Cancer Therapy

22 Pages·2014·0.61 MB·English

Send Orders for Reprints to [email protected]
Current Pharmaceutical Design, 2014, 20, 201-222 201
Cancer Therapy: Targeting Mitochondria and other Sub-cellular Organelles
Obinna C. Ubah1 and Heather M. Wallace2*
1Department of Molecular and Cell Biology, College of Life Sciences and Medicine, University of Aberdeen, AB25 2ZP, UK;
2Division 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- hydrolytic enzymes and these are the cellular organelles responsible
for degrading extracellular and transmembrane proteins, whereas
tate malignant transformation: growth signalling autonomy, insensi-
tivity to anti-proliferating signals, limitless proliferative potential, proteasomes degrade intracellular proteins [7]. It is the compart-
sustained angiogenesis, adjacent tissue invasion and metastasis, and mentalisation of these proteolytic enzymes that prevent them from
producing uncontrolled protein degradation [5]. For tumourigenesis
avoidance of cell death. This intrinsic lack of cell death remains a
major cause of therapeutic failure and may itself contribute to car- to be successful, an effective suppression of both the classical apop-
cinogenesis and tumour progression [1, 2]. Therefore, activation of tosis and lysosomal death pathways must occur, thus supporting the
crucial involvement of these organelles in tumorigenesis [8].
the cell death machinery via drugs designed rationally may signify
a more effective cancer treatment option. The cell death machinery
MITOCHONDRIA
is composed of catabolic hydrolases which are modulated by spe-
cific inhibitors or via sequestration of their activators. Numerous i. Structure and Roles
cell death cascades (apoptotic and necrotic) converge on mitochon-
The rationale for targeting the mitochondria for therapeutic
dria to cause deregulation of function and permeabilisation with the
benefit is based on their critical involvement in bioenergetics, redox
activators of the cell death machinery being liberated following the
balancing and vitally their active regulation of several cell sur-
permeability change in the mitochondrial outer membrane [3]. To
vival/death pathways. Other important roles include thermogenesis,
remove abnormal malignant cells, their death is an important event
Ca2+ homeostasisand essential anabolic cascades. Mitochondrial
which also provides a critical mechanism of innate tumour suppres-
dysfunction such as impaired oxidative phosphorylation and/or
sion. Aberrations blocking these tightly regulated processes tilt the
increased oxidative stress coupled with deregulation of apoptosis
balance in favour of cancer cells successfully evading cell death
has been described recently as a vital pathophysiological mecha-
from either therapeutic means or physiological control systems.
nism not only in human mitochondrial diseases but also in the
Although the clearance of malignant cells often relies on manipulat-
pathogenesis of other congenital and acquired pathologies such as
ing the classical apoptotic pathways (mitochondrial and/or death
cancer [9], neurodegenerative disorders [10], diabetes [11], and
receptor cascades), evidence is emerging that other apoptotic and
cardiovascular diseases [12].
non-apoptotic pathways may play vital roles in tumour cell death.
Experimental evidence has suggested that the endoplasmic reticu- Mitochondria were regarded exclusively as the power house of
lum (ER) and Golgi apparatus can activate both pro-survival cas- the cell throughout the second-half of the 20th century. It was not
cades as well as cell suicide programmes if the stress-signalling until around 1995 that it became clear that they are not only a site
threshold is exceeded [4]. The proteasome is an abundant multi- for energy production via oxidative phosphorylation, but also have
enzyme complex providing the focal pathway for the degradation of crucial role in cell death regulation. This latter discovery which
intracellular proteins in eukaryotes, thus it is the modulator of the revitalised the field of mitochondrial and cell death research ap-
protein pool and is important for cell-cycle progression and apop- peared counterintuitive because experts doubted whether the meta-
totic cell death in both healthy and malignant cells [5, 6]. Cells use bolic functionality of the cell which converge oin the mitochondria
an exceptional assortment of mechanisms to regulate intracellular could be manipulated to cause cell death. The other argument was
cytosolic protein stability and degradation, lysosomes and protea- that the mitochondria during apoptosis do not suffer significant
somes remain the two main routes amongst other mechanisms re- ultra-structural changes thus may not be controlling the fate of the
sponsible for protein integrity. Lysosomes are cytoplasmic 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].
*Address correspondence to this author at the Division of Applied Medicine
and Therapeutics, School of Medicine and Dentistry, University of Aber- In healthy cells, the inner mitochondrial membrane (IM) is
deen, Aberdeen AB25 2ZD, UK; Tel/Fax: [phone]; almost impermeable to all ions and including protons (H+). This
E-mail: [email protected] allows respiratory chain complexes I - IV to accumulate proton
1873-4286/14 $58.00+.00 © 2014 Bentham Science Publishers
202 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace
gradient across the IM which is obligatory for oxidative phosphory- off the Bcl2-homology domain 3 (BH3) into the active form tBid
lation [14, 15]. Ions and small molecules crossing the inner mem- (truncated BID), the latter providing a cross-talk between the ex-
brane do so via specific transporters. The inner mitochondrial trinsic and intrinsic death pathways. tBid then translocates to the
transmembrane potential ((cid:2)(cid:3) ) is generated from the charge imbal- mitochondria where it promotes the mitochondrial permeability
m
ance resulting from the creation of an electrochemical gradient transition (elevated OM permeability) causing cytochrome c (cyt c)
across the inner membrane. Finally, ATP synthesis is driven by the release. The mitochondrial pathway is initiated by the release of
complex V (mitochondrial ATP synthase) of the respiratory chain various apoptogenic factors such as cyt c, apoptosis inducing factor
via the exploitation of the proton gradient. Therefore the mainte- (AIF), second mitochondria-derived activator of caspases
nance of the proton gradient is crucial for cellular bioenergetics [14 (Smac)/direct inhibitor of apoptosis proteins (IAP) binding protein
- 16]. All components of the mitochondrial matrix and those me- with Low PI (DIABLO), Omi/high temperature requirement protein
tabolites crossing the IM are strictly regulated with the aid of highly A (HtrA2) and Endonuclease G (Endo-G) from the IMS into the
selective transport proteins. A permanent dissipation of (cid:2)(cid:3) is cytosol [13, 29 - 31]. The apoptogenic proteins can be released into
m
associated with cell death [17, 18], however cases of transient loss the cytosol using one of the four models hypothesised for the open-
of (cid:2)(cid:3) secondary to functional instability in one or more IM pores ing of the permeability transition pore; [1] Oligomerised Bax/Bak
m
may occur in physiological conditions [19, 20]. The most vital generates channels sufficient for the liberation of the factors [32].
function of the mitochondria is the synthesis of ATP. Dysfunction This hypothesis is sustained by the findings that the structure of
of this process is associated with large number of mitochondrial Bcl-2 family proteins is similar to that of the pore-forming helices
pathologies. of bacterial toxins, and that Bak can form channels in artificial
membranes and liberate cyt c from liposomes with large complexes
In 1972-1973, Boveris and Chance described mitochondria,
isolated from rat liver and pigeon heart, as a source of hydrogen containing Bax and Bak, as seen by electron microscopy [33]. [2]
peroxide (H O ) that diffuses to the cytosol as a major supplier of The proapoptotic proteins may form complexes with mitochondrial
2 2
membrane proteins such as the voltage-dependent anion channel
superoxide anions [21, 22]. Although the cytochrome oxidase
(complex IV) mediates the four-electron reduction of molecular (VDAC) or adenine nucleotide translocator (ANT) to assemble
oxygen to form H O which is the end product of the respiratory pores [34]. This is supported by findings that microinjection of
2
VDAC antibodies into the entrance of the channel inhibited the
chain, a minor fraction of molecular oxygen takes part in one-
electron reduction processes generating reactive oxygen species release of cyt c and prevented apoptosis [35, 36]. [3] Permeability
(ROS) and they include; O -, H O and the highly reactive hydroxyl transition pores are regulated by misfolded proteins and chaperones
2 2 2,
radical (HO-). such as Hsp25. These permeability transition pores are formed by
aggregates of misfolded membrane proteins [37, 38]. [4] Membrane
The intramitochondrial metabolites O -, H O NO and HO- are
2 2 2, damage resulting from the matrix swelling may induce cristae struc-
pro-oxidants with potential to cause oxidative stress and tissue
ture distortion and rupture of the outer membrane thus releasing cyt
damage. These are the chemical species that are linkedto the mo-
c [39, 40].
lecular mechanism of tissue dysfunction in aging, ischemia-
Also the mitochondrial pathways can be triggered by various
reperfusion, neurological diseases, and inflammation [16, 23].
