Oncoscience 01 0777

www.impactjournals.com/oncoscience Oncoscience 2014, Vol.1, No.
12
Glycolysis, tumor metabolism, cancer growth and dissemination.

A new pH-based etiopathogenic perspective and therapeutic
approach to an old cancer question
Khalid O. Alfarouk1, Daniel Verduzco2, Cyril Rauch3, Abdel Khalig Muddathir1,
Adil H. H. Bashir1, Gamal O. Elhassan4,5, Muntaser E. Ibrahim1, Julian David Polo
Orozco6, Rosa Angela Cardone7, Stephan J. Reshkin7 and Salvador Harguindey6
1
University of Khartoum, Khartoum, Sudan
2
H. Lee Moffitt Cancer Center, Tampa, FL, USA
3
University of Nottingham, Sutton Bonington, Leicestershire, Nottingham, UK
4
Unizah Pharmacy College, Qassim University, Unizah, AL-Qassim, King of Saudi Arabia
5
Omdurman Islamic University, Omdurman, Sudan
6
Institute of Clinical Biology and Metabolism, Vitoria, Spain
7
University of Bari, Bari, Italy
Correspondence to: Stephan J. Reshkin, email: stephanjoel.reshkin@uniba.it
Correspondence to: Salvador Harguindey, email: salvaszh@telefonica.net
Keywords: Tumor glycolysis, pH and glycolysis, pH and cancer, proton transport inhibitors, cancer growth, metastatic process,
cancer treatment, new paradigm in oncology
Received: November 19, 2014 Accepted: December 14, 2014 Published: December 18, 2014
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
Cancer cells acquire an unusual glycolytic behavior relative, to a large extent,
to their intracellular alkaline pH (pHi). This effect is part of the metabolic alterations
found in most, if not all, cancer cells to deal with unfavorable conditions, mainly
hypoxia and low nutrient supply, in order to preserve its evolutionary trajectory
with the production of lactate after ten steps of glycolysis. Thus, cancer cells
reprogram their cellular metabolism in a way that gives them their evolutionary
and thermodynamic advantage. Tumors exist within a highly heterogeneous
microenvironment and cancer cells survive within any of the different habitats that
lie within tumors thanks to the overexpression of different membrane-bound proton
transporters. This creates a highly abnormal and selective proton reversal in cancer
cells and tissues that is involved in local cancer growth and in the metastatic process.
Because of this environmental heterogeneity, cancer cells within one part of the
tumor may have a different genotype and phenotype than within another part. This
phenomenon has frustrated the potential of single-target therapy of this type of
reductionist therapeutic approach over the last decades. Here, we present a detailed
biochemical framework on every step of tumor glycolysis and then propose a new
paradigm and therapeutic strategy based upon the dynamics of the hydrogen ion in
cancer cells and tissues in order to overcome the old paradigm of one enzyme-one
target approach to cancer treatment. Finally, a new and integral explanation of the
Warburg effect is advanced.
INTRODUCTION AND PERTINENT cancer cells were highly fermentative. He hypothesized

HISTORY that it was due to a metabolic injury [1,2]. Since the
discovery that cancer cells produced large quantities of
In the early 1920s, Otto Warburg observed that lactic acid and that extracellular/intratumoral acidification
has recently been shown to be a major and fundamental
www.impactjournals.com/oncoscience 777 Oncoscience

factor in local growth and in the metastatic process, transformation resulting in the Warburg Effect include:
NaHCO3 and other alkalinizing agents have been proposed (i) adaptation to transient hypoxia, (ii) insulin resistance
for the treatment of cancer almost a century later [3]. [9,17], (iii) abnormal enzyme content, (iv) abnormal
Later on, while at Roswell Park Memorial Institute enzyme activity or isoenzymatic alterations, (v) problems
(RPMI), Carl and Gerty Cori continued the work on in of compartmental transport translocation of pyruvate to
vivo carbohydrates in cancer [4]. Further along the same the mithocondria), (vi) abnormal content in the number or
line, during the late 70s and early 80s, and also at RMPI, quality of mitochondria, (vii) abnormal electron transport
we continued studying the dynamics of in vivo glycolysis and ATP production, and (viii) oncogenes and suppressor
and tumor secretion of lactic and pyruvic acids in rats genes [18]. Recently, intracellular alkalinity have been
with transplanted tumors as well as the effects of systemic gaining increasing importance as a simple and integral
acidification in dogs as an antyglycolitic therapeutic approach to explain the Warburg phenomenon [8,19].
measure and on tumor regressions in mice [5,6]. In this review we will first outline in detail the
More recently, PET technology has resuscitated different steps of glycolysis and then interrelate them with
the interest of the scientific community on Warburg cancer growth and progression.
initial findings up to the point that a few years ago a
new International Society to study tumor metabolism Glycolysis
and its anticancer therapeutic possibilities was created,
the International Society of Proton Dynamics of Cancer
(ISPDC), that has recently evolved to The International Glycolysis is the metabolic pathway that converts
Society of Cancer Metabolism (ISCaM) (www.ispdc. glucose, C6H12O6, into pyruvate, lactate and hydrogen
eu). In the same line, although tumors have a unique ions (protons). The free energy released in this process is
metabolic system and a concerted strategy to survive, used to form the high-energy compounds, ATP (adenosine
grow and metastasize, a phenomenon we have called triphosphate) and NADH (reduced nicotinamide adenine
the neostrategy of cancer cells and tissues [7,8], the dinucleotide). Glycolysis takes place in the cytoplasm. It
glycolytic metabolism of cancer was under-appreciated for can be directly represented by the following equation [20]:
almost a century until a recent rebirth of the fundamental C6H12O6 + 2NAD+ + 2ADPMg- + 2HxPO43x-
role of tumor microenvironment and glycolysis in cancer 2CH3CCOCO2- + 2 NADH + 2 ATPMg2- + 2 H2O + 2xH+.
growth and progression [912]. This has led the scientific Many allosteric factors of the most varied natures
community to adopt the differential tumor metabolism (hormonal, ionic, viral physical, chemical, genetic and
as an additional hallmark of cancer [13]. This review metabolic) have been described that regulate glycolysis
and integrated new perspective will first consider a and/or glucose consumption (Table 1). For more than 30
detailed study of every single step of glycolysis, mainly years, mainly from the sixties to the nineties, an epidemic
in the cancer context, followed by a unitarian approach of allosteria predominated in glycolysis research in the
to the pathogenesis of glycolysis and pH-related cancer many attempts to find an explanation to Warburgs aerobic
growth and metastasis and a proposal for a new integrated glycolysis [21].
approach to the treatment of malignancy. The whole process of glycolysis takes place through
two phases: a preparatory phase and a harvesting phase.
Classical view of metabolism as either anabolic or
1. Investment, preparatory phase
catabolic
In this regard, glycolysis is the cytoplasmic 1.1 First step. Glucose fixation
utilization of glucose, which is an example of a catabolic In this step, glucose is fixed intracellularly by the
pathway. Normally, glycolysis finishes with the entrance addition of a phosphate group to form glucose-6-phosphate
of pyruvate into the Krebs cycle and the mitochondrion and such phosphorylation prevents glucose efflux. This
in the presence of oxygen. Under certain circumstances, requires the addition of a phosphate group from ATP and
such as an insufficient supply of oxygen, pyruvate is needs hexokinase or glucokinase as a catalyst for this
converted to lactate and pumped out of the cell. In cancer reaction. This step requires Mg+2 as co-factor. It requires
cells, the conversion of pyruvate into lactate takes place about -17 KJ/mole under normal conditions [22] (Figure 1)
even in the presence of oxygen (aerobic glycolysis), (where G0 is the free energy of a reaction and the minus
and this was called the Warburg Effect after it was so (-) sign shows this reaction to be exothermic i.e. releases
termed by Racker [14] and has also been known through energy) (Figure 1). It is most likely that hexokinase has
the years as the first law of cancer biochemistry [15]. an anti-apoptotic function, which is why it might explain
Warburg defended all his life that the aerobic glycolysis its overexpression in tumors [23]. Precisely, this enzyme
of tumors was the primary cause of cancer. However, could be a glucokinase rather than hexokinase because it
time has proven this not to be true [8,16]. Among the is not inhibited allosterically [24].
many proposed mechanisms to explain the metabolic

