cytosol and mitochondria of tumor and normal cells
because the elevated electrical potential gradient (Dw, nega-
tive inside) generated across both the plasma membrane
and inner mitochondrial membrane (reviewed in ref. 56).
However, it is documented that mitochondria from human
colon CX-1 tumor; cells and mitochondria from MCF-7 breast
adenocarcinoma; neu-, v-Va-ras-, b-catenin, and c-myc-initi-
ated mouse tumor cell lines; and A549 (nonsmall cell lung
cancer), and glioblastoma M059K cells, develop a Dw of a
higher magnitude (15–50 mV) than that of normal cells (epi-
thelial cells from spleen, breast, kidney, small airway epithe-
lial cells, fibroblasts, and pulmonary artery smooth muscle
cells) (reviewed in refs. 35,56). Therefore, a significant
higher lipophilic cation drug accumulation is achieved in tu-
mor mitochondria. However, further studies on side effects
of the lipophilic cations are required as the rhodocyanine
MKT-077 showed severe nephrotoxicity in phase I trial and
was discontinued [130].
The reasons for the higher Dw in tumor mitochondria
might be related to the higher content of cardiolipin
(increasing the density of membrane negative charges) and
cholesterol (which may decrease the Hþ and other ions pas-
sive diffusion) in the plasma and inner mitochondrial mem-
branes [66,131]. In rho osteosarcoma cells, 50 times more
rhodamine 123 is required to inhibit proliferation [132].
Recently, it was determined that the vitamin E ana-
logue a-tocopheryl succinate (a-TOS) may specifically target
(and freely diffuse) tumor cells by virtue of the more acidic
extra and intracellular pH. Once inside the cell, a-TOS indu-
ces apoptosis in mesothelioma and other tumors, without
affecting normal cells, by apparently promoting an
increased oxidative stress through the selective inhibition
of SDH (Fig. 3) [133].
We recently reported that the growth of HeLa cells in
multicellular spheroids involves changes in their energy
metabolism in comparison with their counterpart in mono-
layer culture [118]. In the spheroid initial formation, mito-
chondria support more than 80% of the cellular ATP sup-
ply. However, at later spheroid stages, mitochondrial
metabolism fails and glycolysis becomes predominant. In
this mature stage, tumor spheroids become highly sensi-
tive to glycolytic inhibitors (2DOG, gossypol) [118]. Inter-
estingly, casiopeina II-gly and rhodamine 123, laherradurin
(an acetogenin extracted from Annonaceae plants that
potently inhibits respiratory chain site 1) [118], and a-TOS
block the initial cell cluster formation and growth of HeLa
spheroids by directly inhibiting OxPhos (S. Rodrı́guez-Enrı́-
quez, unpublished data). In multispheroids of human
U118MG colon cancer, changing the prevalent metabolism
from glycolytic to oxidative, by adding oxamate a LDH in-
hibitor provokes an increase in the tumor sensitivity to
radiation [91].
Energy metabolism of cancer cells 219
7. Tumor cell metabolism and
positron emission tomography (PET)
In recent years, PET combined with computed tomography
(CT) has been applied for diagnosis, monitoring and treat-
ment of cancer. PET has mainly used 18fluoro-deoxyglucose
(FDG) as tracer under the assumption that tumors have a
higher glycolytic capacity than normal cells (reviewed in ref.
2). FDG is transported into the cells by the glucose trans-
porter, and then phosphorylated to FDG-6-phosphate (FDG-6-
P) by HK [134]. FDG-6-P cannot be isomerized by HPI due to
the C-2 fluor and it is slowly expelled, dephosphorylated, by
the action of glucose-6-phosphatase. In consequence, it is
trapped and accumulated in the neoplastic (and in some
nontumorigenic) cells. Thus, by using FDG/PET it has been
found that the vast majority of metastatic tumors (>90%)
are highly glycolytic, in other words, there is a strict correla-
tion between glycolytic activity and FDG uptake; it has also
allowed the accurate and earlier detection (>90%) of differ-
ent types of cancer [135].
7.1. Limitations of the FDG/PET scan
(A) The accuracy of the FDG/PET scan depends on the tumor
stage. The FDG/PET may identify pancreatic cancer in
advance stage with high sensibility (>85%) and moderate
specifity (>53%), whereas in patients with early tumor
stages (small tumor size) the sensibility decreases [136].
(B) False positives of FDG/PET scan have been frequently
reported in infectious diseases, inflammatory cells,
fibrotic lesions and other diseases [137,138]. Moreover,
tumors with low glycolytic activity [137], carcinoid tumors,
and bronchioalveolar lung carcinoma [139], as well as
aggressive B-cell non-Hodgkin’s lymphoma treated with
rituximab [140], reveal false negative results on FDG/PET
scan. Bladder and prostate tumors are FDG-avid cancers
but PET scan does not provide a clear diagnosis of these
neoplasias [141]. In hepatocellular carcinoma, highly dif-
ferentiated cancer cells are FDG-negative and poorly dif-
ferentiated cancer cells are FDG-positive [142].
(C) OxPhos dependent tumors are not identified. In thyroid
carcinoma, FDG is not concentrated, despite administra-
tion of high and repetitive doses [143], suggesting that
the relevant energy pathway in thyroid carcinoma is not
glycolysis. Although FDG-PET serves an important role in
the follow-up of patients with thyroid cancer, accurate
localization of FDG-positive tissues is often difficult. The
normal brain cells have a high glucose uptake, whereas
the uptake of FDG in most brain tumors is similar or
lower than in the normal tissue.