Normally, the native defense systems consisting primarily of mito- intracellular and extracellular stress signals which ultimately lead to
activation of pro-apoptotic proteins such as Bcl2 antagonist/killer
chondrial (manganese-containing) and cytosolic (copper-zinc-
(Bak), Bcl2-associated X protein (Bax), or inactivation of anti-
containing) superoxide dismutases, glutathione peroxidase, and
phospholipid hydroperoxide neutralise these ROS and their peroxi- apoptotic Bcl2 family members such as Bcl2 or Bcl-xL[41, 42].The
release of cyt c from the mitochondria causes activation of down-
dation products respectively [23 - 25]. Antioxidant supplements and
stream effector pro-caspases such as caspases-3 and 7 via formation
drugs have been used as mitochondria-generated free radical scav-
engers. These drugs inhibit mitochondria in tumour cells, thereby 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
killing these cancer cells; they also protect cells from oxidative
of inducing caspase-independent cell death and produce DNA
destruction, and aid in the repair of defects.
fragmentation [31, 37]. Although the mitochondrial membrane
In healthy cells and during cell death, the outer mitochondrial
permeabilisation (MMP) is vital for caspase-dependent and inde-
membrane (OM) permeability is also altered. Due to the presence of
pendent apoptotic cell death pathways, the cell death cascade can
the voltage dependent anion channel diffusion of metabolites and
also be induced by triggers found in other organelles such as cal-
solutes of ~5kDa across the OM occurs. Although the assumption cium (Ca2+) in the endoplasmic reticulum (ER) and cathepsins in
that the OM is freely permeable by such solutes has been chal-
lysosomes in response to ROS or ceramide [37, 43]. The apoptosis
lenged recently because real-time quantification of mitochondrial
pathways are strictly controlled by a variety of pro- and anti- apop-
Ca2+ concentrations revealed the existence of Ca2+ microdomains in
totic regulators under physiological conditions, since any form of
which the voltage dependent anion channel and a variety of addi-
uncontrolled stimulation of the apoptotic machinery would poten-
tional OM proteins regulate and limit Ca2+ diffusion across the OM
tially translate to a detrimental effects on cell survival. In human
[26, 27]. The OM permeability often increases during cell death
cancers, the anti-apoptotic systems are often abnormally upregu-
allowing for the liberation of soluble proteins into the intermem-
lated thus enabling the cancer cells to evade apoptotic cell death
brane space (IMS), these proteins are otherwise situated and re-
[44].
tained within the mitochondria in healthy cell conditions. More so,
the death associated outer membrane permeabilisation can be an iii. Mitochondria in Cancer Cells
accidental process as well as a finely controlled process with major
Metabolic imbalances and improved resistance to mitochondrial
implications for health and disease [13, 23].
cell death are considered as characteristic features of cancer cells.
Tumours relying on glycolysis to meet their metabolic burdens have
ii. Mitochondria and Cell Death
been acknowledged since the twentieth century [45, 46]. Cancer
In general, two semi-interdependent apoptotic signalling path-
cells are inclined to synthesise ATP mainly through aerobic glyco-
ways have been delineated; ligand binding to the death receptors at
lysis (the so-called Warburg effect), a metabolic state that is associ-
the cell surface (extrinsic) pathway, and the mitochondrial (intrin-
ated with elevated glucose uptake and local acidification owing to
sic) pathway [28]. Activation of either signalling pathway will lead
lactate production. The Warburg effect is actually the principle
to the activation of caspases, a family of cysteine proteases that
behind the development of the positron emission tomography in
function as death effector molecules. However, active caspase-8 which a glucose analogue tracer (2-18fluoro-2-deoxy-D-glucose) is
can cleave the protein BID (BH3-interacting-domain death agonist)
utilised to differentiate between normal and malignant tissue. How-
Cancer Therapy Current Pharmaceutical Design, 2014, Vol. 20, No. 2 203
ever, a comprehensive clarification of the so-called Warburg effect (cid:2) Putatively, in a one-step process cancer cells might repress the
has not been achieved. Some of the non-exclusive hypotheses mitochondrial apoptotic programme via mechanisms that in
which have been put forward to explain this phenotype are de- concert stimulate the Warburg phenomenon. The observation
scribed below: that mitochondrial DNA mutations may induce, through un-
(cid:2) Cancer cells are deficient in mitochondrial respiration and known mechanisms, a decrease of anti-cancer-induced or
ATP generation as a result of accumulation of defects in the spontaneous apoptosis in cancer cells is in favour of this hy-
pothesis [48]. Moreover, hexokinase II which increases in gly-
mitochondrial genome. Also, mitochondrial germline muta-
tions have been implicated as a predisposing factor in cancer colysis and inhibits MOMP when it is in association with
development. In most cases, such mitochondrial mutations are VDAC in the OM provides a possible link between MOMP
suppression and aerobic glycolysis. This interaction between
acquired during and after tumourigenesis. Brandon et al
grouped these acquired mitochondrial DNA mutations in two hexokinase and VDAC is favoured by activation of the Akt
different classes; the first class involves severe mutations that pathway [53] or by the cancer-specific overexpression of mi-
tochondrion-binding hexokinase isoenzymes [48, 62].
impede oxidative phosphorylation, thus causing an increased
and enhanced ROS production and tumour cells proliferation (cid:2) During multistep carcinogenesis, metabolic alteration and
while the second category is of milder mutations permitting MOMP inhibition could be autonomously acquired by cancer
tumour cells adaptation to their new microenvironment, espe- cells. Increased ROS and respiratory dysfunction resulting
cially during progression and metastases [47 - 49]. from mitochondrial mutations could increase the probability of
(cid:2) Mitochondrial enzymes can in specific cases act as tumour cancer cells committing to apoptotic MOMP, and MOMP in-
suppressors and mutations of these nuclear encoded mitochon- hibition would be necessary to maintain carcinogenesis. Oth-
erwise, suppression of the apoptotic pathway might be permis-
drial proteins have been linked to cancer and will indirectly
sive for genomic instability [63], and the Darwinian selection
encourage aerobic glycolysis. For instance, the inactivating
mutation of two enzymes involved in the TCA cycle; mito- of genetically variant cells would then favour the accumulation
of cells expressing the Warburg phenotype.
chondria succinate dehydrogenase (SDH subunits B, C or D)
or fumarate dehydrogenase is considered an oncogenic event In the last ten years, there has been substantial interest in the
causing phaeochromocytoma and leiomyoma, leiomyosacr- possibility that mtDNA mutations might predispose or at least play
coma or renal carcinoma respectively. Also accumulation of a role in human malignancies and other common diseases. Al-
succinate and fumarate in the cytosol as a result of loss of though the mechanism involved in the initiation and progression of
function of their respective dehydrogenases favours the activa- mtDNA mutations, and their involvement in tumourigenesis and
tion of the hypoxia-inducible transcription factor (HIF) and other pathogenesis awaits comprehensive understanding. Interest-
generate a pseudohypoxic state together with HIF-dependent ingly, a recent report showed that the high heterogeneity of human
reprogramming of the metabolism towards aerobic glycolysis mtDNA was significantly amplified in tumours [64]. Polyak et al.
[50]. A mutation in the third TCA cycle enzyme, isocitrate de- demonstrated a consistent presence of somatic mtDNA mutations in
hydrogenase has recently been linked to the majority of grade human tumours. The authors provided evidence of the presence of
II and III gliomas and secondary glioblastomas [51, 52]. homoplasmic mtDNA mutations in 7 out of 10 cell lines from pa-
(cid:2) As the pre-malignant lesion grows progressively further away tients with colorectal carcinomas which were neither found in nor-
from the blood supply, cancer cells (if not all solid tumours) mal colon nor in other tissues from the same patients [65]. Recent
papers report somatic mutations in the mitochondrial genome in
possess the characteristic ability to adapt to decreased oxygen
tension (hypoxia). One way in which these cells adapt to de- nearly one out of every four gastric cancer specimens and stress the
creased oxygen tension is to shut down mitochondrial respira- potential role of those mutations in the evolution of the disease
[66]. Kulawiec et al. [67] demonstrated that mtDNA mutations
tion and simultaneously switch on glycolysis [50, 53].
were not associated with ROS generation in some samples of breast
(cid:2) Cancer cells often upregulate the enzymes and rate-limiting
cancer cells, but constitutively activated the PI3K/AKT pathway
processes of glycolysis, including glucose transporters, for in-
contributing to enhanced metastasis. More over, the PI3K/AKT
stance as a result of the expression of oncogenic factors in-
pathway is strictly linked and activated in association with the ser-
cluding Ras, Src, or Bcl-Abl or as a result of the constitutive
ine/threonine kinase target of rapamycin (mTOR) that regulates key
signalling through the Akt pathway [48, 54].
cellular processes such as cell survival, growth and proliferation.