Table 1: Allosteric factors regulating glucose consumption by normal and/or cancer cells
insulin, adrenal steroids, epinephrine, androgens, estrogens, parathyroid hormone, human
Hormonal growth hormone, glucagon, melatonin
Ionic Pi, Mg++, K+, Ca++, H+.
Viral reovirus, Rous sarcoma virus, Human papilloma virus E16
Physical O2, temperature
Chemical Iodoacetic acid, sacaric acid, sodium folluoride, NH4+, HIF-1
Genetic Chromosome 21
ATP, ADP, AMP, citrate, Krebs cycle intermediates, Ketone bodies, Thiamine, Fatty acids,
2-4-dinitrophenol, glucose-5-phosphate, fructose 1-6-biphosphate, bioflavonoids, dietetic
Metabolic sugars, folic acid, phosphocreatine, 3-phosphoglycerate phosphoenolpyruvate, fructosebi-
phosphatase, 3-5 cyclic AMP, methylglyoxal
Oncogenes tumor supressor genes
Therapeutic drugs Methotrexate, clotrimazole
***
Modified from Harguindey S. [317]
Therapeutic targeting of this step: Mannoheptulose [36]. However, another main concern in
1. Glucose transport inhibitors: glucose is taken up solid tumors is the problem of those enzymatic inhibitors
by the glucose transporters Glut-1 or the sodium-dependent reaching all tumor areas as they probably reach only the
glucose transporters e.g. sodium-dependent glucose tumors outer layers because of low O2 tension and low
cotransporter-1 or sodium-glucose linked transporter 1 blood supply, so impairing drug diffusion [37]. Although
(SGLT1). Glut-1 and SGLT-1 are overexpressed in cancer the following step is not reversible, the glucose-6-
[25]. Targeting the Glut-1 transporter [26] represents a phosphatase enzyme exports glucose extracellularly [38].
potential anti-tumor strategy directed towards glucose Vanadium is also a potential inhibitor of the phosphatase
deprivation [27]. Also, it sensitizes cancer cells to death enzyme [39]. However, targeting this enzyme remains of
receptors by arresting them in G0-G1 phase [28], so re- doubtful value due to its variable expression in tumors
sensitizing them to a death ligand like TRAIL. [40,41].
2. At the substrate level, therapeutic attempts have 1.2 Second step: Gluco-Fructose isomerization
been tried by administering fake substrates which are
not normally reversed by insulin activity; e.g. 2-Deoxy- In this step Anti-AMF (Autocrine Motility factor)
D-glucose (2-DG) [2931]. Glucosamine also inhibits antibodies are correlated with arthritis and considered
hexokinase [32]. Furthermore, metrizamide also inhibits arthritogenic [4244]. Its role in chronic inflammation
hexokinase [33] but to a lesser degree in comparison to might collaborate in inflammation-related carcinogenicity
2-DG and glucosamine [32]. [45,46] since it is known that glucose-6-phosphate is
3. Chromosomal amplification also is another isomerized (intramolecular reaction, rearrangement) into
strategy to stimulate glycolysis [34] which could be fructose-6-phosphate by Glucose-6-phosphate isomerase
targeted through siRNA [35]. (phosphoglucose isomerase or phosphohexose isomerase)
In summary, targeting hexokinase represents (Figure 2). This is a reversible reaction following Le
a seductive strategy in treating cancer, e.g. with Chateliers principle. This principle determines that in a
reversible reaction, when the concentration of a reactant/s
Figure 1: Conversion of Glucose into Glucose 6 Phosphate.

exceeds the concentration of the product/s, the reaction Fructose -1, 6-Bisphosphate
goes in the direction that produce more product/s and
decrease the concentration of the reactant/s (it goes to
Fructose-1,6-biphosphate, also known as
the right). Parallel to the context, if the concentration of
Harden-Young ester, is formed during glycolysis by
the product/s exceeds the concentration of the reactant/s,
phosphofructokinase-1 (PFK-1) and requires Mg+2 and an
the reaction goes in the reverse direction to produce
ATP molecule.
more reactant/s (it goes to the left). This is, when found
Fructose -1,6-biphosphate activates pyruvate kinase
extracellularly, Glucose-6-phosphate isomerase is a
allosterically [59,60]. Fructose-1,6-biphosphate has a
synonym of (I) Neuroleukin (neurotrophic factor) [47],
cytoprotective activity by chelation of iron [61] and
and (II) AMF [48]. Therefore, Glucose-6-phosphate
also behaves as a free radical scavenger [6265]. In this
isomerase is an example of an ectoenzyme (exoenzyme)
regard, fructose -1,6-biphosphate may play a critical role
[49]. Early studies using chromatography suggested that
in the prevention of programmed cell death (apoptosis).
AMF has two active peaks at different pH values [50] that
Therefore, it is possible that this glycolytic intermediate
might be compatible with the uniqueness of the cancer
could be involved in certain cases of de novo multiple
cell reversed pH gradient [51], a selective hallmark of
drug resistance (MDR) and be functionally equivalent
they having an alkaline cytosol and an acidic extracellular
to glutathione in that its absence might promote
microenvironment [37,51]. Therefore, AMF facilitates
carcinogenesis but its elevation can confer chemotherapy
glycolysis inside the cells as a downhill reaction to produce
resistance [66]. Also, both fructose-1,6-biphosphate
lactate and H+, which facilitates its role outside the cells
and glutathione are interlinked together as a survival
as a tool for invasiveness; i.e. AMF has a dual role, first by
strategy against hypoxia [67]. Furthermore, fructose-
activating glycolysis and then by collaborating in inducing
1,6-biphosphate inhibits T-cell proliferation and has anti-
an extracellular acidity that promotes tumor invasiveness
inflammatory properties through inhibition of interleukin
and metastasis [11,52,53]. So, AMF plays a critical role in
1,6 and beta-catenin [68]. Therefore, on one hand, it
neoplastic transformation [54], invasiveness [55,56], and
inhibits immune response, which supports tumors fitness,
metastasis [57,58].
while on the other hand it attenuates the inflammatory
1.3 Third step. Commitment to glycolysis environment which generally alters tumor progression
Fructose-6-phosphate is further phosphorylated into [69]. All in all, its precise role remains mostly undefined.
unstable molecules termed fructose-1,6-biphosphate Glucose-6-phosphate isomerase is an inflammation-
(Figure 3), and fructose-2,6-biphosphate (relatively inducing agent while fructose-1,6-biphosphate is a free
stable than the other ones due to less steric hindrance) radical scavenger and an anti-inflammatory enzyme.
respectively by phosphofructokinase-1 (PFK-1) and
phosphofructokinase-2 (PFK-2).
Figure 2: Shows isomerization of Glucose-6-phosphate into fructose-6-phosphate.
Figure 3: Shows phosphorylation of Fructose 6-phosphate into Fructose 1, 6 bisphosphate.

