1 s2.0 S0169409X0900252X Main PDF

Advanced Drug Delivery Reviews 61 (2009) 12501275
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r
Opportunities in discovery and delivery of anticancer drugs targeting mitochondria

and cancer cell metabolism
Divya Pathania 1, Melissa Millard 1, Nouri Neamati
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles, CA 90089, USA
a r t i c l e
i n f o
Article history:
Received 14 May 2009
Accepted 18 May 2009
Available online 27 August 2009
Keywords:
Warburg effect
Aerobic glycolysis
Glycolytic inhibitors
Targeted drug delivery to cancer
Delocalized lipophilic cations
Inhibitors of mitochondrial electron
transport chain
Biosynthetic alterations in cancer cells
Mitochondrial redox system
Mitochondrial apoptotic machinery
Triphenylphosphonium compounds
a b s t r a c t
Cancer cells are characterized by self-sufciency in the absence of growth signals, their ability to evade apoptosis,
resistance to anti-growth signals, sustained angiogenesis, uncontrolled proliferation, and invasion and
metastasis. Alterations in cellular bioenergetics are an emerging hallmark of cancer. The mitochondrion is the
major organelle implicated in the cellular bioenergetic and biosynthetic changes accompanying cancer. These
bioenergetic modications contribute to the invasive, metastatic and adaptive properties typical in most tumors.
Moreover, mitochondrial DNA mutations complement the bioenergetic changes in cancer. Several cancer
management therapies have been proposed that target tumor cell metabolism and mitochondria. Glycolytic
inhibitors serve as a classical example of cancer metabolism targeting agents. Several TCA cycle and OXPHOS
inhibitors are being tested for their anticancer potential. Moreover, agents targeting the PDC/PDK (pyruvate
dehydrogenase complex/pyruvate dehydrogenase kinase) interaction are being studied for reversal of Warburg
effect. Targeting of the apoptotic regulatory machinery of mitochondria is another potential anticancer eld in
need of exploration. Additionally, oxidative phosphorylation uncouplers, potassium channel modulators, and
mitochondrial redox are under investigation for their anticancer potential. To this end there is an increased
demand for agents that specically hit their target. Delocalized lipophilic cations have shown tremendous
potential in delivering anticancer agents selectively to tumor cells. This review provides an overview of the
potential anticancer agents that act by targeting cancer cell metabolism and mitochondria, and also brings us face
to face with the emerging opportunities in cancer therapy.
2009 Elsevier B.V. All rights reserved.
Abbreviations: AIF, apoptosis inducing factor; AVPI, alanine valine proline isoleucine; BAD, Bcl-2 antagonist of cell death; BAX, Bcl-2 associated protein X; BAK, Bcl-2 homologous
antagonist/killer; BH, Bcl-2 homology domain; BIR, baculovirus IAP repeat; 1,3-BPG, 1,3-bisphosphoglycerate; AMF, autocrine motility factor; ANT, adenine nucleotide transporter;
ARD1, arrest-defective 1 protein; BCNU, bis-chloronitrosourea; 3-BrP, 3-bromopyruvate; CAD, c-terminal activation domain; CARD, caspase recruitment domain; CBP, CREB binding
protein; CCCP, carbonylcyanide-3-chlorophenylhydrazone; COX4/2, cyotochrome oxidase isoform2; CT, computed tomography; DCA, dichloroacetate; 2-DG, 2-deoxyglucose; DHAP,
dihydroxy acetone phosphate; DIABLO, Direct Inhibitor of Apoptosis Binding protein with a Low pI; DLC, delocalized lipophilic cation; DNP, 2,4-dinitrophenol; EGFR, epidermal
growth factor receptor; ERK, extracellular signal regulated kinase; ETC, electron transport chain; FAD, avin adenine dinucleotide (oxidized); FADH2, avin adenine dinucleotide
(reduced); 18FBzTPP, 4-(18F-benzyl) triphenylphosphonium; FCCP, p-triuoromethoxyphenylhydrazone; FDA, Food and Drug Administration; 18F-FDG, 18F-deoxygluxose; FH,
fumarate hydratase; FIH, factor inhibiting HIF-1; FNQ, furanonapthoquinone; F-1,6-BP, fructose-1,6-bisphosphate; F-6-P, fructose-6-phosphate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GPI, glucose phosphate isomerase; Glut, glucose transporter; G-6-P, glucose-6-phosphate; Glyc-3-P, glyceraldehydes-3-phosphate; HBD, hydrogen
bonding donor; HBA, hydrogen bonding acceptor; HDAC, histone deacetylase; HIF-1, hypoxia inducible factor-1; HK, hexokinase; HLRCC, hereditary leiomyomatosis/renal cell
cancer; PGL, hereditary paraganglioma; Hsp90, heat shock protein 90; 3H-TPP, 3H-tetraphenylphosphonium; IAP, inhibitor of apoptosis protein; IMM, inner mitochondrial
membrane; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; MDR, multidrug resistance; MEK, MAPK-ERK kinase; M2-PK,
pyruvate kinase M2; MMP, matrix metalloproteinase; MOM, mitochondrial outer membrane; MPP+, 1-methyl-4-phenylpyridinium cation; MPT, membrane permeability
transition; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 99m-Tc-MIBI, 99m-Tc-Sestamibi; MTD, maximum tolerated dose; mTOR, mammalian target of rapamycin; NAD+,
nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); NCI,
National Cancer Institute; NFAT, nuclear factor of activated T cells; NO, nitric oxide; OXPHOS, oxidative phosphorylation; PCD, programmed cell death; PDC, pyruvate dehydrogenase
complex; PDT, photodynamic therapy; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; PEP, phosphoenol pyruvate; PET, positron emission
tomography; PHD, prolyl hydroxylase; PI3K, phosphoinositide 3-kinase; PFK, phosphofructokinase; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; 2-PG, 2phosphoglycerate; 3-PG, 3-phosphoglcerate; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; Pgp, p-glycoprotein; PK, pyruvate kinase; PKC, protein kinase C delta;
PS, photosensitizer; PSA, prostate specic antigen; PTP, permeability transition pore; pVHL, von HippelLindau protein; ROS, reactive oxygen species; SAR, structure activity
relationship; SCO2, synthesis of cytochrome c oxidase2; SDH, succinate dehydrogenase; Smac, Second mitochondria derived activator of caspases; SPECT, single photon emission
computed tomography; TIGAR, tp53 induced glycolysis and apoptosis regulator; TCA cycle, tricarboxyclic acid cycle; TNFR, tumor necrosis factor receptor; TPA, triphenylarsonium;
TPP, triphenylphosphonium cation; TRAF, TNF receptor associated family of proteins; TKTL1, transketolase like enzyme1; TOS, -tocopheryl succinate; TPI, triosephosphate
isomerase; Trx/TrxR, thioredoxin/thioredoxin reductase; VDAC, voltage dependent anion channel; XIAP, X-linked inhibitor of apoptosis protein.
This review is part of the Advanced Drug Delivery Reviews theme issue on Mitochondrial Medicine and Therapeutics, Part II.
Corresponding author. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, PSC304, 1985 Zonal Avenue, Los
Angeles, CA 90089, USA. Tel.: +1 323 442 2341; fax: +1 323 442 1390.
E-mail address: neamati@usc.edu (N. Neamati).
1
First two authors contributed equally.
0169-409X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.05.010
D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275
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Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rationale for targeting mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glycolytic inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Hexokinase (HK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
2-Deoxyglucose (2-DG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
3-Bromopyruvate (3-BrP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Lonidamine (TH-070) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4.
Methyl jasmonate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Glucose-6-phosphate isomerase (GPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Phosphofructokinase (PFK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Aldolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.
Triosephosphate isomerase (TPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.1.
Iodoacetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2.
Koningic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.
Phosphoglycerate kinase (PGK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8.
Phosphoglycerate mutase (PGM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9.
Enolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.10. Pyruvate kinase (PK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.11. Lactate dehydrogenase (LDH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.12. Transketolase like enzyme (TKTL1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
Role of HIF-1 in promoting the Warburg effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Mitochondrial bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
The Citric Acid Cycle is the entry point for aerobic respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Enzymes of the Electron Transport Chain (ETC) create proton motive force needed to produce ATP . . . . . . . . . . .
5.3.
Mechanisms of respiratory control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Targeting OXPHOS enzymes to induce mitochondrial bioenergetic failure . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1.
Targeting Complex I, mitochondrial NADH ubiquinone reductase . . . . . . . . . . . . . . . . . . . . . . .
5.4.2.
Succinate dehydrogenase links TCA cycle and OXPHOS . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.3.
Cytochrome c reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.4.
Cytochrome c oxidase is the nal electron acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.5.
Targeting the F0F1 ATPase prevents ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.
The other side of the coin: biosynthetic drive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Biosynthesis of nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Biosynthesis of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Biosynthesis of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.
Anaplerosis and NADPH production: role of glutaminolysis in biosynthesis . . . . . . . . . . . . . . . . . . . . . . .
7.
Critical switch between glycolysis and TCA cycle: pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) .
7.1.
Dicholoroacetate (DCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
Radicicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.
Nov3r and AZD7545 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
Mitochondrial OXPHOS uncouplers and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.
Mitochondrial DNA mutations in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.
TCA cycle enzymes as tumor suppressors: succinate dehydrogenase (SDH) and fumarate hydratase (FH) . . . . . . . . . . . .
11.
Targeting mitochondrial redox status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.1. Inhibition of mitochondrial thioredoxin reductase disrupts mitochondrial redox balance and induces tumor cell apoptosis.
12.
Cancer therapy with mitochondrial potassium channel modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.
Targeting mitochondrial apoptotic machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.1. Targeting membrane permeability transition (MPT) to induce apoptosis . . . . . . . . . . . . . . . . . . . . . . . .
13.2. Bcl-2 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3. Smac/DIABLO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.
Small molecule delivery to mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.1. Delocalized lipophilic cations selectively target the mitochondria of tumor cells . . . . . . . . . . . . . . . . . . . . .
14.2. Lipophilic triphenylphosphonium cations rapidly accumulate within mitochondria and have been utilized to target a wide
variety of molecules into the mitochondria of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3. Lipophilic cations as tumor selective radiotracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4. Lipophilic triphenylphosphonium cations as mitochondrial drug carriers . . . . . . . . . . . . . . . . . . . . . . . .
15.
Mitochondrial targeted anti-cancer photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.
Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The mitochondrion is the powerhouse of the cell and serves as the
major energy source. The mitochondrion consists of two membranes
that separate it from the cytosol. The outer and inner mitochondrial
membranes divide the mitochondrion into two compartments, the
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intermembrane space (the space between the outer and inner