Cancer cells have been shown to be resistant to the induction of The mTOR pathway is regularly hyperactivated in many human
mitochondrial outer membrane permeabilisation (MOMP), a proc- tumours and this is consistent with its role in cell proliferation [68].
ess which mediates the intrinsic apoptotic pathway [55]. MOMP
In cancer, oxidative stress has been linked to DNA instability,
being a complex phenomenon is tightly regulated by Bcl-2 family
hypermethylation and mutation in the DNA repair machinery, point
of proteins [41, 42, 56], proteins resident in the permeability transi-
mutations and loss of heterozygosity in DNA microsatellites. In
tion pore complex [57], proteins that influence mitochondrial fusion
addition, cell cycle deregulation coupled with a shortened cycle
and fission dynamics [58] and even transcription factors with the
resulting in inadequate opportunity to repair acquired DNA muta-
capacity to translocate from the nucleus to mitochondria to encour-
tions prior to cells entering into S phase from G phase [69 - 71].
age MOMP. The tumour-suppressor protein p53 is an example of 1
Reports to date indicate that cancer cells exhibit huge varieties
such transcription factors [59]. Inhibition of MOMP disabling the
apoptotic system is vital for the development of solid tumours, and of metabolic change which are associated with alterations in the
also haematological cancers as evident in the switch from pre- mitochondrial structures, dynamics and function, and with tumour
growth and survival. Mitochondria regulate tumour growth through
neoplasia (e.g., low-grade myelodysplastic syndrome) in which
cells spontaneously commit to apoptotic MOMP, to overt neoplasia modulation of the TCA cycle and oxidative phosphorylation, and
(such as acute myeloid leukaemia developing from a myelodysplas- also they are vital in regulating redox homeostasis in the cells, in-
ducing them to either commit to or evade apoptotic cell death sig-
tic syndrome) in which MOMP is hindered [48, 60, 61].
nals. The mitochondrion has been identified as key organelle to
The mechanistic link between the Warburg effect (aerobic gly-
understanding the molecular basis of tumour proliferation and also
colysis) and MOMP resistance in cancer cells has not been fully
rational design of chemotherapeutic agents [52].
elucidated; however the following hypothetical explanations have
been put forward to explain the concurrent occurrence of aerobic
glycolysis and MOMP inhibition in cancer:
204 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace
MITOCHONDRIA AS CANCER THERAPEUTIC TARGETS damine with the anthracycline, epirubicin, increased by 9% the
The underlying principle for targeting the mitochondria for response rate of patients with solid tumours in comparison with
therapeutic benefits lies in the knowledge that this organelle plays a epirubicin alone [89, 90]. In another phase II study designed to
examine the efficacy of cisplatin, paclitaxel and lonidamine co-
vital role in the regulation of bioenergetics, ROS production and
apoptotic cell death. Preferentially targeting the energy metabolism administration in patients with advanced ovarian cancer, an 80 %
of tumour cells and not the healthy cells which also rely upon the overall response rate was reported, including 40% complete re-
sponses and 40% partial responses [91].
essential pathway for ATP supply poses a major challenge. How-
ever, the cancer cell mitochondria are structurally and functionally Betulinic acid (BetA) is a plant-derived pentacyclic triterpe-
different from their healthy counterparts [72, 73]. Cancer cells dis- noid that exerts potent anti-cancer effects in vitro and in vivo with
play an extensive metabolic reprogramming that makes them more remarkable selectivity for tumour cells over non-transformed cells.
susceptible to mitochondrial perturbations than healthy cells [74]. Apoptosis induced by BetA has been shown to involve the activa-
In a broader perspective, cancer-specific mitochondrial alteration tion of caspases, PARP cleavage and DNA fragmentation. In addi-
and energy metabolism can be exploited for the development of two tion, the production of ROS and decrease in the mitochondrial
novel classes of anti-tumour agents. These novel agents will target membrane potential has been described, thus suggesting a reliance
glycolysis and/or reverse the Warburg phenomenon or aim at induc- on the mitochondrial cell death pathway [92 - 94]. BetA triggers
ing apoptosis via targeting mitochondrial proteins and membranes MOMP directly in association with mitochondrial membrane poten-
[48]. As stated earlier, mitochondrially-targeted agents emerged as tial dissipation and cyt c release when added to isolated mitochon-
a means of selectively targeting tumour cells as a result of these dria in a cell-free system [95]. This cytotoxicity induced by BetA
differences that exist between them and non-immortalised cells. We could not be blocked by the pan-caspase inhibitor zVAD.fmk [96].
discuss below a number of mitochondrial features which has been This would imply that the cytotoxic effects of BetA are mediated by
exploited or may be considered as therapeutic target in human can- the MPT which may be induced by ROS overproduction [3]. BetA
cer treatment. modulates the expression levels of Bcl-2 family proteins which
includes the upregulation of the pro-apoptotic mewmbers such as
i. Targeting Mitochondrial Permeability Transition and Ade- the BAX and BCL-X (also known as the BCL2L1). Moreover,
5
nine Nucleotide Translocase (ANT) BetA mediated apoptosis has been shown not to be associated with
The permeability transition pore complex (PTPC) is a highly p53 accumulation and occurred in a p53-indepndent fashion [97].
dynamic supra-molecular complex with a poorly understood struc- GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide),
tural identity. This is probably as a result of the existence of multi- a glutathione-coupled trivalent arsenical compound which is a hy-
ple isoforms of its constituents and also a number of distinct but drophilic derivative of the protein tyrosine phosphates inhibitor
functionally related solute carrier proteins that can substitute for phenylarsine oxide (PAO). This small synthetic mitochondrial poi-
each other within the PTPC. Prototypical PTPC is composed of the son targets angiogenic endothelial cells. The trivalent arsenical of
VDAC in the outer membrane, the adenine nucleotide translocase GSAO interacts with and disrupts ANT of the inner mitochondrial
(ANT) in the mitochondrial inner membrane and cyclophilin D membrane of endothelial cells thus causing growth arrest [98, 99].
(CYPD) in the mitochondrial matrix [13], however studies showing It has been shown to cross-link critical cysteine residues of ANT
that the mitochondrial permeability transition (MPT) occurred in (Cys160 and Cys257) resulting in the inhibition of the ATP/ADP
mouse liver mitochondria with ANT1 and ANT2 knock-out mouse antiporter activity, cytosolic ATP depletion, ROS overproduction,
suggests that both VDAC and ANT (but not CYPD) may not be mitochondrial depolarisation and apoptosis [100]. GSAO would
essential for the lethal functions of the PTPC [75 - 77]. Other stud- preferentially target proliferating cells due to their high mitochon-
ies have also demonstrated that CYPD was also not essential for the drial Ca2+ levels and elevated respiration which renders them more
permeability transition [78, 79], thus leading to the assessment of susceptible to PTPC opening than normal cells [100]. PENAO (4-
the MPT (and ANT) as being relevant for necrotic rather than apop- [N-[S-penicillaminylacetyl]amino] phenylarsonous acid) a second
totic cell death [80]. Despite these findings, the suggestion that the generation ANT inhibitor was designed to evade the pro-drug proc-
ANT isoforms may play a vital role in apoptosis is credible; ANT1 essing and metabolism of GSAO [101]. It is a cysteine mimetic of
and ANT3 are pro-apoptotic, whereas ANT2 (which is often over active metabolite of GSAO, CAO (4-[N-[S-cysteinylacetyl] amino]
expressed in proliferating cells) is anti-apoptotic [81 - 84]. Compo- phenylarsenoxide). PENAO accumulates in cells 85-fold faster than
nents of the PTPC can be targeted by several compounds to induce GSAO, resulting in a 44-fold enhanced anti-proliferative activity
MPT and apoptosis. By depleting the endogenous inhibitors of and a ~20-fold increased anti-tumour efficacy in mice. PENAO
PTPC opening such as glucose, phosphocreatine and glutathione, an targets both proliferating endothelial and tumour cells, unlike
indirect permeabilisation effect can be achieved. Similarly, agents GSAO [102]. A phase 1/IIa dose escalation study of PENAO in
that cause increase in cytosolic Ca2+ or stimulate ROS production patients with solid tumours resistant to standard chemotherapy is
can trigger MPT [85]. Although several ANT ligands have been currently recruiting.
shown to induce intrinsic cell death, they lack ANT isoform-
Retinoid-related compounds such as CD437 (6-[3-(-1-
specific targeting.
adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) and
Lonidamine, 1-(2,4-dichlorobenzyl)-1-H-indazole-3- all trans-retinoic acid are well-known for their ability to induce the
carboxylic acid is a putative ANT ligand that triggers the mito- expression of retinoic acid receptor-responsive genes resulting in
chondrial apoptotic pathway [86]. It is a glycolysis-targeting com- complete clinical remission in a high proportion of patients with
pound which has exited clinical trials and gained approval in acute promyelocytic leukaemia [103]. Interestingly, Belzacq et al.,
Europe for the treatment of solid tumours. Lonidamine acts as a and Notario et al reported that these retinoids (CD437 and the all-
hexokinase inhibitor with selectivity for the mitochondria-bound trans-retinoic acid, 9-cis-retinoic acid and 13-cis-retinoic acids)
form of hexokinase which seems to play a key role in enhancing also trigger ANT-dependent MPT and apoptosis independent from
glycolytic flux in both proliferating normal and transformed cells nuclear receptor binding [88, 104].
