Biochimica et Biophysica Acta 1866 (2016) 87–105

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbacan

Review

Glycolysis inhibition as a cancer treatment and its role in an anti-tumour

immune response
Kheshwant S. Gill a,b, Philana Fernandes a, Tracey R. O'Donovan a, Sharon L. McKenna a, Kishore K. Doddakula b,
Derek G. Power a,c, Declan M. Soden a, Patrick F. Forde a,⁎
a
Cork Cancer Research Centre, Western Gateway Building, University College Cork, Cork, Ireland
b
Cardiothoracic Surgery Department, Cork University Hospital, Cork, Ireland
c
Department of Medical Oncology, Mercy University Hospital, Grenville Place, Cork, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for ATP
Received 15 March 2016 generation. Altered energy metabolism is a biochemical fingerprint of cancer cells that represents one of the
Received in revised form 29 June 2016 “hallmarks of cancer”. The immune system can prevent tumour growth by eliminating cancer cells but this
Accepted 30 June 2016
editing process ultimately results in poorly immunogenic cells remaining allowing for unchallenged tumour
Available online xxxx
growth. In this review we look at the glycolysis pathway as a target for cancer treatments. We also examine
Keywords:
the interplay between the glycolysis modulation and the immune response as an anti-cancer therapy.
Glycolysis © 2016 Elsevier B.V. All rights reserved.
Glycolytic modulator
Immune-metabolic interaction
Electroporation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2. Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.1. Warburg hypothesis/effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.2. Cellular metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.3. Glycolysis in cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3. Rationale behind targeting glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1. Glycolytic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1.1. Hexokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1.2. Phosphofructokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
3.1.3. Glucose transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
3.1.4. Pyruvate dehydrogenase kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
3.1.5. Lactate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.1.6. Pyruvate kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.1.7. Fatty acid synthesis/fatty acid oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4. Relationship between cell glycolysis and immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.1. Immune cell metabolism and tumour microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.1.1. Innate immune cell metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.1.2. Adaptive immune cell metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5. Immune signaling and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6. Metabolic modulators and immune-glycolytic therapeutic implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.1. Targeting tumour hypoxia-inducible factor-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.2. Targeting uncoupling proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

⁎ Corresponding author.
E-mail address: p.forde@ucc.ie (P.F. Forde).

http://dx.doi.org/10.1016/j.bbcan.2016.06.005
0304-419X/© 2016 Elsevier B.V. All rights reserved.
88 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105

6.3. Targeting tumour glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

6.4. Targeting the mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7. Delivery methods of glycolytic modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7.1. Viral drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7.2. Non-viral drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.2.1. Synthetic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.2.2. Natural polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.3. Physical methods of drug-delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.3.1. Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.3.2. Other methods of physically facilitating drug-delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

1. Introduction
Intracellular chemical transformations sustain life and this is re-
ferred to as metabolism. Organisms grow and reproduce, respond to
their environment, and maintain their structure as a result of enzyme-
catalyzed metabolism. Biochemical reactions in cells ensure daily oper-
ations of a cell are accomplished. These reactions are either up- or
downregulated depending on the cell's needs and functions. Monitoring
of the numerous cellular anabolic (breaking down of organic matter)
and catabolic (metabolic component construction) pathways is crucial
to ensure they are balanced in a coordinated fashion at any given
time. Cells organise these reactions into different enzyme-catalyzed
pathways to achieve this goal.
Metabolic pathways are organised chemical reactions of metabolism
where a chemical compound is converted into another one by a series of
enzymatic activity. Enzymes are vital to metabolism. This is because
they allow the organism to drive energy-consuming reactions that will
not occur independently. This is achieved by coupling reactions with
spontaneous reactions which release energy. Enzymes speed up reac-
tion rates as they act as catalysts besides allowing the metabolic path-
way regulation secondary to changes in the cell's environment or to
other cellular signaling.
Basic metabolic pathways and their components are similar across
vast different species [1]. This is exemplified by a set of carboxylic acid
(citric acid intermediates) which is present in all known organisms.
The early appearance in evolutionary history and efficiency in retention
have likely led to the similarities in metabolism [2,3]. The process of
catabolism harvests energy by means of cellular respiration, and in
anabolism, cellular components are constructed using energy e.g.
nucleic acids and proteins. An example of a metabolic pathway which
is common in virtually all cells, both prokaryotic and eukaryotic, is
glycolysis.
2. Glycolysis
and (2) pathway that does not involve the mitochondria (glycolysis;
less energy-efficient and a shorter pathway). Oxidative phosphorylation
is a pathway involving mitochondrial respiration. Depending on intra-
cellular requirements and available resources, cells undergo either gly-
colysis or oxidative phosphorylation to metabolise glucose. Glycolysis
is inhibited (‘Pasteur effect’) by the presence of oxygen in most mam-
malian cells. Oxygen allows the mitochondria to oxidise pyruvate to
carbon dioxide and water. The maintenance of energy production in
various conditions of oxygen concentrations is met by the mammalian
cells' metabolic adaptability.
‘Anaerobic glycolysis’ is whereby glucose is metabolised via glycoly-
sis instead of mitochondrial oxidation in low levels of oxygen. This
results in the conversion of pyruvate into lactate, which is then
exported. Interestingly, ‘aerobic glycolysis’ (glycolysis in the presence
of oxygen) is also seen in cancer cells. This almost a century-old phe-
nomenon in cancer cells is known as the ‘Warburg effect’, and was
hypothesised in 1924 by the German scientist Otto Heinrich Warburg.
2.1. Warburg hypothesis/effect
Cancer cells mainly produce energy by an increased rate of glycolysis
(200 times more as compared to normal tissues of origin) followed by
fermentation of lactate in the cytosol of the cell, even if oxygen is plen-
tiful [5]. This is to fulfill their bioenergetic and biosynthetic demands to
support rapid proliferation. This observation in oncology is called the
‘Warburg effect’. In normal cells however the rate of aerobic glycolysis
is lower, and is followed by oxidative phosphorylation in the mitochon-
dria, where pyruvate is oxidated [6–8]. It was postulated by Otto War-
burg that this metabolic change is the fundamental cause of cancer
[9], a claim now known as the Warburg hypothesis. However, it was
then discovered that mutations in oncogenes and tumour suppressor
genes are responsible for malignant transformation. Instead of a cause,
the Warburg effect is considered to be more of a result of these genetic
mutations [10,11].