mitochondrial membranes) and the matrix lled inner compartment.
The inner mitochondrial membrane (IMM) consists of several folds
known as cristae. The VDAC (voltage dependent anion channel)
present on the outer membrane regulates the entry of different
cellular components into the mitochondria. All the enzymes for the
1252
TCA cycle (tricarboxylic acid cycle/Krebs cycle/citric acid cycle) reside

in the mitochondrial matrix, whereas the respiratory chain and ATP
synthase/ATPase are present on the inner membrane [1].
Besides serving as the cell's primary energy source, mitochondria
are implicated in the regulation of programmed cell death (PCD, the
intrinsic pathway of apoptosis), reactive oxygen species (ROS) generation, and maintenance of calcium homeostasis [24]. Mitochondria
play an important role in cell survival and cell death, and dysregulation of any form leads to diseases. Mitochondrial dysfunction has been
implicated in neurodegenerative and neuromuscular disorders,
ischemia-reperfusion injury, diabetes, obesity, inherited mitochondrial diseases and most importantly, cancer [57].
A change in cellular bioenergetics is one of the key hallmarks of
cancer. The concept of aerobic glycolysis was introduced by Warburg
[8]. Since then research has been carried out with the goal of targeting
the sweet tooth of cancer [9,10]. Aerobic glycolysis provides cancer
cells with several growth advantages. The various advantages include
growth of cells in adverse microenvironment, generation of substrates
for glycosylation reactions, and supply of precursors for biosynthetic
reactions [11,12]. Moreover, metabolism independent functions of the
glycolytic enzymes provide additional benets such as resistance to
apoptosis, invasiveness, malignancy and transcriptional regulation
[13].
In addition to aerobic glycolysis, the malignant phenotype is
characterized by additional metabolic changes, de novo lipid and
nucleotide biosynthesis, and glutamine dependent anaplerosis (replenishment of TCA cycle intermediates). These metabolic changes
are necessary in order to produce sufcient biomass required for rapid
cell proliferation. Various TCA cycle intermediates serve as precursors
for the biosynthetic reactions. Citrate derived from the TCA cycle acts
as a precursor for fatty acid synthesis. Oxaloacteate and -ketoglutarate provide nonessential amino acids required for protein and
nucleotide synthesis. Moreover, cancer cells have higher uptake of
glutamine for replenishing the TCA cycle intermediates through
glutaminolysis. The entire metabolic prole of cancer cells is regulated
by PI3K/Akt/mTOR(PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin), HIF-1 (Hypoxia-inducible factor-1),
and c-Myc. Activation of the PI3K pathway increases glucose and
nutrient uptake, and increases the expression of enzymes involved
in glycolysis and lipid synthesis. Expression of c-Myc is known to
regulate glutamine uptake in cells. c-Myc regulates transcription
machinery components and plays an important role in protein synthesis. The activity of c-Myc modulates nucleotide, amino acid, fatty
acid and polyamine synthesis. The expression of HIF-1 inhibits
translation initiation in addition to induction of glycolysis. HIF-1
inhibits the entry of glycolytic end products in the TCA cycle via its
effect on pyruvate dehydrogenase kinases (PDKs). The metabolic
changes in the cancer cells complement tumor cell requirements
for increased biogenesis, increased energy demand and an adaptive
response to tumor microenvironment [11].
We have provided a summary of approaches for targeting the
metabolism of cancer cells for therapeutic benet. The focus of this
review is on cancer cell metabolism and mitochondrial targeted anticancer approaches. The purpose of this review is to examine the
current knowledge of potential targets in this eld. We propose that
combination therapies can be advantageous for the treatment of
cancer. Moreover, targeting several different functions can potentiate
the therapeutic benets.
2. Rationale for targeting mitochondria
Otto Warburg was the rst to demonstrate that even in ample
supply of oxygen cancer cells rely on glycolysis for energy [8].
According to Warburg the reliance of cancer cells on aerobic glycolysis
was due to defects in oxidative phosphorylation (OXPHOS) in cells.
However, it has now been proved that the defect in OXPHOS is not the
cause of the Warburg effect. Several tumor types have functional