Phosphofructokinase-1 (PFK-1) Fructose 1,6-bisphosphatase (FBPase-1)
PFK-1 is a tetramer enzyme that consists of three Fructose-1,6-biphosphatase is one of the key
subunit types: muscle (M or A), liver (L or B) and platelets enzymes that mediates gluconeogenesis. It has two
(P or C) [70,71]. Phosphofructokinase of muscle is isoforms: Liver (L-FBPase) and muscle (M-FBPase)
composed of homotetramer 4M, the liver predominantly [96100]. Insulin decreases expression of FBPase-1 [101].
contains the L subunit in addition to M and P subunits. Although cAMP increases expression of FBPase-1 [101],
Brain and heart exhibit three subunits [72,73], which AMP strongly inhibits FBPase-1 [102,103]. Fructose-1,6
correlates with differences in tissue specialization. It biphosphate inhibits FBPase-1 [104] by acting
has been shown that tumors over-express PFK-1 and synergistically with Fructose-2,6biphosphate [105].
preferentially its L-subunit [74]. PFK-1 might be useful Also, Fructose-2,6biphosphate acts synergistically with
for monitoring of progression of some cancers and also to AMP to inhibit FBPase-1, and this inhibition is decreased
identify tumor stage [75]. at higher substrate concentrations [105]. The same study
In 1986, it was demonstrated in some strains of rats pointed out that alkaline pH decreases the inhibitory
that they only have M subunits in their muscles but that effect of Fructose-2,6 biphosphate [105] while FBPase-1
other organs differ drastically in tissue/organ proportion activity is increased at higher, alkaline pHi [106].
in PFK-1 subunit expression [72]. From this study a Role of FBPase-1 in mediating resistance
question was raised concerning if there is any possibility
that human ethnicity could lead to differences in tissue/ It has been shown that phosphofructokinase is
proportion of PFK-1. If this diversity is present it could down-regulated while FBPase-1 is up-regulated in
affect tumor behavior in certain cases. This could help radiation resistant cell lines. Both features together
to study tumor progression in terms of ethnicity and suppress apoptosis through increasing glutathione levels
management of cancer is different human populations. [107]. In this context, the PFK-1/PFBase ratio plays a
critical role in tumor proliferation and/or tumor resistance
PFK-1 regulation ratio while FBPase over-expression could be considered to
Interestingly, PFK-1 has the same kinetic be one important adaptive strategy of resistance. In other
characteristics in both aerobic and anaerobic conditions words, by providing more lactate, glycolysis supplies
[76]. Also, a slightly alkaline pHi is the optimum to an evolutionary advantage [108] as well as a metabolic
maximize PFK-1 activity [52,7780]. On one hand, it resource [9]. However, over-expression of gluconeogenic
has been known for decades that an alkaline pHi even enzymes during resistance might support the decrease
slightly above steady-state levels stimulates the activity of in glycolysis and so a reduction in proliferation rates,
this key glycolytic enzyme and inhibits gluconeogenesis. which is an adaptative cost of resistance. Expression of
Indeed, in cancer cells a high pHi situation can increase FBPase-1 leads to the formation of glucose-6-phosphate
the allosteric regulation of PFK-1 more than a 100-fold and therefore feeds the pentose phosphate pathway (PPP)
and even a raise of 0.2 pH units can convert this enzyme [107,109].
from an inactive form to a fully active quaternary structure
[5,8083]. This post-Warburg, H+-related approach to Fructose - 2, 6-bisphosphate
glycolysis and tumour metabolism has originated during
the last few years a completely and integral new paradigm
in approaching oncological metabolic research and cancer Studying the mechanism of action of glucagon
treatment based upon the hydrogen ion dynamics of cancer on gluconeogenesis led to the discovery of Fructose-
cells and tissues [10,11,52,53,8489] 2,6-bisphosphate [110,111]. This molecule is crucial in
Furthermore, phosphocreatine inhibits PFK-1 [90] maintaining the glycolysis downward chain reaction and
while 3-phosphoglycerate and phosphoenolpyruvate act increasing commitment to glycolysis, especially when
synergistically with ATP to inhibit PFK-1 [90,91]. Finally, ATP levels are raised [112]. This step is irreversible. Since,
ADP, among other factors, activates PFK-1 allosterically as described above, PFK-1 is inactive under physiological
[92,93] (see Table 1). PFK-2 also activates PFK-1 through conditions and is activated by Fructose 2,6 bisphosphate
fructose 1,6 biphosphate, but during persistent exercise in synergistically with AMP [113], it seems that Fructose
frog muscle, fructose-2,6 biphosphate levels drop, while 2,6-bisphosphate has evolved in order to enhance insulin
Pi, AMP and ADP all activate PFK-1 during exercise [94]. activity [114] and so increase glucose uptake. Finally,
Thus, in persistent exercise, normal cell physiology relies palmitate decreases the level of Fructose-2, 6-bisphosphate
on endogenous activators in order to maintain energetic [115].
requirements and not only on fructose 2,6-bisphosphate
activity. Finally, clotrimazole has anti-PFK-1 activity in
vitro [95].

PFK-2 2. PPP completes glycolysis by consuming 1 ATP
instead of 2 ATP molecules and, therefore, it is more
energy-efficient than glycolysis. Each molecule of
When the glucose level is decreased, glucagon
glucose uses 2 ATP and produces 4 ATP molecules. So,
is secreted to activate cAMP as a consequence of
the net energy is 2 molecules. Pentose Phosphate pathway
adenylcyclase activation [116,117]. cAMP activates
does consume 1 ATP molecule and does produce 4 ATP
FBPase-2 [118] and activation of FBPase-2 leads to
molecules. Therefore, the net result is 3 ATP molecules.
a decrease of fructose 2,6-biphosphate [119]. In turn,
In this regard, PPP provides more energy, in addition
reduction of fructose 2,6-biphosphate leads to inactivation
activation of PPP results in the biosynthesis of nucleic
of PFK-1 and, thus, stimulates gluconeogenesis [120].
acids. So, cancer cells might decide whether to get
On the other hand, insulin has a reverse effect. Glucose
pyruvate through the complete glycolysis pathway or rely
fixation is increased and also up-regulation of glycolysis as
partially on PPP. Until that decision takes place, PFK-2
a result of the binding of glucokinase to PFK-2/FBPase-1
might also be an endogenous ROS-sensor to determine
[121], which indicates enzymatic interactions.
whether F-1,2-BP is at appropriate levels or decide to
In cardiac muscle, PFK-2 is a cellular defense
finish glycolysis through PPP and so prevent formation
strategy against hypoxia [122] and PFK-2 deficiency is
of F-1,6-BP.
correlated to insulin resistance [123]. Also, citrate inhibits
PFK-2 to a greater extent than PFK-1 [124]. Therefore,
the citrate that results from the Krebs cycle inhibits PFK-
FBPase-2
2 [125], which suggests a negative feedback mechanism.
Phosphoenolpyruvate also inhibits PFK-2 [126]. PFK-2/FBPase-2 is a bifunctional enzyme and
Although it is a commitment step, production of energy FBPase is reciprocal to PFK-2. Insulin inhibits it in both
is a key determinant factor to finish this step. In this way, liver and muscle while epinephrine activates it in muscle
glycolysis is tightly regulated internally. It has also been and inhibits it on liver [135,136]. Therefore, the metabolic
demonstrated that protons inhibit PFK-2 [78,127] while an and/or oxidative stress status of the cell could play a role
alkaline pHi increases its activity [128]. in regulating the relative PFK-2/FBPase-2 cellular activity.
PFK-2 has 4 isoenzymes: PFKFB 1, 2, 3, 4 These relationships raise the question of why is
[129,130]. PFKFB3 and 4 are correlated with cancer it that normal cells do not have the isomerase enzyme
and their expression is higher in metastasis as compared instead of A kinase and phosphatase, so that they can save
to primary tumors [131,132]. Interestingly, Hypoxia one molecule of ATP. In other words, since developing the
Inducible Factor-1 alpha (HIF-1 alpha) increases isomerase enzyme that can translocate a phosphate group
transcription of PFKFB4 [133]. Altogether, these data is less expensive than developing a set of enzymes that
suggest that they could potentially become important consume additional ATP molecules, why do normal cells
antimetastatic targets [131]. However, the targeting of acquire kinase/phosphatase enzyme?
PFK-2 should be carefully monitored because it might Potential answers are as follows:
shift to complete glycolysis through PPP; i.e. as a provider 1. One of the proposed answers is that fructose
of energy with anti-oxidant capacity (energy plus anti- 2,6-biphosphate acts as cellular regulator as well as
apoptosis). a reservoir. Further, bifunctional enzymes are tightly
As PFK-1 is inactive under physiological conditions correlated with function and isomerases require a
[113] and it is activated synergistically by Fructose-2,6- reversible reaction that is not controlled by external
biphosphate together with AMP, one could expect that signaling. In this context, many basic researchers and
PFK-2 should be activated in the first place followed clinicans might consider Fructose 2,6-biphosphate as an
by PFK-1 activation. There is a great deal of literature Achilles heel; however, it is not as simple as that since
describing that the PPP serves cancer cells to produce Fructose 2,6-biphosphate is a component of various
nucleic bases; i.e. via the formation of cellular building essential cycles.
blocks. Although this is an acceptable hypothesis, yet we 2. Fructose 1,6-bisphosphate is an unstable molecule
will now follow a different approach to complement the due to steric hindrance (negative charge of phosphate as a
significance of the PPP pathway. bulky group at carbons 1 and 6. This molecule is reversibly
1.PPP is a very critical pathway because it is fragmented into D-glyceraldehyde-3-phosphate and
capable of forming Glutathione-S-Transferase (GST). dihydroxyacetone phosphate in addition to the remaining
GST is a Reactive Oxygen Species (ROS) scavenger. steps of glycolysis which are reversible too (except the last
ROS are carcinogens that induce DNA-mutations but upon one). Therefore, shifting from the product into fructose
carcinogenesis ROS prevents the induction of apoptosis. 2,6-bisphosphate prevents fructose 1,6-bisphosphate
Therefore, PPP is essential for tumor cell immortality. accumulation and, consequently pushes glycolysis to
Inhibition of Glucose-6-Phospahte Dehydrogenase move downward instead of reversing to a gluconeogenic
(G6PD), which is a key determinant step of PPP, results in direction. This answer is highly compatible with a role of
the prevention or slowing of carcinogenesis [134]. fructose-2,6-bisphosphate as a glycolytic directing agent.