Glycolysis pathway of cellular respiration is a series of reactions that
constitute the first phase of most carbohydrate catabolism. The word
glycolysis is derived from two Greek words and means the breakdown
of something sweet. Glycolysis is an oxygen independent metabolic
pathway, meaning it does not use molecular oxygen for any of its
reactions. It occurs in the cellular cytosol of most organisms. Embden–
Meyerhof–Parnas (EMP pathway) [4] is the most common type of glycol-
ysis. Other types include the Entner-Doudoroff pathway and various
hetero- and homofermentative pathways.
The glycolytic pathway can be divided into two phases. Firstly, the
preparatory phase (or investment phase) is where ATP is utilised and
followed by production of ATP in the second Pay Off phase. In the cell,
glucose catabolism occurs via two main pathways, (1) one which in-
volves mitochondrial respiration (longer but energy-efficient pathway)
2.2. Cellular metabolism
Normal cell metabolism derives about 70% of its energy from the
Krebs cycle and only 20% from glycolysis. Most of its energy production
is via oxidative phosphorylation. In contrast, cancer cells exhibit a defec-
tive tricarboxylic acid (TCA) cycle and derive little or no energy from it.
Instead, they derive almost all their energy from glycolysis (fermenta-
tion phosphorylation) and in the absence of oxygen. Normal cells oper-
ate at normal metabolic levels and reproduce themselves at a regulated
phase by possessing hormones and enzymes behaving in balanced
manner. Normal functioning cells orderly-divide to proliferate only
when in demand. Cancer cells on the other hand are overactive and re-
produce themselves in requiring more nutrients. These cells have over-
active or underactive enzymes and hormones, and develop an aberrant
 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
DNA or gene structure or acquire abnormal number of chromosomes.
This continues to be created uncontrollably and ultimately these
surpluses of cells form a mass. Tumour cells possess a high rate of
glucose uptake and glycolytic metabolism. The majority of their en-
ergy is supplied by glycolysis, whilst maintaining increased rates of
lactic acid production. This is important for cancer cell survival in
hypoxic environments.
2.3. Glycolysis in cancer cells
Only about 10% of normal cells energy is produced by glycolysis. Mi-
tochondrial respiration contributes to the remaining 90%. A glucose
molecule is metabolised into two molecules of pyruvate and NADH+
each, and gains 2 molecules of ATP. This is a markedly smaller amount
of energy produced as compared to 38 molecules of ATP produced via
mitochondrial respiration. Therefore, to obtain sufficient energy to
grow and proliferate, cancer cells require an increased glucose uptake.
In hypoxic conditions, glucose via glycolysis is catabolised to lactate to
produce ATP. The mitochondriae are unable to produce ATP in oxidative
phosphorylation during hypoxia because oxygen is crucial for electron
transport. Also, in hypoxic conditions, the monocarboxylate trans-
porters MCT4 (plasma membrane lactate transporter) are up regulated
via a HIF-1α mediated mechanism. This enables cells to dispose the
accumulation of lactic acid rapidly [12].
Given the small yield of energy via glycolysis, it is understood that
not only the metabolic switch to glycolysis from oxidative phosphoryla-
tion, but also mitochondrial dysfunction [13,14] is important for cancer
cell growth. Cancer cells develop a high glucose uptake rate and glycol-
ysis to produce energy. This increased rate of glycolysis in hypoxia leads
to higher levels of lactate, which is sufficient for cancer cell survival.
Mitochondrial defects are commonly seen in cancer. The enzymes
succinate dehydrogenase (SDH), isocitrate dehydrogenase (IDH) and
fumarate dehydrogenase (FDH) in the TCA cycle are affected by mito-
chondrial DNA (mtDNA) mutations [15]. Besides that, the bioenergetics
of cancer cells are affected by gene mutations in nuclear DNA (nDNA) of
the mitochondria [15]. Thus it is evident that a shift in cancer cell metab-
olism can be caused by mitochondrial dysfunction. Having said that,
there are several benefits of aerobic glycolysis that drive cancer cells
to favour it over mitochondrial oxidation [16]. The glycolytic rate in can-
cer cells are accelerated to about 100 fold thereby resulting in higher
ATP production [17,18], required for cellular maintenance and sufficient
for cancer growth. Besides ATP production, the biosynthesis of macro-
molecules is essential for tumour cell proliferation [19]. Ribose-5-
phosphate and NADPH (essential for nucleic acid and lipid synthesis)
are produced via the Pentose Phosphate Pathway (PPP) promoted by
the accumulation of these glycolytic intermediates. The antioxidant glu-
tathione (GSH; produced in presence of NADPH) maintains the redox
levels and counteracts chemotherapeutic agents thus protecting cancer
cells [20,21]. The transketolase (TKTL1) enzyme involved in the PPP has
been shown to improve cell survival in stressful times by affecting
chemosensitivity of cancer cells to chemotherapeutic drugs e.g. imatinib
[22] and cetuximab [23]. In summary, aerobic glycolysis and the PPP
provide multiple advantages to cancer cells and help tumour to progress
besides resisting them from therapy. And this key signature of cancer
metabolism (glycolysis) caters therapeutic intervention targets.
3. Rationale behind targeting glycolysis
Targeting the glycolytic pathway has the potential to be utilised as a
cancer treatment strategy. Tumour cells have a higher uptake of glucose
not only to meet the manifold requirements but also the high-energy
demand. Apart from providing energy, intermediates of the glycolytic
pathway are also used for anabolic reactions such as glucose-6-phos-
phate used to synthesise ribose-5-phosphate. This is then further used
in the synthesis of nucleic acids. Dihydroxyacetone is utilised in lipid
synthesis. Tumour cells proliferate rapidly and in an uncontrollable
89
fashion, therefore the increased glucose uptake is not merely a biochem-
ical switch but also a metabolic transformation that is indispensible. On-
cogenic activation (e.g. c-myc, Akt) up-regulates glycolytic activity in
cancer cells [24] via HIF-1α activation. Akt activates aerobic glycolysis
and more this makes cancer cells dependent on glycolysis to survive
[25]. The anabolic and catabolic processes in cancer cells are coordinated
and regulated by networks of signaling pathways such as the mammali-
an Target of Rapamycin (mTOR). AMPK (energy sensing molecule) is
able to influence the activation mechanism of mTOR complex 1
(mTORC1). This in turn either delays or stops the energy consuming
synthetic processes [26]. Activation of c-myc and HIF-1α genes regulates
the expression of glycolytic enzymes, which in turn are regulated by
mTORC1 [26–28].
It is evident that cancer cells benefit from aerobic glycolysis with the
PPP not only to promote tumour growth but also offering its therapy re-
sistance. Hence, tumour glycolysis provides the best possible targets for
therapeutic interventions. Fig. 1 is a pictorial representation of glycolytic
pathway targets.
3.1. Glycolytic targets
In addition to providing sufficient energy, the environment of cancer
cell glycolysis also promotes proliferation by providing its building
blocks, whilst supressing apoptosis at the same time. Cell death (ATP-in-
dependent) will occur from rapid depletion of ATP as a result of direct
glycolytic inhibition. This strategy will also likely damage normal
healthy tissues. Non-malignant tissues e.g. smooth and skeletal muscle
and normal viscera will be affected as glycolytic inhibition is non-
selective. Translational potential of glycolytic inhibitors such as Hexoki-
nase inhibitors and 3-Bromopyruvate are limited because of this reason.
Their clinical trials have not been successfully completed. Pre-clinical
data suggests the induction of necrosis in cancer and has also shown sig-
nificant toxicities in animal models at even slightly-above therapeutic
doses.
3.1.1. Hexokinase
Hexokinase (HK) is a tissue-specific isoenzyme which catalyzes the
phosphorylation of glucose to glucose-6-phosphate (G6P), after glucose
enters the cell via glucose transporters (GLUT). This ATP-dependent re-
action is the first step in glycolysis, and is also rate limiting. G6P serves
as the starting point for the sugar to enter the glycolic pathway or the
PPP, or for glycogen synthesis. HK has four isoforms, and they are desig-
nated HK I, II, III, and IV (glucokinase found in hepatocytes) or A, B, C,
and D. Among these isoforms, HK-II plays a key role in the development
of the Warburg phenotype exhibited by most types of cancer. HK-II iso-
form is found mainly in skeletal muscle and adipose tissues. Overex-
pression of HK-II is also seen in some tumour tissues and associated
with poor prognosis [29,30]. Phosphorylated HK-IIs are able to bind to
the voltage-dependent anion channels (VDACs) found on the outer mi-
tochondrial membrane, besides its glucose phosphorylation activity.
Negative feedback inhibition of G6P is prevented by this VDAC-HK in-
teraction to ensure tumour cells entrap enough glucose. VDAC-HK inter-
action also confers HK direct access to ATP generated by mitochondriae.
Binding of BAX (protein apoptotic regulator which binds to VDAC in
normal cells) is interfered by VDAC-HKII interaction [31]. This VDAC-
HKII association also affects the pro-apoptotic functions of BAX in nor-
mal cells, where cytochrome C (located in the inter-membrane mito-
chondria space) escapes from the mitochondria thus triggering
apoptosis. This ultimately leads to tumour cellular apoptosis being
prevented.
HKs maintain the downhill concentration gradient that favours the
facilitated transport of glucose into cells by catalyzing the first step in
glycolysis. ‘Trapping’ of glucose and 2-deoxyhexoe glucose analogs
(e.g. 2-deoxyglucose, and 2-fluoro-2-deoxyglucose) within cells is due
to the addition of a charged phosphate group at the 6-position of hex-
ose. This is because charged hexose phosphates cannot easily cross the
90 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
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cell membrane. HKs, besides GLUT1, exert the main control (71%) of gly-
colytic flux in hepatocellular and cervical carcinoma cell lines [32].
Many anti-cancer strategies implicating HK-II as a target have been of
big interest recently. Publication of work on identifying HK-I–HK-IV
inhibitors goes back to the mid-20th century e.g. dehydroascorbic
acid, alloxan and other compounds were found to inhibit liver HK [33].
After phosphorylation by HK, 2-deoxyglucose (2DG), a structural an-
alog of glucose, enters cells through GLUTs and selectively accumulates
in cancer cells. This is because they are not able to progress through the
glycolytic pathway. Cellular energy metabolism and its survival are like-
ly to be profoundly affected by inhibition of HK. Thus, HK is an attractive
