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Current Pharmaceutical Design, 2014, 20, 201-222 201

Cancer Therapy: Targeting Mitochondria and other Sub-cellular Organelles

Obinna C. Ubah1 and Heather M. Wallace2*

1
Department of Molecular and Cell Biology, College of Life Sciences and Medicine, University of Aberdeen, AB25 2ZP, UK;
2
Division of Applied Medicine and Therapeutics, School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK

Abstract: Tumour cell death is required for the clearance of malignant cells and is a vital part of the mechanism of natural tumour sup-
pression. Cancer cells, having acquired multiple deregulated pathways involving several cellular oragenelles, are capable of disrupting
these normally finely tuned processes thereby evading both physiological and therapeutic intervention. Although current available data
indicate the dependence of successful tumour cell clearance on classical apoptotic pathways (intrinsic and/or extrinsic pathways), there is
now evidence suggesting that alternative apoptotic and non-apoptotic pathways may effectively contribute to tumour cell death. The mi-
tochondria, proteasomes, endoplasmic reticulum, Golgi apparatus, lysosomes and lysosome-related organelles of tumour cells exhibit a
number of deregulations which have been identified as potential druggable targets for successful rational drug design and therapy. In this
review, we summarise the roles of these cellular organelles in tumour initiation and establishment as well as current trends in develop-
ment of agents that target deregulations in these organelles.

Keywords: Cancer therapy, polyamines, apoptosis, Endoplasmic reticulum, Golgi apparatus, Lysosome, Proteasome, , ROS, MOMP,
Warburg effect.

INTRODUCTION membrane-bound vesicles that encapsulate proteases and other

There are six established alterations in cell physiology that dic-
tate malignant transformation: growth signalling autonomy, insensi-
tivity to anti-proliferating signals, limitless proliferative potential,
sustained angiogenesis, adjacent tissue invasion and metastasis, and
avoidance of cell death. This intrinsic lack of cell death remains a
major cause of therapeutic failure and may itself contribute to car-
cinogenesis and tumour progression [1, 2]. Therefore, activation of
the cell death machinery via drugs designed rationally may signify
a more effective cancer treatment option. The cell death machinery
is composed of catabolic hydrolases which are modulated by spe-
cific inhibitors or via sequestration of their activators. Numerous
cell death cascades (apoptotic and necrotic) converge on mitochon-
dria to cause deregulation of function and permeabilisation with the
activators of the cell death machinery being liberated following the
permeability change in the mitochondrial outer membrane [3]. To
remove abnormal malignant cells, their death is an important event
which also provides a critical mechanism of innate tumour suppres-
sion. Aberrations blocking these tightly regulated processes tilt the
balance in favour of cancer cells successfully evading cell death
from either therapeutic means or physiological control systems.
Although the clearance of malignant cells often relies on manipulat-
ing the classical apoptotic pathways (mitochondrial and/or death
receptor cascades), evidence is emerging that other apoptotic and
non-apoptotic pathways may play vital roles in tumour cell death.
Experimental evidence has suggested that the endoplasmic reticu-
lum (ER) and Golgi apparatus can activate both pro-survival cas-
cades as well as cell suicide programmes if the stress-signalling
threshold is exceeded [4]. The proteasome is an abundant multi-
enzyme complex providing the focal pathway for the degradation of
intracellular proteins in eukaryotes, thus it is the modulator of the
protein pool and is important for cell-cycle progression and apop-
totic cell death in both healthy and malignant cells [5, 6]. Cells use
an exceptional assortment of mechanisms to regulate intracellular
cytosolic protein stability and degradation, lysosomes and protea-
somes remain the two main routes amongst other mechanisms re-
sponsible for protein integrity. Lysosomes are cytoplasmic
*Address correspondence to this author at the Division of Applied Medicine
and Therapeutics, School of Medicine and Dentistry, University of Aber-
deen, Aberdeen AB25 2ZD, UK; Tel/Fax: +44 1224 437956;
E-mail: h.m.wallace@abdn.ac.uk
hydrolytic enzymes and these are the cellular organelles responsible
for degrading extracellular and transmembrane proteins, whereas
proteasomes degrade intracellular proteins [7]. It is the compart-
mentalisation of these proteolytic enzymes that prevent them from
producing uncontrolled protein degradation [5]. For tumourigenesis
to be successful, an effective suppression of both the classical apop-
tosis and lysosomal death pathways must occur, thus supporting the
crucial involvement of these organelles in tumorigenesis [8].
MITOCHONDRIA
i. Structure and Roles
The rationale for targeting the mitochondria for therapeutic
benefit is based on their critical involvement in bioenergetics, redox
balancing and vitally their active regulation of several cell sur-
vival/death pathways. Other important roles include thermogenesis,
Ca2+ homeostasisand essential anabolic cascades. Mitochondrial
dysfunction such as impaired oxidative phosphorylation and/or
increased oxidative stress coupled with deregulation of apoptosis
has been described recently as a vital pathophysiological mecha-
nism not only in human mitochondrial diseases but also in the
pathogenesis of other congenital and acquired pathologies such as
cancer [9], neurodegenerative disorders [10], diabetes [11], and
cardiovascular diseases [12].
Mitochondria were regarded exclusively as the power house of
the cell throughout the second-half of the 20th century. It was not
until around 1995 that it became clear that they are not only a site
for energy production via oxidative phosphorylation, but also have
crucial role in cell death regulation. This latter discovery which
revitalised the field of mitochondrial and cell death research ap-
peared counterintuitive because experts doubted whether the meta-
bolic functionality of the cell which converge oin the mitochondria
could be manipulated to cause cell death. The other argument was
that the mitochondria during apoptosis do not suffer significant
ultra-structural changes thus may not be controlling the fate of the
cell. Since 2001, over 13,000 articles have been published linking
apoptosis and mitochondria, thus the initial opposition to the con-
cept of mitochondrial cell death control has been overcome [13].
In healthy cells, the inner mitochondrial membrane (IM) is
almost impermeable to all ions and including protons (H+). This
allows respiratory chain complexes I - IV to accumulate proton

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202 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
gradient across the IM which is obligatory for oxidative phosphory-
lation [14, 15]. Ions and small molecules crossing the inner mem-
brane do so via specific transporters. The inner mitochondrial
transmembrane potential (m) is generated from the charge imbal-
ance resulting from the creation of an electrochemical gradient
across the inner membrane. Finally, ATP synthesis is driven by the
complex V (mitochondrial ATP synthase) of the respiratory chain
via the exploitation of the proton gradient. Therefore the mainte-
nance of the proton gradient is crucial for cellular bioenergetics [14
- 16]. All components of the mitochondrial matrix and those me-
tabolites crossing the IM are strictly regulated with the aid of highly
selective transport proteins. A permanent dissipation of m is
associated with cell death [17, 18], however cases of transient loss
of m secondary to functional instability in one or more IM pores
may occur in physiological conditions [19, 20]. The most vital
function of the mitochondria is the synthesis of ATP. Dysfunction
of this process is associated with large number of mitochondrial
pathologies.
In 1972-1973, Boveris and Chance described mitochondria,
isolated from rat liver and pigeon heart, as a source of hydrogen
peroxide (H2O2) that diffuses to the cytosol as a major supplier of
superoxide anions [21, 22]. Although the cytochrome oxidase
(complex IV) mediates the four-electron reduction of molecular
oxygen to form H2O which is the end product of the respiratory
chain, a minor fraction of molecular oxygen takes part in one-
electron reduction processes generating reactive oxygen species
(ROS) and they include; O2-, H2O2, and the highly reactive hydroxyl
radical (HO-).
The intramitochondrial metabolites O2-, H2O2, NO and HO - are
pro-oxidants with potential to cause oxidative stress and tissue
damage. These are the chemical species that are linkedto the mo-
lecular mechanism of tissue dysfunction in aging, ischemia-
reperfusion, neurological diseases, and inflammation [16, 23].
Normally, the native defense systems consisting primarily of mito-
chondrial (manganese-containing) and cytosolic (copper-zinc-
containing) superoxide dismutases, glutathione peroxidase, and
phospholipid hydroperoxide neutralise these ROS and their peroxi-
dation products respectively [23 - 25]. Antioxidant supplements and
drugs have been used as mitochondria-generated free radical scav-
engers. These drugs inhibit mitochondria in tumour cells, thereby
killing these cancer cells; they also protect cells from oxidative
destruction, and aid in the repair of defects.
In healthy cells and during cell death, the outer mitochondrial
membrane (OM) permeability is also altered. Due to the presence of
the voltage dependent anion channel diffusion of metabolites and
solutes of ~5kDa across the OM occurs. Although the assumption
that the OM is freely permeable by such solutes has been chal-
lenged recently because real-time quantification of mitochondrial
Ca2+ concentrations revealed the existence of Ca2+ microdomains in
which the voltage dependent anion channel and a variety of addi-
tional OM proteins regulate and limit Ca2+ diffusion across the OM
[26, 27]. The OM permeability often increases during cell death
allowing for the liberation of soluble proteins into the intermem-
brane space (IMS), these proteins are otherwise situated and re-
tained within the mitochondria in healthy cell conditions. More so,
the death associated outer membrane permeabilisation can be an
accidental process as well as a finely controlled process with major
implications for health and disease [13, 23].
ii. Mitochondria and Cell Death
In general, two semi-interdependent apoptotic signalling path-
ways have been delineated; ligand binding to the death receptors at
the cell surface (extrinsic) pathway, and the mitochondrial (intrin-
sic) pathway [28]. Activation of either signalling pathway will lead
to the activation of caspases, a family of cysteine proteases that
function as death effector molecules. However, active caspase-8
can cleave the protein BID (BH3-interacting-domain death agonist)
Ubah and Wallace
off the Bcl2-homology domain 3 (BH3) into the active form tBid
(truncated BID), the latter providing a cross-talk between the ex-
trinsic and intrinsic death pathways. tBid then translocates to the
mitochondria where it promotes the mitochondrial permeability
transition (elevated OM permeability) causing cytochrome c (cyt c)
release. The mitochondrial pathway is initiated by the release of
various apoptogenic factors such as cyt c, apoptosis inducing factor
(AIF), second mitochondria-derived activator of caspases
(Smac)/direct inhibitor of apoptosis proteins (IAP) binding protein
with Low PI (DIABLO), Omi/high temperature requirement protein
A (HtrA2) and Endonuclease G (Endo-G) from the IMS into the
cytosol [13, 29 - 31]. The apoptogenic proteins can be released into
the cytosol using one of the four models hypothesised for the open-
ing of the permeability transition pore; [1] Oligomerised Bax/Bak
generates channels sufficient for the liberation of the factors [32].
This hypothesis is sustained by the findings that the structure of
Bcl-2 family proteins is similar to that of the pore-forming helices
of bacterial toxins, and that Bak can form channels in artificial
membranes and liberate cyt c from liposomes with large complexes
containing Bax and Bak, as seen by electron microscopy [33]. [2]
The proapoptotic proteins may form complexes with mitochondrial
membrane proteins such as the voltage-dependent anion channel
(VDAC) or adenine nucleotide translocator (ANT) to assemble
pores [34]. This is supported by findings that microinjection of
VDAC antibodies into the entrance of the channel inhibited the
release of cyt c and prevented apoptosis [35, 36]. [3] Permeability
transition pores are regulated by misfolded proteins and chaperones
such as Hsp25. These permeability transition pores are formed by
aggregates of misfolded membrane proteins [37, 38]. [4] Membrane
damage resulting from the matrix swelling may induce cristae struc-
ture distortion and rupture of the outer membrane thus releasing cyt
c [39, 40].
Also the mitochondrial pathways can be triggered by various
intracellular and extracellular stress signals which ultimately lead to
activation of pro-apoptotic proteins such as Bcl2 antagonist/killer
(Bak), Bcl2-associated X protein (Bax), or inactivation of anti-
apoptotic Bcl2 family members such as Bcl2 or Bcl-xL [41, 42]. The
release of cyt c from the mitochondria causes activation of down-
stream effector pro-caspases such as caspases-3 and 7 via formation
of a large cytosolic complex, the cyt c/Apaf-1/caspases-9 complex
[2, 13, 42]. Endo-G and apoptosis inducing factor (AIF) are capable
of inducing caspase-independent cell death and produce DNA
fragmentation [31, 37]. Although the mitochondrial membrane
permeabilisation (MMP) is vital for caspase-dependent and inde-
pendent apoptotic cell death pathways, the cell death cascade can
also be induced by triggers found in other organelles such as cal-
cium (Ca2+) in the endoplasmic reticulum (ER) and cathepsins in
lysosomes in response to ROS or ceramide [37, 43]. The apoptosis
pathways are strictly controlled by a variety of pro- and anti- apop-
totic regulators under physiological conditions, since any form of
uncontrolled stimulation of the apoptotic machinery would poten-
tially translate to a detrimental effects on cell survival. In human
cancers, the anti-apoptotic systems are often abnormally upregu-
lated thus enabling the cancer cells to evade apoptotic cell death
[44].
iii. Mitochondria in Cancer Cells
Metabolic imbalances and improved resistance to mitochondrial
cell death are considered as characteristic features of cancer cells.
Tumours relying on glycolysis to meet their metabolic burdens have
been acknowledged since the twentieth century [45, 46]. Cancer
cells are inclined to synthesise ATP mainly through aerobic glyco-
lysis (the so-called Warburg effect), a metabolic state that is associ-
ated with elevated glucose uptake and local acidification owing to
lactate production. The Warburg effect is actually the principle
behind the development of the positron emission tomography in
which a glucose analogue tracer (2-18fluoro-2-deoxy-D-glucose) is
utilised to differentiate between normal and malignant tissue. How-
Cancer Therapy
ever, a comprehensive clarification of the so-called Warburg effect
has not been achieved. Some of the non-exclusive hypotheses
which have been put forward to explain this phenotype are de-
scribed below:

Cancer cells are deficient in mitochondrial respiration and
ATP generation as a result of accumulation of defects in the
mitochondrial genome. Also, mitochondrial germline muta-
tions have been implicated as a predisposing factor in cancer
development. In most cases, such mitochondrial mutations are
acquired during and after tumourigenesis. Brandon et al
grouped these acquired mitochondrial DNA mutations in two
different classes; the first class involves severe mutations that
impede oxidative phosphorylation, thus causing an increased
and enhanced ROS production and tumour cells proliferation
while the second category is of milder mutations permitting
tumour cells adaptation to their new microenvironment, espe-
cially during progression and metastases [47 - 49].

Mitochondrial enzymes can in specific cases act as tumour
suppressors and mutations of these nuclear encoded mitochon-
drial proteins have been linked to cancer and will indirectly
encourage aerobic glycolysis. For instance, the inactivating
mutation of two enzymes involved in the TCA cycle; mito-
chondria succinate dehydrogenase (SDH subunits B, C or D)
or fumarate dehydrogenase is considered an oncogenic event
causing phaeochromocytoma and leiomyoma, leiomyosacr-
coma or renal carcinoma respectively. Also accumulation of
succinate and fumarate in the cytosol as a result of loss of
function of their respective dehydrogenases favours the activa-
tion of the hypoxia-inducible transcription factor (HIF) and
generate a pseudohypoxic state together with HIF-dependent
reprogramming of the metabolism towards aerobic glycolysis
[50]. A mutation in the third TCA cycle enzyme, isocitrate de-
hydrogenase has recently been linked to the majority of grade
II and III gliomas and secondary glioblastomas [51, 52].

As the pre-malignant lesion grows progressively further away
from the blood supply, cancer cells (if not all solid tumours)
possess the characteristic ability to adapt to decreased oxygen
tension (hypoxia). One way in which these cells adapt to de-
creased oxygen tension is to shut down mitochondrial respira-
tion and simultaneously switch on glycolysis [50, 53].

Cancer cells often upregulate the enzymes and rate-limiting
processes of glycolysis, including glucose transporters, for in-
stance as a result of the expression of oncogenic factors in-
cluding Ras, Src, or Bcl-Abl or as a result of the constitutive
signalling through the Akt pathway [48, 54].
Cancer cells have been shown to be resistant to the induction of
mitochondrial outer membrane permeabilisation (MOMP), a proc-
ess which mediates the intrinsic apoptotic pathway [55]. MOMP
being a complex phenomenon is tightly regulated by Bcl-2 family
of proteins [41, 42, 56], proteins resident in the permeability transi-
tion pore complex [57], proteins that influence mitochondrial fusion
and fission dynamics [58] and even transcription factors with the
capacity to translocate from the nucleus to mitochondria to encour-
age MOMP. The tumour-suppressor protein p53 is an example of
such transcription factors [59]. Inhibition of MOMP disabling the
apoptotic system is vital for the development of solid tumours, and
also haematological cancers as evident in the switch from pre-
neoplasia (e.g., low-grade myelodysplastic syndrome) in which
cells spontaneously commit to apoptotic MOMP, to overt neoplasia
(such as acute myeloid leukaemia developing from a myelodysplas-
tic syndrome) in which MOMP is hindered [48, 60, 61].
The mechanistic link between the Warburg effect (aerobic gly-
colysis) and MOMP resistance in cancer cells has not been fully
elucidated; however the following hypothetical explanations have
been put forward to explain the concurrent occurrence of aerobic
glycolysis and MOMP inhibition in cancer:
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 203

Putatively, in a one-step process cancer cells might repress the
mitochondrial apoptotic programme via mechanisms that in
concert stimulate the Warburg phenomenon. The observation
that mitochondrial DNA mutations may induce, through un-
known mechanisms, a decrease of anti-cancer-induced or
spontaneous apoptosis in cancer cells is in favour of this hy-
pothesis [48]. Moreover, hexokinase II which increases in gly-
colysis and inhibits MOMP when it is in association with
VDAC in the OM provides a possible link between MOMP
suppression and aerobic glycolysis. This interaction between
hexokinase and VDAC is favoured by activation of the Akt
pathway [53] or by the cancer-specific overexpression of mi-
tochondrion-binding hexokinase isoenzymes [48, 62].