[87]. Lonidamine in combination with the peripheral benzodi-
azepine receptor (PBR) ligand diazepam showed a cytostatic effect ii. Agents that Induce ROS Overproduction and Targeting Mi-
on tumour growth in patients with recurrent glioblastoma multi- tochondrial Peroxiredoxin III
forme in a phase II clinical study. 50 % of patients exhibited disease
Motexafin gadolinium (gadolinium texaphyrin) is an aromatic
stabilisation following treatment including one case in which pro- macrocylic compound that exhibits an elevated oxidising potential,
gression occurred after 12 months [86, 88]. The inclusion of loni-
thus triggering excess production of ROS and interfering with the
Cancer Therapy Current Pharmaceutical Design, 2014, Vol. 20, No. 2 205
antioxidant machinery. Motexafin gadolinium has been shown to apoptotic and autophagic death is initiated by production of ROS
preferentially accumulate in malignant cells, probably due to their resulting from inhibiton of oxidative phosphorylation[118,119].
metabolic perturbations, and to improve in vivo sensitisation to PEITC in combination with metformin demonstrated a significant
radiation and chemotherapy of xenotransplanted tumours [105]. In a anti-proliferative and cytotoxic effect in ovarian cancer cells in
phase III study based on motexafin gadolinium and brain radiother- vitro. This novel combination was effective in cisplatin resistant
apy for the treatment of patients with lung cancer and brain metas- cell lines [120].
tasis reported a motexafin gadolinium-mediated delayed neurologi-
2-methoxyoestradiol is selectively cytotoxic to human leu-
cal progression. In addition, paediatric glioblastoma patients toler- kaemia cells but not normal lymphocytes. It inhibits angiogenesis
ated the same regimen [106], suggesting a further investigation of by reducing endothelial cell proliferation and causing apoptotic cell
this combination therapy is warranted.
death. There is accumulation of superoxide via its ability to inhibit
Menadione (2-methyl-1,4-naphthoquinone) and thiol cross- SOD [121, 122]. 2-methoxyoestradiol has shown disease stabilisa-
linking agents such as diamide (diazenediacarboxylic acid bis tion and it is also well tolerated according to reports from several
5N,N- dimethylamide),bismaleimido-hexane (BMH) and dithio- phase I/II studies in patients with solid tumours or multiple mye-
dipyridine (DTDP) cause oxidation of a critical cysteine residue loma [123, 124]. ANT-224, an orally bioavailable second-
(Cys 56) of ANT and can bypass Bcl-2 mediated cytoprotection. generation copper-chelating tetrathiomolybdate analogue showed
Recombinant Bcl-2 fails to prevent thiol modification of ANT. similar effects as to the oestradiol analogue with potential antiangi-
Moreover, these thiol cross-linkers induce mitochondrial membrane ogenic and antineoplastic activities [125].
permeabilization and cell death irrespective of the expression level
The synthesis of ATP requires large amounts of oxygen, which
of Bcl-2 [107]. This suggests that thiol crosslinking agents cause a usually leads to the generation of ROS. Cellular damage ensues if
covalent modification of ANT which supercedes the control by Bcl-
these ROS are not detoxified by the antioxidant systems. Elevated
2 thus commits the cells to apoptosis.
pools of mitochondrial ROS and the disturbance of peroxiredoxin
(cid:2)-lapachone (ARQ 501) which undergoes futile redox cycles (Prx) generation in malignant cells may cause oxidative stress and
catalysed by NAD(P)H:quinone oxidoreducatse 1, thereby inducing the induction of apoptosis. Peroxiredoxins are family of enzymes
the overproduction of ROS, poly(ADP-ribose) polymerase 1 that catalyze the reduction of H O and hydroperoxides to water
2 2
(PARP1) hyperactivation and apoptosis. (cid:4)-lapachone is in clinical and alcohol respectively and there are six isoforms of the enzyme
trial as a monotherapy and in combination therapy with gemcitabine (Prx I - VI). The Prx system is a cellular defence system against
in the management of patients with cancer of the pancreas and also oxidative stress, and Prx III and V have been reported to be ele-
head and neck malignancy [108]. vated in the mitochondria of cancer cells [126, 127]. Also elevated
Accumulation of ROS can be achieved by inhibiting the anti- expression of Prx I has been reported in several human cancers
oxidant systems, thus suggesting an alternative approach. including non-small cell lung, oral, breast and liver cancer. Over-
expression of Prx II has also been reported in breast cancer, meso-
Buthionine sulphoximine causes an accumulation of ROS by in-
hibiting the synthesis of reduced gluthatione (GSH) whereas thelioma, and head and neck cancers [128]. Elevated Prx II is asso-
imexon, an aziridine-containing small pro-oxidant molecule, de- ciated with the chemotherapeutic resistance of leukemia and stom-
ach cancers. Downregulation of Prx II sensitised head-and neck
pletes the GSH pool due to its thiol-binding activity [109, 110]. The
combination therapy of buthionine sulphoximine and mephalan, an cancers to radiation and gastric carcinoma to cisplatin [128, 129].
alkylating agent, is being investigated in a phase I clinical study As mentioned above, recent studies have suggested that the overex-
pression of Prx III and its electron donors might provide a primary
involving patients with neuroblastoma and melanoma. Also,
imexon alone and in combination with docetaxel demonstrated line of defence against hydrogen peroxide produced by the mito-
some efficacy in another phase I clinical study in patients with chondrial respiratory chain, thus agents targeting Prx III and the
mitochondrion-specific electron suppliers Trx2, Trx reductase
breast, prostate and lung cancer [110, 111].
(TrxR)2 and sulfiredoxin (Srx) may potentially be administered in
Elesclomol sodium (STA-4783) induces oxidative stress with a
combination with various chemotherapeutic agents. Srx plays a
consequent increase in ROS in both transformed and healthy cells.
vital role by reducing hyperoxidised Prx III via translocation into
Since tumour cells have elevated ROS levels compared to healthy
mitochondria. Overexpression of the mitochondrion-targeted Srx
cells, STA-4783 will induce oxidative stress beyond baseline levels
efficiently enhances the restoration of Prx III and leads to cellular
with a resultant elevated ROS levels overwhelming the tumour cell
resistance to apoptotic cell death achieved by an enhanced elimina-
antioxidant systems. This may result in the induction of the apop-
tion of mitochondrial H O and decreased rates of (cid:2)(cid:3) collapse
totic cell death pathway [112, 113]. STA-4783 alone and in combi- 2 2 m
[130].
nation with paclitaxel showed promising data in phase I/II clinical
studies in patients with refractory solid tumours, however a recent iii. MOMP as a Target
phase III study in patients with melanoma has been discontinued on
As discussed earlier, MOMP can cause MPT to proceed, how-
safety grounds [114, 115].
ever MPT can also result from events originating from the outer
Mangafodipir is a superoxide dismutase (SOD) mimetic pos- membrane. Pro-apoptotic proteins such as BAX and BAK can me-
sessing both catalase and gluthatione reductase activities. In non- diate MOMP via their pore-forming activity [33, 41]. BAX is cyto-
transformed cells, Mangafodipir acts as an antioxidant whereas in solic in healthy cells whereas BAK is an integral OM protein. BAX
malignant cells it has been shown to elevate H2O2 pool and also translocates to the OM in response to pro-apoptotic triggers and
potentiate the anticancer activity of paclitaxel against xenotrans- BAK undergoes conformational modifications locally. These two
plants of colon cancer in mice [116]. Mangafodipir is in phase II processes consequently result in the assembly of homo-oligomers
trial in subjects with colon cancer. and/or hetero-oligomers that form protein-permeable conduits
Isothiocyanates such as (cid:2)-phenylethyl isothiocyanate through which toxic intermembrane space proteins are liberated
(PEITC), a consitutuent of edible cruciferous vegetables such as into the cytosol [33, 131]. Bcl-2 and Bcl-XL can cause the seques-
watercress, is a thiol modifier that interacts with redox regulatory tration of BAX and BAK thus displaying their strong cytoprotective
proteins [117]. PEITC does not only provide significant protection ability. Moreover, BAX can be directly activated or liberated from
against chemically induced oncogenesis in experimental rodents but inhibitory interactions with anti-apoptotic Bcl-2 family proteins by
also inhibits proliferation of human cancer cells via induction of cytoplasmic p53 [59]. The binding groove of BAX that mediates its
apoptosis and autophagy. Although the underlying mechanism is interaction with anti-apoptotic proteins has been known for many
not fully understood, studies demonstrate that PEITC-induced years, but the site for direct activation by pro-apoptotic BH3-only
206 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace
proteins of the Bcl-2 family has only recently been identified with clinical activity and it is being evaluated currently as a monother-
the help of nuclear magnetic resonance analysis. This represents a apy or incombination with temozolomide or topotecan in the
novel druggable target for therapeutic modulation of apoptosis treatement of patients with SCLC, leukaemia, chronic lymphoma or
[132]. TOM22 which is a core component of the OM protein trans- advanced B-cell malignancies [148]. Apogossypol (NSC736630) is
location pore has been reported as the mitochondrial receptor for a semi-synthetic derivative of gossypol with reported superior anti-
BAX [133], although a recent study outcome has suggested that tumour activity and reduced toxicity compared to the parent com-
BAX and BAK oligomerisation occur independent of TOM22 pound. Daily oral dosing studies showed that mice tolerated doses
[134]. Endophilin B1 is another BAX- interacting protein which has of the derivative 2 - to - 4 times higher than gossypol. Hepatotoxic-
been reported to mediate BAX-dependent MOMP [135]. ity and gastrointestinal toxicity represent the major adverse effects
of gossypol, with the derivative apogossypol showing far less toxic-
MOMP can also be initiated via mitochondrial lipids destabili-
sation by pro-apoptotic stimuli, with a consequent formation of ity. Efficacy tested both in vitro and in vivo, apogossypol displayed
transient gaps in the outer membrane that permits leakage of inter- superior activity [149].