target for anticancer agents. Lonidamide is an inhibitor of HK-II and has
been shown to induce apoptosis and treat multidrug resistance in mul-
tiple cell lines [34–36]. Phase II clinical trials of Lonidamide for benign
prostatic hyperplasia treatment have been suspended due to its toxicity
on the liver [36,37]. Combination of Lonidamide with Paclitaxel (che-
motherapeutic agent) is undergoing clinical trials for cancer treatment
[7,38].
2-DG and 3-bromopyruvate (3-BrPA) are also well known HKII in-
hibitors [39,40], which have shown to interrupt binding of HKII to the
mitochondrion besides inducing cell death by depleting ATP and
inhibiting the cell cycle progression. 3-BrPA inhibition of glycolysis
causes ATP depletion and this suppresses ATP-binding cassette (ABC)
transporter activity and drug efflux leading to more drug retention.
This results in the ability of 3-BrPA in overcoming chemoresistance
and improving cancer therapeutics.
2-DG induces cancer cell death by depriving intracellular glucose
levels. This results in induction of stress-related proteins [41,42], the
generation of free radicals [43], and inhibition of energy metabolism
[42–44]. Combination of 2-DG with ionizing radiation (IR) to treat ma-
lignant gliomas in recent clinical trials showed it to be well tolerated
with low toxicity levels and surrounding normal tissues being un-
harmed[45–47]. 2-DG was shown to activate pro-survival pathways in
cancer cells contrary to the wide believe as an anticancer agent [48].
Hypoxic cells have also demonstrated chemoresistance against 2-DG
[49]. This challenges the success of 2-DG as a single agent for
antiglycolytic therapy. However, 2-DG showed good outcomes when
combined with other treatments [50,51]. Clinical trials for 2DG as a gly-
colytic inhibitor for prostate cancer (NCT00633087) and intracranial
neoplasms (NCT00247403) have been suspended, mainly due to non-
selectivity of the drug thus causing damage to non-cancerous cells too.
3.1.2. Phosphofructokinase
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the
irreversible transfer of a phosphate from ATP to fructose-6-phosphate.
PFK is considered to be a key regulatory enzyme for glycolysis because
of the irreversible nature of this step in glycolysis, and its activity is ex-
tremely sensitive to small changes in pH, with its activity dropping as
pH decreases [52,53]. PFKs activity is inhibited by allosteric regulation
by ATP itself when ATP levels are high in the cell. High intracellular
levels of citrate also inhibit PFK activity. The enzyme ATP citrate lyase
converts citrate into acetyl-CoA in the cytosol. PFK activity is inhibited
because the cell can get enough energy from the citric acid cycle and
thus not depend on glycolysis to supply carbons into the citric acid
cycle. The two types of PFKs are PFK-1 and PFK-2. Translation of four
mRNAs produces the minimum six isoenzymes of 6-phosphofructo-2-
kinase/fructose-2.6-biphosphatase (PFK-2/FBPase). Concentration of
F2,6BP determines PFK-1 activity and thus promoting glycolysis
when levels are high. PFK-1 activity is stimulated by fructose-2,6-
bisphosphate (F-2,6-BP) and is often overexpressed in various primary
cancers, metastases and in transformed cells [54]. PFK-2/FBPase
Fig. 1. Schematic representation of the glycolytic pathway. Following uptake of glucose from the bloodstream into the tumour microenvironment, it is delivered intracellularly by GLUT
transporters. In glycolysis the glucose is processed by a sequence of reactions, which lead to the production of pyruvate (aerobic conditions) and lactate (anaerobic conditions). HIF-1
affects the expression of enzymes and transporters in the glycolysis pathway.
91
expression pattern is often elevated in colon, breast, ovarian and thyroid
cancer with a particular preference for PFK-2/FBPase [55].
The PFK inhibitor, chalcone-like 3-(3-pyridinyl)-1-(4-pyrinidyl)-2-
propene-1-one (3PO), is a PFKFB3-specific inhibitor. 3-PO has been
found to decrease intracellular F2,6BP and glycolytic flux in transformed
cells and to selectively affect ras-transformed cells and decrease
tumourigenic growth in several adenocarcinomas in vivo [56]. In non-
small cell lung carcinoma (NSCLC), 3-PO has been used in combination
with ascorbic acid and was shown to have a synergistic activity. Apopto-
sis was induced with this combination strategy by an ROS-dependent
mechanism [57]. The effects of this compound are yet to be explored
in clinical trials.
3.1.3. Glucose transporters
Glucose transporters (GLUTs) are a wide group of membrane
proteins that facilitate the transport of glucose and other substrates
over a plasma membrane and entering into cells as nutrients. 12
GLUTS are encoded by human genome. A sodium-linked transporter
system (SGLT) and facilitative GLUTs [58] are the two different types
of membrane transporter proteins. These assist the transport of glucose
and other sugars through the lipid bilayer of the cell membrane down
its concentration gradient. GLUT-1 is related to chemo- or radiotherapy
responses and rectal cancer stages [59]. Human cell lines of breast and
lung cancer (NSCLC) were shown to reduce in cellular proliferation by
50% and 75% respectively by inhibiting GLUT-1, and also mediated
apoptosis [60].
Overexpression of GLUT-1 and/or GLUT-3 is associated with poor
prognosis of several types of human tumours [61,62]. GLUT-1 transport-
er is a potential target for anticancer therapy as inhibition of its expres-
sion is associated with reduction in tumour growth [63]. Phloretin, a
natural type of phenol found in the leaves of apples and pears, is a com-
petitive GLUT inhibitor. It has been shown to inhibit tumour growth
and/or trigger apoptosis in leukaemia, melanoma, liver, colon, and
breast cancers in vitro and in xenograft models. Another flavonoid,
silybin (flavonolignan) was tested in men with prostate cancer [64]
and liver cancer (trials NCT00487721 and NCT01129570). It showed
this drug to have low tissue penetration and therefore needed high
doses for effectiveness [64].
In contrast to flavonoids, fasentin, WZB117 and STF-31 are promi-
nent inhibitors of GLUT-1 that induce glucose deprivation, starvation
and subsequent death in renal cell carcinoma cell lines and animal
models [65]. Overexpressed GLUTs have been explored as receptors to
carry nanoparticles loaded with drugs across the blood-brain barrier
(BBB). Jiang et al. demonstrated that 2-DG-modified polymer nanopar-
ticles showed efficient GLUT-mediated transcytosis across BBB as well
as endocytosis into glioma cells enabling targeted delivery of paclitaxel
for brain glioma therapy [66]. These results show that the Warburg ef-
fect confers distinct characteristics to tumour cells that can be selective-
ly targeted for anti-cancer therapy [65].
3.1.4. Pyruvate dehydrogenase kinase
Mitochondrial activators increase mitochondrial influx and subse-
quently increasing mitochondrial function. They have proven to be a
strategy towards inhibiting cancer growth. For example, dichloroacetate
(DCA)–pyruvate dehydrogenase kinase (PDK) inhibitor acutely in-
creases glucose oxidation (GO). Dromparis P et al. showed that the
mitochondriae of cancer cells are not permanently damaged but rather
suppressed, because GO occurs exclusively in the mitochondria. There
is a solid reason for developing similar inhibiting-molecules as antican-
cer therapies. A study showed that DCA depolarised the hyperpolarised
membrane potential of non-small cell, Glioblastoma multiforme (GBM)
and breast cancer cell mitochondriae. This suggests the restoration of
92 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
mitochondrial function [67]. By enhancing GO and lowering the produc-
tion of lactate, DCA decreases the glycolysis to GO ratio. Xenotransplant
tumour size was shown to reduce in size when treated with DCA and it
also lessened the aggressiveness of breast cancer [68]. Non-cancerous
cell mitochondriae were not affected with DCA treatment but cancer
cells treated with DCA showed to have activated Kv-channels, increased
mROS and inhibited NFAT. Many other studies in colon [69], prostate
[70], and endometrial [71] also showed regression in tumour size
when treated with DCA. Mitaplatin (cisplatin + DCA) is a cancer selec-
tive drug and treatment which induces mitochondrial-dependent apo-
ptosis. Furthermore, it also overcame the resistance towards cisplatin
initially thus reducing proliferation of tumour.
3.1.5. Lactate dehydrogenase
Lactate dehydrogenase (LDH) is a tetrameric enzyme with two sub-
types: LDH-A (skeletal muscle type or LDH-M) and LDH-B (heart type or
LDH-H). In hypoxic cells, conversion of pyruvate into lactate (final step
in the glycolytic pathway) is catalyzed by LDH-A whilst LDH-B catalyzes
conversion of lactate into pyruvate. Lactate accumulation substantially
reduces intracellular pH and this is detrimental to the cell. Production
of NAD+ from oxidation of the cofactor NADH is necessary from the re-
duction step of pyruvate into lactate to continue glycolysis. Evidence
suggests that LDH-A, which is upregulated in invasive glycolytic can-
cers, plays a critical role in cell proliferation. This allows the tumours
to survive even in low oxygen levels. Elevated serum LDH levels are as-
sociated with poor prognosis in pancreatic [72], renal [73], nasopharyn-
geal [74] and lung cancers [75].
LDH activity is not needed for pyruvate metabolism through the TCA
cycle, and therefore inhibitors of this enzyme should spare glucose me-
tabolism of normal non-proliferating cells. Several studies have already
found that the inhibition of LDH-A in cancer cells could stimulate mito-
chondrial pyruvate metabolism (in a sense mimicking the effects of
DCA), decrease mitochondrial membrane potentials and finally leading
to cancer cell death [76,77]. Oxidative stress is increased when intracel-
lular ATP depletion occurs as a result of treatment with FX11 (LDH in-
hibitor and antimalarial drug). Tumour cell death both in vitro and
in vivo has been shown with FX11 treatment [76]. FX11 inhibits pro-
gression of sizable human lymphoma and pancreatic cancer [76]. An-
other LDH inhibitor, oxamate, sensitizes resistant cancer cells to
chemotherapeutic agents. A synergistic inhibitory effect was shown in
combinational treatment of paclitaxel and oxamate in paclitaxel-
resistant breast cancer cells, which had higher apoptotic levels [78].
Lactate suppresses the function of tumour-specific T cells and in-
hibits tumour specific cytotoxic cell activity [79]. It also induces defect
in signal transduction [80]. Dendritic cells' (DC) Ag-presenting ability
is altered by low pH levels [81]. Furthermore, high lactate levels also in-
hibit cytotoxic activity [82] and promote IL-23/IL-17 pro-inflammatory
pathway [83]. Inhibition of LDH has demonstrated promising effects in
preclinical investigations and its further progress depends on the out-
come of clinical trials. However, in order to translate into clinical prac-
tice, further studies are warranted to demonstrate target specificity
and therapeutic efficacy of experimental agents.