OXPHOS and but rely on aerobic glycolysis due to invasive and
adaptive benets [9,14]. Aerobic glycolysis has been observed in
almost all cancers. The bioenergetic switch from a higher energy
yielding process (TCA cycle) to a lower energy yielding process
(glycolysis) is a hallmark of most cancer cells. The increased glycolytic
dependency of cancer cells is attributable to mitochondrial defects
and malfunctions, nuclear DNA mutations, abnormal expression of
metabolic enzymes (fumarate hydratase and succinate dehydrogenase), adaptation to the tumor microenvironment (HIF-1), and
disruption in oncogenic/tumor suppressor signaling (Ras, Akt, p53,
pVHL) (Fig. 1).
Many forms of cancer exhibit mitochondrial DNA (mtDNA) mutations (Fig. 2). MtDNA mutations can cause inhibition of OXPHOS,
increase ROS generation, and aid in tumor adaptation under adverse
conditions [15]. Abnormalities in nuclear DNA can also lead to increased aerobic glycolysis in cancer cells. Mutations in nuclear DNA
can give rise to abnormal succinate dehydrogenase (SDH) and
fumarate hydratase (FH), as well as overexpression of glycolytic, and
pentose phosphate pathway enzymes [16]. Transketolase like enzyme,
TKTL1 is a pentose phosphate pathway enzyme which is overexpressed in many human tumors. Overexpression leads to increased
production of gylceraldehyde-3-phosphate which is utilized for the
energy yielding phase of glycolysis [17]. Two TCA cycle enzymes, SDH
and FH act as tumor suppressors. SDH catalyzes succinate to fumarate
in the TCA cycle. Fumarate is further hydrolyzed to malate by FH. SDH
mutations give rise to hereditary paraganglioma (PGL) and phaeochromocytomas. Impaired FH function is implicated in hereditary
leiomyomatosis/renal cell cancer (HLRCC) syndromes [18].
The adaptive response to tumor hypoxia leads to stabilization of
HIF-1 and/or HIF-2. HIF activation leads to transcriptional activation of
the genes for glucose transporters, glycolytic enzymes, and angiogenic
factors. HIF-1 may also be elevated under normoxic conditions in
tumor cells (Table 1) [19].
Several factors such as pVHL (von HippelLindau protein) inactivation or Ras, Src, or PI3K/Akt activation can lead to stabilization of
HIF-1 under normoxia. Oncogenes can also directly activate glycolysis
independent of HIF-1. c-Myc binds the promoters of several glycolytic
genes (Hexokinase II, Enolase1 and Lactate dehydrogenaseA), glucose
transporters, and activates them even under normoxia. Constitutive
activation of Akt increases surface expression of the Glut1 glucose
transporter. Akt also maintains the mitochondrial association of
hexokinase. This association prevents changes in the permeability of
the outer mitochondrial membrane and cytochrome c release upon
apoptotic stimulation. Akt phosphorylates BAD (Bcl-2 antagonist of cell
death) and inhibits the association of pro-apoptotic BAX (Bcl-2 associated protein X) and BAK (Bcl-2 homologous antagonist/killer) [19,20].
Tumor suppressor p53 affects mitochondrial respiration as well.
p53 can transactivate SCO2 (synthesis of cytochrome c oxidase2).
SCO2 is required for the assembly of the mitochondrial cytochrome c
oxidase complex. Moreover p53 stimulates TIGAR (tp53 induced
glycolysis and apoptosis regulator), which decreases fructose-2,6bisphosphate and hence suppresses glycolysis [21].
Recent reports have shown the involvement of mitochondrial
uncouplers in aerobic glycolysis. It has been suggested that aerobic
glycolysis may represent a shift to the oxidative metabolism of
nonglucose carbon sources (fatty acid oxidation). Additionally, mitochondrial uncoupling may be responsible for increased resistance to
chemotherapy [22].Thus, designing therapeutic strategies for specifically killing cancer cells by exploiting their metabolic alterations
seems to be a promising approach.
3. Glycolytic inhibitors
Glycolysis refers to the series of enzymatic reactions which convert a molecule of glucose into lactate with the generation of two
1253
Fig. 1. Opportunities in treating cancer by targeting cancer energy metabolism and mitochondria. Cancer energy metabolism and mitochondria play a crucial role in tumor
development. Increased glycolysis is a hallmark of most cancer cells. Various factors contribute to the phenomenon of the Warburg effect seen in tumors. Oncogenic alterations
(PI3K/Akt) and HIF-1 stabilization result in increased expression of glucose transporters and glycolytic enzymes. Moreover, glycolysis aids in increasing the cellular anabolic
processes by shunting intermediates to the pentose phosphate pathway. Glutaminolysis serves as a mode of replenishing the TCA cycle intermediates that are used up in fatty acid
biosynthesis. Glutaminolysis also promotes nucleotide and protein synthesis in the cells. Glutamine uptake is controlled by c-Myc, via regulating the cell surface expression of AST2
and SN2 glutamine transporters [339]. Besides the bioenergetic changes, the apoptotic machinery of the mitochondria also serves as a remarkable target for anti-cancer therapy.
Caspases are the key players of apoptosis. Activation of caspases occurs as a consequence of cytochrome c release from the mitochondria and formation of apoptosome. The proapoptotic Bcl-2 proteins inhibit the formation of apoptosome. XIAP (X-linked inhibitor of apoptosis protein) inhibits the activation of caspases. Agents mimicking Smac, DIABLO, the
natural inhibitor of XIAP are being studied for their anticancer potential. Furthermore, oncogenic proteins (Akt) promote the association of hexokinase and VDAC (voltage dependent
anion channel) exerting an anti-apoptotic effect on cells.
molecules of ATP. Overexpression of glycolytic genes has been seen

in numerous cancer cell lines [16]. Glycolytic inhibitors are being
designed that target the enzymes involved in the glycolysis pathway.
Some glycolytic enzymes have functions in addition to their enzymatic

roles. Hexokinase, glyceraldehydes-3-phosphate dehydrogenase
(GAPDH), enolase and LDH are known to function in transcriptional
regulation. Hexokinase and GAPDH have also been implicated in
the regulation of apoptosis. Glucose-6-phosphate isomerase plays a
crucial role in cellular motility and invasion [13]. Therefore inhibiting
glycolytic enzymes can provide multifold benet in cancer therapy.
Several glycolytic inhibitors (Fig. 3) are in various stages of preclinical
and clinical studies. Table 1 provides a brief overview of glycolytic
enzymes implicated in various types of cancer, their inhibitors and the
clinical status of these inhibitors.
3.1. Hexokinase (HK)
Fig. 2. Rationale for targeting mitochondria for anticancer therapy. Various factors
affect mitochondrial function, and make it an exciting target for anticancer therapy.
Mitochondrial DNA mutations result in inhibition of oxidative phosphorylation, increased ROS, and help cancer cells to adapt to adverse conditions. Nuclear DNA mutations can give rise to malfunctional succinate dehydrogenase and fumarate hydratase
resulting in PGL (hereditary paraganglioma) and HLRCC (hereditary leiomyomatosis/
renal cell cancer). Moreover, there is an increased expression of glycolytic enzymes as a
consequence of nuclear DNA mutations. Additionally, HIF-1 promotes the expression
the glycolytic enzymes leading to Warburg effect. Oncogenic and tumor suppressor
proteins also function to cause alterations in cellular bioenergetics. Lastly, dysregulation of mitochondrial machinery of apoptosis aids in tumor progression.
Hexokinase catalyzes the rst step of glycolysis, phosphorylating