During multistep carcinogenesis, metabolic alteration and
MOMP inhibition could be autonomously acquired by cancer
cells. Increased ROS and respiratory dysfunction resulting
from mitochondrial mutations could increase the probability of
cancer cells committing to apoptotic MOMP, and MOMP in-
hibition would be necessary to maintain carcinogenesis. Oth-
erwise, suppression of the apoptotic pathway might be permis-
sive for genomic instability [63], and the Darwinian selection
of genetically variant cells would then favour the accumulation
of cells expressing the Warburg phenotype.
In the last ten years, there has been substantial interest in the
possibility that mtDNA mutations might predispose or at least play
a role in human malignancies and other common diseases. Al-
though the mechanism involved in the initiation and progression of
mtDNA mutations, and their involvement in tumourigenesis and
other pathogenesis awaits comprehensive understanding. Interest-
ingly, a recent report showed that the high heterogeneity of human
mtDNA was significantly amplified in tumours [64]. Polyak et al.
demonstrated a consistent presence of somatic mtDNA mutations in
human tumours. The authors provided evidence of the presence of
homoplasmic mtDNA mutations in 7 out of 10 cell lines from pa-
tients with colorectal carcinomas which were neither found in nor-
mal colon nor in other tissues from the same patients [65]. Recent
papers report somatic mutations in the mitochondrial genome in
nearly one out of every four gastric cancer specimens and stress the
potential role of those mutations in the evolution of the disease
[66]. Kulawiec et al. [67] demonstrated that mtDNA mutations
were not associated with ROS generation in some samples of breast
cancer cells, but constitutively activated the PI3K/AKT pathway
contributing to enhanced metastasis. More over, the PI3K/AKT
pathway is strictly linked and activated in association with the ser-
ine/threonine kinase target of rapamycin (mTOR) that regulates key
cellular processes such as cell survival, growth and proliferation.
The mTOR pathway is regularly hyperactivated in many human
tumours and this is consistent with its role in cell proliferation [68].
In cancer, oxidative stress has been linked to DNA instability,
hypermethylation and mutation in the DNA repair machinery, point
mutations and loss of heterozygosity in DNA microsatellites. In
addition, cell cycle deregulation coupled with a shortened cycle
resulting in inadequate opportunity to repair acquired DNA muta-
tions prior to cells entering into S phase from G1 phase [69 - 71].
Reports to date indicate that cancer cells exhibit huge varieties
of metabolic change which are associated with alterations in the
mitochondrial structures, dynamics and function, and with tumour
growth and survival. Mitochondria regulate tumour growth through
modulation of the TCA cycle and oxidative phosphorylation, and
also they are vital in regulating redox homeostasis in the cells, in-
ducing them to either commit to or evade apoptotic cell death sig-
nals. The mitochondrion has been identified as key organelle to
understanding the molecular basis of tumour proliferation and also
rational design of chemotherapeutic agents [52].
204 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
MITOCHONDRIA AS CANCER THERAPEUTIC TARGETS
The underlying principle for targeting the mitochondria for
therapeutic benefits lies in the knowledge that this organelle plays a
vital role in the regulation of bioenergetics, ROS production and
apoptotic cell death. Preferentially targeting the energy metabolism
of tumour cells and not the healthy cells which also rely upon the
essential pathway for ATP supply poses a major challenge. How-
ever, the cancer cell mitochondria are structurally and functionally
different from their healthy counterparts [72, 73]. Cancer cells dis-
play an extensive metabolic reprogramming that makes them more
susceptible to mitochondrial perturbations than healthy cells [74].
In a broader perspective, cancer-specific mitochondrial alteration
and energy metabolism can be exploited for the development of two
novel classes of anti-tumour agents. These novel agents will target
glycolysis and/or reverse the Warburg phenomenon or aim at induc-
ing apoptosis via targeting mitochondrial proteins and membranes
[48]. As stated earlier, mitochondrially-targeted agents emerged as
a means of selectively targeting tumour cells as a result of these
differences that exist between them and non-immortalised cells. We
discuss below a number of mitochondrial features which has been
exploited or may be considered as therapeutic target in human can-
cer treatment.
i. Targeting Mitochondrial Permeability Transition and Ade-
nine Nucleotide Translocase (ANT)
The permeability transition pore complex (PTPC) is a highly
dynamic supra-molecular complex with a poorly understood struc-
tural identity. This is probably as a result of the existence of multi-
ple isoforms of its constituents and also a number of distinct but
functionally related solute carrier proteins that can substitute for
each other within the PTPC. Prototypical PTPC is composed of the
VDAC in the outer membrane, the adenine nucleotide translocase
(ANT) in the mitochondrial inner membrane and cyclophilin D
(CYPD) in the mitochondrial matrix [13], however studies showing
that the mitochondrial permeability transition (MPT) occurred in
mouse liver mitochondria with ANT1 and ANT2 knock-out mouse
suggests that both VDAC and ANT (but not CYPD) may not be
essential for the lethal functions of the PTPC [75 - 77]. Other stud-
ies have also demonstrated that CYPD was also not essential for the
permeability transition [78, 79], thus leading to the assessment of
the MPT (and ANT) as being relevant for necrotic rather than apop-
totic cell death [80]. Despite these findings, the suggestion that the
ANT isoforms may play a vital role in apoptosis is credible; ANT1
and ANT3 are pro-apoptotic, whereas ANT2 (which is often over
expressed in proliferating cells) is anti-apoptotic [81 - 84]. Compo-
nents of the PTPC can be targeted by several compounds to induce
MPT and apoptosis. By depleting the endogenous inhibitors of
PTPC opening such as glucose, phosphocreatine and glutathione, an
indirect permeabilisation effect can be achieved. Similarly, agents
that cause increase in cytosolic Ca2+ or stimulate ROS production
can trigger MPT [85]. Although several ANT ligands have been
shown to induce intrinsic cell death, they lack ANT isoform-
specific targeting.
Lonidamine, 1-(2,4-dichlorobenzyl)-1-H-indazole-3-
carboxylic acid is a putative ANT ligand that triggers the mito-
chondrial apoptotic pathway [86]. It is a glycolysis-targeting com-
pound which has exited clinical trials and gained approval in
Europe for the treatment of solid tumours. Lonidamine acts as a
hexokinase inhibitor with selectivity for the mitochondria-bound
form of hexokinase which seems to play a key role in enhancing
glycolytic flux in both proliferating normal and transformed cells
[87]. Lonidamine in combination with the peripheral benzodi-
azepine receptor (PBR) ligand diazepam showed a cytostatic effect
on tumour growth in patients with recurrent glioblastoma multi-
forme in a phase II clinical study. 50 % of patients exhibited disease
stabilisation following treatment including one case in which pro-
gression occurred after 12 months [86, 88]. The inclusion of loni-
Ubah and Wallace
damine with the anthracycline, epirubicin, increased by 9% the
response rate of patients with solid tumours in comparison with
epirubicin alone [89, 90]. In another phase II study designed to
examine the efficacy of cisplatin, paclitaxel and lonidamine co-
administration in patients with advanced ovarian cancer, an 80 %
overall response rate was reported, including 40% complete re-
sponses and 40% partial responses [91].
Betulinic acid (BetA) is a plant-derived pentacyclic triterpe-
noid that exerts potent anti-cancer effects in vitro and in vivo with
remarkable selectivity for tumour cells over non-transformed cells.
Apoptosis induced by BetA has been shown to involve the activa-
tion of caspases, PARP cleavage and DNA fragmentation. In addi-
tion, the production of ROS and decrease in the mitochondrial
membrane potential has been described, thus suggesting a reliance
on the mitochondrial cell death pathway [92 - 94]. BetA triggers
MOMP directly in association with mitochondrial membrane poten-
tial dissipation and cyt c release when added to isolated mitochon-
dria in a cell-free system [95]. This cytotoxicity induced by BetA
could not be blocked by the pan-caspase inhibitor zVAD.fmk [96].
This would imply that the cytotoxic effects of BetA are mediated by
the MPT which may be induced by ROS overproduction [3]. BetA
modulates the expression levels of Bcl-2 family proteins which
includes the upregulation of the pro-apoptotic mewmbers such as
the BAX and BCL-X5 (also known as the BCL2L1). Moreover,
BetA mediated apoptosis has been shown not to be associated with
p53 accumulation and occurred in a p53-indepndent fashion [97].
GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide),
a glutathione-coupled trivalent arsenical compound which is a hy-
drophilic derivative of the protein tyrosine phosphates inhibitor
phenylarsine oxide (PAO). This small synthetic mitochondrial poi-
son targets angiogenic endothelial cells. The trivalent arsenical of
GSAO interacts with and disrupts ANT of the inner mitochondrial
membrane of endothelial cells thus causing growth arrest [98, 99].
It has been shown to cross-link critical cysteine residues of ANT
(Cys160 and Cys257) resulting in the inhibition of the ATP/ADP
antiporter activity, cytosolic ATP depletion, ROS overproduction,
mitochondrial depolarisation and apoptosis [100]. GSAO would
preferentially target proliferating cells due to their high mitochon-
drial Ca2+ levels and elevated respiration which renders them more
susceptible to PTPC opening than normal cells [100]. PENAO (4-
[N-[S-penicillaminylacetyl]amino] phenylarsonous acid) a second
generation ANT inhibitor was designed to evade the pro-drug proc-
essing and metabolism of GSAO [101]. It is a cysteine mimetic of
active metabolite of GSAO, CAO (4-[N-[S-cysteinylacetyl] amino]
phenylarsenoxide). PENAO accumulates in cells 85-fold faster than
GSAO, resulting in a 44-fold enhanced anti-proliferative activity
and a ~20-fold increased anti-tumour efficacy in mice. PENAO
targets both proliferating endothelial and tumour cells, unlike
GSAO [102]. A phase 1/IIa dose escalation study of PENAO in
patients with solid tumours resistant to standard chemotherapy is
currently recruiting.
Retinoid-related compounds such as CD437 (6-[3-(-1-
adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) and
all trans-retinoic acid are well-known for their ability to induce the
expression of retinoic acid receptor-responsive genes resulting in
complete clinical remission in a high proportion of patients with
acute promyelocytic leukaemia [103]. Interestingly, Belzacq et al.,
and Notario et al reported that these retinoids (CD437 and the all-
trans-retinoic acid, 9-cis-retinoic acid and 13-cis-retinoic acids)
also trigger ANT-dependent MPT and apoptosis independent from
nuclear receptor binding [88, 104].
ii. Agents that Induce ROS Overproduction and Targeting Mi-
tochondrial Peroxiredoxin III
Motexafin gadolinium (gadolinium texaphyrin) is an aromatic
macrocylic compound that exhibits an elevated oxidising potential,
thus triggering excess production of ROS and interfering with the
Cancer Therapy
antioxidant machinery. Motexafin gadolinium has been shown to
preferentially accumulate in malignant cells, probably due to their
metabolic perturbations, and to improve in vivo sensitisation to
radiation and chemotherapy of xenotransplanted tumours [105]. In a
phase III study based on motexafin gadolinium and brain radiother-
apy for the treatment of patients with lung cancer and brain metas-
tasis reported a motexafin gadolinium-mediated delayed neurologi-
cal progression. In addition, paediatric glioblastoma patients toler-
ated the same regimen [106], suggesting a further investigation of
this combination therapy is warranted.
Menadione (2-methyl-1,4-naphthoquinone) and thiol cross-
linking agents such as diamide (diazenediacarboxylic acid bis
5N,N- dimethylamide), bismaleimido-hexane (BMH) and dithio-
dipyridine (DTDP) cause oxidation of a critical cysteine residue
(Cys 56) of ANT and can bypass Bcl-2 mediated cytoprotection.
Recombinant Bcl-2 fails to prevent thiol modification of ANT.
Moreover, these thiol cross-linkers induce mitochondrial membrane
permeabilization and cell death irrespective of the expression level
of Bcl-2 [107]. This suggests that thiol crosslinking agents cause a
covalent modification of ANT which supercedes the control by Bcl-
2 thus commits the cells to apoptosis.
-lapachone (ARQ 501) which undergoes futile redox cycles
catalysed by NAD(P)H:quinone oxidoreducatse 1, thereby inducing
the overproduction of ROS, poly(ADP-ribose) polymerase 1
(PARP1) hyperactivation and apoptosis. -lapachone is in clinical
trial as a monotherapy and in combination therapy with gemcitabine
in the management of patients with cancer of the pancreas and also
head and neck malignancy [108].
Accumulation of ROS can be achieved by inhibiting the anti-
oxidant systems, thus suggesting an alternative approach.
Buthionine sulphoximine causes an accumulation of ROS by in-
hibiting the synthesis of reduced gluthatione (GSH) whereas
imexon, an aziridine-containing small pro-oxidant molecule, de-
pletes the GSH pool due to its thiol-binding activity [109, 110]. The
combination therapy of buthionine sulphoximine and mephalan, an
alkylating agent, is being investigated in a phase I clinical study
involving patients with neuroblastoma and melanoma. Also,
imexon alone and in combination with docetaxel demonstrated
some efficacy in another phase I clinical study in patients with
breast, prostate and lung cancer [110, 111].
Elesclomol sodium (STA-4783) induces oxidative stress with a
consequent increase in ROS in both transformed and healthy cells.
Since tumour cells have elevated ROS levels compared to healthy
cells, STA-4783 will induce oxidative stress beyond baseline levels
with a resultant elevated ROS levels overwhelming the tumour cell
antioxidant systems. This may result in the induction of the apop-
totic cell death pathway [112, 113]. STA-4783 alone and in combi-
nation with paclitaxel showed promising data in phase I/II clinical
studies in patients with refractory solid tumours, however a recent
phase III study in patients with melanoma has been discontinued on
safety grounds [114, 115].
Mangafodipir is a superoxide dismutase (SOD) mimetic pos-
sessing both catalase and gluthatione reductase activities. In non-
transformed cells, Mangafodipir acts as an antioxidant whereas in
malignant cells it has been shown to elevate H2O2 pool and also
potentiate the anticancer activity of paclitaxel against xenotrans-
plants of colon cancer in mice [116]. Mangafodipir is in phase II
trial in subjects with colon cancer.
Isothiocyanates such as -phenylethyl isothiocyanate
(PEITC), a consitutuent of edible cruciferous vegetables such as
watercress, is a thiol modifier that interacts with redox regulatory
proteins [117]. PEITC does not only provide significant protection
against chemically induced oncogenesis in experimental rodents but
also inhibits proliferation of human cancer cells via induction of
apoptosis and autophagy. Although the underlying mechanism is
not fully understood, studies demonstrate that PEITC-induced
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 205
apoptotic and autophagic death is initiated by production of ROS
resulting from inhibiton of oxidative phosphorylation[118,119].