3.1.6. Pyruvate kinase
Pyruvate kinase (PK) is a critical enzyme in the glycolysis pathway.
PK has two isoenzymes, encoded by the PKM gene: PKM1 and PKM2.
PK converts phosphoenolpyruvate (PEP) to pyruvate at the final irre-
versible rate-limiting glycolytic step, and produces one molecule of
ATP in the process. The fate of pyruvate depends on local oxygen con-
centration levels in normal cells. In the presence of oxygen, pyruvate en-
ters the mitochondria where it is converted into acetyl-Co-A by
pyruvate dehydrogenase complex (PDC) or into alanine by transamina-
tion. However, in low oxygen levels pyruvate is converted into lactic
acid by LDH [84].
The PKM2 is highly expressed in cancer cells [85,86] and the glyco-
lytic flux regresses to limit the generation of ATP in cancer cells by
inhibiting PKM2. This results in the opposite to detrimental effects to tu-
mour growth. Both normal and cancer cells need to generate macromol-
ecules such as proteins, lipids and nucleic acids. Cell proliferation will
not occur in the absence or low levels of these macromolecules even
in the presence of ATPs. Shuttling of PEP from slowing down glycolysis
will result in the production of macromolecules necessary for cellular
proliferation [87–89]. However, there is still interest in inhibiting
PKM2 as it is important to tumour metabolism. A combination of mod-
ulation of both, the transfer of an amine from glutamine during mito-
chondrial respiration and PK, has been suggested as a method for
metabolic targeting of tumours. This is because glutamine metabolism
is important for tumour metabolism. Many steps in glutamine metabo-
lism have been shown to be regulated by Myc [90]. Novel inhibitors and
nucleic acid therapies were designed to target PKM2 to achieve the de-
sired anticancer activity. Thallion Pharmaceuticals started a Phase II
clinical trials in 2007 with PKM2 inhibitor called TLN-232/CAP-232, ad-
ministered to patients with refractory metastatic renal cell carcinoma
[91] and it was found to be well tolerated, but Phase III of this study
was terminated due to legal reasons.
PKM2 interacts with suppressor of cytokines signaling 3 (SOCS3)
making it highly immunomodulatory. Antigen-presenting ability of
DCs was disrupted with the PKM2-SOCS3 interaction [92]. T-cell upreg-
ulation of glucose uptake and glycolysis during interaction with
antigen-presenting cells (APC) produces instant ATP to ensure a
sustained immunogenic response to an antigen [93,94]. Furthermore,
PKM2 has also been shown to interact with IgE receptor on the cells
resulting in the inhibition of its activity [95].
3.1.7. Fatty acid synthesis/fatty acid oxidation
In the cytoplasm, fatty acids are produced from acetyl-CoA, via
malonyl-CoA, and catalyzed by fatty acid synthase (FAS). The synthesis
of cancer cell membrane requires a high amount of fatty acids and this
demand is fed from the Krebs cycle intermediate citrate [19] rather
than external sources. The metabolite citrate accumulates when en-
zymes of the Krebs cycle are mutated. This has been shown to be as-
sociated with cancer. In pre-clinical breast cancer model, inhibition
of fatty acid synthesis by the compound C75 also showed increased
apoptotic levels and inhibition of tumour growth [96,97]. The en-
zyme malonyl-CoA decarboxylase (MCD) catalyzes the conversion
of malonyl-CoA into acetyl-CoA and carbon dioxide. Inhibition of
FAS results in increased levels of its substrate malonyl-CoA, which
allows FAS inhibitors to display their anti-tumour effects. Therefore,
inhibition of MCD can be used to enhance the efficacy of FAS inhibi-
tors as anti-cancer agents [98]. Through the Randle cycle, fatty
acid oxidation (FAO) inhibition indirectly promotes GO and thus
inhibiting growth of cancer.
4. Relationship between cell glycolysis and immune cells
Like other cells, immune cells use up the nutrients through cellular
metabolism and thus modulating its growth, volume and ion integrity
[99]. Immune cells utilise the ATPs produced to perform various
functional activities and its proliferation, in addition to housekeep-
ing proliferation and sustenance [99,100]. Notable and rapid changes
in metabolism are required as most of these actions are thermody-
namically taxing [99,100]. Besides that, immune cells must also be
able to increase ion signaling and the turnover of phospholipids,
macromolecule manufacturing and ultimately facilitate changes in
the cytoskeleton [101]. Resting immune cells depend on imported
glucose to meet their metabolic needs as they contain little amounts
of stored glycogen [93,102,103].
4.1. Immune cell metabolism and tumour microenvironment
The diverse functions of immune cells give rise to the differences in
metabolism between the immune and other cells in the body. To
 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
generate ATP, alveolar cells depend on oxidative phosphorylation [104]
whereas those always in contact with oxygenated blood use mitochon-
drial respiration. APCs migrate and present the foreign antigens detect-
ed by travelling immune cells, to lymphocytes. There are epidermal DCs
[105] in the deep tissue layers of the skin (lower oxygen tension com-
pared to dermis layer [106]). To survive and function properly in hypox-
ic conditions, immune cells must be able to use different forms of
metabolism [107]. Lymphocytes traverse the cell wall of the endotheli-
um into the targeted area upon activation within the secondary lym-
phoid organs [108], resulting in a slightly hypoxic environment too
and the loss of dependence on oxidative phosphorylation. Inflammatory
sites are also hypoxic due to the blocking of blood vessels by innate
phagocytes [109–111]. To promote more glycolytic pathways, immune
cells need to localise and adapt to different oxygen concentration levels
[112,113].
Protecting the body from pathogens is important and both innate
and adaptive immune cell activation is vital for this. Metabolic choices
within immune cells are not solely dictated by the tumour microenvi-
ronment. Immune cells must be efficient in their nutrient metabolism.
Here it would seem like immune cells should utilise the oxidative phos-
phorylation pathway which generates most energy, but however this is
not the case. The immune cell specificity and reactivity-state dictates
the amount of mitochondrial respiration used. To synthesise macromol-
ecules and proliferate, activated immune cells prefer using the much
faster glycolysis [18].
4.1.1. Innate immune cell metabolism
Foreign body substances in the body are constantly monitored by
naive APCs. Myeloid cells and granulocytes favour glycolysis at resting
state [114,115]. As mentioned above, APCs immediately upregulate
co-stimulatory molecules and process and present antigens on their
cell surface once an antigen is phagocytosed, requiring a greater level
of ATP. However, APCs continue to depend on glycolysis [114,115].
When stimulated by toll-like receptors (TLRs), DCs undergo metabolic
changes and increase their glycolytic rate [116]. Classically activated
macrophages (M1; proinflammatory) are regulated by glycolysis where-
as alternatively activated macrophages (M2; anti-inflammatory), rely
more mitochondrial respiration [117,118]. Granulocytes e.g. neutrophils
also favour glycolysis [114,119]. As the first mediators at foreign body
sites, neutrophils' rapid degranulation results to cell death [120]. Neutro-
phils need to act quickly but it is not necessary to depend on greater ATP
production to survive. This makes more sense for granulocytes and APCs
(fast turnover rates) to use the glycolytic pathway to generate the high
demands of ATP. Lymphocytes switch their metabolic need to glycolysis
when activated, whilst rely heavily on oxidative phosphorylation in qui-
escence. Memory is generated by lymphocytes (in oxidative phosphory-
lation state) after clearance of an antigen. This shows that they fluctuate
their metabolic pathways [121].
4.1.2. Adaptive immune cell metabolism
Plentiful supply of nutrients and T-cell receptor (TCR) stimulation at
resting state is necessary for T cells to function adequately. To generate
ATP reserves (via catabolic metabolism), naïve lymphocytes are sub-
jected to oxidative phosphorylation [103]. Following stimulation by an
antigen, quiescent immune cells quickly mobilise to the source [122].
Quiescence is not only preserved by the high-ATP turnover of cell
cycle proteins [123,124] but also by the upregulation of kinase inhib-
itors. Quiescent cells, via autophagy, are able to extract nutrients
from degraded proteins [99,125,126]. Shortage of TCR interaction
downregulates the transport of glucose and ATP. The mitochondrial
potential will also be reduced [127]. BAX induction followed by
apoptosis results from the lack of glucose uptake and the naïve T
cell population can be controlled by this stringency.
Upon antigen stimulation, lymphocytes (B and T cells) need to be-
come rapidly activated, like the innate cells. Lymphocytes considerably
enlarge their size within the first 24 h post-mitogen stimulation [121].
93
New nucleotides and proteins are also synthesised during this time. Ef-
fector cells result from the division and differentiation of T cells. It is im-
portant to obtain sufficient amounts of nutrients and therefore driving
glycolysis [103] to maintain the quick change in cellular size and
function.
T cells heavily depend on glycolysis, and their proliferation is halted
even in the presence of glutamine abundance [128]. They generate ATP
and lactate via glycolysis by converting glucose to pyruvate [18]. This in
turn converts NADH back to NAD+ thus retaining glycolysis. More lac-
tate means more NADH production [93], which are needed as macro-
molecular precursors by T cells. T cells have high energy requirement
and are not able to enhance mitochondrial respiration to meet their
needs. Anabolic metabolism is driven efficiently by glycolysis and this
is important to maintain cellular function and longevity and especially
during times of pathogen clearance [103,129] e.g. seen in T cell activity.
However, in situations of glucose shortage [130], some oxygen is still
consumed [131]. Upregulation of mitochondrial enzymatic activity is
driven by calcium flux from lymphocytic activation resulting in in-
creased oxidative phosphorylation [132,133]. Unstable Teff cells under-
go apoptosis in conditions of low IL-2 and low nutrient levels. Some
clone cells have higher mitochondrial concentration and therefore are
able to switch to oxidative phosphorylation. On the contrary, constantly
activated T cells rely on oxidative phosphorylation too in autoreactivity
and in graft-versus host diseases [134,135].
T regulator cells (Tregs) [136] and memory CD8+ T cell [137] gener-
ation relies on lipid oxidation. Memory is generated after antigen-
clearance post T cell activation and apoptosis of T effector cells (Teff) oc-
curs secondary to shortage of growth factors and metabolic activity
[138]. Metabolism of both memory T cells and naïve cells requires oxi-
dative phosphorylation. To ensure the survival of T cells, some impor-