hexose (e.g. glucose) to hexose-6-phosphate. There are four tissue
specic hexokinase isozymes (IIV). Hexokinase is physically associated with the outer surface of external membrane of mitochondria
through specic binding to VDAC. Akt signaling promotes this association between VDAC and hexokinase. Mitochondrial hexokinase is
known to be upregulated in malignant tumor cells. Hexokinase is also
known to inuence PCD. It can inhibit PCD in cancer cells by affecting
the pro- and anti-apoptotic Bcl-2 family proteins. Akt signaling activates mitochondrial hexokinase, which inhibits cytochrome c release
and thus apoptosis. Hexokinase inhibition affects cellular metabolism
as well as cell survival [23,24]. Several hexokinase inhibitors have
been designed for cancer therapy.
3.1.1. 2-Deoxyglucose (2-DG)
Hexokinase phosphorylates 2-DG to 2-DG phosphate, so that it
cannot participate in the downstream glycolytic steps and in effect
1254
Table 1
Expression of metabolic enzymes in cancer and their inhibitors.a
Target
Status in cancer
Inhibitor
Clinical status of inhibitor
References
Hexokinase
Colorectal cancer, hepatocellular

carcinoma, breast cancer
2-Deoxyglucose
Clinical trials terminated
[29,30,3739,
267270]
3-Bromopyruvate
Lonidamine
Showed efcacy in animal studies

Clinical trials terminated in US.
Approved in Europe for use.
Tested in animals
Tested in mitochondrial fractions
from cancer cell lines
Experimental drugs
[41,267]
Tested in animals
[44,271]
Under development
[271273]
Antiprotozoal agents
[41,272,274]
Experimental inhibitor
Tested in animal models
Under development
[41,267,274278]
Experimental inhibitors
[41,49,272]
Effective against breast

cancer cells
[41,267,271,272]
[51,267,271]
Mannoheptulose
Methyl jasmonate
Glucose-6-phosphate
isomerase
Colorectal cancer
Phospho-fructokinase
HLRCCb uterine tumor
D-fructose-6-phosphate,
Aldolase
Triosephosphate isomerase
Glyceraldehyde-3-phosphate
dehydrogenase
Phosphoglycerate kinase
Phosphoglycerate mutase
HLRCC uterine tumor,

lung squamous carcinoma
Lung squamous carcinoma,
pancreatic cancer
Colorectal cancer, prostate
cancer, pancreatic cancer
Colorectal cancer, HLRCCb
uterine tumor, pancreatic
ductal adenocarcinoma
Lung squamous carcinoma
Enolase
Colorectal cancer, HLRCC uterine

tumor, lung squamous carcinoma
Pyruvate kinase
Colorectal cancer, HLRCCb

uterine tumor
Lactate dehydrogenase
Pyruvate dehydrogenase
kinase
HLRCCb, B cell non-Hodgkin lymphoma

Head and neck squamous cancer
Transketolase like enzyme
Uterine, cervix cancer, non small

cell lung carcinoma, breast cancer
a
b
6-phospho-gluconic acid,
N-bromoacetyl aminoethyl phosphate
3-(3-pyridinyl)-1-(4-pyridinyl)-2propen-1-one (3-PO)
3-Fluoro-D-glucose/3-deoxy-D-glucose,
4-deoxy-D-glucose/4-uoro-D-glucose
2-Carboxyethylphsophonic acid,
N-hydroxy-4-phosphono-butanamide,
2-phospho-glyceric acid
Iodoacetate
Koningic acid
1,3-bisphosphoglyceric acid analogs
Benzene hexacarboxylic acid,

3-phosphoglyceric acid
MJE3
Sodium uoride,
phosphonoacetohydoxamic acid,
2-phospho-D-glyceric acid
Fluorophosphates, pyridoxal-5'-phosphate,
creatine phosphate, L-phospholactate
CAP-232/TLN-232
Oxalate, oxamate
Dicholoroacetate
Oxythiamine
Completed Phase II clinical trials

Phase I trials for metastatic
solid tumor;
Phase II trials for brain cancer
Tested in animals
[41,267,271,279]
[149,280,281]
[131,282]
[52,283285]
Consult reference [16] for a more detailed overview on the expression of glycolytic enzymes in cancer.
HLRCC, hereditary leiomyomatosis/renal cell cancer.
competitively inhibits the enzyme. 2-DG decreases the mitochondrial

association of hexokinase [25]. Additionally, 2-DG causes abnormal
GlcNAcylation of proteins leading to accumulation of misfolded
proteins and endoplasmic reticulum stress response [26]. Studies
have shown that 2-DG enhances the anticancer activity of drugs like
adriamycin and paclitaxel [27]. 2-DG exhibits synergistic effects with