In essence, fructose 2,6-biphosphate could be considered is also expressed in kidney [146,147]. The expression of
as as guardian of glycolysis. The most striking example most aldolase isoforms fluctuate from one tissue to another
is insulin. Insulin increases glucokinase (hexokinase), and in the same tissue from one time to another and in
PFK-2 and Pyruvate Dehdyrogenase (PDH), so facilitating certain tissues at the moment when the tissue acquires
its entrance into the Krebs cycle. Therefore, insulin diseases. For example, aldolase B is predominant in the
promotes the irreversible steps of glycolysis. Insulin also liver of neonates, aldolase A increases in the fetal stage
stimulates the Na+/H+ exchanger isoform 1 (NHE1) and and returns back to aldolase B at adulthood [148]. It was
increases pHi [137]. reported early on that aldolase content is increased in
cancer [149]. Further, aldolase A becomes dominant in
1.4 Fourth step. Fission step hepatoma while aldolase B decreases in hepatoma and
gastric cancer [150]. Once again, intracellular alkalinity is
the optimum environmental condition for aldolase activity
Fructose-1,6-bisphosphate is degraded [151].
into D-glyceraldehyde 3-phosphate (GADP) and Aldolase B is also necessary for fructolysis, which
Dihydroxyacetone phosphate (DHAP) by the activity of is responsible for the formation of glyceraldehyde-
fructose-bisphosphate aldolase (Figure 4). DHAP can be 3-phosphate from fructose-1-phosphate. Therefore,
reversed into GADP through activity of triose phosphate Aldolase shows a difference in the ratio of FDP (fructose-
isomerase. Again, the presence of a negative charge and 1-6-biphosphate) and F1P (fructose 1-phosphate) between
the double bond at carbon 2 leads to steric hindrance that liver and spleen [152] which may reflect the following:
isomerizes it into GADP by triose phosphate isomerase. 1. Tumor behavior differs from one organ to another.
DHAP is an intermediate of several biochemical pathways, So, there is no unifying glycolytic microenvironment.
including the glycerol phosphate shuttle (the glycerol 2. It might also reflect the presence of fructolysis in
phosphate shuttle is a mechanism that regenerates NAD+ certain tumors (conversion of fructose 1 phosphate into
from NADH). Importantly, triose phosphate isomerase is glyceraldehyde-3phosphate) as some tumors over-express
overexpressed in tumors and is correlated with hepatic GLUT-5 [153] as the key transporter of glucose [154]. In
metastasis [138,139] but its expression is decreased when this regard, tumor cells uptake fructose and phosphorylate
cancer overexpresses drug resistance proteins; e.g. MDR it. Conversely, the source of fructose1-phosphate could
[140]. Triose phosphate isomerase also shows a higher form from PPP too. This raises the question if the Warburg
activity at alkaline pH [141]. Effect should be reappraised as to whether glycolysis in
Furthermore, in the next step, DHAP is converted tumors occurs through the preparatory phase and/or the
into glyceraldehyde-3 phosphate (GADP). GADP is a node PPP that meets pH-dependant glycolysis in GADP node
for several biochemical pathways including glycolysis, or is there a ratio between them.
gluconeogenesis, PPP, tryptophan biosynthesis and the
glycerol-3 phosphate shunt. GADP inhibits PFK-2 and,
2. Pay-off phase (Harvesting Phase)
in this way, might trigger gluconeogenesis. GADP also
inhibits caspase-3 activity (anti-apoptotic effect) [142].
Aldolase (fructose diphosphate aldolase) is the enzyme that 2.1 Fifth step. Rearrangement process
converts fructose 1, 6 biphosphate into GADP and DHAP
and has 3 isoforms: A, B and C [143,144]. Generally, Glyceraldehyde 3-phosphate (GADP) is
aldolase C is found in the brain [145], aldolase A is the converted into 1, 3-biphosphoglycerate by the activity
ubiquitous form and the predominant isozyme in muscle of glyceraldehyde phosphate dehydrogenase (GADPH)
while aldolase B is the predominant isozyme in liver and (Figure 5). This reaction needs NAD+ and inorganic
Figure 4: hydrolysis of Fructose 1, 6 bisphosphate into D-glyceraldehyde 3-phosphate (GADP) and Dihydroxyacetone
phosphate (DHAP).

phosphate Pi. Finally, GAPDH is inhibited by Monochloroacetate
GAPDH has been shown to have apoptotic (MCA) [165]. Indeed, intracellular alkalinity is once again
properties as well as preventing mutagenicity [155] . the optimum pH for GADPH activity (pH=8.5) [166].
This raises the question as to what is the evolutionary 2.2 Sixth step. First Energy Releasing step
advantage of its tumor over-expression if it initiates
apoptosis? In this respect, HIF1-alpha increases 1,3-bisphosphoglycerate is converted to
expression of GAPDH [156], raising the question: does 3-phosphoglycerate by phosphoglycerate kinase (PGK)
HIF1-alpha induce cell death? It is well documented that and produces ATP at substrate level phosphorylation
GAPDH has an apoptotic property, which is why its (Figure 6). PGK has several isozymes [167,168] and its
over-expression plays a critical role in neurodegenerative expression rises during anoxia [169] which reflects its
diseases and its inhibition serves as a promising strategy preferential expression at anoxic areas of solid tumors
for treating such diseases [157160]. So, the precise [9,108]. However, while some data has shown that PGK-
question is how does GADPH promote carcinogenesis, overexpression is correlated with disseminated cancer
as well as inducing neurodegeneration? One possible [170], other data shows an inverse correlation with tumor
convincing answer could be that the higher rate of NAD+ incidence and consider that PGK enhances an anti-tumor
production will reduce GADPH capacity by converting it effect because of its anti-inflammatory and anti-angiogenic
from a tetramer into a dimer [161]. Therefore, glycerol activity [171].
- 3 - phosphate dehydrogenase might play an essential Regardless of our recent criticism on the anti-
role in producing such an apoptotic reduction. Moreover, angiogenic approach and its possible correlation with
PPP reduces ROS that deactivates GADPH [162] and so a damaging selection of a hypoxic/anoxic phenotype
maintains GADPH levels. In this regard, PPP continues [108], such contradictory evidence could be solved by
GADPH activity while the glycerol phosphate shuttle considering it as a delicate balance between plasminogen
attenuates its apoptotic capacity. In the end, tumor cells activation and inhibition in extracellular matrix
modulate the dialectic of the contraries of metabolism (ECM) turnover [172], especially as PGK regulates
in an extremely coordinated manner in order to maintain urokinase receptor expression, which is involved in
its cellular integrity. Furthermore, it is not sure whether ECM remodeling, cell proliferation and migration as
precise modulation comes from lactate dehydrogenase well as in modulating cell adhesion [173]. Therefore, in
or glycerol3phosphate dehydrogenase. In conclusion, targeting PGK the exact determination of the timing of
Alzheimers Disease and other neurodegenerative diseases administration of the inhibitory compound is required,
have an opposing pathogenesis as compared to cancer, e.g. depending on whether the goal is to activate or inhibit
intracellular acidity might aggravate neurodegenerative ECM turnover, quantify oxygenated/hypoxic areas, or
capacity as it has been previously suggested [7,163,164]. manipulate the anoxic ratio of the tumor population.
Figure 5: conversion of D-glyceraldehyde 3-phosphate (GADP) into into 1, 3-bisphosphoglycerate.
Figure 6: conversion of 1, 3 - bisphosphoglycerate into 3-phosphoglycerate.