PEITC in combination with metformin demonstrated a significant
anti-proliferative and cytotoxic effect in ovarian cancer cells in
vitro. This novel combination was effective in cisplatin resistant
cell lines [120].
2-methoxyoestradiol is selectively cytotoxic to human leu-
kaemia cells but not normal lymphocytes. It inhibits angiogenesis
by reducing endothelial cell proliferation and causing apoptotic cell
death. There is accumulation of superoxide via its ability to inhibit
SOD [121, 122]. 2-methoxyoestradiol has shown disease stabilisa-
tion and it is also well tolerated according to reports from several
phase I/II studies in patients with solid tumours or multiple mye-
loma [123, 124]. ANT-224, an orally bioavailable second-
generation copper-chelating tetrathiomolybdate analogue showed
similar effects as to the oestradiol analogue with potential antiangi-
ogenic and antineoplastic activities [125].
The synthesis of ATP requires large amounts of oxygen, which
usually leads to the generation of ROS. Cellular damage ensues if
these ROS are not detoxified by the antioxidant systems. Elevated
pools of mitochondrial ROS and the disturbance of peroxiredoxin
(Prx) generation in malignant cells may cause oxidative stress and
the induction of apoptosis. Peroxiredoxins are family of enzymes
that catalyze the reduction of H2O2 and hydroperoxides to water
and alcohol respectively and there are six isoforms of the enzyme
(Prx I - VI). The Prx system is a cellular defence system against
oxidative stress, and Prx III and V have been reported to be ele-
vated in the mitochondria of cancer cells [126, 127]. Also elevated
expression of Prx I has been reported in several human cancers
including non-small cell lung, oral, breast and liver cancer. Over-
expression of Prx II has also been reported in breast cancer, meso-
thelioma, and head and neck cancers [128]. Elevated Prx II is asso-
ciated with the chemotherapeutic resistance of leukemia and stom-
ach cancers. Downregulation of Prx II sensitised head-and neck
cancers to radiation and gastric carcinoma to cisplatin [128, 129].
As mentioned above, recent studies have suggested that the overex-
pression of Prx III and its electron donors might provide a primary
line of defence against hydrogen peroxide produced by the mito-
chondrial respiratory chain, thus agents targeting Prx III and the
mitochondrion-specific electron suppliers Trx2, Trx reductase
(TrxR)2 and sulfiredoxin (Srx) may potentially be administered in
combination with various chemotherapeutic agents. Srx plays a
vital role by reducing hyperoxidised Prx III via translocation into
mitochondria. Overexpression of the mitochondrion-targeted Srx
efficiently enhances the restoration of Prx III and leads to cellular
resistance to apoptotic cell death achieved by an enhanced elimina-
tion of mitochondrial H2O2 and decreased rates of m collapse
[130].
iii. MOMP as a Target
As discussed earlier, MOMP can cause MPT to proceed, how-
ever MPT can also result from events originating from the outer
membrane. Pro-apoptotic proteins such as BAX and BAK can me-
diate MOMP via their pore-forming activity [33, 41]. BAX is cyto-
solic in healthy cells whereas BAK is an integral OM protein. BAX
translocates to the OM in response to pro-apoptotic triggers and
BAK undergoes conformational modifications locally. These two
processes consequently result in the assembly of homo-oligomers
and/or hetero-oligomers that form protein-permeable conduits
through which toxic intermembrane space proteins are liberated
into the cytosol [33, 131]. Bcl-2 and Bcl-XL can cause the seques-
tration of BAX and BAK thus displaying their strong cytoprotective
ability. Moreover, BAX can be directly activated or liberated from
inhibitory interactions with anti-apoptotic Bcl-2 family proteins by
cytoplasmic p53 [59]. The binding groove of BAX that mediates its
interaction with anti-apoptotic proteins has been known for many
years, but the site for direct activation by pro-apoptotic BH3-only
206 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
proteins of the Bcl-2 family has only recently been identified with
the help of nuclear magnetic resonance analysis. This represents a
novel druggable target for therapeutic modulation of apoptosis
[132]. TOM22 which is a core component of the OM protein trans-
location pore has been reported as the mitochondrial receptor for
BAX [133], although a recent study outcome has suggested that
BAX and BAK oligomerisation occur independent of TOM22
[134]. Endophilin B1 is another BAX- interacting protein which has
been reported to mediate BAX-dependent MOMP [135].
MOMP can also be initiated via mitochondrial lipids destabili-
sation by pro-apoptotic stimuli, with a consequent formation of
transient gaps in the outer membrane that permits leakage of inter-
membrane space proteins. Inner membrane proteases such as prese-
nilin-associated rhomboid-like (PARL) also regulate the release of
cyt c from the mitochondria by cleaving the dynamin-related pro-
tein optic atrophy 1 (OPA1), in this way directing the remodelling
of cristae independent of mitochondrial fusion [136, 137]. PARL-
deficient cells have been shown to display low levels of OPA1 and
were more susceptible to mitochondrial apoptotic triggers [136].
Therefore inhibition of PARL seems a promising strategy to induce
MOMP and commit transformed cells to apoptosis.
The susceptibility of cancer cells to undergo apoptotic cell
death has repeatedly been demonstrated to be determined by the
ratio of pro-apoptotic versus anti-apoptotic Bcl-2 proteins. Shifting
the balance of the “so-called” Bcl-2 rheostat in favour of the pro-
apoptotic protein members, such as the BH3-only proteins gener-
ates a powerful means to initiate MOMP-dependent apoptosis [3].
Several inhibitors of Bcl-2 anti-apoptotic proteins have been identi-
fied recently as being capable of inducing apoptosis in a variety of
tumour cells, thus indicating their potential in cancer therapy. How-
ever, following the investigation of some of the putative Bcl-2 in-
hibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine
and ABT-737), it appears that only ABT-737 specifically targeted
Bcl-2 proteins and induced apoptosis via caspase-9 activation.
These BH3 mimetics currently under preclinical and clinical testing
have shown that they also interact with cellular targets that are un-
related to Bcl-2 with the exception of ABT-737 and ABT-263, and
this “unwanted” interaction might mitigate further development of
this class of drugs due to potential toxicity concerns [138, 139].
ABT-737, a small-molecule inhibitor of the anti-apoptotic pro-
teins Bcl-2, Bcl-X L and Bcl-W, with affinity two to three orders of
magnitude more potent than the previously mentioned counterparts.
Data obtained from mechanistic studies suggests that ABT-737
does not directly initiate the apoptotic cell death process; rather it
enhances the effects of death signals [140]. It has shown a synergis-
tic cytotoxicity profile with conventional chemotherapeutics and
radiotherapy against haematological malignancies and solid tu-
mours [141, 142]. ABT-737 exhibits single-agent-mechanism-based
killing of small-cell lung cancer and lymphoma cell lines, as well as
in primary tumours and animal models. ABT-737 improves sur-
vival, regresses established tumours and is associated with high
cure rates of tumours in mice [140]. Furthermore, ABT-737 re-
versed the chemoresistance of cancer cells against conventional
chemotherapeutic agents, and ABT-737 resistance could be over-
come by using any of the classical cytotoxic agents [143, 144].
ABT-263, is an orally bioavailable derivative of ABT-737 with
improved clinical potential. It mimics the mode of action of ABT-
737 and has shown promising preclinical outcome. It is currently in
Phase I/II clinical trials for chronic lymphocytic leukaemia, lym-
phoma and small cell lung cancer, as monotherapy or in combina-
tion with conventional chemotherapeutic agents, or with mono-
clonal antibodies [145].
Gossypol (AT-101) is a natural phenol derived from the cotton
plant (genus Gossypium) [146] that simultaneously inhibits the
anti-apoptotic Bcl-2 proteins, BCL-2, Bcl-XL, Bcl-W and Mcl1
[138, 147]. In a phase I study against prostate cancer, it showed
Ubah and Wallace
clinical activity and it is being evaluated currently as a monother-
apy or incombination with temozolomide or topotecan in the
treatement of patients with SCLC, leukaemia, chronic lymphoma or
advanced B-cell malignancies [148]. Apogossypol (NSC736630) is
a semi-synthetic derivative of gossypol with reported superior anti-
tumour activity and reduced toxicity compared to the parent com-
pound. Daily oral dosing studies showed that mice tolerated doses
of the derivative 2 - to - 4 times higher than gossypol. Hepatotoxic-
ity and gastrointestinal toxicity represent the major adverse effects
of gossypol, with the derivative apogossypol showing far less toxic-
ity. Efficacy tested both in vitro and in vivo, apogossypol displayed
superior activity [149].
A-385358 is a small molecule with some selectivity for binding
to Bcl-X L versus Bcl-2 [150]. It enters the cells efficiently and co-
localises with the mitochondrial membrane. As a monotherapy, A-
385358 shows a modest cytotoxicity towards tumour cell lines, and
enhances the in vitro cytotoxic effects of other chemotherapeutic
agents like paclitaxel, etoposide, cisplatin and doxorubicin in sev-
eral tumour types. In A549, non-small-cell lung cancer, A-385358
enhanced the activity of paclitaxel by about 25-fold, and this poten-
tiation of paclitaxel by A-385358 is reproducible in vivo. Combina-
tion of A-385358 with the maximally or half maximally tolerated
dose of paclitaxel showed a significant growth inhibition in A-549
xenograft model.more so, significant increase in mitotic arrest and
consequent apoptosis have been reported for the combination ther-
apy compared to paclitaxel monotherapy [150].
Obatoclax (GX15-070) is a small-molecule indole bipyrrole
compound which is predicted to occupy a hydrophobic pocket
within the BH3 binding groove of Bcl-2 anti-apoptotic proteins thus
antagonising their actions with a consequent induction of apoptosis
dependent on BAX and BAK [151, 152]. Unlike ABT-737, obato-
clax efficiently disrupts the direct interaction between BAK and
MCL-1 in intact mitochondrial outer membrane and also inhibits
the association between MCL-1 and BAK in intact cells thereby
overcoming the MCL-1 dependent resistance to ABT-737 and
bortezomib, a proteasome inhibitor and the gold standard treatment
for patients with multiple myeloma [151]. Obatoclax has demon-
strated a modest activity as a monotherapy in a phase I clinical
study involving patients with advanced chronic lymphocytic leu-
kaemia [153], however it is currently undergoing clinical evaluation
as a monotherapy and in combination regimen in phase I/II studies
for the treatment of haematological malignancies and solid tumours
[154]. In another phase II clinical study using obatoclax for the
treatment of myelofibrosis (MF) the molecule had no significant
clinical activity in the MF patients at the dose and schedule evalu-
ated [155].
Oblimersen (G3139) is an antisense oligonucleotide compound
rationally designed to specifically bind to the first 6 codons of the
human bcl-2 mRNA sequence resulting in the degradation of the
bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation
[156]. Oblimersen in combination with chemotherapeutic agents
such as doxorubicin, docetaxel, cyclophosphamide and fludarabine
has been clinically evaluated in various cancer types (157, 158]. In
a phase III study involving patients with relapsed or refractory
chronic lymphocytic leukaemia, there was a significant increase in
response rate and its duration with the addition of oblimersen to
fludarabine plus cyclophosphamide therapy [158]. In a phase II
multicentre study, oblimersen in combination with rituximab
showed clinical benefit and safety in relapsed/refractory B-cell non-
Hodgkin lymphoma patients [159]. However, another phase III
clinical study in patients with multiple myeloma showed no signifi-
cant differences in response rate between both groups treated with
either dexamethasone plus oblimersen or dexamethasone alone
[160]. Despite extensive data supporting a critical role for Bcl-2 in
chemoresistance in small cell lung cancer, addition of oblimersen to
a standard regimen for the tumour (carboplatin and etoposide) did
not enhance any clinical outcome measure [161]. It has been sug-
Cancer Therapy
gested that the reported lack of efficacy may be due to insufficient
suppression of Bcl-2 in vivo, however further evaluation of
oblimersen in small cell lung cancer is not necessary.
HA14-1 (2-amino-6-bromo-4-[1-cyano-2-ethoxy-2-oxoethyl)-
4H-chromene-3-carboxylate), a small organic molecule (molecular
weight = 409) and nonpeptidic ligand of a bcl-2 surface pocket. In
vitro binding studies demonstrated the interaction of HA14-1 with
this bcl-2 surface pocket that is vital for bcl-2 biological function
[162]. HA14-1 enhances the sensitivity of cultured glioblastoma
cells to chemotherapy or radiotherapy [163]. It effectively and se-
lectively induces apoptosis in human acute myeloid leukaemia (HL-
60) cells overexpressing Bcl-2 anti-apoptotic proteins and its activ-
ity is not altered by the expression of multidrug-resistance pheno-
type [162, 164].
Polyamine analogues and conjugates, the naturally occurring
polyamines spermidine and spermine and their diamine precursor
putrescine are vital for eukaryotic cell growth, differentiation and
death [165]. Although the targeted inhibition of specific biosyn-
thetic enzymes in the polyamine metabolic pathway has yet to show
significant clinical success in the treatment of cancer, there is a
large body of evidence in favour of the rational for targeting the
polyamine function and metabolism in cancer [165 - 167]. Poly-
amine analogues and conjugates have been developed as a strategy
to exploit the self-regulatory nature of polyamine metabolism. The
macrocyclic polyamine analogues, symmetrically substituted
bis(alkyl) and asymmetrically substituted polyamine analogues, and
the novel oligoamine analogues have been associated with mito-
chondrial DNA as their target for cytotoxicity activity [168 - 170].
Cyt c release from the mitochondria of treated cells also suggested
some involvement of the mitochondria in unleashing the apoptotic
cell death response to these compounds. Xie et al showed that an-
tharcenylmethyl homospermidine (ANTMHspd), a polyamine con-
jugate induced mitochondrial membrane potential loss followed by
the release of cytochrome c and apoptotic cell death in promyelo-
cytic leukemia (HL-60) cells [171]. This group further demon-
strated that the combination therapy of ANTMHspd with alpha-
difluoromethylornithine (DFMO), an irreversible inhibitor of or-
nithine decarboxylase enzyme (ODC) was synergistic in inducing
the classical apoptosis in HL-60 cells via mitochondria/caspase-
9/caspase-3 dependent pathway. Ant 4, a putrescine-anthracene
conjugate, also demonstrated similar cytotoxicity profile in HL-60
cell line. Ant 4 also induced oxidative stress in the cells; the latter
discovery provides a possible mechanism for the triggering of apop-
tosis via the mitochondrial pathway [172].
iv. Targeting the Warburg Effect
Elevated glycolytic rate and improved glucose uptake can pro-
vide several benefits to a proliferating tumour cell. Although oxida-
tive phosphorylation generates more ATP per molecule of glucose,
glycolysis can provide ATP at a higher rate provided glucose sup-
ply is unlimited [173]. While Warburg believed that the preferential
use of glycolysis even in the presence of oxygen by tumour cells
was as a result of defects in their mitochondrial respiratory path-
ways , recent studies have suggested that tumour cells do contain