tant changes happen during an immune response. Memory cells have
a higher affinity towards TCRs. This ensures survival in scarce-nutrient
conditions and low homeostatic engagements [139]. CD8 T cells have
extra respiratory capacities (necessary for ATP production in stressful
times), and they will be memory cells [140]. This also ensures cellular
survival [141,142]. Noxa gene blocking generates CD4 T cell memory
[143,144].
5. Immune signaling and metabolism
Almost every immune cell type such as T cells, B cells, NK cells, baso-
phils, DCs, neutrophils, myeloid-derived suppressor cells (MCDCs) and
NKT cells is well recognised in cancer progression [145–147]. Cells com-
municate with their environment through a variety of cell-surface re-
ceptors that recognise and bind molecules present in the extracellular
environment. Immune cells are activated when ligand binds with cell
membrane receptors and a reaction known as signal transduction is
triggered when this binding happens. In response to cellular signaling,
immune cells secrete cytokines (often produced in cascades) that bind
to specific membrane receptors. These membrane receptors then signal
the cell via second messengers, often tyrosine kinases, to change the
activity of the cells. Interleukins are manufactured by one leukocyte to
act on other leukocytes as signaling ligands. They are the biggest class
of cytokines.
Immune cells possess clear metabolic characteristics that influence
their immunological functions. For example, when DCs and T cells are
activated, the metabolism of glucose and amino acids alters [148].
Also, energy, iron, and lipid metabolisms distinct metabolic characteris-
tics are related to macrophage polarisation [146]. The engagement of
the Major Histocompatibility Complex-peptide on APC cells is required
for the stimulation of T cells via the T-Cell Receptor (TCR). Also, down-
stream signaling and effector function depend on co-stimulation during
T cell activation. However, T cell anergy and deletion results from a lack
of co-stimulation [149,150]. The archetypal co-stimulatory molecule for
T cell activation is the CD28 and it promotes T cell differentiation via its
ability to bind CD80/CD86 on APCs [151,152]. The ability of CD28 to
94 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
trigger a build-up of glycolytic intermediates and thus enhancing glu-
cose metabolism [93], stimulating glycolysis and increasing the expres-
sion of glucose transporters [94] is similar to insulin-receptor signaling.
In the hypoxic tumour microenvironment, the glycolytic shift is re-
quired by cancer cells to survive. This glycolytic shift is triggered by mul-
tiple mechanisms and is discussed elsewhere [153,154]. The effects of
CD28 are nullified by CTLA-4 and this reduces glycolysis and making
cells dormant [155]. The PI3K-Akt-mTOR pathway is crucial for bridging
the gap between metabolism and T cell activation. CD28, IL-2 and TCR
engagement leads to PI3K-dependent Akt activation. This then increases
activities of enzymes of glycolysis secondary to the surplus of glucose
transporters on the plasma membrane of cells [103,156,157]. Myc (reg-
ulator gene that codes for a transcription factor) induces glucose trans-
porters and glycolytic enzymes, and it is activated by PI3K, MAPK and
NF-κB [158,159]. In resting T cells, AKT together with STAT5 influence
glucose uptake into cells [160]. Lymphocytes only express the GLUT1
glucose transporter unlike other cell types. GLUT1 will be internalized
in the absence of sufficient cytokine and/or TCR stimulation. This leads
to lower glucose uptake across the plasma membrane secondary to
down regulation of surface expression, and eventually decreasing the
viability of the cell. CD28-mediated Akt signaling is vital for expression
and trafficking of GLUT1 to the surface of the cell that in turn increases
glucose uptake [93,161]. Following B cell receptor (BCR) engagement, B
cells also increase GLUT1 expression [102]. PI3K regulates glycolysis and
GLUT1 and is involved critically in immunoglobulin synthesis and B cell
proliferation [162]. Even though critical for lymphocyte activation,
GLUT1 overexpression can result in hyperactive lymphocytes and
other pathologies [161]. Therefore, a balance must exist.
Another important regulator of metabolism in immune cells is
mTOR [163]. They couple nutrient sensing to metabolic outcomes like
glycolysis, fatty-acid and protein synthesis. mTOR enhances the expres-
sion of HIF1α [164,165] and thus mediating the glycolytic process. In all
mammalian cells, and not only DCs and T cells, mTOR is a downstream
target of AKT signaling [166]. Protein synthesis [167] and mRNA trans-
lation are induced by the ability of mTOR to sense nutrient availability
[168,169]. T cell proliferation will be blocked [170] without proper
mTOR signaling and anabolic storage processes will be decreased [171,
172]. T cell anergy [173,174] results from inhibition of the PI3K-Akt-
mTOR pathway. T cells that are deficient of mTOR differentiate into reg-
ulatory T cells (Treg) and not effector T cells (Teff) [175]. The role of PI3K-
Akt in glycolysis is reported for immune cells like macrophages, DCs and
T cells is reported elsewhere [164,166,176]. T cell migration results from
links between mTOR and chemokine-dependent signaling [177–179].
This link also results in cancer metastasis [180,181]. To initiate an effec-
tive adaptive immune response, it is important to have proper metabol-
ic signaling. This can also reveal targets for therapeutic intervention.
Oxidative phosphorylation (lower activity rate) is still necessary
for functionality although immune cells mainly use glycolysis to
metabolise. Cytokine binding and cytokine receptor expression
have also been connected with metabolism. TNF binding to specific
cellular receptors [182] and IL-2R expression on lymphocytes are
reduced [183] if the electron transport chain in mitochondrial respi-
ration is blocked. The importance of proinflammatory cytokines in
metabolic signaling is shown in a study where TNFα-deficient
mice were protected from insulin resistance secondary to obesity
[184,185]. Cytokines of the innate immune system e.g. IL-1, IL-6, IL-3
and IL-7 also play a role in cellular metabolism. Each cytokine binds to
a specific receptor on the cell surface, and is involved in the coordina-
tion of T cell function with metabolic needs. IL-1 can prevent fatty
acid synthesis [186], whereas IL-6 can both increase the levels of lipid
and glucose metabolism [187]. A metabolic shift to glycolysis from oxi-
dative phosphorylation is caused by IL-3 [188]. Besides that, IL-3 is also
important in supporting GLUT1 on lymphocyte surfaces [157] and to aid
in the growth of lymphoid and myeloid cells. Lastly, IL-7 must be pres-
ent to sustain uptake of glucose by activated AKT in T cells (both resting
and activated states) [128].
To link metabolism and immunity, adipocytes release adipokines,
functioning similarly to macrophages [94,189,190]. These adipokines
include IL-1, IL-6, IFNγ, TNFα, and MCP1. Lymphocytes and monocytes
are recruited into the adipose tissue by adipokines, which in turn pro-
motes both pro- and anti-inflammatory functions. Macrophage entry
into the adipose tissue is induced by adipocyte hypertrophy [191].
This hypertrophy leads to MCP-1 production and a core with low levels
of oxygen and is secondary to overnutrition. These infiltrating macro-
phages are modulated by lymphocytes which are associated with
adipose tissue [192]. An anti-inflammatory macrophage phenotype in
seen in lean mice with higher levels of Treg cells [193]. Also, recruitment
of more proinflammatory macrophages is associated with proinflam-
matory Teff cells seen in the fat of obese mice [194]. This contributes to
insulin resistance [195,196].
6. Metabolic modulators and immune-glycolytic
therapeutic implications
The immunology of tumour microenvironment is very complex and
makes the development of modulators of metabolism challenging. The
main reasons of limited success of cancer immunotherapy include
poor infiltration of effector cells into solid tumours and poor host re-
sponses towards tumour antigens. Furthermore, the intrinsically immu-
nosuppressive nature of the tumour microenvironment adds to this
poor success rate. The effectivity of cancer immunotherapy depends
on the balance of immune responses from tumour protection towards
its rejection [197–199]. The microenvironment of tumours is therefore
also to be considered in order to achieve better antitumour responses
in future. Spontaneous regression has been witnessed in metastatic
melanoma treated with adoptive cell therapy (ACT) using autologous
tumour infiltrating lymphocytes (TIL) [200–202]. (Table 1.)
6.1. Targeting tumour hypoxia-inducible factor-1
Hypoxia inducible factors (HIF) are comprised of an oxygen labile α
subunit (HIF-α) and a β-subunit, expressed throughout various cell
types irrespective of oxygen levels [203]. HIF-1α (one of three isoforms)
expression in tissues is universal and was the first to be discovered [204]
whereas HIF-2αs are expressed in the vasculature endothelium. Tu-
mour microenvironment is hypoxic (≤2% oxygen) and HIF-1 modulates
metabolism in these conditions. HIF-1α levels are not only regulated by
oxygen levels but also by cytokines IL-1β and TNF-α [205]. Further-
more, DNA binding of HIF-1 stimulated by p44/42 MAP kinase can
also increase HIF-1 levels [206]. It is an important signaling molecule
for proliferation and growth of tumours. Aerobic glycolysis is promoted
in increased pyruvate levels [207]. HIF-1α binds to GLUT1 [208] and gly-
colytic enzymes to facilitate this metabolic switch. For example, cancer
cell dependence on oxidative phosphorylation is reduced due to low
levels of acetyl-CoA (converted from pyruvate) because of increased
PDK activity; secondary to HIF-1α mediated hypoxia responsive ele-
ments [209]. This leads to lactate build-up in cells. This switch in metab-
olism preserves cellular viability in hypoxic times. Increased levels of
HIF-1 activation promote tumour growth, angiogenesis and metastasis
by reducing cytochrome c levels causing caspase-9 and -3 inactivation
that prevents cell death [210] suggesting poorer prognosis, whilst low
activity levels of HIF are associated with decreased tumour metabolism,
growth and vascularity [211].
Hypoxic macrophages and DCs also switch their metabolism to gly-
colysis by HIF-1α and mTOR. Activated lymphocytes undergo similar
metabolic changes to hypoxic cells. This questions the role of HIF-1s in
tweaking T cell activation. Immune cells need to survive and function
properly in hypoxic conditions too, and this highlights the importance
of HIF-1 activation [107]. In activated T cells, GLUT1 expressions are in-
creased secondary to HIF-1 induced gene expressions that improve via-
bility and survival of immune cells during aerobic glycolysis [212,213].
Furthermore, T cell differentiation is also regulated by HIF-1. However,
 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105 95