Table 2: Distribution of Pyruvate Kinase isoforms among tissues
Pyruvate Kinase Isoforms Tissue localization
PKL RBCs
PKR Liver
PKM1 Muscle
Kidney, brain, heart, thymus, spleen, lung, adipose tissue, testis and
PKM2
ovary
***
For further details, see text
PGK also shows maximum activity at a cellular pH range of enolase and attenuates that of MBP-1 [195]. Moreover,
similar to that of cancer cells [174,175], which is alkaline the presence of hypoxia increases the production of ROS
[176]. and the expression of c-myc [195]. C-myc increases
2.3 Seventh Step production of mitochondrial ROS and it has been shown
to stabilize HIF1-alpha [196,197] suggesting a delegate
3Phosphoglycerate undergoes structural balance between C-myc, ROS and HIF1-alpha in
isomerization and yields 2-phosphoglycerate by maintaining cellular survival and tumor progression. In
phosphoglycerate mutase (PGM) (Figure 7). PGM is conclusion, ENO-1 expresses different proteins that can
overexpressed in cancer and is correlated with poor be localized either in the nucleus as a tumor repressor
prognosis [177]. The maximum activity of PGM also [191] or in the cytosol as a glycolytic enzyme or, finally,
occurs at alkaline pHs [178]. at the cell surface, where it promotes invasiveness and
2.4 Eight Step metastasis [198]. It has a higher activity at pH 7.5 when in
phosphate buffer [199].
2Phosphoglycerate is converted into
Phosphoenolpyruvate by the enolase enzyme (Figure 8). In 2.5 Ninth Step (Formation of Pyruvate)
mammalian cells, there are three independent genetic loci: Phosphoenolpyruvate (PEP) is converted into
, and . They code and express three different isozymes pyruvate through pyruvate kinase enzymes (Figure
according to tissue specificity. Alpha enolase (ENO1) is 9). This second step produces ATP at substrate level
found in most adult tissues, beta enolase (ENO3) is found phosphorylation. Pyruvate kinase has four isozymes
in muscle and gamma enolase (ENO2) is found in the PK L, R, M1, and M2 (Table 2) [200,201]. L-alanine is
brain [179]. a strong inhibitor of PK-L and hepatoma and has little
It has also been postulated that the enolase has effect on PK-M while phenylalanine inhibits PKM [202].
tumor suppressive properties [180] as it is absent in Phosphoenolpyruvate activates pyruvate kinase and
some tumors [181] while other data supported that it is fructose 1,6-biphosphate [60]. Epinephrine and glucagon
overexpressed in some malignant tumors [182]. One of phosphorylate and deactivate PK-L [203] while insulin
the possible answers for this inconsistency comes through dephosphorylates enzymes and activates it [204,205].
the understanding of the subcellular localization of enolase The ATP/AMP ratio is very important in determining PK
and its translational process. Enolase has been found at activity.
the cell surface as a plasminogen binding protein, which Although rabbit PEP binding with PKL is increased
was found to promote tumor invasiveness and metastasis with increasing pH from 6 to 8.5 [206], the effect of
[183], boosting immunization to prevent bacterial pH on PK activity is very intricate and is dependent on
virulence [184189]. Besides, at the cellular membrane the concentration of allosteric activators, ATP levels
enolase has also been found at the cytoplasm and nucleus and species variation [200,207,208]. This is why small
[190]. The ENO-1 gene is responsible for expression of changes in intracellular pH alters PK activity [209]. At
enolase-1 as well as for Myc-binding protein-1 (MBP-1); least in yeast, protons facilitate PEP binding but weaken
that is, the same gene provides two different proteins at the binding with Mg+2 and ADP [210].
translational level [191,192]. PKM2 can be translocated into the nucleus and
Enolase is very crucial in completing glycolysis induces cellular proliferation [211] unless it binds with
and, thus, so might promote tumorigenesis while MBP-1 other agents to induce apoptosis [211]. One can therefore
blocks the activity of c-myc expressing protein [193,194]. ask, what is the role of PKM in cells that are undergoing
Therefore, the key determinant that instigates either apoptosis? The most convincing answer is that apoptosis is
tumor growth or tumor regression is translation of ENO-1 an active process that needs energy [212214]. Therefore,
gene towards Enolase or MBP-1 expression respectively further work should be done to differentiate how and
(Enolase/MBP-1 ratio). Such an evolutionary fate might when the energy produced could be invested for tumor
be determined through microenvironmental selection, e.g. cell proliferation or exploited to undergo programmed cell
hypoxia. Tumor hypoxia preferentially selects translation death.

Malignant progression is accompanied by a decrease system (OXPHOS), might not indicate that mitochondria
in the PKM1/PKM2 ratio, both being different splicing have re-activated their role as a cell death machinery.
products of the M-gene (exon 9 for PKM1 and exon However, activation of the Krebs cycle produces huge
10 for PKM2) [215]. Only PKM2 is active when tissue amounts of ATP in comparison to glycolysis and when the
becomes cancerous [216,217]. PKM2 can be found in ATP level is raised, PFK2 is inhibited leading to inhibition
two forms, dimer and tetramer [218]. The tetramer form of fructose 1, 6-biphosphate. So, it is a negative feedback
has a high affinity for PEP even below physiological mechanism.
concentrations of PEP [218]. In contrast, the dimer has a In conclusion, the dimer/tetramer ratio acts as an
low affinity for PEP and is nearly inactive at physiological auto-sensor that drives the synthesis of nucleic acid and
PEP concentrations, being mainly expressed in tumors other components at certain times while at other times
[219]. The fact that cancer cells can preferentially express cancer cells generate energy from mitochondria. So,
the less active form of PKM2 could be due to various it will not be a surprise if this ratio is coordinated and
possibilities: synchronized with the cell cycle. Any alteration in this
A decrease in the conversion of PEP into pyruvate sensor pathway might lead to catastrophic events in cancer
leads to accumulation of glycolytic intermediates at at levels of either the individual cell selection or at group
upstream pathways that facilities and encourages the selection (tumor population). Endogenous agents that
formation of other building blocks such as nucleic acids. alter this cycle include: (i) tyrosine kinase, that leads to a
PKM2 encourages lactate formation rather than the release of fructose 1,6-biphosphate [216] and (ii) thyroxin,
entrance into the Krebs cycle; that is to say that the PKM2/ that might disturb or induce sensor noise [222] because
PKM1 ratio is directly proportional to the LDH/PDH cytosolic thyroxin hormone-binding protein (P58) is a
(Lactate dehydrogenase/Pyruvate dehydrogenase ratio) monomer of PK subtype M2v [222]. Interestingly, this
[220]. The provision of lactate (i) supports regeneration of last possibility might explain the thermogenic activity
another NAD+ and (ii) increases tumor acidity. In this way, of thyroxin and its delicate balance and correlation with
PKM2 promotes tumor fitness, which becomes another adaptation of cancer to hypo/hyperthermia. If so, we
evolutionary advantage [32]. should look again at the impact of thyroxin diffusion
Fructose 1,6-biphosphate encourages re-association across or through a tumor colony in the same way as for
of the dimer into a tetramer [221]; i.e. fructose estrogen diffusion [223].
1,6-biphosphate decreases the dimer/tetramer ratio and PKM1 has less impact on in vivo proliferation than
so facilitates entry into the Krebs cycle which promotes PKM2 [219]. Therefore, again this ratio will not only
mitochondrial activity as well as decreases lactate support our hypothesis of synchronization with the cell
production. However, activation of the Krebs cycle cycle but also this ratio is very important in maintaining
might not necessarily induce apoptosis. In other words, tumor population density especially under low nutrient
production of ATP through the oxidative phosphorylation conditions, e.g. improper blood supply that leads to
Figure 7: Shows conversion of into 3-phosphoglycerate into 2-phosphoglycerate.
Figure 8: shows conversion of 2-phosphoglycerate into Phosphoenolpyruvate.