functional mitochondria and that the enhanced glycolytic flux con-
fer a growth advantage [45, 46].
In tumour cells much of the carbon entering the TCA cycle is
extruded as citrate resulting in a truncated TCA cycle that is used
for lipid and fatty acids synthesis [174]. Glucose plays a key role in
maintaining mitochondrial integrity by promoting the association of
hexokinase II with the mitochondria, thus preventing the liberation
of cyt c [62], as well as regulating various cell death effectors such
as the pro-survival Mcl-1, pro-apoptotic BAD and Bax [139]. Lac-
tate, the by-product of glycolysis is believed to promote tumour
invasion and metastasis via degradation of extracellular matrices
[175]. The understanding of the role of glucose in sustaining tu-
mour cell proliferation formed the basis for the development of
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 207
therapeutic strategies that preferentially target glycolysis and con-
sequently kill tumour cells (see Fig. 1).
3-bromopyruvate (3-BrPA), a lactate/pyruvate analogue is an
alkylating agent and a potent inhibitor of glycolysis. Its anticancer
effect is mediated by its capacity to inhibit hexokinase thus causing
a depletion of ATP in cancer cells. This effect is most severe in
cells with mitochondrial DNA deletion and respiratory defects,
leading to massive apoptotic cell death [176, 177]. Inhibition of
hexokinase also results in a rapid dephosphorylation of Bcl-2 asso-
ciated death promoter homologue (BAD), a molecule known to be
vital in both apoptosis and glycolysis. Dephosphorylation of BAD
at Ser-112 is associated with re-localisation of BAX to mitochon-
dria, cyt c release and apoptotic cell death [177]. Ganapathy-
Kanniappan et al. (2010) have described that the primary mecha-
nism of 3-BrPA is via preferential alkylation of GAPDH and that 3-
BrPA mediated cell death is linked to free radicals generation. Fur-
thermore, research from their laboratory also revealed that 3-BrPA
induced endoplasmic reticulum stress, inhibited global protein syn-
thesis which contributed to cancer cell death [178]. 3-BrPA has
been shown to have antitumour effects when injected intraarterially
in the hepatic artery of rabbits with VX-2 tumours [178]. The glu-
cose transporter isoform 1 (GLUT1) gene encodes a key factor for
glucose transport into cancerous tissue [179, 180]. Although the
expression and functional significance of GLUT1 in neuroblastoma
have not been fully described, 3-BrPA significantly suppressed the
proliferation of neuroblastoma cells with high GLUT1 gene expres-
sion compared to those with low expression [181], suggesting that
glycolysis inhibitors are a potential therapeutic option for treatment
of tumours expressing GLUT1. Combination of 3-BrPA with an
mTOR inhibitor demonstrated synergistic effects on leukemia and
lymphoma cells [182]. 3-BrPA may interact with other cellular
components owing to its alkylating properties suggesting that ex-
tensive toxicity study needs to be carried out. The interaction be-
tween HK and VDAC offers another fascinating target to selec-
tively commit cancer cells to apoptotic cell death. Disrupting this
interaction has been shown to preferentially kill tumour cells both
in vitro and in vivo via the promotion of PTPC and MPT [183, 184].
3-BrPA and Methyl Jasmonate, a plant hormone has been shown
to disrupt this HK-VDAC interaction and selectively trigger cell
death in cancer cells. It binds to HK thereby disrupting its associa-
tion with the mitochondria and apoptotic cell death is initiated
[184]. Methyl jasmonate disrupts the HK-mitochondrial interaction
at a concentration as high as 1 mM, thus it may serve as a lead
compound in the development of more potent and specific agents.
2-deoxy-D-glucose (2-DG) is a glucose analogue and has long
been known to act as a competitive inhibitor of glucose metabolism
[185]. 2-DG is transported into cells and is phosphorylated by
hexokinase to 2-DG-P. However, unlike its analogue metabolite
glucose-6-phosphate (G-6-P), 2-DG-P cannot be metabolised by
phosphohexose isomerase which usually converts G-6-P to fruc-
tose-6-phosphate. This leads to the accumulation of 2-DG-P in the
cells with a consequent inhibition of glycolysis at the step of
hexokinase mediated phosphorylation of glucose. Cellular ATP
depletion leading to cell cycle arrest and cell death occurs following
the inhibition of the rate-limiting step involving hexokinase medi-
ated glucose phosphorylation [54]. Nonetheless, the efficacy of 2-
DG is significantly affected by the presence of glucose and appears
to only partially reduce glucose availability for glycolysis. 2-DG
causes endoplasmic reticulum (ER) stress because of its ability to
affect protein glycosylation causing aberrant GlcNAcylation of
proteins and induces accumulation of misfolded proteins in the ER
[186]. In vitro studies demonstrated that 2-DG exhibits cytotxic
effect in cancer cells especially in those harbouring mitochondrial
defects or cells in hypoxic environment [187]. Inhibition of glyco-
lysis by 2-DG significantly increased the cytotoxicity of cisplatin in
human head and neck cancers via enhancing oxidative stress [188].
There has been concern that 2-DG might compromise the glycolytic
208 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
metabolism of the brain and heart, however discussions centre on
whether the therapeutic window of the 2-DG will be broad enough
to justify its clinical development and also to develop specific in-
hibitors of some glucose transporter isoforms that are frequently
upregulated in cancer. Data from a clinical study suggests that 2-
DG at doses up to 250 mg/kg seems safe for use in combination
with radiotherapy in glioblastoma multiforme patients [189]. 2-DG
has been shown to stimulate Akt phosphorylation in vitro and an-
tagonise the anti-tumour action of a radio-immunotherapeutic inter-
vention in vivo. These effects undermines the prospective therapeu-
tic value of 2-DG [190]. The pursuit of further clinical development
of this compound has recently been halted according to Threshold
Pharmaceuticals [61].
Dichloroacetate (DCA) is a small molecule inhibitor of pyru-
vate dehydrogenase kinase (PDK) which has the capacity to induce
a metabolic switch from aerobic glycolysis to glucose oxidation,
thus decreasing mitochondrial hyperpolarisation and rendering
tumour cells more sensitive to apoptotic signals [191]. Pyruvate
dehydrogenase is a mitochondrial enzyme which converts pyruvate
to acetyl CoA, and its activity is negatively regulated by PDK thus
DCA indirectly stimulates the pyruvate to acetyl CoA conversion.
DCA also upregulates the expression of the K+ channel Kv1.5,
which is often underexpressed in tumour cells [191]. DCA has pre-
viously been utilised in the clinic for the management of lactic aci-
dosis and showed a promising safety profile even when used
chronically [192]. A phase I study for DCA in currently under way
in Canada and is accepting patients with recurrent or metastatic
solid tumours [3, 61].
ATP citrate lyase (ACL) is a key enzyme that associates glu-
cose metabolism with lipid biosynthesis via catalysing the conver-
sion of citric acid to cytosolic acetyl-CoA has become a promising
potential drug target in cancer treatment. Frequently malignant cells
redirect pyruvate towards lipid synthesis which is a key component
required to support the elevated demand for membrane generation
in highly proliferating cells [193]. Tumour growth was shown to be
suppressed in vitro following the inhibition of ACL by RNAi or
using a pharmacological inhibitor, SB-204990 [193]. It has been
demonstrated that ACL is essential for the provision of adequate
acetyl CoA required for histone acetylation and hence gene expres-
sion [194]. The implication regarding this recent discovery is that
ACL inhibition might not only normalise tumour metabolism at the
lipid synthesis level but it will also play a part in the epigenetic
reprogramming of malignant cells.
PROTEASOMES
i. Structure, Localisation and Function
The functionally active 26S proteasome is a very large 2.4 MDa
ATP-dependent proteolytic complex that is situated in the cyto-
plasm and nucleus of eukaryotic cells. The 26S proteasome consists
of a 20S core catalytic cylindrical complex capped at both ends by
19S regulatory subunits [195]. Although ubiquitinylation is not a
prerequisite for the degradation of every protein, the 26S protea-
some identifies proteins and degrades proteins that have been
marked for degradation for their ubiquitin ‘tag’ [6]. The 20S protea-
some is a complex of 28 protein subunits arranged into 4 stacked
rings to create a cylindrical structure. The
-subunits made up of
seven polypeptides form the top and bottom rings of the 20S cylin-
drical structure. The enzyme active site of the proteasome complex
is in the two inner rings which consist of seven -subunits that form
the central chamber [196]. Proteasome enzymatic function is per-
formed by three (1, 2 and 5) of the seven -subunits, while the
function of the other four subunits is unclear [6]. For the 20S pro-
teasome to function in vivo, it will require the association of a regu-
latory unit that partially determine the specificity in function. 19S
regulatory complex is an example of such regulatory unit [6]. The
mammalian cells contain another regulatory complex that associates
with the 20S proteasome: the 11S regulator or PA28. It is made up
Ubah and Wallace
of a 28-KDa
and -subunits, and also a third sequence homolo-
gous -subunit [197]. The -subunit has different enzymatic fea-
tures and seems not to interact with the
- and -subunits to form
the 11S regulatory complex [198].
Proteasomes are situated in the both the nucleus and cytoplasm
of eukaryotic cells, however their distribution varies with regards to
cell or tissue type. It is now known that they undergo cell-cycle-
specific redistribution between the nucleus and cytoplasm. In the
cytoplasm, proteasomes localise near centrosomes, on the outer
surface of the endoplasmic reticulum and in cytoskeletal network
[199].
Proteasomes regulate the levels of proteins such as cyclins, Bcl-
2, caspases, nuclear factor B (NF-B) that are crucial in cell-cycle
progression and cell death [5, 6], and many of these proteasome
substrates are known mediators of deregulated systems in cancer.
There is evidence that proteasome affects cell-cycle progression via
its regulatory action on the cyclins. Also, it can cause an elevation
or attenuation in apoptotic events through its action on the caspases,
Bcl-2, and NF-B [6]. Suffice to say that specific defects in protea-
some function or structure are yet to be linked to neoplasia although
abnormalities of the aforementioned regulatory pathways are linked
to tumourigenesis.
ii. Targeting the Proteasome for Cancer Therapy (Table 1)
The dilemma associated with targeting the proteasome as a
means of chemotherapeutic intervention is that this organelle plays
a crucial role in the execution of many cellular functions, thus mak-
ing it difficult to target selectively and at the same time maintain a
tolerable therapeutic window. However, studies have shown that
many types of actively proliferating cancer cells display a higher
sensitivity to proteasome inhibition than the non-transformed cells
[6].
The proteasome inhibitors N-carbobenzoxy-L-leucyl-L-leucyl-
norvalinal (LLnV) and N-carbobenzoxy-L-isoleucyl-L-
-t-butyl-
L-glutamyl-L-alanyl-L-leucinal (PSI) demonstrated a selective
p53-dependent apoptotic cell death induction in the proliferating
human leukemic HL60 cells compared with the quiescent HL-60
cells [200]. Also compared with actively proliferating primary en-
dothelial cells, a 340-fold higher concentration of PSI was required
to induce apoptosis in contact inhibited quiescent cells [6]. Another
proteasome inhibitor (PI), Lactacystin, which is an irreversible
inhibitor of the catalytic -subunit of the proteasome induces apop-
totic cell death in human chronic lymphocytic leukaemia sensitive
or insensitive to radiation at doses that do not affect normal lym-
phocytes [201]. Apoptotic cell death was also observed in oral
squamous-cell carcinoma cells, but not in normal oral epithelial
cells or gingival fibroblasts following treatment with LLnV and
Lactacystin [201]. It is fascinating that treatment with LLnV or
lactacystin was associated with an accumulation of KIP1 and re-
duced expression of the anti-apoptotic protein, Bcl-2 thus leading to
cell-cycle arrest and consequent apoptosis [6]. Several studies have
provided proof of principle that the proteasome is a valid target for
anticancer therapy; however the available inhibitors lack specificity
[202]. This led to the design and development of boronic acid-
derived compounds that inhibited the proteasome pathway in a
highly specific fashion. Most of these compounds developed by
Adam et al showed activity across the 60 panel of cell lines from
the National Cancer Institute. Bortezomib was identified from these
compounds as the best candidate on the basis of its potency and
cytotoxicity [203].
Bortezomib (N-pyrazinecarbonyl-L-phenylalanine-L-
leucineboronic acid; previously known as PS-341 or MLN-341) a
boronic acid dipeptide was the first PI to enter clinical trials and
was granted accelerated approval by the FDA in 2003 after demon-
strating remarkable single-agent responses in patients with relapsed
and refractory myeloma. It has since been approved for the treat-
ment of newly diagnosed multiple myeloma and also mantle cell
Cancer Therapy
Table 1. Proteins affected by the Ubiquitin-Proteasome Pathway inhibition
lymphoma [202]. It reversibly and rapidly inhibits the proteasome
pathway by directly binding to the 20S proteasome complex and
blocking its enzymatic activity [203]. The underlying mechanism
leading to cell-cycle arrest and apoptotic cell death include NF-
B
inhibition, upregulation of various apoptotic pathways and effects
on tumour micro-environment. It is unlikely that proteasome inhibi-
tion with bortezomib will be observed in tumours resident in the
brain or the testes unless the blood-brain or blood-testes barrier has
been disrupted by the tumour. This was observed in tumour tissues
isolated from brain and testes showing no proteasome inhibition
[203]. Preclinical studies demonstrated that the cytotoxic and anti-
proliferating actions of bortezomib are correlated with the inhibi-
tion of proteasome in a p53 independent manner and also non-
overlapping with other chemotherapeutics [203]. Tumour resistance
to conventional anticancer agents can be overcome by the addition
of bortezomib as shown in two phase II multiple myeloma studies
[204, 205]. There is strong evidence suggesting that the activity of
bortezomib in solid tumours is significantly enhanced when used in
combination with chemotherapeutic agents such as doxorubicin,
gemcitabine, docetaxel, paclitaxel, and irinotecan [202, 206, 207].
Phase II clinical trials of bortezomib with gemcitabine, docetaxel
and other conventional cytotoxic agents are on-going.
SECOND-GENERATION PROTEASOME INHIBITORS
(PIs)
Following an improvement in our understanding of the ubiq-
uitin-proteasome pathway, the development of new PIs with distinct
efficacy and safety profiles was embarked upon. More so the sec-
ond generation PIs provide potential options for patients with borte-
zomib resistant tumours.
Carfilzomib (PR-171) has in vitro anti-proliferative effect
across a range of tumour cell types including multiple myeloma
cells resistant to bortezomib. PR-171 is more active than borte-
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 209
zomib when cells are exposed for short duration mimicking in vivo
single-dose exposure; however both compounds have superior ac-
tivity when cells are exposed for prolonged periods of time [208,
209]. To achieve a prolonged inhibition in vivo, PR-171 was admin-