membrane space proteins. Inner membrane proteases such as prese- A-385358 isa small molecule with some selectivity for binding
nilin-associated rhomboid-like (PARL) also regulate the release of to Bcl-X versus Bcl-2 [150]. It enters the cells efficiently and co-
L
cyt c from the mitochondria by cleaving the dynamin-related pro- localises with the mitochondrial membrane. As a monotherapy, A-
tein optic atrophy 1 (OPA1), in this way directing the remodelling 385358 shows a modest cytotoxicity towards tumour cell lines, and
of cristae independent of mitochondrial fusion [136, 137]. PARL- enhances the in vitro cytotoxic effects of other chemotherapeutic
deficient cells have been shown to display low levels of OPA1 and agents like paclitaxel, etoposide, cisplatin and doxorubicin in sev-
were more susceptible to mitochondrial apoptotic triggers [136]. eral tumour types. In A549, non-small-cell lung cancer, A-385358
Therefore inhibition of PARL seems a promising strategy to induce enhanced the activity of paclitaxel by about 25-fold, and this poten-
MOMP and commit transformed cells to apoptosis. tiation of paclitaxel by A-385358 is reproducible in vivo. Combina-
The susceptibility of cancer cells to undergo apoptotic cell tion of A-385358 with the maximally or half maximally tolerated
dose of paclitaxel showed a significant growth inhibition in A-549
death has repeatedly been demonstrated to be determined by the
xenograft model.more so, significant increase in mitotic arrest and
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- consequent apoptosis have been reported for the combination ther-
apy compared to paclitaxel monotherapy [150].
apoptotic protein members, such as the BH3-only proteins gener-
ates a powerful means to initiate MOMP-dependent apoptosis [3]. Obatoclax (GX15-070) is a small-molecule indole bipyrrole
Several inhibitors of Bcl-2 anti-apoptotic proteins have been identi- compound which is predicted to occupy a hydrophobic pocket
fied recently as being capable of inducing apoptosis in a variety of within the BH3 binding groove of Bcl-2 anti-apoptotic proteins thus
tumour cells, thus indicating their potential in cancer therapy. How- antagonising their actions with a consequent induction of apoptosis
ever, following the investigation of some of the putative Bcl-2 in- dependent on BAX and BAK [151, 152]. Unlike ABT-737, obato-
hibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine clax efficiently disrupts the direct interaction between BAK and
and ABT-737), it appears that only ABT-737 specifically targeted MCL-1 in intact mitochondrial outer membrane and also inhibits
Bcl-2 proteins and induced apoptosis via caspase-9 activation. the association between MCL-1 and BAK in intact cells thereby
These BH3 mimetics currently under preclinical and clinical testing overcoming the MCL-1 dependent resistance to ABT-737 and
have shown that they also interact with cellular targets that are un- bortezomib, a proteasome inhibitor and the gold standard treatment
related to Bcl-2 with the exception of ABT-737 and ABT-263, and for patients with multiple myeloma [151]. Obatoclax has demon-
this “unwanted” interaction might mitigate further development of strated a modest activity as a monotherapy in a phase I clinical
this class of drugs due to potential toxicity concerns [138, 139]. study involving patients with advanced chronic lymphocytic leu-
kaemia [153], however it is currently undergoing clinical evaluation
ABT-737, a small-molecule inhibitor of the anti-apoptotic pro-
teins Bcl-2, Bcl-X and Bcl- , with affinity two to three orders of as a monotherapy and in combination regimen in phase I/II studies
L W
magnitude more potent than the previously mentioned counterparts. for the treatment of haematological malignancies and solid tumours
[154]. In another phase II clinical study using obatoclax for the
Data obtained from mechanistic studies suggests that ABT-737
does not directly initiate the apoptotic cell death process; rather it treatment of myelofibrosis (MF) the molecule had no significant
enhances the effects of death signals [140]. It has shown a synergis- clinical activity in the MF patients at the dose and schedule evalu-
ated [155].
tic cytotoxicity profile with conventional chemotherapeutics and
radiotherapy against haematological malignancies and solid tu- Oblimersen (G3139) is an antisense oligonucleotide compound
mours [141, 142]. ABT-737 exhibits single-agent-mechanism-based rationally designed to specifically bind to the first 6 codons of the
killing of small-cell lung cancer and lymphoma cell lines, as well as human bcl-2 mRNA sequence resulting in the degradation of the
in primary tumours and animal models. ABT-737 improves sur- bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation
vival, regresses established tumours and is associated with high [156]. Oblimersen in combination with chemotherapeutic agents
cure rates of tumours in mice [140]. Furthermore, ABT-737 re- such as doxorubicin, docetaxel, cyclophosphamide and fludarabine
versed the chemoresistance of cancer cells against conventional has been clinically evaluated in various cancer types (157, 158]. In
chemotherapeutic agents, and ABT-737 resistance could be over- a phase III study involving patients with relapsed or refractory
come by using any of the classical cytotoxic agents [143, 144]. chronic lymphocytic leukaemia, there was a significant increase in
response rate and its duration with the addition of oblimersen to
ABT-263, is an orally bioavailable derivative of ABT-737 with
fludarabine plus cyclophosphamide therapy [158]. In a phase II
improved clinical potential. It mimics the mode of action of ABT-
737 and has shown promising preclinical outcome. It is currently in multicentre study, oblimersen in combination with rituximab
showed clinical benefit and safety in relapsed/refractory B-cell non-
Phase I/II clinical trials for chronic lymphocytic leukaemia, lym-
Hodgkin lymphoma patients [159]. However, another phase III
phoma and small cell lung cancer, as monotherapy or in combina-
tion with conventional chemotherapeutic agents, or with mono- clinical study in patients with multiple myeloma showed no signifi-
cant differences in response rate between both groups treated with
clonal antibodies [145].
either dexamethasone plus oblimersen or dexamethasone alone
Gossypol (AT-101) is a natural phenol derived from the cotton
[160]. Despite extensive data supporting a critical role for Bcl-2 in
plant (genus Gossypium) [146] that simultaneously inhibits the
chemoresistance in small cell lung cancer, addition of oblimersen to
anti-apoptotic Bcl-2 proteins, BCL-2, Bcl-X , Bcl- and Mcl1
L W a standard regimen for the tumour (carboplatin and etoposide) did
[138, 147]. In a phase I study against prostate cancer, it showed
not enhance any clinical outcome measure [161]. It has been sug-
Cancer Therapy Current Pharmaceutical Design, 2014, Vol. 20, No. 2 207
gested that the reported lack of efficacy may be due to insufficient therapeutic strategies that preferentially target glycolysis and con-
suppression of Bcl-2 in vivo, however further evaluation of sequently kill tumour cells (see Fig. 1).