adaptation of hypoxia that might increases c-myc and this appearance of the Warburg Effect) [10,19,176,234].
later might be correlated with PKM2 expression too [224] Thus, among all the many allosteric factors
as well as with enolase. Moreover, even expression of controlling glycolysis, the (H+), hydrogen ion
PKM2 is not enough to generate lactate as a driving fuel concentration and/or pH, has become the most significant
of carcinogenesis and tumor progression, because it seems factor, even overwhelming all others (Table 1). In the
that PKM2 is dependent on other cell signaling pathways presence of adequate oxygen levels, the intracellular pH
through serine such as mTOR1 [225,226]. plays a key role in determining the way cancer cells obtain
energy: an alkaline pHi driving aerobic glycolysis and an
3. Lactate production acidic pH driving oxidative phosphorylation [233]. An
explanation for this phenomenon derives from the fact
that both the processes of OXPHOS and glycolysis are
The tricarboxylic acid cycle (TCA) produces high exquisitely but oppositely pH-sensitive, and a rapid shift
amounts of protons that may decrease pHi. However, of cell metabolic patterns follows either acidification or
molecular oxygen interacts with protons to produce H2O. alkalinisation. In this vein, it has been known for decades
So, oxygen acts as a detoxifying agent. Therefore, in that an alkaline pHi even slightly above steady-state levels
the absence of oxygen, pyruvate is converted to lactate stimulates the activity of key glycolytic enzymes such as
and takes NADH to produce NAD+ through lactate phosphofructokinase (PFK-1) and at the same time inhibits
dehydrogenase activity, and so conversion into lactate may gluconeogenesis [5,8083]. Indeed, the steady high pHi
be a compensation to overcome cellular death. characteristic of cancer cells can increase the allosteric
Cancer cells adapt to hypoxia. Over-expression regulation of PFK-1 more than 100-fold [52,82]. Indeed, it
of HIF1-alpha induces pyruvate dehydrogenase kinase can now be considered that the high pHi of tumor cells, the
expression and that activates pyruvate dehydrogenase Warburg effect and the steady-state and selective hallmark
[227,228]; i.e. a shut-down of the mitochondrias role of all cancer cells proton reversal may very well represent
in glucose utilization. Thus, over-expression of proton one and the same phenomenon observed from different
transporters and over-expression of HIF1-alpha function perspectives, at different historical times and through less
as a strategic defense to prevent H+ accumulation. integral perspectives [8].
Although oxygen antagonizes hypoxic-induced proteins
[229231], cancer cells have a high rate of glycolysis
The errors and limitations of Otto Warburgs
even in the presence of oxygen [9]. Therefore, cancer cells
prefer to increase LDH/PDH ratio. theory. A further insight into the primary cause
of cancer.
An integral perspective on the pH of cancer cells
and the Warburg Effect: a synthetic explanation To understand the meaning of the most recent and
dynamic observations and interpretations on glycolysis
we need to go back to the postulated origin of cancer
Beyond the in-depth and dynamic consideration
cells by Warburg [1,235]. In doing so, we can realize that
of each of the glycolytic steps, the idea that the
a fundamental confusion in the entire field of metabolic
shift to glycolytic metabolism relative to oxidative
and biochemical cancer research was created from its very
phosphorylation under aerobic conditions could be
beginning.
explained by an increase in the intracellular pH has been
Presently, it is clear that Otto Warburg was wrong
increasingly gaining weight with the passing of time
on perhaps the main and most obscure point of his
[19,8183,232,233]. Nagata et al., as well as our group
famous theory during his time, namely, the levels of
have recently reached the conclusion that the Warburg
cancer cell pHi and, consequently, on its relationship to
effect can perhaps be fully explained by the simple
glycolysis. Indeed, Warburg always believed that the
elevation of pHi in cancer cells [19,84]. These groups
intracellular pH of cancer cells was acid because of their
and others have also shown that malignant alkalinisation
high production rates of lactic acid [236238]. Probably,
drives the initial activation of aerobic glycolysis (first
Figure 9: shows conversion of Phosphoenolpyruvate into Pyruvate.

the main reason for overlooking the fundamental pH/ [5,8,10,11,82,84,88,176,255]. Finally, it is interesting to
glycolysis relationship, or at least for being given a note that some recent and otherwise complete reviews
secondary role at that time was that, during the 60s and dealing with Warburgs contributions to modern concepts
70s, the necessary technology to measure pHi was not in cancer metabolism, tumor glycolysis, the initiation
available [239]. This situation, however, started to turn of cancer and oxidative phosphorylation have not even
around just after Warburgs death in 1970, when different considered the tight cause-effect interrelationships
reports began to emphasize that the pHi of cancer cells between intracellular and extracellular pH, glycolysis, the
was the opposite from what was generally thought during Warburg effect and cancer proton reversal [15,248,250].
Warburgs life [83,232,239241]. Thus, Warburg could
not have been aware of the essential fact that cellular Anticancer and antimetastatic potential of the
alkalosis not only activates glycolysis but at the same new and potent NHE1 inhibitors
time hinders oxidative phosphorylation and the entrance
of pyruvate in the Krebs cycle [81,242]. While this simple
consideration may turn his theory completely upside The development and maintenance of a reversed pH
down, at the same time it allows a further insight into the gradient in cancer cells of all malignant tumors (high pH
reasons behind decades of confusion and disagreements inside/low pH outside), which is the opposite to the normal
on his theory of the abnormal respiratory mechanisms situation, is accepted to be directly due to the ability of the
of cancer cells, that he defended until his death in tumor cells to secrete protons (H+) [11,84,255,256]. This
1970 [2,81,82,233,235,242,243]. It is also important proton secretion depends on the buffering capacity of the
to remember that at Warburgs time there were not cell and is driven by a series of membrane-bound proton
techniques permitting the discrimination between the pH transporters (MBPT), mainly the Na+/H+ exchangers
of the cytosol and of the internal organelles. Today we are but also carbonic anhydrases (CAs, mainly CA IX and
able to show that within tumor cells the cytosol is alkaline XII), vacuolar H+-ATPases, the H+/Cl- symporter, the
while the cytoplasmic vesicles are very acidic [244246]. monocarboxylate transporter (MCT, mainly MCT1), also
This is possible thanks to proton pumps and transporters, known as the lactate-proton symporter, the Na+-dependent
on one side eliminating protons outside the tumor cell Cl-/HCO3- exchanger or bicarbonate transporter and the
when expressed on the plasma membrane, while pumping ATP synthase [11,85,256260], each of them having its
them from the cytosol into the internal lumen of the acidic specific inhibitors [261].
vacuoles in order to avoid internal acidification [247]. Among them, the most important, functionally
Most importantly, any consideration concerning the active, cancer-selective and better studied proton
intimate relationship of high pHi and glycolysis was also transporter is the Na+/H+ exchanger isoform 1, NHE1
fully missed during the famous arguments mainly between [262264]. The NHE1 is specifically involved in
Otto Warburg and Sidney Weinhouse published in Science cellular acid-base balance and is the predominant
in 1956 [2,16]. Indeed, all those heated discussions could isoform expressed in tumors, where it has been shown
have been obviated if the true effect of pH on anaerobic that it contributes to cellular pH homeostasis, cell
and/or aerobic glycolysis and oxidative phosphorylation transformation, proliferation, motility, migration, tumor
(parahypoxia) [89] could have been taken into account. growth, invasion, activation of the metastatic process,
Probably, this is also the main reason behind the fact that resistance to chemotherapy and probably also for at least
the search for the real cause underlying the Warburg effect certain cases of spontaneous regression of cancer [84,265
has created many disagreements during the last decades 269]. An elevated NHE1 activity is considered to be the
[2,15,53,238,248254]. All in all, it can now be said that major factor in promoting tumor extracellular/interstitial
Warburg was right up to a certain point but that his critics acidity from even the earliest pre-cancer stage of
were also partially right. However, all of them missed the oncogene-driven neoplastic transformation [176,270,271].
main point. Aerobic glycolysis or damaged respiration was Regarding NHE-related malignant angiogenesis, the
not the primary cause of cancer, as Warburg defended all activity of a significant number of proangiogenic factors
his life. Indeed, the primary cause of cancer appears to and oncogenes has been shown to positively affect NHE1
be, precisely, the main cause behind the aerobic glycolysis expression while, on the contrary, a wide array of anti-
of tumors: a profound disruption of the homeostatic acid- angiogenic drugs inhibit NHE1 [272,273]. Conversely,
balance of the cell represented by an abnormally high pHi decreasing NHE1 expression or inhibiting NHE1 activity
induced and maintained by an extremely varied number leads to acidification of the intracellular space and so to the
of etiological factors of different natures (for a review, see inhibition of glycolysis, thus to tumour cell growth arrest
ref. No. 8). and, finally, to selective apoptosis [234,267,274,275].
In summary, cellular alkalosis represents a common Consequently, the new, potent and highly selective NHE1
final pathway in cell transformation induced by a myriad inhibitors - mainly Cariporide, Phx-3 and Compound 9t -
of different stimuli, from oncogenes to virus to mitogens appear predestined to be taken advantage of as a new and
to growth factors and hormones to gene products highly selective therapeutic magic bullets in probably
most types of human cancer [51,263,276278].