istered daily and well tolerated at doses that inhibited over 80 % of
proteasome chymotrypsin-like-5 subunit (CT-L) activity in blood
and tissues. This observation contrasts that of bortezomib in which
a twice-weekly clinical-dosing schedule that allows full recovery of
proteasome function between doses was preferred because of ex-
cessive toxicity associated with more frequent dosing schedule
[210, 211]. Carfilzomib has also been investigated in combination
with lenalidomide and low-dose dexamethasone in patients with
relapsed/refractory multiple myeloma [212]. The FOCUS trial
(N=302) is comparing carfilzomibmonotherapy with best suppor-
tive care in the relapsed/refractory stage. Moreover, other early-
phase studies are investigating carfilzomib in frontline multiple
myeloma in combination with either thalidomide or lenalidomide
and dexamethasone [213].
Marizomib (NPI-0052; salinosporamide A) is a structurally
and pharmacologically unique -lactone--lactam proteasome in-
hibitor. Its potent and sustained inhibition of all three proteolytic
activities of the proteasome has generated a renewed motivation
inspiring the extensive preclinical evaluation in multiple myeloma,
mantle cell lymphoma, acute and chronic lymphocytic leukemia,
Waldenstrom’s macroglobulinemia, glioma, colorectal and pancre-
atic cancer models. It has shown efficacy as monotherapy, and in
combination with chemotherapeutics, biologics and targeted thera-
peutics [214]. Clinical trials in patients with multiple myeloma,
lymphomas, leukemias and solid tumours, and also patients with
bortezomib treatment failure, as well as in patients with diagnoses
where other PIs have been shown to be significantly efficacious is
underway [214 - 216].
210 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
MLN9708/MLN2238 is a selective, orally bioavailable second-
generation PI that is currently in a phase I clinical development
[217]. It has a shorter proteasome dissociation half-life with an
enhanced PK-PD, and anticancer activity compared with borte-
zomib. Treatment of multiple myeloma cells with MLN9708 pre-
dominantly blocked CT-L activity of the proteasome and induced
accumulation of ubiquitinated proteins. Multiple myeloma cells
resistant to bortezomib and other conventional therapies were
shown to respond to anti-proliferative and apoptotic effects of
MLN9708 and these activities are selective for tumour cells rather
than non-transformed cells [218]. Mechanistic studies showed that
MLN9708-mediated apoptosis is linked with the activation of
caspases-3, 8, and 9; elevated p53, p21, NOXA, PUMA, and E2F;
induction of endoplasmic reticulum stress response proteins Bip,
phosphor-eIF2-
, and CHOP; inhibition of NF-B [218]. MLN9708
in combination with lenalidomide, dexamethasone or histone deace-
tylase inhibitor suberoylanilidehydroxamic acid triggers synergistic
activity in the treatment of multiple myeloma [210, 218].
ONX-0912, is an orally bioactive tripeptideepoxyketone with
anti-proliferative effects and also induces apoptosis multiple mye-
loma cells resistant to conventional and bortezomib therapies. Its
anti-myeloma activity is associated with activation of caspases-3, 8,
and 9, and poly(ADP) ribose polymerase, also the suppression of
multiple myeloma cells migration [219]. In animal tumour models,
ONX-0912 significantly reduced tumour progression and improved
survival, and it improved the anti-tumour activity of bortezomib,
lenalidomide, histone deacetylase inhibitors or dexamethasone in
multiple myeloma [210, 219]. A phase I study with ONX-0912 in
patients with solid tumours is on-going, and an interim results from
the study showed that at doses that were tolerated, more that 80%
proteasome inhibition in blood could be achieved on a once-daily
dosing regimen for 5 consecutive days [220].
ENDOPLASMIC RETICULUM (ER) - GOLGI NETWORK
Interestingly a growing number of reports recognise proteins in
control of ER and Golgi homoeostasis as potential therapeutic tar-
gets in the treatment of malignancies. Eukaryotic cells consist of a
set of organelles with a tightly and dynamically regulated abun-
dance with regards to cellular demand. These organelles are
equipped with specific cellular functions. The ER is an organelle in
which secretory and membrane proteins are synthesised, and cor-
rectly folded proteins by the ER chaperones are transported to the
Golgi apparatus [221]. The ER retains the unfolded or misfolded
proteins and they are subsequently degraded by the ER-associated
degradation (ERAD) [222, 223]. In cases of accumulation of un-
folded proteins in the ER, an upregulated expression of the ER
chaperone and components of ER-associated degradation machin-
ery occurs in an effort to enhance the protein folding capacity and
degradation of unfolded or misfolded proteins. This process occurs
through the cytoprotective mechanism referred to as the ER stress
response or unfolded protein response [224]. Large amounts of
secretory proteins are transported to the Golgi apparatus when the
ER stress response enhances the capacity of ER function, thus caus-
ing inadequacy of Golgi function. This is referred to as Golgi stress,
which initiates the Golgi stress response to cope with the situation
[221]. To ensure the fidelity of protein folding and to sustain the
accumulation of unfolded or misfolded proteins, the ER has devel-
oped a group of signal transduction pathways, the unfolded protein
response (UPR) signalling pathways to alter transcriptional and
translational programs [225]. The basic mammalian cell UPR path-
ways is composed of three major signalling pathways initiated by
three primary ER-localised protein stress sensors; IRE1
(inositol-
requiring enzyme 1 alpha), PERK (double-strand RNA-activated
protein kinase-like ER kinase), and ATF6 (activating transcription
factor 6). The primary function of the UPR is to prevent the cell
from ER stress by limiting the amount of proteins translocated into
the ER lumen, increasing retrotranslocation and degradation of ER-
localised proteins, and enhancing the protein-folding capacity of the
Ubah and Wallace
ER. Nonetheless, the UPR will induce cell death programmes to
eliminate stressed cells in situations where attempts to recover the
cells from ER stress fails [226]. During carcinogenesis, there is a
high demand for an increased activity of ER protein folding, as-
sembly, and transport to support the elevated proliferation rate, and
this condition can induce physiological ER stress. Moreover, there
is an increased nutrient starvation and hypoxia as tumour cells
grow, and these are strong inducers of the accumulation of unfolded
or misfolded proteins in the ER and the subsequent activation of the
UPR pathways [227]. There is valid and accumulating evidence
showing that the UPR is a key mechanism required for cancer cells
to maintain malignancy and resistance to therapy [226].
Experimental evidence suggests that ER and Golgi apparatus
can activate both pro-survival and apoptotic mechanisms if the
stress-signalling threshold is exceeded [4]. The Golgi apparatus is
known to dissemble during apoptosis in a fashion resembling that
observed during mitosis [225]. It is plausible that the subtle balance
of protein trafficking between various subcellular compartments
afford an extremely sensitive damage sensor. It is worth mentioning
the interaction between the mitochondrion with the Bcl-2 family
proteins regulating various cell death signals and the ER. Thus,
overexpression of Bax has recently been shown to induce Ca2+ mo-
bilisation from ER stores [228, 229]. There is substantial evidence
supporting the Bcl-2 co-localisation with the ER and protects cells
from thapsigargin-mediated apoptosis [225]. More so, evidence is
mounting that signals originating from the ER-Golgi network may
bypass the mitochondrion and efficiently execute apoptotic cell
death [4, 225].
Targeting the UPR/ER Stress Components
The role of UPR in tumour maintenance has stimulated great
interest in exploring therapeutic potential by targeting the UPR
components. The understanding that healthy cells are not subjected
to stress and the UPR pathway remains in an inactive state while
that of transformed cells rely on activated UPR for survival creates
a discrepancy between tumour cells and their healthy counterpart
and thus offer an advantage for the agents targeting these compo-
nents to achieve specificity (Table 2).
PROTEASOME INHIBITORS
The degradation of the majority of cellular proteins is mediated
by the 26S proteasome. The role of proteasome in regulating cell
growth, survival and metastasis of malignant cells is well reported
leading to the development of PIs (e.g., Bortezomib; see previous
section for discussion on PIs). It is believed that PIs cause a build-
up of misfolded proteins which overwhelms the ERAD pathway
resulting in ER stress [230].
Multiple myeloma cells constitutively express ER stress sur-
vival factors in order to function as secretory cells, and it is also
assumed that the UPR components are required for B-cell differen-
tiation into antibody-secreting cells [231]. Thus agents that interfere
with the UPR and ER-stress induced apoptosis will show greater
effect in myeloma cells and other secretory cells as a result of the
reliance of these cells on the UPR pathway. As discussed in the
previous section, multiple myeloma cells are very sensitive to the PI
bortezomib which rapidly induces components of the pro-apoptotic
/terminal UPR, including PERK ultimately resulting in increased
cell death [230]. Since multiple myeloma cells are dependent on an
activated UPR for survival suggesting that these cells have a lower
threshold for ER stress. Thus, any additional stress can slant the
balance in favour of cell death.
HSP90 INHIBITORS
Heat shock protein 90 (HSP90) is involved in regulating the
proper folding, stability and function of a wide variety of signal-
transducing proteins that regulate cell proliferation and differentia-
tion. These proteins include steroid hormone receptors and protein
Cancer Therapy
Table 2. A representative list of ER stress-aggravating agents
kinases and most notably are Akt, Bcr-Abl, Apaf-1, surviving and
cyclin dependent kinases with established roles in tumorigenesis
[230].
17-Allylamino-17-demethoxygeldanamycin (17-AAG) and 17
(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-
DMAG) are analogues of geldanamycin, they are currently in clini-
cal trials. 17-DMAG was found to activate all three components of
the UPR in myeloma plasma cells leading to the suggestion that
HSP90 inhibitors induce apoptotic cell death at least in part via the
ER stress and the UPR death pathway [230, 232].
HIV PROTEASE INHIBITORS
Nelfinavir, is an HIV protease inhibitor which has recently
been shown to induce ER stress, autophagy and apoptotic cell death
in vitro and in vivo in non-small cell lung cancer [233]. Ata-
zanavir, another HIV protease inhibitor alongside Nelfinavir
showed in a separate report to trigger ER stress and cause cell death
in various malignant glioma cell lines in vitro due to their ability to
induce UPR [234]. As outlined earlier, PIs appear to be more effec-
tive in secretory cells however solid tumours frequently display
reduced sensitivity to bortezomib. Kraus et al considered utilising a
dual therapeutic approach to achieve a more effective tumour kill-
ing using agents that will enhance ER protein load and simultane-
ously blocking the proteasome degradation pathway using borte-
zomib [235]. In this setting, another HIV protease inhibitor Rito-
navir was used to interfere with the ERAD machinery with a con-
sequent decrease in the degradation of newly synthesised proteins
thus inducing the UPR in sarcoma cells previously resistant to
bortezomib. This combination of ritonavir and bortezomib resulted
in the activation of PERK, IRE1
, and ATF. This dual approach
showed synergistic actions in the induction of JNK, caspases - 4
and 9, and apoptotic cell death greater than 90 % [230, 235].
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 211
INHIBITORS OF PRO-SURVIVAL MODULE, GPR78
A number of studies have presented strategies to incapacitate
GPR78 function in vitro and in vivo, although few have made it to
clinical testing. There are several naturally occurring compounds
that interfere with GPR78 expression or activity at pharmacological
concentrations, for example salicylic acid from plants [236]; the
polyphenolic green tea component a (-) -epigallocatechin-3-gallate
(EGCG) [237]; genistein, an active ingredient of soy [238]; the
microbial metabolites verispelostatin [239], prunustatin A [240],
verrucosidin [241], piericidin A [242], deoxyverrucosidin [243];
synthetic members of the biguanide class of compounds (met-
formin, phenformin and buformin) [244]; the cyanine dye pyrv-
inium [245]; and the plant product arctigenin [246]. Since GPR78
has been located outside the ER lumen; it has been identified in the
cytosol, mitochondria, nucleus and tumour cells surface distinctly
suggesting that this protein exerts functions beyond its ER trans-
membrane signalling function [244, 247]. This discovery has lim-
ited the progress of some of the GPR78 inhibitors since they seem
to have a non-specific GPR78 inhibition.
Versipelostatin (VST) is the first compound reported to spe-
cifically inhibit GPR78 expression with a unique selective cytotox-
icity in glucose-deprived tumor cells by preventing the UPR [239].
Under stress conditions, it causes a robust cell death by inhibiting
GPR78 and GPR94 induction and repression of ATF4 and XBP1,
which are the UPR transcription activators [230, 239]. In glucose-
deprivation conditions, VST exerts actions similar to those of ra-
pamycin, thus suggesting that it may have dual activity as antican-
cer agent, inhibiting the UPR and modulating the mTOR signalling
pathway. The translational control during UPR is complex, thus
further studies on the mechanism of action of VST is required to
elucidate this translational events.
212 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
Other Therapeutic Agents that Trigger ER Stress
Brefeldin A (BFA), a fungal macrolytic lactone with a wide
range of antibiotic activity show anti-tumor activity by disrupting
the vesicular coating process involved in intracellular protein trans-
port thereby inhibiting intracellular transport of proteins from the
ER to the Golgi thus causing disintegration of the Golgi complex.
BFA has been shown to induce p53-independent apoptosis in di-
verse human cancer cell lines [230, 248, 249]. BFA acts by inhibit-
ing directly the ADP-ribosylation factor (ARF) activity which is
essential for coatamer assembly onto Golgi membranes and results
in the activation of the UPR [230]. The critical role of ER stress in
mediating BFA cytotoxicity was demonstrated using the flavones
baicalein which prevented BFA-induced ER stress with a resultant
significantly reduced BFA cytotoxicity [250]. Breflate, a water-
soluble pro-drug analogue of BFA was developed as a parenterally
administered agent; unfortunately this agent did not progress to
clinical trials [244].
Thapsigargin, is a sesquiterpene lactone of natural origin that
potently inhibits sarcoplasmic/endoplasmic reticulum calcium AT-
Pase (SERCA). The inhibition of SERCA resulted in significant
calcium leakage from the ER store into the cytosol causing a pow-
erful ER stress induction. The clinical use of this agent was never
achieved because of poor tolerance in experimental animals. Be-
sides its potent anti-tumour activity, it also caused histamine release
and stimulates arachidonic acid metabolism [244]. Overall, these
features were perceived as shortcomings in the context of systemic
therapy.
To circumvent systemic toxicity, a pro-drug of thapsigargin
with tumour-targeting potential was developed. It is a chemically
modified thapsigargin coupled to a peptide carrier that is a substrate