HDAC (histone deacetylase) inhibitors [28].
Phase I clinical trials have been conducted by Threshold Pharmaceuticals to evaluate the safety and efcacy of 2-DG for the treatment
of solid tumors. The aim of the trial was to calculate the MTD
Fig. 3. Potential glycolytic inhibitors for anti-cancer therapies. Several glycolytic inhibitors are being evaluated for their anticancer effects.
(maximum tolerated dose) for 2-DG, and to determine the pharmacokinetic parameters of 2-DG and 2-DG/docetaxel combination. The
trial was conducted on thirty-four patients having solid tumors that
relapsed after chemotherapy. The 2-DG/docetaxel combination
showed no pharmacokinetic interactions. Anti-tumor activity was
reported for breast cancer, and head and neck tumors. However,
further clinical trials with 2-DG have been terminated by Threshold
pharmaceuticals [29]. 5-Thioglucose and Mannoheptulose are other
examples of glucose analogs that competitively inhibit hexokinase,
and also affect the glucose uptake in cells [30,31].
3.1.2. 3-Bromopyruvate (3-BrP)
3-Bromopyruvate is a synthetic bromo-derivative of pyruvic acid
that inhibits hexokinase by alkylation of the sulfhydryl groups of
hexokinase [23]. The strong alkylating capacity of 3-BrP brings its
selectively towards hexokinase into question [32]. 3-BrP in combination with mTOR inhibitors displays synergistic effects on leukemia
and lymphoma cells [33].
3.1.3. Lonidamine (TH-070)
This derivative of indazole-3-carboxylic acid inhibits glycolysis in
relatively hypoxic conditions [34]. Lonidamine increases the intracellular content of doxorubicin due to reduced ATP availability [35].
In combination, lonidamine enhances the cytotoxicity of alkylating
agents such as cisplatin, melphalan, and BCNU (bis-chloronitrosourea) [36]. Lonidamine has been approved for use in Europe for
cancer therapy (450900 mg daily in three divided doses) [37].
However, phase III clinical trials for lonidamine have been terminated
in the U.S. Phase III trials were being carried out by Threshold
Pharmaceuticals to investigate the safety and efcacy of lonidamine in
treating benign prostatic hyperplasia [38].
3.1.4. Methyl jasmonate
Methyl jasmonate binds to mitochondrial hexokinase and detaches
it from VDAC. This causes hexokinase to dissociate from the mitochondria with a concomitant release of cytochrome c. These effects of
methyl jasmonate have been validated in mitochondrial fractions
isolated from murine melanoma B16, murine colon carcinoma CT26,
murine B cell leukemia BCL1, and human T lymphoblastic leukemia
Molt-4 cells. It has been proposed that hydrophobic jasmonates
interfere with the hydrophobic interactions between hexokinase and
VDAC leading to the disruption of the mitochondrial hexokinase-VDAC
interaction [39].
3.2. Glucose-6-phosphate isomerase (GPI)
The second enzyme of glycolytic pathway catalyzes the conversion
of glucose-6-phosphate into fructose-6-phosphate. GPI promotes
proliferation and motility of cancer cells in an autocrine manner, and
is also known as autocrine motility factor (AMF). Moreover, it plays a
role in invasiveness of tumors by inducing matrix metalloproteinase-3
(MMP-3) [40]. Chemical compounds such as D-fructose-6-phosphate,
6-phosphogluconic acid, and N-bromoacetylethanolamine phosphate
are being developed as GPI inhibitors [41].
1255
sion of these enzymes lead to a higher abundance of fructose-1,6bisphosphate which is shunted into the pentose phosphate pathway
for ribose 5 phosphate synthesis [43]. 3PO [3-(3-pyridinyl)-1-(4pyridinyl)-2-propen-1-one] is a small molecule that has shown
inhibitory activity against PFKFB3 and thus inuences the activity of
PFK1 [44].
3.4. Aldolase
Fructose-1,6-bisphosphate is converted into dihydroxy acetone
phosphate (DHAP) and glyceraldehyde-3-phosphate by an aldolase
catalyzed reaction. There are three isozymes of aldolase, AC. Small
molecules such as 3-deoxy or 3-uoro-D-glucose and 4-deoxy or 4uoro-D-glucose are being investigated for their inhibitory potential
against this enzyme [41].
3.5. Triosephosphate isomerase (TPI)
This enzyme catalyzes the reversible isomeric conversion of DHAP and
glyceraldehyde-3-phosphate. TPI inhibitors are being developed as antiprotozoal drugs. Examples of TPI inhibitors are 2-carboxyethylphosphonic acid, N-hydroxy-4-phosphono-butanamide, 2-phosphoglyceric
acid, and ornidazole [41,45].
3.6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
GAPDH converts glyceraldehydes-3-phosphate to 1,3-bisphosphoglycerate with a simultaneous reduction of NAD+ to NADH (nicotinamide adenine dinucleotide; oxidized and reduced respectively). There
are several GAPDH inhibitors currently under development.
3.6.1. Iodoacetate
Iodoacetate reacts with cysteine sulfhydryl groups in the active
site of GAPDH [46]. Being a strong alkylating agent, the selectivity of
iodoacetate towards GAPDH is debatable.
3.6.2. Koningic acid
Koningic acid is a sesquiterpene antibiotic that binds to the sulfhydryl
group within the active site of GAPDH [47].
-Chlorohydrin, ornidazole and 1-chloro-3-hydroxypropanone
have also shown anti-GAPDH activity [32].
3.7. Phosphoglycerate kinase (PGK)
PGK catalyzes the rst ATP yielding reaction in glycolysis, transferring a phosphate group from 1,3-bisphosphoglycerate onto ADP to
generate ATP and 3-phosphoglycerate. Two isozymes of PGK, PGK1 and
PGK2 have been identied. PGK has been reported to show disulde
reductase activity. It is secreted by tumor cells and it aids in angiogenic
processes. PGK targets the disulde bonds in plasmin, and initiates the
proteolytic release of angiostatin (angiogenesis inhibitor) [48]. Bisphosphonate analogs of 1,3-bisphosphoglyceric acid have been designed
and studied as PGK inhibitors [41].
3.8. Phosphoglycerate mutase (PGM)
3.3. Phosphofructokinase (PFK)