(D) Variable sensitivity in hypoxic regions. Accumulation of
FDG in malignant tumors is related to regional hypoxia,
but a poor correlation between hypoxia and FDG uptake
may appear. Therefore, FDG/PET cannot reliably differen-
tiate hypoxic (i.e., glycolytic) from normoxic tumors in fre-
quently hypoxic (head and neck cancer and glioblastoma
multiforme) and less frequently hypoxic tumors (breast
cancers) [144]. For rectal carcinoma, there is not signifi-
cant difference in FDG uptake between patients with
hypoxic tumors and those with normoxic tumors [145].
Knowing that not all tumors are glycolytic, but that
some of them have a predominant oxidative metabolism or at
least they have low glucose uptake (mediastinal lymph node,
metastases of lung cancer, and prostate cancer) (see Table 3),
it seems as a reasonable alternative to applying PET-CT with
tracers directed to mitochondria. Copper (II)-pyruvaldehyde-
bis (N4-methylthiosemicarbazone) (Cu-PTSM) was used for
monitoring electron transport chain in normal brain mitochon-
dria and Ehrlich ascites [146], and (60)Cu-diacetyl-bis (N(4)-
methylthiosemicarbazone) ((60)Cu-ATSM) for detecting rectal
carcinoma and uterine cervix cancer. Moreover, in prostate
and hepatocellular carcinoma, the use of 11C-acetate/PET has
been successfully validated byboth clinical and experimental
studies [147], probably because the predominant energy
pathway in these cancers is not glycolysis, but fatty acid oxi-
dation, and hence, OxPhos [148]; 11C- and 18F-labeled choline
analogues are also successful tracers for prostate cancer, he-
patocellular carcinoma, and primary brain tumors.
The use of mitochondrial tracers in PET analysis may en-
counter the same difficulties described for FDG, regarding the
detection of false negatives in low oxidative tumors, and false
positives in normal tissues with high oxidative activity. The
question of whether mitochondrial PET tracers may be more
specific for tumors than for normal, healthy cells will be
answered when more data become available, but it is worth
noting that mitochondria of oxidative tumors develop a higher
electrical membrane potential than mitochondria of normal tis-
sue, thus favoring the accumulation of lipophilic, cationic
drugs and facilitating the detection of oxidative tumors.
8. Conclusions
All tumor cell types show an enhanced glycolytic flux; how-
ever, not all of them have a diminished mitochondrial meta-
bolic capacity. Therefore, not all tumor cell types depend
exclusively on glycolysis for ATP supply; some of them may
equally or predominantly rely on OxPhos (Table 3). We think
that both energy pathways are not mutually excluding each
other in cancer cells. In consequence, the driving force for
the enhanced glycolysis in tumor cells cannot be an energy
deficiency induced only by a damaged OxPhos. The acceler-
ated cellular proliferation may also impose an energy defi-
ciency (as well as a higher demand for glycolytic and Krebs
cycle biosynthetic intermediaries), which can only be covered
by an increased glycolysis, and an unperturbed OxPhos.
Certainly, there is genetic, biochemical, and morpho-
logical heterogeneity in cancer cells, but all of them do
depend only on glycolysis and OxPhos for ATP supply. The
enhanced tumor glycolysis results from an over-expression
of most of the enzymes and transporters of the pathway,
with HK and PFK-1 being markedly overexpressed in different
isoforms (with different kinetic properties to that of the tis-
sue of origin). At the genetic level, the enhanced glycolysis
220 BioFactors
is primarily modulated by HIF-1a, although other transcrip-
tion factors such as c-Myc and p53 may also been involved.
These last two factors might also affect the mitochondrial
function.
In tumor types in which glycolysis is not the predomi-
nant energy pathway, that is, in oxidative tumors (Fig. 2; Ta-
ble 3), the application of mitochondria-directed drugs such
as 11C-acetate or glutamine or 11C-rhodamines or casiopeinas
in PET-CT analysis may be considered as alternative detec-
tion and therapeutic strategies. These observations empha-
size the necessity in advancing the understanding of tumor
energy metabolism for improvement in diagnosis, drug
design and chemotherapy of cancer.
Traditional chemotherapy currently offers little long-
term benefit for most malignant gliomas and is often associ-
ated with adverse side effects that diminish the length or
quality of life. Hence, new approaches are required that can
provide long-term management of malignant brain tumors
while permitting a better quality of life [149]. On this regard,
it seems more rational for drug design to gather information
by applying the metabolic control analysis, which allows the
quantitative identification of the main controlling steps in a
pathway, along with providing understanding of the underly-
ing regulatory mechanisms and faciliting the prediction of
the system behavior.
The encouraging results with some energy-metabolism
drugs indicate that, to successfully block growth of oxidative
or partially oxidative tumors (Table 3), it is required to use
simultaneously specific permeable drugs for glycolysis and
OxPhos (reviewed in ref. 56). It may be argued that cancer
cells are genetic and phenotypically heterogeneous from tu-
mor to tumor. However, all tumor cell lines do depend on
these two pathways for ATP supply. The metabolic therapy
searches for physico- and biochemical differences between
tumor and normal cells to improve the design of strategies
that preferentially affect tumor metabolism and growth,
without altering drastically the host tissue and organ func-
tionality. This approach may complement the existent che-
motherapeutic treatments, so that in combination may suc-
cessfully stop tumor growth, invasiveness, and drug
resistance.
Acknowledgements
The present work was partially supported by grants No.
80534 and 83084 from CONACYT-México.
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