oblimersen in small cell lung cancer is not necessary. 3-bromopyruvate (3-BrPA), a lactate/pyruvate analogue is an
HA14-1 (2-amino-6-bromo-4-[1-cyano-2-ethoxy-2-oxoethyl)- alkylating agent and a potent inhibitor of glycolysis. Its anticancer
4H-chromene-3-carboxylate), a small organic molecule (molecular effect is mediated by its capacity to inhibit hexokinase thus causing
weight = 409) and nonpeptidic ligand of a bcl-2 surface pocket. In a depletion of ATP in cancer cells. This effect is most severe in
vitro binding studies demonstrated the interaction of HA14-1 with cells with mitochondrial DNA deletion and respiratory defects,
this bcl-2 surface pocket that is vital for bcl-2 biological function leading to massive apoptotic cell death [176, 177]. Inhibition of
[162]. HA14-1 enhances the sensitivity of cultured glioblastoma hexokinase also results in a rapid dephosphorylation of Bcl-2 asso-
cells to chemotherapy or radiotherapy [163]. It effectively and se- ciated death promoter homologue (BAD), a molecule known to be
lectively induces apoptosis in human acute myeloid leukaemia (HL- vital in both apoptosis and glycolysis. Dephosphorylation of BAD
60) cells overexpressing Bcl-2 anti-apoptotic proteins and its activ- at Ser-112 is associated with re-localisation of BAX to mitochon-
ity is not altered by the expression of multidrug-resistance pheno- dria, cyt c release and apoptotic cell death [177]. Ganapathy-
type [162, 164]. Kanniappan et al. (2010) have described that the primary mecha-
nism of 3-BrPA is via preferential alkylation of GAPDH and that 3-
Polyamine analogues and conjugates, the naturally occurring
polyamines spermidine and spermine and their diamine precursor BrPA mediated cell death is linked to free radicals generation. Fur-
thermore, research from their laboratory also revealed that 3-BrPA
putrescine are vital for eukaryotic cell growth, differentiation and
induced endoplasmic reticulum stress, inhibited global protein syn-
death [165]. Although the targeted inhibition of specific biosyn-
thetic enzymes in the polyamine metabolic pathway has yet to show thesis which contributed to cancer cell death [178]. 3-BrPA has
been shown to have antitumour effects when injected intraarterially
significant clinical success in the treatment of cancer, there is a
in the hepatic artery of rabbits with VX-2 tumours [178]. The glu-
large body of evidence in favour of the rational for targeting the
polyamine function and metabolism in cancer [165 - 167]. Poly- cose transporter isoform 1 (GLUT1) gene encodes a key factor for
glucose transport into cancerous tissue [179, 180]. Although the
amine analogues and conjugates have been developed as a strategy
expression and functional significance of GLUT1 in neuroblastoma
to exploit the self-regulatory nature of polyamine metabolism. The
macrocyclic polyamine analogues, symmetrically substituted have not been fully described, 3-BrPA significantly suppressed the
proliferation of neuroblastoma cells with high GLUT1 gene expres-
bis(alkyl) and asymmetrically substituted polyamine analogues, and
sion compared to those with low expression [181], suggesting that
the novel oligoamine analogues have been associated with mito-
chondrial DNA as their target for cytotoxicity activity [168 - 170]. glycolysis inhibitors are a potential therapeutic option for treatment
of tumours expressing GLUT1. Combination of 3-BrPA with an
Cyt c release from the mitochondria of treated cells also suggested
mTOR inhibitor demonstrated synergistic effects on leukemia and
some involvement of the mitochondria in unleashing the apoptotic
cell death response to these compounds. Xie et al showed that an- lymphoma cells [182]. 3-BrPA may interact with other cellular
components owing to its alkylating properties suggesting that ex-
tharcenylmethyl homospermidine (ANTMHspd), a polyamine con-
tensive toxicity study needs to be carried out. The interaction be-
jugate induced mitochondrial membrane potential loss followed by
the release of cytochrome c and apoptotic cell death in promyelo- tween HK and VDAC offers another fascinating target to selec-
tively commit cancer cells to apoptotic cell death. Disrupting this
cytic leukemia (HL-60) cells [171]. This group further demon-
interaction has been shown to preferentially kill tumour cells both
strated that the combination therapy of ANTMHspd with alpha-
difluoromethylornithine (DFMO), an irreversible inhibitor of or- 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
nithine decarboxylase enzyme (ODC) was synergistic in inducing
to disrupt this HK-VDAC interaction and selectively trigger cell
the classical apoptosis in HL-60 cells via mitochondria/caspase-
9/caspase-3 dependent pathway. Ant 4, a putrescine-anthracene death in cancer cells. It binds to HK thereby disrupting its associa-
tion with the mitochondria and apoptotic cell death is initiated
conjugate, also demonstrated similar cytotoxicity profile in HL-60
[184]. Methyl jasmonate disrupts the HK-mitochondrial interaction
cell line. Ant 4 also induced oxidative stress in the cells; the latter
discovery provides a possible mechanism for the triggering of apop- 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.
tosis via the mitochondrial pathway [172].
2-deoxy-D-glucose (2-DG) is a glucose analogue and has long
iv. Targeting the Warburg Effect been known to act as a competitive inhibitor of glucose metabolism
Elevated glycolytic rate and improved glucose uptake can pro- [185]. 2-DG is transported into cells and is phosphorylated by
vide several benefits to a proliferating tumour cell. Although oxida- hexokinase to 2-DG-P. However, unlike its analogue metabolite
tive phosphorylation generates more ATP per molecule of glucose, glucose-6-phosphate (G-6-P), 2-DG-P cannot be metabolised by
glycolysis can provide ATP at a higher rate provided glucose sup- phosphohexose isomerase which usually converts G-6-P to fruc-
ply is unlimited [173]. While Warburg believed that the preferential tose-6-phosphate. This leads to the accumulation of 2-DG-P in the
use of glycolysis even in the presence of oxygen by tumour cells cells with a consequent inhibition of glycolysis at the step of
was as a result of defects in their mitochondrial respiratory path- hexokinase mediated phosphorylation of glucose. Cellular ATP
ways , recent studies have suggested that tumour cells do contain depletion leading to cell cycle arrest and cell death occurs following
functional mitochondria and that the enhanced glycolytic flux con- the inhibition of the rate-limiting step involving hexokinase medi-
fer a growth advantage [45, 46]. ated glucose phosphorylation [54]. Nonetheless, the efficacy of 2-
DG is significantly affected by the presence of glucose and appears
In tumour cells much of the carbon entering the TCA cycle is
to only partially reduce glucose availability for glycolysis. 2-DG
extruded as citrate resulting in a truncated TCA cycle that is used
causes endoplasmic reticulum (ER) stress because of its ability to
for lipid and fatty acids synthesis [174]. Glucose plays a key role in
affect protein glycosylation causing aberrant GlcNAcylation of
maintaining mitochondrial integrity by promoting the association of
proteins and induces accumulation of misfolded proteins in the ER
hexokinase II with the mitochondria, thus preventing the liberation
[186]. In vitro studies demonstrated that 2-DG exhibits cytotxic
of cyt c [62], as well as regulating various cell death effectors such
effect in cancer cells especially in those harbouring mitochondrial
as the pro-survival Mcl-1, pro-apoptotic BAD and Bax [139]. Lac-
defects or cells in hypoxic environment [187]. Inhibition of glyco-
tate, the by-product of glycolysis is believed to promote tumour
lysis by 2-DG significantly increased the cytotoxicity of cisplatin in
invasion and metastasis via degradation of extracellular matrices
human head and neck cancers via enhancing oxidative stress [188].
[175]. The understanding of the role of glucose in sustaining tu-
There has been concern that 2-DG might compromise the glycolytic
mour cell proliferation formed the basis for the development of
208 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace
metabolism of the brain and heart, however discussions centre on of a 28-KDa (cid:2) and (cid:4)-subunits, and also a third sequence homolo-
whether the therapeutic window of the 2-DG will be broad enough gous (cid:5)-subunit [197]. The (cid:5)-subunit has different enzymatic fea-
to justify its clinical development and also to develop specific in- tures and seems not to interact with the (cid:2)- and (cid:4)-subunits to form
hibitors of some glucose transporter isoforms that are frequently the 11S regulatory complex [198].
upregulated in cancer. Data from a clinical study suggests that 2- Proteasomes are situated in the both the nucleus and cytoplasm
DG at doses up to 250 mg/kg seems safe for use in combination of eukaryotic cells, however their distribution varies with regards to
with radiotherapy in glioblastoma multiforme patients [189]. 2-DG
cell or tissue type. It is now known that they undergo cell-cycle-
has been shown to stimulate Akt phosphorylation in vitro and an- specific redistribution between the nucleus and cytoplasm. In the
tagonise the anti-tumour action of a radio-immunotherapeutic inter- cytoplasm, proteasomes localise near centrosomes, on the outer
vention in vivo. These effects undermines the prospective therapeu-
surface of the endoplasmic reticulum and in cytoskeletal network
tic value of 2-DG [190]. The pursuit of further clinical development [199].
of this compound has recently been halted according to Threshold
Proteasomes regulate the levels of proteins such as cyclins, Bcl-
Pharmaceuticals [61].