The phosphorylation of fructose-6-phosphate to fructose-1,6bisphosphate is catalyzed by PFK1. It is present in three tissue specic
isozymes (liver L, muscle M, and platelet P). The activity of PFK1 is an
important point of regulation in glycolysis. PFKFB3 (6-phosphofructo2-kinase/fructose-2,6-bisphosphatase 3) is the key enzyme regulating
the activity of PFK1. PFKFB3 regulates the synthesis/degradation of
fructose-2,6-bisphosphate, the main positive allosteric modulator of
PFK1 [42]. PFK1 and PFKFB3 expression has also been implicated in
the biosynthetic changes observed in tumor cells. The overexpres-
The internal transfer of phosphate from C-3 to C-2, to generate 2phosphoglycerate from 3-phosphoglycerate is catalyzed by PGM.
Benzene hexacarboxylic acid and 3-phosphoglyceric acid are being
developed as PGM inhibitors [41]. MJE3 is another small molecule that
is under development as a PGM inhibitor [49].
3.9. Enolase
Enolase catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenol pyruvate. There are several tissue specic
1256
isozymatic forms of this enzyme. Sodium uoride, phosphonoacetohydroxamic acid, and 2-phospho-d-glyceric acid are known to inhibit
the action of enolase [41].
3.10. Pyruvate kinase (PK)
PK catalyzes the second ATP generating step in glycolysis. PK
facilitates the reversible transfer of a phosphate group from phosphoenol pyruvate to ADP, giving ATP and pyruvate as products. There
are several isozymes of pyruvate kinase. Tumor cells express the M2PK form of this enzyme. Fluorophosphates, pyridoxal 5-phosphate,
creatine phosphate, oxalate, and L-phospholactate inhibit PK [41].
There are also phosphoenolpyruvate (PEP) analogs with modied
phosphate and carboxylate groups being designed to inhibit PK [50].
CAP-232/TLN-232 is a synthetic cyclic heptapeptide which targets PK.
A phase II clinical trial in patients with refractory metastatic renal cell
carcinoma has been successfully completed for CAP-232. Currently,
phase II trials for studying the efcacy of TLN-232 in patients with
recurring metastatic melanoma are being conducted [51].
3.11. Lactate dehydrogenase (LDH)
LDH catalyzes the conversion of pyruvate to lactate. A molecule of
NADH is oxidized to NAD+ in the course of this reaction. Inhibitors for
LDH are being developed as antiprotozoal agents. Oxalate is known to
inhibit LDH activity [32].
3.12. Transketolase like enzyme (TKTL1)
TKTL1 is a pentose phosphate pathway enzyme overexpressed in
many cancers. Overexpression increases the activity of the pentose
phosphate pathway, leading to augmented production of glyceraldehde-3-phosphate. Oxythiamine is a potent inhibitor of TKTL1 [52].
4. Role of HIF-1 in promoting the Warburg effect
HIF-1 is a transcription factor that regulates transcription of several
genes involved in cancer metabolism (Table 2). HIF-1 activates gene for
glucose metabolism, cell survival/proliferation, angiogenesis, and
metastases [53,54]. HIF-1 is a heterodimer of HIF-1 and HIF-1. The
HIF-1 subunit of the heterodimer is constitutively expressed in
cells [55]. HIF-1 can also dimerize with HIF-2 and regulate gene
activation [56,57]. Another HIF protein, HIF-3 acts as an inhibitor of
HIF-1 [58].
The expression of HIF-1 is regulated via oxygen independent and
dependent mechanisms. The protein synthesis of HIF-1 is under the
control of the PI3K and ERK-MAPK (ERK, extracellular signal regulated
kinase; MAPK, mitogen activated protein kinase) signaling pathways.
These pathways are activated via signaling of G-protein coupled
receptors, receptor tyrosine kinases, and non-receptor tyrosine
kinases [59]. Degradation of HIF-1 is mediated via oxygen dependent
mechanisms. Proline hydroxylation of Pro 402 and 564 of HIF-1 by
PHD 1-3 (prolyl hydroxylase) targets it to the pVHL tumor suppressor.
pVHL acts as recognition marker for E3 ubiquitin protein ligase and
thus leads to proteosomal degradation of HIF-1. PHDs utilize oxygen
and -ketoglutarate, and generate prolyl hydroxylated HIF-1 and
succinate. Increased levels of succinate in cells promote HIF-1
stabilization [60]. Acetylation can also regulate the levels of HIF-1.
Lysine-532 of HIF-1 is acetylated by ARD1 (arrest defective1 protein)
acetyltransferase. Acetylated HIF-1 interacts with pVHL, which causes
its ubiquitination and proteosomal degradation [61]. Furthermore
hydroxylation of asparagine-803 by FIH-1 (factor inhibiting HIF-1)
inhibits the interaction of HIF-1 with its co-activators p300 and CBP
(CREB binding protein) [62]. HIF-1 can also be regulated by gain of
function of oncogenes (PI3K etc), and loss of function of tumor suppressors (pVHL).
HIF-1 activation has been shown to play an important role in
altering the metabolism of cancer cells. It causes increased glycolysis
and decreased mitochondrial function in tumors by regulating several
genes involved in these processes (Table 2). Several anticancer agents
that affect activity or levels of HIF-1 in cells inuence HIF-1 without
directly targeting it. The agents that target the HIF-1 regulatory
systems include inhibitors of PI3K/Akt/mTOR and ERK-MAPK pathways, Hsp90 complexes (Heat shock protein90 complexes), thioredoxin-1, topoisomerase-1, and molecules that cause disruption of
microtubules. Recently interest has developed in designing inhibitors
that target the interactions of HIF-1 with DNA or its coactivators [63].
Moreover, high throughput screens are being conducted to nd small
molecule inhibitors of HIF-1. A list of these compounds is mentioned
in Table 3. These agents target HIF-1 and HIF-1 proteinprotein
interactions, the HIF-1/coactivator interaction or the DNA binding
ability of HIF-1 (Table 3).
Table 2
Role of HIF-1 in cancer energy metabolism.
Effect of HIF-1 on metabolism
HIF-1 target gene product
Role in metabolism
Ref.
Increase in glycolysis
Glucose transporters Glut1 and Glut3

Hexokinase 2
Phosphoglucose isomerase
(autocrine motility factor)
Phosphofructokinase1
Aldolase
Glucose uptake in cells

Phosphorylation of glucose
Conversion of glucose-6-phosphate to fructose-6-phosphate;
motility of cancer cells
Converts fructose-6-phosphate into fructose-1,6-bisphosphate
Catalyses conversion of fructose-1,6-bisphosphate to dihydroxy acetone
phosphate (DHAP) and glyceraldehydes-3-phosphate
Isomeric conversion of DHAP and glyceraldehydes-3-phosphate
Converts glyceraldehydes-3-phosphate into 1,3-bisphosphoglycerate
Transfers a phosphate group from 1,3-bisphosphoglycerate onto ADP and
generates ATP and 3-phosphoglucerate
Converts 3-phophoglycerate into 2-phosphoglcerate
Causes dehydration of 2-phosphoglcerate into phosphoenol pyruvate
Leads to reversible transfer of phosphate from phosphoenol pyruvate to
ADP and produces ATP and pyruvate
Reversible conversion of fructose-6-phosphate and
fructose-2,6 bisphosphate
Conversion of pyruvate to lactate
Removal of lactate from the cell
Decreases the entry of pyruvate into TCA CYCLE
Decrease mitochondrial activity
Increased oxygen consumption in hypoxia
Increased oxygen consumption in hypoxia
[286288]
[289]
[290]
Triosephosphate isomerase
Glyceraldehde-3-phosphate dehydrogenase
Phosphoglycerate kinase
Phosphoglycerate mutase
Enolase
Pyruvate kinase
Decrease rate of TCA cycle and

oxidative phosphorylation
6-phosphofructo-2-kinase/fructose-2,6bisphosphatase 1-4
Lactate dehydrogenase A
Monocarboxylate transporter4
Pyruvate dehydrogenase kinase 1
Max interactor 1
Cytochrome oxidase isoform2 (COX4/2)
LON protease
[54,288]
[54,288]
[291]
[292]
[54,288]
[54]
[54,288]
[293]
[294]
[54,288,295]
[296]
[297,298]
[299]
[300]
[300]
1257
Table 3
Table 3 HIF-1 pathway inhibitors.
Mechanism
Inhibitors
Ref.
Inhibit HIF-1 binding to DNA

Inhibit HIF-1 mediated transactivation by binding to p300
Antisense that reduces the expression of HIF-1 mRNA and protein
Prevents p300 binding to CAD of HIF-1 by promoting FIH binding to CAD
Inhibits deubiquitination of HIF-1 and increase its polyubiquitination; inhibits HIF-1 translation
Reduces HIF-1 protein synthesis
Inhibition of HIF-1 transcriptional activity
Echinomycin/NSC-13502, synthetic polyamides

Chetomin
RX0047
YC-1
PX-478
103D5R
DX-52-1/NSC-607097
NSC-609699,NSC-639174, NSC-606985
NSC-134754,NSC-643735
NSC-50352
[301,302]
[303]
[304]
[305]
[306]
[307]
[308]
Inhibit HIF-1 activity