for prostate-specific antigen (PSA) protease. The protease acts on
the pro-drug to liberate the active agent within metastatic prostate
cancer sites [251]. G202, a related pro-drug construct whose anti-
tumour activity is activated by carboxypeptidase prostate-specific
membrane antigen (PSMA) showed a significant anti-tumour activ-
ity against a panel of human cancer xenografts in vivo with minimal
host toxicity [252]. A phase 1 dose escalation clinical study with
G202 has commenced in patients with advanced tumours [244,
253].
Celecoxib, is a selective cyclooxygenase 2 (COX-2) inhibitor
and a non-steroidal anti-inflammatory drug (NSAID) developed and
originally approved for management of acute pain and inflamma-
tion, and later as an adjunct for the management of familial adeno-
matous polyposis (FAP) [254]. In the last decade, novel pharmacol-
ogical activity and targets most likely independent of its COX-2
inhibition emerged. They include ER stress aggravation and car-
bonic anhydrases inhibition [255, 256]. Its ability to induce ER
stress is associated with its inhibition of SERCA function causing
calcium efflux from the ER [257]. Generally, the clinical therapeu-
tic outcome for celecoxib in advanced cancer management has been
unimpressive. It has been suggested that the poor outcome observed
may be due to the fact that celecoxib is optimised towards COX-2
inhibition rather than to its subsequently recognised ESRA activity,
thus celecoxib might not accomplish adequately high levels of ER
stress in tumour cells [244]. Although this current assumption re-
mains unresolved, efforts are geared towards developing celecoxib
analogues with optimised ERSA activity and minimised COX-2
inhibitory activity.
OSU-03012 and 2, 5 - dimethyl - celecoxib (DMC) are ana-
logues of celecoxib with no COX-2 inhibitory activity and en-
hanced ERSA potency [258]. DMC contains a 2,5-dimethyl sub-
stituent at its terminal phenyl ring compared with celecoxib with a
4-methyl moiety. This chemical modification resulted in the loss of
COX-2 inhibitory function with a simultaneous anti-tumour activity
enhancement. DMC is a stronger ER stress inducer than its parent
compound. It inhibits SERCA activity with a consequent induction
Ubah and Wallace
of ER stress markers in vitro and also in vivo xenograft tumour
models [257]. DMC is yet to be tested in the clinic. OSU-03012 is a
product of an extensive structural adaptation of the celecoxib mole-
cule which also shows no inhibitory activity on COX-2, and has
demonstrated promising preclinical antitumour outcomes as mono-
therapy and in combination with other conventional chemothera-
peutics such as imatinib, tamoxifen, trastuzumab and many others
[259 - 261]. A phase 1 clinical study of OSU-03012 (as AR-12) in
patients with advanced or recurrent solid tumours or lymphoma is
in progress.
Chloroquine, the classical antimalarial agent is now causing
excitement in cancer research field because of its capacity to block
autophagy. Since autophagy blockade is associated with aggrava-
tion of ER stress, it was not completely unexpected that chloroquine
may induce ERSA activity [262]. In a clinical trial in Mexico,
chloroquine when used as an adjuvant sensitised glioblastoma mul-
tiforme to temozolamide [263]. Temozolomide is the standard che-
motherapeutic care for patients with glioblastoma multiforme, how-
ever it was shown to activate autophagy as a defense mechanism of
the tumour cells [264]. The greatest benefit from chloroquine in
cancer therapy has been linked to its ability to chemosensitise and
radiosensitise tumor cells [265, 266].Clinical trials are underway in
the U.S. to evaluate the anticancer potentials of chloroquine in
breast (NCT01023477) and lung (NCT00969306) cancer patients
[244].
LYSOSOMES AND LYSOSOME-RELATED ORGANELLES
Lysosomes were first described in 1955 by De Duve et al as
organelles enriched with acidic hydrolases and potentially harmful
to cells [267]. They were viewed as ‘waste-disposal’ compartment
of the cell, however this view have been challenged following the
discovery of other related organelles and the finding that lysosomes
are dynamic organelles that move along the cytoskeleton and asso-
ciate with other cellular compartments. There are well documented
evidence showing that the acid vacuolar compartment in all eukary-
otic cells participate in a wide range of cellular processes (Fig. See
Castino et al., 2003, Destination “Lysosomes”) such as partial or
extensive degradation of various substrates, trafficking and recy-
cling of molecules among internal organelles to and from the exte-
rior of the cell, post-translational maturation of secretory products
and storage of undigested material [268]. For the purpose of this
review, we will focus on the roles of lysosomes that are more rele-
vant in tumorigenesis, although the lysosomes or lysosome-related
organelles have other functions such as the enlargement of the
membrane surface and resealing of plasma membrane following
calcium-dependent exocytosis [269, 270]. Lysosomes participate in
various events associated with neoplastic phenotype, namely the
loss of cell mass homeostasis, loss of function of ‘programmed cell
death’, acquisition of chemo-resistance and evading immune sur-
veillance system, and the acquisition of invasive and metastatic
potentials.
Lysosomes being the organelles with the highest concentration
of proteases and other hydrolytic enzymes inside the cell are largely
involved in the maintenance of cellular homeostasis by realizing the
degradation of autologous material. In malignancy, lysosome-
mediated protein degradation is compromised [268, 271]. Both
heterophagic and autophagic cargos find their final destiny in
lysosomes, where they are acted upon by many hydrolytic enzymes.
Amongst the most studied lysosomal hydrolases are the proteases of
the cathepsin family. Cathepsins are optimally active in acidic pH
of lysosomes (pH 4-5), although they can function with reduced
efficacy in neutral pH outside the lysosomes [272]. Besides their
function in protein turnover, certain cathepsins participate in the
control of cell cycle, antigen presentation, hair follicle morpho-
genesis and epidermal homeostasis [273]. Emerging evidence sug-
gests that cathepsins degrade the extracellular matrix when secreted
to the extracellular space and also execute apoptosis when released
Cancer Therapy
to the cytosol [272]. The most implicated cathepsins in cancer are
the cysteine cathepsins B and L, and the aspartate cathepsin D, and
their elevated expression is a reliable diagnostic marker of poor
disease outcome [274]. The altered lysosomal function found in
murine fibroblasts and breast epithelial cells transformed with ras
oncogene was an influence from the oncogene suggesting that its
activation may play a role in the alteration of lysosomal functions
[275]. Together with matrix metalloproteases and the plasminogens
activator system, cathepsins secreted into the cytosol have been
suggested to participate in the degradation of extracellular matrix,
thus empowering cellular motility, invasion and angiogenesis [276].
This hypothesis has been recently supported by reports from two in
vivo mouse model of pancreatic cancer and glioblastoma where
pharmacological inhibition of cysteine cathepsin activity impaired
angiogenic switching in progenitor lesions and repressed tumour
growth, vascularity and invasiveness [277, 278].
Parallel to tumour progression, cathepsins B, L, and D have
been implicated as mediators of the lysosomal cell death pathway,
and death stimuli such as death-receptor activation, p53 activation,
oxidative stress, growth factor deprivation and microtubule-
stabilising agents can as induce partial lysosomal membrane per-
meabilisation and the release of cathepsins into the cytosol [8].
From the cancer therapy point of view, it was exciting to note that
cathepsins are able to mediate caspase- and mitochondrion-
independent apoptotic cell death, allowing cell death to ensue even
in malignant cells with multiple defects in their apoptosis pathway.
It is believed that they activate the classical apoptosis pathway by
cleaving the proapoptotic Bcl-2 family proteins [8, 279].
Targeting Lysosomes as Cancer Therapy
For tumourigenesis to be successful, an effective suppression of
both the classical apoptosis and lysosomal death pathways must
occur [8]. Many malignant cells display characteristics of increased
activity of phosphatidylinositol-3'-kinase (PI3K) and they contrib-
ute to the stability of tumour cell lysosomes [280]. PI3K inhibition
in certain settings tends to shift the TNF-induced death pathway
from caspase dependent to cathepsin dependent [281]. The translo-
cation of heat shock protein 70 (HSP 70) into the lysosomal mem-
brane confers protection on tumour cells against lysosomal leakage.
It has been shown that cells with HSP 70 on their lysosomal mem-
branes contain larger and more stable lysosomes and the depletion
of these HSP 70 triggers a tumour cell-specific lysosomal death
programme [282]. Thus, agents that inhibit the PI3K pathway or the
lysosomal localisation of HSP 70 have great potential in cancer
therapy as tumour sensitisers to conventional chemotherapeutics by
facilitating lysosomal membrane permeabilisation. In addition,
antiblastic agents such as the C1311, a topoisomerase II-inhibitor
[268]; the synthetic retinoid CD437 [283]; and the
-tocopheryl
succinate [284] have been shown to induce apoptotic cell death via
the induction of primary lysosomal leakage. One ultimate goal in
cancer medicine research is geared towards identifying a carrier that
would allow site-specific and delivery of cytotoxic cargo into tu-
mour tissue. Over-expression of cell surface molecules in cancer
cells is a regular occurrence and these over-expressed molecules
can be exploited as Trojan Horses to internalise anti-cancer agents.
For instance, the peroxidised low-density lipoproteins (LDL) can
be internalised via LDL receptors and consequently accumulate
within the lysosomes, where they induce lysosomal destabilisation,
acid hydrolases leakage into the cytosol with an attendant cell death
pathway induction [268, 285].
Liposomes and Nanocarriers
Using mitochondrially targeted carriers such as liposomes or
nanoparticles, active molecules can be selectively delivered to the
mitochondria instead of directing the molecules themselves to mi-
tochondria. This research area of cancer-selective nanoparticles and
carriers is currently highly active [286]. Cellular internalisation of
Current Pharmaceutical Design, 2014, Vol. 20, No. 2 213
small molecule drugs and macromolecules requires transporters,
thus shifting the burden of selectively targeting the mitochondria of
cancer cells to the transporters. However the major obstacle facing
this approach is the rapid elimination of liposomes and nanoparti-
cles from systemic circulation due to the elevated clearance rate by
the reticuloendothelial system (RES) and other organs. This limits
the use of intravenously administered liposomes and nanoparticles
in cancer therapy [287], although conjugation to polyethyleneglycol
(PEG) chain enhances half-life and affords longer-lasting particles
in the systemic circulation [288, 289]. Carrier-size reduction to size
below 200 nm allows for efficient accumulation in tumour tissue
due to permeability and retention effect enhancement [286]. We
discuss below some of the cancer therapies based on liposome and
nanoparticle delivery to mitochondria of malignant cells. Mito-
chondriotropic liposomes made of positively charged amphiphilic
molecules have been produced. One member which have been ex-
tensively studied is the DQAsomes formed by dequalinium, a
symmetrical molecule in which a relatively long aliphatic chain
seperates two charged moieties. DQAsomes was investigated for
mitochondrial delivery of mitochondrial leader sequence-DNA
conjugates and paclitaxel, and it showed positive outcome in initial
experiments studying the growth of a human colon tumour in nude
mice [290, 291]. However systemic toxicity associated with de-
qualinium poses a major challenge. Triphenylphosphonium head
carried on fatty acid chains have been used to bequeath liposomes
with mitochondriotropic feature, and enhance internalisation by
cultured cells. Rh123 and ceramide have been successfully deliv-
ered into the mitochondria using this system, and also the latter
showed growth inhibitory efficacy in an in vivo murine model of
mammary carcinoma [292, 293].
The novel Mito-Porter; a nano-carrier system for delivery of
macromolecules into mitochondria via membrane fusion was de-
veloped by the Harashima group. The “Mito-Porter”, a DOPE (1,2-
dioleoylsn-glycero-3-phosphatidyl ethanolamine; found to be opti-
mal for membrane fusion) liposome carrying a high density of fu-
sogenic R8 (octaArg) or similar surface peptide was developed as a
variation of the “multifunctional envelope-type nanodevices”
(MEND) [294, 295]. Oligo-Arg peptides are known to act in TAT-
like fashion enhancing membrane fusion as well as exhibiting mito-
chondrotropic functions. In cultured cells, the Mito-porter appeared
to be internalised via macropinocytosis and the resulting vesicles
deliver their cargo to the mitochondrial inter-membrane space
[296].
SUMMARY
The successful activation of the cell death machinery of cancer
cells cannot be overemphasised. Cancer cells exhibit a significant
number of metabolic aberrations associated with the mitochondria
and other sub-cellular organelles. These aberrations promote tu-
mour growth and survival, and at the same time exhibit unique
properties which enhances their vulnerability to certain anticancer
agents. By these unique features cancer cells have been successfully
targeted selectively. All the organelles discussed display some sort
of deregulation in cancer cells as compared with their counterparts
in healthy cells, this being one of the reasons for the recorded suc-
cess in drug development and cancer therapy (Tables 1-3).
Although significant progress has been made towards elucidat-
ing the mechanism and role of these alterations in oncogenesis and
resistance to chemotherapy, a better understanding of the key
pathophysiological differences between organelles in normal and
cancer cells will in no doubt complement effort towards improving
selectivity of targeted anticancer agents. For instance, a conclusive
elucidation of the association involving the Warburg effect and
MOMP resistance will be clinically important in reversing related
chemoresistance.
Most of the agents discussed in this review, e.g. ABT-737 and
its orally bioavailable derivative, ABT-263 possess single-agent
214 Current Pharmaceutical Design, 2014, Vol. 20, No. 2
Table 3. Mechanism of action and targets of some compounds with anticancer activity
mechanism based cytotoxicity as well as capapcity to reverse
chemoresistance in cancer cells. Interestingly, their cytotoxicity is
enhanced in combination with other chemotherapeutic agents or
monoclonal antibodies. Other compounds with enhanced cytotoxic-
ity resulting from combination therapy were lonidamine (with PBR,
anthracycline, epirubicin);
- lapachone (with gemcitabine); Borte-
zomib (with doxorubicin, paclitaxel, gemcitabine). The use of “in-
hibitory” analogues and their conjugates such as the ANTMHspd
and Ant-4 in the form of “Trojan horses” has shown some promise
as monotherapies and in combination with DFMO.
Ubah and Wallace
An efficient drug delivery system is as crucial as the drug
molecule itself. A balanced effort between research in anticancer
drug discovery and drug delivery systems such as the liposomes,
nanoparticles and conjugates is likely to be the most successful.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
Declared none.
Cancer Therapy Current Pharmaceutical Design, 2014, Vol. 20, No. 2 215