Table 1
Current glycolytic and immune modulator clinical trials.

Modulator Target Effect Tumour type targeted Stage Study number

Vemurafenib plus BRAF BRAF/MEK inhibitor Metastatic melanoma Phase 2 NCT02414750

Cobimetinib
Response to neoadjuvant Determine the correlation between histopathological response Breast cancer NCT01440413
CT based on AT IR in (pCR) and induction of tumour immunity in response to
localized breast cancer neoadjuvant chemotherapy
2-deoxy-2-(18)fluoro-D-- Immunotherapy for FDG PET measure the response to immune checkpoint Melanoma, renal cell Phase 0 NCT01666353
glucose (FDG) melanoma blockade therapy carcinoma, non-small
lung cancer
Abraxane with or without Gene expression profile Humanised monoclonal antibody combined with Abraxane to Breast cancer Phase 2 NCT01307891
Tigatuzumab and the serve as targeting agent for triple negative breast cancer
proliferation/apoptotic patients
markers
K562-GM allogene Cancer testis antigen Patients with allogenic tumour express high levels of multiple Sarcoma, melanoma, Phase 1 NCT01313429
(CTA) CTA in combination with depletion with T regulatory cells germ cell tumour
TGFB2-Antisense-GMCSF TGGB2 Expression of TGFB2/GMCSF following transcription by Advance and Phase 1 NCT00684294
electroporation and irradiation of autologous cancer tissue non-curable solid
tumour
EMD 273066 EpCAM EMD 273066 is an experimental biological drug that may Ovarian, prostate, Phase 1 NCT00132522
increase the immune response to certain cancers colorectal or
non-small cell lung
cancers
MVA-brachyury-TRICOM Brachyury protein Vaccine it teaches immune cell to target which protein is Lung, breast, prostate, Phase 1 NCT02179515
present in some tumour cells, and it can help tumour cells ovarian
spread to other parts of the body.
Sipuleucel-T PA2024 Anti-androgen treatment started before or after sipuleucel-T Prostate cancer Phase 2 NCT01431391
leads to superior augmentation of immune response to
sipuleucel-T
2-Deoxy-D-glucose Hexokinase Inhibits the metabolism of glucose in the cells of the body Lung, breast, prostate Phase 1/2 NCT00633087
AP23573 mTORC1 Inhibits mTORC1 Solid tumours Phase 2 NCT00110188
Ipilimumab and Anti-CTLA-4 monoclonal Ipilimumab augments the effects of chemotherapy in animal Malignant melanoma Phase 2 NCT01654692
Fotemustrin antibody models relies on the ability of the cytotoxic agent to induce
apoptosis of tumour cells