Inhibit PAS-A mediated interaction of HIF-1 and HIF-1
5. Mitochondrial bioenergetics
5.1. The Citric Acid Cycle is the entry point for aerobic respiration
In non-transformed cells, the three-step process of aerobic
respiration takes places in the mitochondria beginning with the citric
acid cycle. TCA cycle is a series of oxidation and reduction reactions
that results in the transfer of hydride ions (2 electrons) from carbon
precursors to NAD+ and FAD (avin adenine dinucleotide, oxidized)
resulting in the formation of FADH2 (avin adenine dinucleotide,
reduced), 3 NADH, 2 molecules of carbon dioxide and the regeneration of intermediates to complete the cycle. The NADH and FADH2
produced in TCA cycle stores energy as electron motive force. These
electron carriers are shunted to the electron transport chain where
they interact with complexes I and II respectively.
The unconstrained replicative potential of transformed cells
requires the constant production of large amounts of biomaterial in
the form of nucleotides and fatty acids and sufcient energy to drive
the biosynthetic reaction. Normal metabolic processes simply cannot
meet the increased biosynthetic and energetic demands of DNA
replication and membrane formation and therefore, co-option of
metabolic intermediates is often observed in highly proliferative
cancers. Citrate formed by the condensation of acetyl-CoA and
oxaloacetate is shunted from TCA cycle into the cytoplasm where it
is used in the formation of fatty acids and NADPH (nicotinamide
adenine dinucleotide phosphate, reduced) via conversion to lactate.
To compensate for the loss of this TCA cycle intermediate, cancer cells
consume excess glutamine. Once in the mitochondria, glutamine is
converted to the TCA cycle intermediate -ketoglutarate allowing for
the regeneration of oxaloacetate and the continued production of
citrate as a precursor for downstream TCA cycle intermediates as well
as lipogenesis. Under these circumstances of metabolic re-organization, the citric acid cycle becomes a potent source of citrate for the
biosynthetic pathways relating to growth, replication and energy
production (Fig. 4) [11].
[309]
[310]
and the cytosol. The nal step of aerobic respiration utilizes the proton
gradient established by the presence of hydrogen ions in the
intermembrane space to power the phosphorylation of ADP to ATP
by complex IV (Fig. 5). Aerobic respiration is a slower process than
glycolysis, but yields higher amounts of ATP. A complete cycle of
respiration can generate ~ 30 molecules of ATP from one glucose
molecule.
5.3. Mechanisms of respiratory control
In normal cells the rate of energy production via oxidative phosphorylation is tightly controlled such that energy production and
demand are balanced. OXPHOS is coupled to the consumption of ATP
and this is the primary means of respiratory control. Excess ATP and
intermediary reaction substrates interact with the matrix exposed
portion of Complex IV and allosterically inhibit enzyme function to
slow the rate of respiration [64]. Three additional mechanisms of
control include allosteric inhibition, tissue specic expression of
isozymes and regulation by cell signaling. Allosteric controls have
been reported mainly in Complex IV. ATP binds a pocket formed by
residues of subunit I, II and IV, the matrix exposed portion of Complex
IV, to decrease ADP phosphorylation. In vitro studies have demonstrated that members of the OXPHOS machinery can be phosphorylated [64]. In some cases in vivo studies have validated these results
but the effect of these phosphorylations on function remains to be
fully understood. Multiple cell signaling pathways have been
5.2. Enzymes of the Electron Transport Chain (ETC) create proton motive
force needed to produce ATP
The electron motive force stored as NADH and FADH2 is converted
to a proton motive force by the enzymes of the electron transport chain
in the process of OXPHOS. Complexes I and II are the entry point of
electrons into the citric acid cycle. Complex I oxidizes NADH and
complex II oxidizes FADH2 to NAD+ and FAD respectively. The
electrons liberated by oxidation are transferred to ubiquinol and
carried to complex III. Complex III functions to move electrons across
the inner membrane, where they are transferred to cytochrome c.
Electrons are then carried to Complex IV where they are used to reduce
oxygen to water. Each complex harnesses the energy of electron
movement to pump hydrogen ions into the intermembrane space. The
process of electron transport also generates low levels of ROS,
primarily superoxide O
2 which can readily diffuse into the matrix
Fig. 4. The reliance of cancer cells on TCA intermediates for biosynthetic materials
makes mitochondria an attractive target for anti-cancer therapies. In order to meet the
increased biosynthetic demands of rapid proliferation, the TCA cycle intermediate,
citrate is exported from the mitochondria into the cytosol. Once in the cytosol, it is
utilized in the production of energy and lipids. Cancer cells consume excess glutamine
to compensate for the loss of citrate in TCA. Glutamine is converted to glutamate in the
mitochondria where it enters the TCA in the form of -ketoglutarate.
1258
Fig. 5. Schema depicting electron transport and oxidative phosphorylation. Electrons enter the ETC at complexes I and II in the form of NADH and FADH2 respectively. In the process,
complexes I, III and IV pump protons across the inner mitochondrial membrane. Accumulation of protons in the intermembrane space creates a charge gradient that is utilized by
Complex V (ATP synthase) to drive the synthesis of ATP from ADP and Pi. Specic inhibitors of the OXPHOS machinery are shown in bold.
implicated in the regulation of mitochondrial respiration such as the

PI3K/Akt and p38 MAPK pathways as well as Src and EGFR (epidermal
growth factor receptor) mediated signaling pathways [6568]. These
same kinases that have been implicated in control of mitochondrial
respiration are commonly upregulated in many cancers [69,70]. The
relationship between mitochondrial respiratory control and increased
expression of pro-oncogenic kinases has not been fully investigated.
5.4. Targeting OXPHOS enzymes to induce mitochondrial bioenergetic
failure
Several lines of evidence suggest that selective inhibition of tumor
cell OXPHOS may be a rational approach for the treatment of cancers
(Table 4).
The reliance of cancer cells on glycolysis for the production of
energy has been attributed to mitochondrial dysfunction and the
resultant ROS species. OXPHOS components are the major source of
ROS in cells [71]. ROS have been implicated in HIF1 stabilization,
DNA damage and disturbances in cellular redox status [7274]. The
instability of dysregulated OXPHOS components may impart a greater
sensitivity to antagonists in tumors compared to normal cells and
represents one rationale for targeting these metabolic enzymes.
Cellular respiration via glycolysis is observed in early embryonic
tissue and in stem cells which normally reside in hypoxic niches
[75,76]. De-differentiation to a stem-cell like phenotype of self
renewal and resistance to apoptosis is observed in hypoxic cancers
cells derived from breast, brain and neural origins [7780]. Hypoxic
stabilization allows HIF 1 signaling to maintain the undifferentiated
state [81]. Inhibition of respiratory complexes, presumably via
inhibition of oxygen sensing mechanisms has been shown to prevent
HIF1 stabilization. Increased degradation of HIF1 via inhibition of
respiratory chain components could contribute to the restoration of
the differentiated phenotype.
The metabolic co-option of citrate for the formation of NADPH and
fatty acid synthesis compensated by production of -ketoglutarate via
the breakdown of glutamine by mitochondrial glutaminases suggests
that this pathway is a mechanism critical to fullling the biosynthetic
needs of transformed cells. Evidence in Saccharomyces cerevisiae