2, caspases, nuclear factor (cid:3)B (NF-(cid:3)B) that are crucial in cell-cycle
Dichloroacetate (DCA) is a small molecule inhibitor of pyru-
progression and cell death [5, 6], and many of these proteasome
vate dehydrogenase kinase (PDK) which has the capacity to induce
substrates are known mediators of deregulated systems in cancer.
a metabolic switch from aerobic glycolysis to glucose oxidation, There is evidence that proteasome affects cell-cycle progression via
thus decreasing mitochondrial hyperpolarisation and rendering
its regulatory action on the cyclins. Also, it can cause an elevation
tumour cells more sensitive to apoptotic signals [191]. Pyruvate
or attenuation in apoptotic events through its action on the caspases,
dehydrogenase is a mitochondrial enzyme which converts pyruvate Bcl-2, and NF-(cid:3)B [6]. Suffice to say that specific defects in protea-
to acetyl CoA, and its activity is negatively regulated by PDK thus
some function or structure are yet to be linked to neoplasia although
DCA indirectly stimulates the pyruvate to acetyl CoA conversion.
abnormalities of the aforementioned regulatory pathways are linked
DCA also upregulates the expression of the K+ channel Kv1.5, to tumourigenesis.
which is often underexpressed in tumour cells [191]. DCA has pre-
viously been utilised in the clinic for the management of lactic aci- ii. Targeting the Proteasome for Cancer Therapy (Table 1)
dosis and showed a promising safety profile even when used
The dilemma associated with targeting the proteasome as a
chronically [192]. A phase I study for DCA in currently under way
means of chemotherapeutic intervention is that this organelle plays
in Canada and is accepting patients with recurrent or metastatic
a crucial role in the execution of many cellular functions, thus mak-
solid tumours [3, 61].
ing it difficult to target selectively and at the same time maintain a
ATP citrate lyase (ACL) is a key enzyme that associates glu- tolerable therapeutic window. However, studies have shown that
cose metabolism with lipid biosynthesis via catalysing the conver- many types of actively proliferating cancer cells display a higher
sion of citric acid to cytosolic acetyl-CoA has become a promising sensitivity to proteasome inhibition than the non-transformed cells
potential drug target in cancer treatment. Frequently malignant cells [6].
redirect pyruvate towards lipid synthesis which is a key component
The proteasome inhibitors N-carbobenzoxy-L-leucyl-L-leucyl-
required to support the elevated demand for membrane generation
norvalinal (LLnV) and N-carbobenzoxy-L-isoleucyl-L-(cid:2)-t-butyl-
in highly proliferating cells [193]. Tumour growth was shown to be
L-glutamyl-L-alanyl-L-leucinal (PSI) demonstrated a selective
suppressed in vitro following the inhibition of ACL by RNAi or
p53-dependent apoptotic cell death induction in the proliferating
using a pharmacological inhibitor, SB-204990 [193]. It has been
human leukemic HL60 cells compared with the quiescent HL-60
demonstrated that ACL is essential for the provision of adequate
cells [200]. Also compared with actively proliferating primary en-
acetyl CoA required for histone acetylation and hence gene expres-
dothelial cells, a 340-fold higher concentration of PSI was required
sion [194]. The implication regarding this recent discovery is that
to induce apoptosis in contact inhibited quiescent cells [6]. Another
ACL inhibition might not only normalise tumour metabolism at the
proteasome inhibitor (PI), Lactacystin, which is an irreversible
lipid synthesis level but it will also play a part in the epigenetic
inhibitor of the catalytic (cid:4)-subunit of the proteasome induces apop-
reprogramming of malignant cells.
totic cell death in human chronic lymphocytic leukaemia sensitive
or insensitive to radiation at doses that do not affect normal lym-
PROTEASOMES
phocytes [201]. Apoptotic cell death was also observed in oral
i. Structure, Localisation and Function
squamous-cell carcinoma cells, but not in normal oral epithelial
The functionally active 26S proteasome is a very large 2.4 MDa cells or gingival fibroblasts following treatment with LLnV and
ATP-dependent proteolytic complex that is situated in the cyto- Lactacystin [201]. It is fascinating that treatment with LLnV or
plasm and nucleus of eukaryotic cells. The 26S proteasome consists lactacystin was associated with an accumulation of KIP1 and re-
of a 20S core catalytic cylindrical complex capped at both ends by duced expression of the anti-apoptotic protein, Bcl-2 thus leading to
19S regulatory subunits [195]. Although ubiquitinylation is not a cell-cycle arrest and consequent apoptosis [6]. Several studies have
prerequisite for the degradation of every protein, the 26S protea- provided proof of principle that the proteasome is a valid target for
some identifies proteins and degrades proteins that have been anticancer therapy; however the available inhibitors lack specificity
marked for degradation for their ubiquitin ‘tag’ [6]. The 20S protea- [202]. This led to the design and development of boronic acid-
some is a complex of 28 protein subunits arranged into 4 stacked derived compounds that inhibited the proteasome pathway in a
rings to create a cylindrical structure. The (cid:2)-subunits made up of highly specific fashion. Most of these compounds developed by
seven polypeptides form the top and bottom rings of the 20S cylin- Adam et al showed activity across the 60 panel of cell lines from
drical structure. The enzyme active site of the proteasome complex the National Cancer Institute. Bortezomib was identified from these
is in the two inner rings which consist of seven (cid:4)-subunits that form compounds as the best candidate on the basis of its potency and
the central chamber [196]. Proteasome enzymatic function is per- cytotoxicity [203].
formed by three ((cid:4)1, (cid:4)2 and (cid:4)5) of the seven (cid:4)-subunits, while the Bortezomib (N-pyrazinecarbonyl-L-phenylalanine-L-
function of the other four subunits is unclear [6]. For the 20S pro- leucineboronic acid; previously known as PS-341 or MLN-341) a
teasome to function in vivo, it will require the association of a regu-
boronic acid dipeptide was the first PI to enter clinical trials and
latory unit that partially determine the specificity in function. 19S was granted accelerated approval by the FDA in 2003 after demon-
regulatory complex is an example of such regulatory unit [6]. The strating remarkable single-agent responses in patients with relapsed
mammalian cells contain another regulatory complex that associates
and refractory myeloma. It has since been approved for the treat-
with the 20S proteasome: the 11S regulator or PA28. It is made up ment of newly diagnosed multiple myeloma and also mantle cell
Cancer Therapy Current Pharmaceutical Design, 2014, Vol. 20, No. 2 209
Table 1. Proteins affected by the Ubiquitin-Proteasome Pathway inhibition
lymphoma [202]. It reversibly and rapidly inhibits the proteasome zomib when cells are exposed for short duration mimicking in vivo
pathway by directly binding to the 20S proteasome complex and single-dose exposure; however both compounds have superior ac-
blocking its enzymatic activity [203]. The underlying mechanism tivity when cells are exposed for prolonged periods of time [208,
leading to cell-cycle arrest and apoptotic cell death include NF-(cid:2)B 209]. To achieve a prolonged inhibition in vivo, PR-171 was admin-
inhibition, upregulation of various apoptotic pathways and effects istered daily and well tolerated at doses that inhibited over 80 % of
on tumour micro-environment. It is unlikely that proteasome inhibi- proteasome chymotrypsin-like-(cid:3)5 subunit (CT-L) activity in blood
tion with bortezomib will be observed in tumours resident in the and tissues. This observation contrasts that of bortezomib in which
brain or the testes unless the blood-brain or blood-testes barrier has a twice-weekly clinical-dosing schedule that allows full recovery of
been disrupted by the tumour. This was observed in tumour tissues proteasome function between doses was preferred because of ex-
isolated from brain and testes showing no proteasome inhibition cessive toxicity associated with more frequent dosing schedule
[203]. Preclinical studies demonstrated that the cytotoxic and anti- [210, 211]. Carfilzomib has also been investigated in combination
proliferating actions of bortezomib are correlated with the inhibi- with lenalidomide and low-dose dexamethasone in patients with
tion of proteasome in a p53 independent manner and also non- relapsed/refractory multiple myeloma [212]. The FOCUS trial
overlapping with other chemotherapeutics [203]. Tumour resistance (N=302) is comparing carfilzomibmonotherapy with best suppor-
to conventional anticancer agents can be overcome by the addition tive care in the relapsed/refractory stage. Moreover, other early-
of bortezomib as shown in two phase II multiple myeloma studies phase studies are investigating carfilzomib in frontline multiple
[204, 205]. There is strong evidence suggesting that the activity of myeloma in combination with either thalidomide or lenalidomide
bortezomib in solid tumours is significantly enhanced when used in and dexamethasone [213].