Amiloride: This compound was the first NHE on how to therapeutically target the NHE1-mediated
inhibitor developed and it was shown to decrease metabolic transformations of cancer cells with Cariporide
vasoendothelial growth factor (VEGF) production and [253]. The only non-Amiloride based compounds with
the activity of urokinase-type plasminogen activator NHE1 inhibitory activity that have undergone clinical
(PA), metalloproteinases (MMP) and other proteases, all trials are Cariporide and Eniporide, and, unfortunately,
of which aid in the activation of the metastatic process those trials were not in the field of cancer but in a
[277,279282]. Amiloride alone was shown to achieve cardiological setting and for ischaemic-reperfusion injury
a complete in vivo anti-metastatic effect in transplanted [297301]. Cariporide has been shown to be useful in
tumors in rats [283]. Indeed, there are occasional reports overcoming multiple drug resistance (MDR) and the
of long-term treatment with Amiloride in humans activity of the metastatic process [302]. Besides, it is
achieving remissions of cancer after chemotherapy orally bioavailable and by this route of administration has
had failed to control disease progression [284]. Recent been used but, unfortunately, never to date as an anticancer
publications on the use of amiloride in cancer therapy drug [297,299301,303308]. Cariporide also reduces
discussed the different studies where its utilization had hypoxia-mediated tumor invasion of human tongue
clear anti-neoplastic effects with few side-effects [285]. squamous cell carcinoma by inhibiting NHE1 [309]. In
This potassium-sparing diuretic, apart from having a this study, the authors demonstrated that inhibition of
direct antitumoral, antimetastatic and antiangiogenic NHE1 by Cariporide (HOE-642) suppressed the invasion
effect [283,285,286], at least in part by inhibiting uPA and and migration of Tca8113 cells under hypoxic conditions.
VEGF, has been shown to be well tolerated and safe when In another study pharmacological inhibition of p38 MAPK
used in the chronic situation in pharmacological dosages (mitogen-activated protein kinase) also significantly
in humans, the main side-effect being occasionally suppressed C/EBP expression under hypoxia conditions
increased plasma K+ levels [284,287,288]. Since more after NHE1 inhibition [295]. Indeed, in addition to
selective and powerful NHE inhibitors, like Cariporide, VEGF release and, subsequently, neoangiogenesis, being
Phx-3 and compound 9t are not available for human stimulated by hypoxia, upregulation of VEGF has also
use [19,289,290], amiloride should still be part of new been linked as being secondary to acidic pHe [310,311].
protocols dealing with the concerted use of a cocktail of Also, NHE1-dependent lowering in pHi, apart from
proton transport inhibitors (PTIs) as anticancer agents in deactivating glycolysis at its different enzymatic targets
different human solid tumors [85,284,291]. also reduces the release of VEGF from the tumor cell so
HMA: Along the same line, striking results in hindering motility and invasion [274,312].
different kinds of leukemic cells were reported with the Phx-3: An additional series of NHE1 inhibitors
potent NHE1 inhibitor HMA (5-(N,N-hexametylene)- whose structure is independent of Amiloride have
amiloride), which specifically decreases the pHi well been later developed. Phx-3 (2-Aminophenoxazine-3-
below the survival threshold leading to selective apoptosis one) is highly selective for NHE1 inhibition and was
in a variety of human leukemic cells [274]. This has led shown to selectively stimulate apoptosis in a variety of
to the consideration that inducing a low pHi-mediated cancer cell lines while normal lymphocytes were not
apoptosis as a cancer-specific therapeutic modality for affected [19,234]. Also, Phx-3 also effectively reversed
all cancer cells and tissues could be a new and original a subcutaneously injected adult T-cell leukaemia tumor
approach to clinical therapeutics [19,89,255,257,292]. In growth in animal studies without noticeable toxicity (A.
summary, a great deal of evidence has been accumulating Tomoda, personal communication).
showing that the NHE1, among other MBPT (membrane- Compound 9t: Otherwise, researchers at Bristol-
bound proton transporters) is an important, and possibly Myers synthesized a 5-aryl-4-(4-(5-methyl-1H-imidazol-
selective, anticancer target [89,263,276278,288]. The 4-yl) piperididn-1-yl) pyrimidine analog (compound 9t)
pharmacology and therapeutic possibilities of the rest of that was reported to have an excellent NHE1 inhibitory
the different proton transporters besides NHE1 have been activity 500-fold more potent than cariporide. Besides,
thoroughly reviewed recently and will not be further dealt compound 9 has a reported 52% oral bioavailability, a
with here [84,260,263,269]. plasma half-life of 1.5 hours in rats, low side-effects in
Cariporide: It has been demonstrated that treating mice and may possess a significantly improved safety
various kinds of cancer cells with selective and potent profile over other NHE1 inhibitors [290]. Unfortunately,
inhibitors of NHE1, including Cariporide, suppresses their there have been no further publications utilizing this
invasive capability [268,293295]. Di Sario et al., have compound in any anticancer attempt either in vitro or in
also shown that Cariporide, through its selective inhibition vivo.
of NHE1 and subsequent decrease of intracellular Finally, there are many reasons to think that any
pH reduces proliferation and induces apoptosis in of these new and potent NHE1 inhibitors could have a
cholangiocarcinoma cells [296], leading these authors significant selectivity in the treatment of cancer, since
to suggest the potential therapeutic value of Cariporide even if NHE1 is ubiquitous and plays a fundamental role
against this human tumor. A recent review has also focused in pH housekeeping and volume control, it is also well