Tregs are powered via lipid oxidation [136]. Innate cell functions e.g. ATP
generation, glycolytic enzymes and GLUT1 expressions, are modulated
by HIF-1 levels, as seen in macrophages and neutrophils [214]. When
HIF-1 levels are low, innate cell functions deteriorate and thus resulting
in decreased abilities in T cell activation and its pathogen killing capabil-
ities [115,215]. This is coupled by weakened granulocyte responses and
APC phagocytosis in hypoxic conditions [216,217] leading to poor host
defenses. However by affecting the innate and adaptive immune cells,
both HIF-1 and chronic inflammation can initiate tumour advancement
and autoimmunity. Glycolysis upregulated by HIF-1α has been linked
with tumour-associated-macrophages (TAMs), which are macrophages
containing perinecrotic and hypoxic tumour tissues that aid in tumour
angiogenesis and growth.
Both HIF-1 and HIF-2 levels are raised in malignancy e.g. in bladder,
colon, ovarian, pancreatic, prostate, hepatocellular, and glial cancers.
HIF-1α has been widely studied as a glycolytic target for cancer therapy,
both at pre-clinical and clinical stages [218], given its extensive involve-
ment in regulating glycolysis. Cytotoxic agents are developed with aim
to block HIF-1α expression, transcription and translation and also to in-
terrupt binding of targets. Digoxin for example inhibits HIF-1 gene ex-
pression and tumour proliferation [219], besides blocking RORγt-
dependent TH17 differentiation [220]. HIF-1 represses Treg differentia-
tion and therefore inhibiting HIF may elevate suppressive T cells. This
phenomenon may be advantageous in protecting against self-antigen
recognition. Active CD8 T cells may still be able to kill tumours if HIF-1
is blocked. Other approaches of HIF-1 blockade include induction of
HIF-1α degradation by HSP90 inhibitor and 1-β-2 methoxyestradiol,
HRE-binding HIF-1 inhibition by doxorubicin and daunorubicin, and
also bortezomib which inhibits HIF-1 transcription [221,222]. More re-
cent genetic approaches targeting HIF-1α inhibition include siRNA ther-
apy EZN-2968, tested in hepatic metastasis. However, issue of toxicity
arose in a number of patients with this approach [223]. Micro-RNA
(miRNA)-mediated therapy is also being tested in glycolytic inhibition.
miR100 (mTOR signaling inhibitor) has been identified in clear cell
ovarian cancer [224]. Delivery of these promising HIF-1 inhibitors
should be encapsulated with nanoparticles to ensure targeted delivery
and avoiding nonspecific toxicity, as demonstrated by successful
antitumour siRNA delivery to hypoxic tumour sites [225].
6.2. Targeting uncoupling proteins
Mitochondrial ATPs are produced by coupling the electron transport
chain (ETC) and ADP-ATP phosphorylation. However, this process is not
efficient in the presence of uncoupling proteins (UCP), which are anion
transporters of the mitochondrial inner membrane [226]. Besides that,
UCPs also reduce the production of mitochondrial-free radicals [227]
for example by decreasing pyruvate entry into the TCA cycle [228,
229], and are also involved in thermogenesis e.g. UCP1 involvement in
thermogenesis activity of brown tissue [230]. Three main uncoupling
proteins which are UCP1, UCP2 and UCP3 have different uncoupling ac-
tivities [226,231] and play different roles in mitochondrial physiology
[232]. UCP2 knockout results in resistance to infections [233,234] due
to NF-κB activation and increased levels of reactive oxygen species
(ROS) in immune cells, making UCP2 an immunometabolic target.
High levels of mitochondrial UPC2 are found in activated T cells owing
to its need for high proliferation via glycolysis. Low ROS levels are also
necessary for expressing genes and signaling activity [235]. Uncoupling
the ETC when high levels of ROS are generated resets homeostasis,
particularly in the innate immune and T cell interplay. UPC2 is pro-
glycolytic and is shown by its ability to stabilise the glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) in cancer cell cytoplasm [236].
UCP2 supports the glycolytic flux by its antioxidant effects of GAPDH ox-
idation and nuclear translocation inhibition. With this, they prevent
cancer cells from stimulating cell death mechanisms. UCP2 decreases
glucose-stimulated insulin release [237]. This enables them to control
the autoreactive T cell responses. Increased levels of UCP2 are found in
96 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
leukaemia, kidney, colorectal, lung, breast, prostate, ovarian, bladder,
oesophageal and testicular cancers [238].
Uncouplers have not been utilised in the treatment of autoreactive T
cells. Rottlerin (a chemical mitochondrial uncoupler) has been shown to
decrease apoptosis in autoimmune-diseased alveolar macrophages
[239]. A fatty-liver (non-alcoholic) diseased model showed to have re-
duced insulin resistance when treated with 2,4-dinitrophenol [202].
This drug also causes adhesion build-up particularly seen in post-
peritoneal surgery [240]. Uncouplers are cell-specific making them a
potential therapy target for immune diseases (high oxidative stress).
Uncoupling inhibitors on the other hand can be used to treat standard
chemotherapy-resistant cancers [241].
6.3. Targeting tumour glycolysis
Immune cells are highly dependent on glucose metabolism. Anti-
glycolytics limit immunity e.g. Rapamycin inhibits glucose metabolism
by blocking the mTOR downstream of PI3K-Akt and ultimately lowering
proliferation rate of cells due to reduced T and B cell activity. It can also
lead to hyperglycaemia because of this blockade. Rapamycin can affect T
cell differentiation and memory at the immune cell level. Furthermore,
besides causing a glycolytic switch to oxidative phosphorylation whilst
increasing CD8 T cell memory response quality, rapamycin can also im-
itate dietary restriction and improve longevity [242] in memory T cells.
Rapamycin can initiate Treg development in CD4 T cells[243,244] and
also higher infection rates[245,246] are seen with rapamycin treatment
because of its immunosuppressive capabilities.
3-Bromopyruvate (3BP) (HK inhibitor) is an anti-glycolytic also
used in cancer treatment. Cell death occurs with 3BP treatment as intra-
cellular ATP levels deplete [247]. 3BP is selective to cancer cells and
therefore it is less toxic to healthy surrounding tissues [248,249], and
treatment with it can increase tumour immunity with higher infiltra-
tion of killer cells whilst diminishing the restraining tumour microenvi-
ronment. Localized treatment especially intra-tumoural approach
induces anti-tumour CD8 memory T cells. T cells may be harmed via
systemic treatment with 3BP because of the switch of metabolism to ox-
idative phosphorylation from glycolysis.
6.4. Targeting the mitochondria
The mitochondrial ETC produces ROS and redox-damaged byproducts
that result in cellular death and therefore ETC mutations can decrease the
amount of ROS and eventually prolong life [250,251]. NF-κB deactivation
(reducing cytokine-induced adhesion of endothelial cells [252,253]) and
defects in phagocytes are caused by inhibition of various parts of the
mitochondrial ETC [254]. Metformin is widely used in clinical practice
as an anti-hyperglycaemic. Its mechanism of action includes increasing
skeletal muscle glucose uptake and reducing gloconeogenesis in the
liver in order to diminish circulating insulin levels. UCP2 levels are in-
creased with the use of metformin [255]. In conditions of low oxygen,
metformin is able to block the mTOR function by AMP-activated protein
kinase (AMPK) activation which itself increases glucose uptake. All
these eventually lead to inhibition of cell proliferation [256,257]. Also,
metformin is able to inhibit cancer cell oxygen consumption by hindering
complex I of the ETC. Targeted tumour therapy with metformin causes T
cells to kill cancerous cells and reduce tumour growth. Metformin
can regulate the immune system and the increased levels of memory
CD8 T cells prove this post-administration of the drug on ova-specific T
cells, alongside tumour regression.
Another approach of metabolic control for immune regulation is the
usage of antioxidants across an array of pathologies from autoimmunity,
neurodegeneration and cancer [258–262]. Antioxidants are known to
reduce ROS levels e.g. hydrogen peroxide (H2O2) is metabolised by the
antioxidant glutathione during aerobic respiration [263]. There are
various ways in which antioxidants improve the immune system.
For example, epigallocatechin-3-gallate (antioxidant in green tea)
downregulates cytokine receptors and thus reduces T cell signaling
[264], ameliorates Experimental Autoimmune Encephalomyelitis
(EAE) and enhances Treg cells [265]. Other studies showed the antioxi-
dants' anti-inflammatory ability by restricting NF-κB activation [266–
268] and glycolytic reduction in lymphoma cells through LDH activity
inhibition, by α-tocopherol (an antioxidant found in Vitamin E) [269].
Metalloporphyrins are a group of catalytic antioxidants that can
completely remove toxic ROS. They show no toxicity and are able to
modulate the immune function. It is an alternative metabolic therapy
that may be beneficial in reducing inflammation and potentiating can-
cer regression. They have been shown to reduce incidence of type 1 di-
abetes [258,270],activate NF-κB [268] and reduce CD8 Teff cell functions
[271]. Metalloporphyrins can reduce hypoxia, block HIF-1α activity and
suppress growth of tumour [272]. Early studies suggest administration
of metalloporphyrins decreases aerobic glycolysis, which in turn de-
creased T cell differentiation resulting in quiescence.
7. Delivery methods of glycolytic modulators
Current cancer treatment involves surgery, chemotherapy, radio-
therapy or a combination of these. With the aid of cancer imaging tech-
nologies, the removal becomes more tumour-selective. Tumours may
recur because cancer cells are not completely eliminated from the
body. Disease recurrence is a major challenge in cancer treatment. Nu-
merous anticancer reagents have been developed to suppress the
growth of residual or inoperable tumours. The identification of glycolyt-
ic inhibitors for cancer therapy has led to the specific destruction of neo-
plastic cells in several types of cancer. However, many types of cancer
still require effective anticancer drugs. To enhance anticancer activity
whilst minimizing adverse effects and toxicity to healthy surrounding
tissues, the cancer-selective delivery of therapeutic molecules is de-
sired. Viral or non-viral route is the main category of drug delivery
methods. Each type of vector has their advantages and limitations.
Innovations in micro/nanoparticle designs, new drugs e.g. post-
transcriptional gene silencing nucleotides, and advanced new bio-
materials result in new drug delivery methods.
7.1. Viral drug delivery system
Drug delivery refers to various approaches and formulations, differ-
ent technologies incorporated into developing vectors and different sys-
tems for transporting a pharmaceutical composite. In doing so, the
desired therapeutic effect has to be safely achieved. Viral delivery sys-
tems, virosomes, consist of viral envelope components of inactivated
viral particles. They act as a vehicle for therapeutic molecules.
Virosomes are composed of a phospholipid bilayer. Glycoproteins on
the viral surface protrude from these vesicle surfaces [273–275].
Virosomes do not contain a lot of preservative and their preparation
usually does not involve complicated techniques [276]. Virosomes are
able to transport macromolecules e.g. drugs, nucleic acids, and proteins
to erythrocytes, hepatocytes, immune cells and glioma effectively [277–
279]. In conventional drug delivery systems, the therapeutic molecules
get degraded prior to reaching the target cells. However, virosomes are
able to deliver these drug molecules to target cells by virosome-
lysosome membrane fusion without being affected by the host defense
mechanism. Many recombinant virus based delivery systems have
emerged lately and these have been used for gene therapy and for vac-
cine purposes [280]. By combining fusion peptide from influenza virus
haemagglutinin (HA), gene transfer efficiency of receptor-mediated
gene delivery systems has been enhanced. HVJ liposomes have been
widely used in animal models for cancer treatment. To prevent melano-
ma growth, melanoma-associated antigen gene or RNA has been
injected into spleen or skeletal muscle [281]. This has successfully in-
voked immunity to prevent melanoma growth. Newer viral vaccine sys-
tems offer advantages over conventional vaccines; however, they have
their own limitations and side effects [282,283]. These viral vaccines
 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
include adenovirus types 2 and 5, pox-, lenti-, retro-, and alphaviruses,
adeno-associated virus and lastly the herpes simplex virus (HSV).
They can delivery antigens to specific subsets of immune cells effective-
ly such as the APCs which can potentially act as adjuvants. Viral vectors
can be combined with other vaccines and administered to result in en-
hanced immune responses. Both RNA and DNA viruses have been used
as vaccines to express the antigenic/therapeutic protein in vivo and to
stimulate potent specific humoral and cellular immune responses [284].
7.2. Non-viral drug delivery system
A promising technology of the delivery of variety of compounds to
target sites is liposomes and liposomal-based delivery systems. Lipo-
somes are a non-viral delivery system and are used as a vehicle for ad-
ministration of macromolecules into cells. In liposomes, water-soluble
molecules incorporate into a lipid bilayer-vesicle. They have been
utilised to target tumour tissues by systemic administration. To improve
the transfection efficiency and to reduce the cytotoxicity of the lipoplex,
numerous cationic lipids have been synthesised [285–288]. Lipoplex
with HLA-B7 and b-2 microglobulin genes were found to induce
antitumour immunity in HLA-B7-negative melanoma patients. Also,
Allovectin-7 (a liposomal drug) has been clinically tested to treat meta-
static melanoma [287]. Glioblastoma patients in Japan were treated
with the IFN-b gene. These were delivered by cationic liposomes
which were composed of N-(a-trimethy-lammonioacetyl)-didodecyl-
D-glutamate chloride (TMAG) [289] dilauroyl phosphatidylcholine,
and dioleoyl phosphatidylethanolamine. Doxil which is a liposomal
preparation composed of the high phase-transition temperature phos-
pholipid hydrogenated soy phosphatidylcholine (HSPC) and cholester-
ol, is currently in use for the treatment of recurrent breast cancer [290,
291].
7.2.1. Synthetic polymers
Currently, synthetic polymers are the most actively researched areas
of biomedicine. They have easily adjustable characteristics. Polymer-
based nanoparticle delivery systems e.g. polymeric nanoparticles,
polymeric micelles, and dendrimers are newly innovative candidates
to not only diagnose but also to monitor disease status and treat cancer