combination with chemotherapeutic agents such as doxorubicin, Marizomib (NPI-0052; salinosporamide A) is a structurally
gemcitabine, docetaxel, paclitaxel, and irinotecan [202, 206, 207]. and pharmacologically unique (cid:3)-lactone-(cid:4)-lactam proteasome in-
Phase II clinical trials of bortezomib with gemcitabine, docetaxel
hibitor. Its potent and sustained inhibition of all three proteolytic
and other conventional cytotoxic agents are on-going. activities of the proteasome has generated a renewed motivation
inspiring the extensive preclinical evaluation in multiple myeloma,
SECOND-GENERATION PROTEASOME INHIBITORS
mantle cell lymphoma, acute and chronic lymphocytic leukemia,
(PIs)
Waldenstrom’s macroglobulinemia, glioma, colorectal and pancre-
Following an improvement in our understanding of the ubiq- atic cancer models. It has shown efficacy as monotherapy, and in
uitin-proteasome pathway, the development of new PIs with distinct combination with chemotherapeutics, biologics and targeted thera-
efficacy and safety profiles was embarked upon. More so the sec- peutics [214]. Clinical trials in patients with multiple myeloma,
ond generation PIs provide potential options for patients with borte- lymphomas, leukemias and solid tumours, and also patients with
zomib resistant tumours. bortezomib treatment failure, as well as in patients with diagnoses
Carfilzomib (PR-171) has in vitro anti-proliferative effect where other PIs have been shown to be significantly efficacious is
across a range of tumour cell types including multiple myeloma underway [214 - 216].
cells resistant to bortezomib. PR-171 is more active than borte-
210 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Ubah and Wallace
MLN9708/MLN2238 is a selective, orally bioavailable second- ER. Nonetheless, the UPR will induce cell death programmes to
generation PI that is currently in a phase I clinical development eliminate stressed cells in situations where attempts to recover the
[217]. It has a shorter proteasome dissociation half-life with an cells from ER stress fails [226]. During carcinogenesis, there is a
enhanced PK-PD, and anticancer activity compared with borte- high demand for an increased activity of ER protein folding, as-
zomib. Treatment of multiple myeloma cells with MLN9708 pre- sembly, and transport to support the elevated proliferation rate, and
dominantly blocked CT-L activity of the proteasome and induced this condition can induce physiological ER stress. Moreover, there
accumulation of ubiquitinated proteins. Multiple myeloma cells is an increased nutrient starvation and hypoxia as tumour cells
resistant to bortezomib and other conventional therapies were grow, and these are strong inducers of the accumulation of unfolded
shown to respond to anti-proliferative and apoptotic effects of or misfolded proteins in the ER and the subsequent activation of the
MLN9708 and these activities are selective for tumour cells rather UPR pathways [227]. There is valid and accumulating evidence
than non-transformed cells [218]. Mechanistic studies showed that showing that the UPR is a key mechanism required for cancer cells
MLN9708-mediated apoptosis is linked with the activation of to maintain malignancy and resistance to therapy [226].
caspases-3, 8, and 9; elevated p53, p21, NOXA, PUMA, and E2F;
Experimental evidence suggests that ER and Golgi apparatus
induction of endoplasmic reticulum stress response proteins Bip, can activate both pro-survival and apoptotic mechanisms if the
phosphor-eIF2-(cid:2), and CHOP; inhibition of NF-(cid:3)B [218]. MLN9708 stress-signalling threshold is exceeded [4]. The Golgi apparatus is
in combination with lenalidomide, dexamethasone or histone deace-
known to dissemble during apoptosis in a fashion resembling that
tylase inhibitor suberoylanilidehydroxamic acid triggers synergistic observed during mitosis [225]. It is plausible that the subtle balance
activity in the treatment of multiple myeloma [210, 218]. of protein trafficking between various subcellular compartments
ONX-0912, is an orally bioactive tripeptideepoxyketone with afford an extremely sensitive damage sensor. It is worth mentioning
anti-proliferative effects and also induces apoptosis multiple mye- the interaction between the mitochondrion with the Bcl-2 family
loma cells resistant to conventional and bortezomib therapies. Its proteins regulating various cell death signals and the ER. Thus,
anti-myeloma activity is associated with activation of caspases-3, 8, overexpression of Bax has recently been shown to induce Ca2+ mo-
and 9, and poly(ADP) ribose polymerase, also the suppression of bilisation from ER stores [228, 229]. There is substantial evidence
multiple myeloma cells migration [219]. In animal tumour models, supporting the Bcl-2 co-localisation with the ER and protects cells
ONX-0912 significantly reduced tumour progression and improved from thapsigargin-mediated apoptosis [225]. More so, evidence is
survival, and it improved the anti-tumour activity of bortezomib, mounting that signals originating from the ER-Golgi network may
lenalidomide, histone deacetylase inhibitors or dexamethasone in bypass the mitochondrion and efficiently execute apoptotic cell
multiple myeloma [210, 219]. A phase I study with ONX-0912 in death [4, 225].
patients with solid tumours is on-going, and an interim results from
the study showed that at doses that were tolerated, more that 80% Targeting the UPR/ER Stress Components
proteasome inhibition in blood could be achieved on a once-daily The role of UPR in tumour maintenance has stimulated great
dosing regimen for 5 consecutive days [220]. interest in exploring therapeutic potential by targeting the UPR
components. The understanding that healthy cells are not subjected
ENDOPLASMIC RETICULUM (ER) - GOLGI NETWORK to stress and the UPR pathway remains in an inactive state while
Interestingly a growing number of reports recognise proteins in that of transformed cells rely on activated UPR for survival creates
control of ER and Golgi homoeostasis as potential therapeutic tar- a discrepancy between tumour cells and their healthy counterpart
gets in the treatment of malignancies. Eukaryotic cells consist of a and thus offer an advantage for the agents targeting these compo-
set of organelles with a tightly and dynamically regulated abun- nents to achieve specificity (Table 2).
dance with regards to cellular demand. These organelles are
equipped with specific cellular functions. The ER is an organelle in PROTEASOME INHIBITORS
which secretory and membrane proteins are synthesised, and cor- The degradation of the majority of cellular proteins is mediated
rectly folded proteins by the ER chaperones are transported to the by the 26S proteasome. The role of proteasome in regulating cell
Golgi apparatus [221]. The ER retains the unfolded or misfolded growth, survival and metastasis of malignant cells is well reported
proteins and they are subsequently degraded by the ER-associated leading to the development of PIs (e.g., Bortezomib; see previous
degradation (ERAD) [222, 223]. In cases of accumulation of un- section for discussion on PIs). It is believed that PIs cause a build-
folded proteins in the ER, an upregulated expression of the ER up of misfolded proteins which overwhelms the ERAD pathway
chaperone and components of ER-associated degradation machin- resulting in ER stress [230].
ery occurs in an effort to enhance the protein folding capacity and
Multiple myeloma cells constitutively express ER stress sur-
degradation of unfolded or misfolded proteins. This process occurs
vival factors in order to function as secretory cells, and it is also
through the cytoprotective mechanism referred to as the ER stress
assumed that the UPR components are required for B-cell differen-
response or unfolded protein response [224]. Large amounts of
tiation into antibody-secreting cells [231]. Thus agents that interfere
secretory proteins are transported to the Golgi apparatus when the
with the UPR and ER-stress induced apoptosis will show greater
ER stress response enhances the capacity of ER function, thus caus-
effect in myeloma cells and other secretory cells as a result of the
ing inadequacy of Golgi function. This is referred to as Golgi stress,
reliance of these cells on the UPR pathway. As discussed in the
which initiates the Golgi stress response to cope with the situation
previous section, multiple myeloma cells are very sensitive to the PI
[221]. To ensure the fidelity of protein folding and to sustain the
bortezomib which rapidly induces components of the pro-apoptotic
accumulation of unfolded or misfolded proteins, the ER has devel-
/terminal UPR, including PERK ultimately resulting in increased
oped a group of signal transduction pathways, the unfolded protein
cell death [230]. Since multiple myeloma cells are dependent on an
response (UPR) signalling pathways to alter transcriptional and
activated UPR for survival suggesting that these cells have a lower
translational programs [225]. The basic mammalian cell UPR path-
threshold for ER stress. Thus, any additional stress can slant the
ways is composed of three major signalling pathways initiated by
balance in favour of cell death.
three primary ER-localised protein stress sensors; IRE1(cid:2) (inositol-
requiring enzyme 1 alpha), PERK (double-strand RNA-activated HSP90 INHIBITORS
protein kinase-like ER kinase), and ATF6 (activating transcription
Heat shock protein 90 (HSP90) is involved in regulating the
factor 6). The primary function of the UPR is to prevent the cell
proper folding, stability and function of a wide variety of signal-
from ER stress by limiting the amount of proteins translocated into
transducing proteins that regulate cell proliferation and differentia-
the ER lumen, increasing retrotranslocation and degradation of ER-
tion. These proteins include steroid hormone receptors and protein
localised proteins, and enhancing the protein-folding capacity of the