known that in normal tissues the NHE1 is quiescent and is on their own, in the context of preventing and controlling
activated only during acidosis or cell shrinkage. Therefore, the metastatic process and in any attempts to reverse MDR
blocking it will have very little effect on normal tissues. [8].
This should be an advantage to consider and exploit as an It is expected that the effects of a targeted therapy
important degree of specificity in the anticancer effect of will not be durable when the therapy is designed to target
NHE1 inhibitors, as it has been known from cell studies a single enzyme or biological molecule. This is because
since the year 2000 [19,274]. cellular pathways operate like webs with multiple
However, during the last few years the holding of redundancies or alternate routes that may be activated
international patents on the new, selective and powerful in response to the inhibition of a certain pathway. For
NHE1 inhibitors by different pharmacological companies this reason, combination and concerted therapies with
has made, and it is still making most difficult to achieve PTIs will be often needed to effectively treat many
any real progress along these new and highly promising tumors screened for pertinent pathway dependence.
anticancer therapeutic lines, as it has been recently It can be advanced that the new NHE1 inhibitors show
proposed [51,313] a great promise as a new and selective approach to the
treatment of a wide array of different malignant tumours
Discussion. Proton transport inhibition (PTI) as and even leukaemias and, hopefully, they will help to
a selective tumor antiglycolytic and anticancer overcome the present impasse and flat progress in cancer
therapeutic approach. A new strategy after one treatment [291,315,316]. These strategies have been
hundred years of metabolic cancer research recently discussed in an occasional review [8,84] and in
a perspective [85], and introduce a real paradigm shift in
cancer treatment. At the same time, there is a continuously
The utilization of different proton transport growing interest in this new paradigm as shown by the
inhibitors (PTIs) in cancer therapeutics was originally number of its publications increasingly available in the
suggested by the group of Pouyssgur and our group as a most recent scientific literature [313, 318, 319].
novel approach to the pH-related treatment of malignant
tumors because of its potential as a more selective and CONCLUSIONS
less toxic approach to therapeutics than conventional
chemotherapy [85,252,314]. Pouyssgur has also proposed The Warburg Effect represents an unusual strategy
the use of PTIs as a valid approach to cancer treatment, of cellular defense that reduces the oxidative stress status
advancing that this pH-targeted therapy, perhaps of the cells and so it has certain evolutionary advantages.
combined with anti-angiogenesis in order to increase This has made targeting of glycolytic enzymes a very
hypoxia-mediated acidosis, would synergistically induce appealing approach for decades, but unfortunately so far
the collapse and massive shrinkage of solid tumours [314]. with dissapointing results. Designing any future strategy
Similarly, from the therapeutic point of view, inhibiting should take into account the crossing of drugs across
tumor glycolysis and reverting the Warburg effect by the heterogeneous multi-habitats of cancer cells and
selective intracellular acidification has been advanced as a tissues in order to cover all the tumor cell populations.
treatment of cancer [8,19]. Indeed, in the light of the older In conclusion, before investing in the discovery of new
and the more recent contributions [5,19,84,233,234,242] pathways and introducing new biological techniques, a
it can now be concluded that counteracting the Warburg new approach to cancer therapeutics could be achieved
effect and its aerobic glycolysis through any therapeutic by introducing novel and broadminded perspectives to the
method directed to selectively induce intracellular fight against human malignant tumors and leukaemias.
acidification in cancer cells and/or reverting proton In this vein, even from the times of Walter Cannon
reversal now appears to represent one and the same and Hans Selye cell acid-base balance has been recognized
phenomenon. to be the main parameter to define cellular homeostasis,
In summary, the most potent and promising the life of cells being possible only within a very narrow
Amiloride and non-Amiloride derivatives, such as range of pH (less than one unit). It becomes essential
Cariporide, Phx-3 and compound 9t [19,268,290,298] to recognize that the pH of normal cells and cancer
need to be included in pre-clinical and clinical trials as cells deviate towards opposite ends of a biological and
an important part of the anticancer armamentarium. metabolic spectrum. This energetic abnormality represents
That these compounds have not yet reached translational the largest difference among normal cellular physiology
oncology becomes difficult to understand taking into and cancer pathophysiology and a recently recognized
account the massive theoretical background, available new and selective hallmark of all cancer cells and tissues
preclinical data as well as the results of the molecular, [8,176].
biochemical and metabolic studies already available at
the present time. These anticancer compounds can be
useful either as antitumoral and chemotherapeutic agents

Summary selectively exploited in the treatment of many different
malignancies.
Cariporide, other potent NHE1 inhibitors of the
From an etiological and etiopathogenic perspective,
Amiloride series, as well as powerful and selective
the hydrogen-related dynamics of malignancy has become
NHE1 inhibitors of the non-Amiloride series, like
a new approach to cancer and its dynamics and energetic
Phx-3 and compound 9t, have the potential of being
mechanisms that is helping to reach a better understanding
highly promising, minimally toxic and truly effective
of several, until now disparaged areas of cancer research
anticancer agents in a wide array of malignant tumours
both at basic and clinical levels, as well as of the intimate
and leukaemias, hopefully representing a new paradigm
nature of malignant disease. This unifying thermodynamic
in cancer therapeutics.
view now permits an integration of different cancer fields,
In order to achieve significant progress along
ranging from cell transformation and metabolism, local
these new lines, a radical change of vision is strongly
growth and invasion, neovascularisation and the activation
needed from the pharmacological companies that hold
and progression of the metastatic process (pH centric
international patents on the new and selective NHE1
paradigm).
inhibitors and international legislations in these areas.
From a therapeutic perspective, the primary
This will be a great help furthering preclinical and cancer
aim of this pH-based approach to cancer treatment
clinical research and treatment using proton transport
is to manipulate the selective forces controlling the
inhibitors in modern and less toxic anticancer therapeutics.
dysregulated pH dynamics of all cancer cells and tissues
in-order-to regress tumor growth, control local invasion
and deactivate the metastatic potential of malignant ACKNOWLEDGMENTS
tumors. All available evidence seems to indicate that this
would take place regardless of pathological differences, We thank Mr. Cameron Burton for sending
tissue type or genetic origin. This therapeutic approach necessary materials. SJR was supported by grant #11348
would also provide much less toxicity than present day of the Italian Association for Cancer Research (lAIRC)
treatments, probably also more effective therapies than and his laboratory is part of the Italian network Istituto
any other chemotherapy known to date and it has real Nazionale Biostrutture e Biosistemi (INBB). SH was
possibilities to become a successful strategy in treating supported by the Mercedes Castresana Foundation,
human cancer in general. A pathologically elevated pHi Vitoria, Spain, and SH and JDPO by the Association for
and its associated proton reversal (a reversed pH gradient Proton Cancer Research and Treatment, Madrid, Spain.
in cancer cells and tissues (pHi to pHe, pHi/pHe) CR is supported by Vertex Pharmaceutical Ltd. and
can perhaps be now considered the most specific cancer BBSRC.
abnormality and essential hallmark of all kinds of The authors also want to apologize to all
malignant cells and tissues. investigators in the field, past and present, whose work is
This hydrogen ion-based perspective has also not specifically cited in this review.
permitted the better understanding of the Warburg effect,
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www.impactjournals.com/oncoscience Oncoscience 2014, Vol.1, No.

Glycolysis, tumor metabolism, cancer growth and dissemination.

INTRODUCTION AND PERTINENT cancer cells were highly fermentative. He hypothesized

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Figure 1: Conversion of Glucose into Glucose 6 Phosphate.

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Figure 2: Shows isomerization of Glucose-6-phosphate into fructose-6-phosphate.

Figure 3: Shows phosphorylation of Fructose 6-phosphate into Fructose 1, 6 bisphosphate.

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Figure 5: conversion of D-glyceraldehyde 3-phosphate (GADP) into into 1, 3-bisphosphoglycerate.

Figure 6: conversion of 1, 3 - bisphosphoglycerate into 3-phosphoglycerate.

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Figure 7: Shows conversion of into 3-phosphoglycerate into 2-phosphoglycerate.

Figure 8: shows conversion of 2-phosphoglycerate into Phosphoenolpyruvate.

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Figure 9: shows conversion of Phosphoenolpyruvate into Pyruvate.

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