diseases. This is mainly due to their special features of enhanced perme-
ability, biocompatibility and biodegradability, improved bioavailability
and transport efficiency, low toxicity depot effect, and lastly targeted
delivery [292]. Less toxic synthetic polymers like poly-D,L-lactide-co-
glycolide (PLGA) and polylactic acid (PLA) also provide biological com-
patibility [293]. ‘Smart’ polymers undergo conformational changes in
response to changes in the environment such as the temperature and
surrounding pH [294]. They are able to employ in different fields and
in response to specific physiological triggers they are able to release
the contained drugs. Polymeric micelles are block-copolymers, which
consist of two distinct linear polymers, are conjugated to therapeutic
molecules by an electrostatic interaction, hydrophobic interaction or
metal complex formation. Doxorubicin was encapsulated into PEG-b-
polyaspartic acid copolymer. PEG-b-polyaspartamide with a 1,2-
diaminoethane side chain is used as a block-copolymer for DNA encap-
sulation. This micelle for DNA encapsulation enhanced the penetration
into tumour tissue and enabled gene expression in the hypoxic state
[295]. The amount of doxorubicin in tumours by pH-sensitive micelles
was approximately fourfold higher than that by pH-insensitive micelles,
and more efficient tumour regression was achieved by pH-sensitive
micelles than by conventional micelles [296].
Atelocollagen is a bovine type I collagen that is treated with pepsin
and solubilised by protease. However, its physical properties are virtual-
ly identical to those of natural collagens, which are not solubilised.
Natural substances do not possess the superior characteristics found in
atelocollagen. The atelocollagen (gel biomaterial in clinical use) has a
positive charge and can be a carrier of DNA and siRNA [297]. The
97
siRNA existed in tumour tissue for more than one week when the
siRNA/atelocollagen complex was intravenously injected into tumour-
bearing mice. Intravenously administered SiRNA-atelocollagen complex
into mice with bone metastasis from human prostate cancer [298]
revealed marked suppression of metastatic tumours in both types of
siRNA in an in vivo imaging analysis.
7.2.2. Natural polymers
Other polymer-based delivery systems include cationised gelatins.
These gelatins are prepared by the introduction of amine residues to
the carboxyl groups of gelatin via chemical means. Crosslinking
cationised gelatin with glutaraldehyde result in cationised gelatin
hydrogels. These hydrogels have different in vivo degradabilities as
the carrier of DNA. The plasmid DNA incorporated gelatin prolongs
the duration of gene expression. The antitumour plasmid DNA of NK4s
(protein made up of the NH2-terminal hairpin and the four-kringled do-
mains of hepatocyte growth factor) biological activity is promoted by
the controlled release technology [299]. NK4 is an HGF antagonist that
inhibits the ability of HGF to promote tumour metastasis and angiogen-
esis. The mice with which received subcutaneous injection (controlled
release approach) of hydrogel microspheres had significant prolonged
survival as compared to mice that received NK4 plasmid DNA in
solution.
Chitosan, prepared from chitin by deacetylation, is a positively
charged linear polysaccharide consisting of one to four linked N-
acetyl- D -glucosamine and D -glucosamine subunits, and interacts
strongly with negatively charged DNA [300]. Chitin is a component
of fungi and exoskeleton of crustaceans' cell walls. Chitosan is a non-
toxic polymer and can be digested by enzymes in the blood and
human intestines. These enzymes include chitinases or lysozymes. In-
terest to this polymer as a drug delivery system in pharmaceutical re-
search was based on the above-mentioned special characteristics of
chitosan. Galactosylated, trimethylated and N-dodecylated chitosan
[285] are examples of chitosan derivatives. Chitosan is also used for
the intravenous delivery of siRNA for cancer therapy [301]. Another
form of DNA delivery into cultured cells is polyethylenimine (PEI).
The proton sponge effect causes the escape of DNA from the endosome
to the cytoplasm, enhanced by PEI [302]. Stearic acid modified PEI-
based nanoparticles (more effective combination than PEI/siRNA com-
plex) were developed to deliver siRNA against signal transducer and
activator of transcription 3 (STAT3) to suppress mouse melanoma
in vivo [303].
7.3. Physical methods of drug-delivery
Drug deliveries mediated by different physical methods have grown
significantly over the past decade and a half. Systems of drug delivery
aim to deliver the therapeutic amounts of drug to targeted sites in the
body, and to promptly achieve and maintain the desired drug concen-
trations [304]. Physically facilitating drug-delivery (PFDD) systems can
be classified mainly as photo-, ultrasound-, magnetic-, and electrical-
based modulation systems. This vast literature on PFDD is impossible
to be covered in this review. Physical methods to push the DNA into
cells have been developed to enhance gene delivery based on naked
DNA injection, firstly reported for skeletal muscle [305] and subse-
quently reported for other tissues such as the thyroid, cardiac muscle,
skin and liver [306].
7.3.1. Electroporation
Electroporation (EP) is a technique where electrical fields are
applied to cells to increase the permeability of the cell membrane. The
cell membrane represents a physical barrier to the intracellular transfer
of macromolecules, peptides, hydrophilic drugs, or DNA to be intro-
duced into the cell [307]. The phospholipid bilayer of the plasma
membrane restricts the transmembrane movement of hydrophilic and
polar molecules, other than by specific channel or receptor-mediated
98 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
assistance. EP is the strategy that involves direct application of an elec-
trical field to target tissues or tumours to allow passage of various mol-
ecules including those that destabilise the cell membrane, by surpassing
the cell membrane barrier. Both in vitro and in vivo cellular membranes
become transiently permeable through short high-voltage electric field
pulse application. These voltages are optimised to balance its reversible
and irreversible effects [308,309]. Cellular membrane composition
[310], its size and shape [311,312], and cellular suspension density
[313] are factors that influence the extent of cellular permeabilisation
after EP. EP with high electric currents causes tissue damage but having
said that, malignant tissues are more affected by electrochemotherapy
(ECT) as compared to normal tissues [314]. EP at a lower voltage with
similar transfection efficiency has been developed to solve the problem
of irreversible EP [315]. There are two types of electrodes, the plate type,
which provides more stable results, and the needle type that is more
useful for transfection in various tissues. Newer electrode designs are
being developed and used in clinical trials for internal tumours e.g.
liver metastasis from colorectal cancer [316]. In addition to the use of
drugs, EP can also be used to deliver a wide range of potentially thera-
peutic agents including proteins, dyes, tracers, antibodies, oligonucleo-
tides, RNA and DNA [317]. EP is used in animal models of cancer
treatments for the intramuscular delivery of IL-12 and IL-27 genes and
also for the regional tumour delivery of various therapeutic molecules,
including microRNA against mutant K-ras [318].
In clinical practice, reversible EP e.g. ECT, is an anti-cancer strat-
egy used with combination of chemotherapy drugs (intravenous or
intratumoural bleomycin administration followed by topical EP for
recurrent cutaneous breast cancer) [319]. Irreversible EP is another
anti-cancer strategy where cellular membranes do not reseal when
higher electric fields are applied and ultimately results in cell death.
This approach however does not include combination with other
drugs. The study of melanoma treatment is one of the more relevant
applications of EP. Human dendritic cells were transfected with tumour
mRNA using EP and then injected into melanoma patients in a Phase I/II
study [320]. A Phase I trial of the direct injection of the IL-12 gene by EP
was performed with melanoma patients, and the safety of the method
was shown [321]. Gene therapy (IL-12) has been proven as safe, effec-
tive, reproducible and titratable by means of EP-assisted delivery
(gene electrotransfer) since the initial human trials [321].
7.3.2. Other methods of physically facilitating drug-delivery systems
Since the 1950s, the use of ultrasound has been known to improve
targeted drug delivery in human tissues [322]. Ultrasound is also avail-
able for the delivery of therapeutic molecules to tissue [323]. This trans-
fection method depends on cavitation activity, one of the biological
actions of ultrasound. Contrast reagents such as Optison and Levovist,
which consist of gas-filled lipid or albumin-coated particles, are mixed
with DNA. Ultrasound energies rupture these bubbles and subsequently
enhance the membrane permeability. With these activities, DNA is in-
corporated into target cells and most of the membranes are recovered
in 60 s. Increment in the mechanical index, frequency and exposure
time increase the transfection efficiency. It is essential to determine
the optimum conditions for each case as these parameters are also
closely related to cell damage. Tissue damage caused by sonoporation
seems to be less than the damage caused by EP. However, the delivery
efficacy is comparable to that of EP [324]. Sonoporation is also used
for enhancing the liposomal delivery of the IFN-b gene to tumours
[325] and regional intratumoural delivery of toxic compounds, such as
bleomycin and the cytolethal toxin gene, by sonoporation was success-
ful in mice [326]. Advantages of ultrasound-based delivery system (high
frequency sonoporation) include good-recorded safety usage in clinical
trials, minimally invasive, heterogeneous skin permeability and its abil-
ity to deliver small molecules and transfer genes of interest locally [327,
328].
The application of controlled hydrodynamic pressure in capillaries is
known as hydrodynamic delivery, and this method was incepted in the
late 1990s. It enhances capillary endothelial and parenchymal cell per-
meability. It began with investigations in intravascular injection of plas-
mid DNA solution for gene delivery in whole animals [329–331]. This
delivery method is now a common method for DNA and RNA delivery
in rodents and proven to be a useful method for delivery for a wide
range of applications [332]. For example, the hydrodynamic tail vein in-
jection of plasmid DNA has been used to assess the involvement of spe-
cific genes in the development or regression of pathophysiological
conditions. Gene transfer efficiency is dependent on the elasticity of
the target organ as its delivery to the cells of the organ is achieved by
pressure e.g. a soluble form of the fetal liver kinase-1 gene, a receptor
for vascular endothelial growth factor (VEGF), delivered by hydrody-
namic injection suppressed VEGF-driven angiogenesis and inhibited
the growth of renal cell carcinoma and lung carcinoma in mice [333]. In-
travascular hydrodynamic limb vein delivery of the human gp100
melanoma-associated antigen gene to skeletal muscle was used for mel-
anoma vaccination [334].
8. Conclusion
Increased aerobic glycolysis is commonly exhibited in cancer cells. In
order to generate ATP, malignant cells adapt to metabolic changes mak-
ing them addicted to and dependent on glycolysis. Cancer cell survival is
at an advantage given the alterations in energy metabolism with in-
creased glycolytic enzymatic activity and other molecules promoting
longevity. Increased glycolysis results in an acidic tumour microenvi-
ronment making it conducive for a selection of cancer cells with high
survival capacity.
The immune condition of tumour microenvironments is an impor-
tant factor in tumour prevention, development, and progression. Signif-
icant evidences have been provided in several studies regarding the
correlation of disease outcome with tumour microenvironment status.
The future of cancer immunotherapies depends on recognizing the con-
cept of switching the immune response to a tumour-destructive profile,
instead of a tumour-promoting one. Manipulating immunological pa-
rameters in the tumour microenvironment may be adequate in tipping
the balance of host response towards effective immunity.
The complex metabolic switch, immune-glycolytic interactions and
tumour microenvironment present a major challenge in cancer treat-
ment. Over the years, many potential drugs have been developed,
some of which are currently in preclinical and clinical stages. However,
several such promising drug candidates have been discontinued during
various stages of development due to poor bioavailability, unfavourable
pharmacokinetic profile and associated nonspecific toxicity. Cancer im-
munotherapy has been revived with the successes of immune check-
point blockade. However, a better understanding of immune response
mechanisms and checkpoint blockade resistance needs to be attained
in order to reach the goals of achieving substantial antitumour re-
sponses. The development of combination therapies (e.g. combination
therapy with ipilimumab and electrochemotherapy) [335] which in-
crease therapeutic benefits and reduce its side effects are necessary for
the group of patients who do not benefit from a single checkpoint
blockade.
The burgeoning field of nanodelivery system has shown tremendous
promise in circumventing many of the drawbacks associated with con-
ventional drug delivery approaches. This has opened avenues to revisit
application of several discontinued chemotherapeutic agents for the
treatment of different medical conditions. Recent literature survey
clearly indicates that the advantages of nanocarriers have also led to nu-
merous reports on delivery of drugs targeted against glycolytic path-
way. In solid tumours, drug penetration is a critical aspect to consider
when looking at therapeutic strategies. With the advent of novel tu-
mour-penetrating nanoparticles, these nanodelivery systems open the
possibility of delivering therapeutic agents into tumour parenchyma.
Apart from using small molecule and biologics individually in targeting
aerobic glycolysis, combination therapy with small molecules and
 K.S. Gill et al. / Biochimica et Biophysica Acta 1866 (2016) 87–105
nucleic acids is another potential approach that can be explored. Combi-
nation delivery of therapeutics using nanoparticle-based delivery
systems has been successfully reported to achieve synergistic effects
of therapeutic agents especially in multidrug-resistant cancer.
Conflict of interest
There is no conflict of interest.
Acknowledgements
This work was funded by the Breakthrough Cancer Research,
Glenlee, Western Road, Cork, Ireland.
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