Appendix

Fig. (1). Metabolic alterations and Warburg phenotype in tumour cells and related therapeutic targets. Cancer cells rely mostly on glycolysis through
enhanced expression and activities of glucose transporters and HK. In healthy cells, glucose is converted to pyruvate through series of reactions in the cytosol.
In the mitochondria, Pyruvate is converted to Acetyl-CoA which is a key component of the TCA cycle, and drives the synthesis of ATP via oxidative phos-
phorylation. During malignancy, pyruvate is preferentially converted to lactate which is transported out of the cell. Thus, pyruvate which enters the mitochon-
dria drives the truncated TCA cycle in which citrate is expelled from the mitochondria to stimulate lipid biosynthesis. Lonidamine, 3-BrPA, 2-DG, methyl
jasmonate, DCA, Arsenic, SB-204990 and
- cyano-4-hydroxycinnamate are listed as therapeutic agents while GAPDH, HK, PDK, ACL, and monocarboxy-
late transporters are listed as therapeutic targets.

GPR78 = G-protein coupled receptor 78

ABBREVIATIONS
HIF = Hypoxia inducible transcription factor
ACL = ATP citrate lyase
HIV = Human Immunodeficiency virus
AIF = Apoptotic inducing factor
HK = Hexokinase
ANT = Adenine nucleotide translocator
HSP = Heat shock protein
Ant 4 = Putrescine-anthracene conjugate
I - KB = Nuclear factor kappa B inhibitor
ANTMHspd = anthracenylmethyl homospermidine
IAP = Inhibitor of apoptosis protein
ARF = ADP-ribosylation factor
IM = Inner Mitochondrial membrane
ATF6 = Activating transcription factor 6
IMS = Intermembrane space
BAD = Bcl-2 associated death promoter homologue
IRE1
= Inositol – requiring enzyme 1 alpha
BAK = Bcl-2 antagonist/killer
JNK = c-Jun N-terminal kinase
BAX = Bcl-2 associated X protein
KIP1 = Kinase interacting protein 1
BH3 = Bcl-2 homology domain 3
LLnV = N-carbobenzoxy-L-leucyl-L-leucyl-
BID = BH3 – interacting domain death agonist
norvalinal
CYPD = Cyclophilin D
MEND = Multifunctional envelope-type nanodevices
Cyt c = Cytochrome C
MM = Multiple myeloma
DFMO = Difluoromethyl ornithine
MOMP = Mitochondrial outer membrane permeabilisa-
ER = Endoplasmic reticulum tion
ERSA = Endoplasmic reticulum stress aggravator MPT = Mitochondrial permeability transition
FAP = Familial adenomatous polyposis mtDNA = mitochondrial DNA
GAPDH = glyceraldehydes -3 – phosphate dehydro- mTOR = serine/threonine kinase target of rapamycin
genase
NF - KB = Nuclear factor kappa B
GLUT1 = Glucose transporter isoform 1
216 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace

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Received: March 11, 2013 Accepted: May 15, 2013
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