demonstrated that dysfunction in components of the electron transport system results in decreased expression of components of both
the electron transport chain and the citric acid cycle [82]. Although
this effect has not been studied extensively in humans it suggests that
targeting mitochondrial components of the OXPHOS machinery could
be benecial on several levels, including the interruption of metabolic
processes required for cancer cell proliferation (Table 4).
5.4.1. Targeting Complex I, mitochondrial NADH ubiquinone reductase
Complex I, NADH ubiquinone reductase, is the largest and most
complex enzyme of the electron transport chain and the main entry
point of electrons into the electron transport chain. Seven of the
fourteen units essential for function are encoded in the mtDNA. The
remainder of the forty-six subunits are encoded in nuclear DNA
[83].
Targeted inhibition of Complex I has the potential to signicantly
curtail the ow of electrons through the transport chain to the nal
electron acceptor oxygen, resulting in bioenergetic depletion. Complex I is embedded in the inner mitochondrial membrane and in the
process of electron transfer to ubiquinol, protons are pumped into the
intermembrane space while ROS species diffuse into the matrix.
Human cell lines derived from patients carrying inherent deciencies
in Complex I activity and number produced higher levels of ROS and
exhibit differences in mitochondrial morphology compared to normal
matched cells [84]. Prolonged inhibition of complex I in human skin
broblasts results in increased ROS production accompanied by
changes in mitochondrial morphology [85]. Based on these results
and that of other studies it can be assumed that pharmacologic
inhibition of Complex I would lead to inhibition of OXPHOS, increased
ROS production and mitochondrial mediated apoptosis.
A wide array of compounds ranging from pesticides and rodent
poisons to antibiotics and antihistamines, have been shown to target
Complex I [86] but most lack acceptable therapeutic proles or reasonable specicity to deem them suitable for development as drugs.
Initial functional studies of the respiratory complexes were
facilitated by the discovery of potent and selective inhibitors. Complex
1259
Table 4
Drugs targeting components of the mitochondrial respiration machinery.
Target
Compound
Complex I
Acetogenins
Activity
Cytotoxic in mammary adenocarcinoma, ovarian and hepatocellular

carcinomas, activity not signicant in mice, toxicity observed
-lac acetogenins
No activity currently reported
Rhein
Induces apoptosis in multiple cancer cell lines.
Diphenyliodium
Induces apoptosis in prostate cancer cell lines
TEMPOL
Chemopreventive in mouse models; antiproliferative activity in mammalian
cell lines. Potentiates effect of temezolimide in human glioblastoma cell lines
MP-6, MP-24
No in vivo or cell based activity currently reported
NSC analogs
Anti-leukemic activity in cell lines
Complex II
Malonate
Derivatives under investigation as clinical imaging agents
-tocopheryl succinate Induces apoptosis in cancer cell lines inhibits tumor growth in animal models
Complex III Stigmatellin
Antibacterial agent
Benzylisothiocyanate
Found in cruciferous plants, chemopreventive in animal models,
induces ROS, cell cycle arrest and apoptosis in cancer cell lines
Myxothiozol
Antibiotic; reversibly blocks cell cycle in Jurkat cells.
Antimycin A
Antifungal, antibiotic; induces apoptosis in cancer cell lines
Complex IV A2E
Endogenously produced; induces apoptosis in retinal pigment cells
Ditercalinium
Antibacterial agent used in commercial preps, anti-tumor intercalating agent
F0F1 ATPase AA1
Prevented the growth of human bladder cancer xenografts in nude mice.
MKT-077
Completed Phase II clinical trials, withdrawn due to nephrotoxicity.
Rhodamine 123
Completed phase I clinical trials for treatment of prostate cancer,
showed reasonable efcacy, low toxicity.
Oligomycin
Shows potent cytotoxicity in NCI60 screen.
Apoptolidin
Shows potent cytotoxicity in NCI60 screen.
Resveratrol
Potent antioxidant, displays both chemopreventive and chemotherapeutic
properties in cancer cell lines and animal xenograft models
Drug-like propertiesa
Ref.
High logP, high number

of rotatable bonds
High number of rotatable bonds
Reasonable drug properties
Lacks HBD or HBA
No rotatable bonds
[311313]
Lacks HBD or HBA

[314]
[315317]
[318]
[319322]
[323]
[106,324]
[325]
[326]
[114,327329]

[330]
[331,332]
[116,333]
May have reasonable drug properties [334,335]
[336]
[123]
[124]
High logP, high HBA
High logP, high HBA
Naturally occurring antioxidant
[337]
[337]
[338]
Drug-like properties refer to adherence to Lipinski's Rule of Five, a rule of thumb measure for predicting druglikeness. HBA-hydrogen bond acceptor, HBD-hydrogen bond donor.
I function was deciphered based on the inhibitory action of rotenone,

a non-competitive binder. Rotenone itself exhibits anti-tumor activity
in animal models [86].
Complex I antagonists share a number of common properties and
thus are classied accordingly. Complex I inhibitors are often grouped
(Fig. 6) as class I or II, depending on their ability to bind in a partially
competitive or non-competitive manner, respectively. Properties such
as kinetics, ubiquinone or semiubiquinone antagonists, and the ability
to induce or repress ROS production have also been considered when
classifying complex I inhibitors [8789].
The acetogenins, such as rollinistatin and bullatacin are potent
Complex I inhibitors that have shown anti-tumor properties. The terminal and unsaturated -methylbutyrolactone imparts potency at
the target site but shows high reactivity with non-target proteins [90].
lac-acetogenins lacking this strong electrophilic moiety are selective
but act on different sites within complex I than previously described
acetogenins. The sensitivity to lac-acetogenins amongst cancer cell
lines varies considerably [91].
Rhein and diphenyliodonium act as competitive inhibitors [92].
Rhein acts to inhibit Complex I by blocking electron input. This is in
contrast to most known complex I antagonists which function at or near
the site of ubiquinone reduction [93]. Diphenyliodonium increases ROS
production resulting cell death via overproduction of superoxide ion
[94].
Complete inhibition of complex I requires binding at two regions,
the hydrophobic site and the hydrophilic site. As their names imply,
the hydrophilic is accessible to hydrophilic antagonists while hydrophobic residues protect the hydrophobic site [95]. Oddly, despite the
differences in the physical properties of both binding sites there does
not seem to be much selectivity amongst the majority of inhibitors,
with the exception of the propylpyridium derivatives, MP-6 and MP24 which target the hydrophilic site preferentially [96,97].
Self organizing map analysis of compounds from the NCI (National
Cancer Institute) public database of anti-tumor drug screening data
revealed a series of ve compounds selective for Complex I. The three
most potent compounds contained pyrimadinediamine head groups
separated by alkyl linkers 1012 carbons in length and showed antileukemic activity when tested in cell models [98].
The prodrug TEMPOL, a piperidine nitroxide, is reduced by ubiquinol

within mitochondria. The resulting hydroxylamine inhibits Complex I
and decreases glutathione levels, mitochondrial membrane potential
and oxygen consumption and induces apoptosis in cancer cells [99].
Interestingly, addition of triphenylphosphonium as a targeting moiety
increased mitochondrial accumulation at the expense of Complex I
specicity. MitoTEMPOL has higher mitochondrial membrane afnity
and thus interacts preferentially with Co-enzyme Q [100].
Caution must be taken in targeting Complex I inhibitors to the
intended site of action (i.e. tumors). Inhibition of complex I as an offtarget affect has been associated with serious drug related toxicities, as
illustrated by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)
and utatminde. MPP+(1-methyl-4-phenylpyridinium cation), the
cytotoxic metabolite of MPTP, a contaminant in narcotic street drugs
induces parkinsonian type symptoms as a result of ROS-induced
dopaminergic neuron cell death. Use of the antiandrogen utamide for
the treatment of prostate cancer, in rare instances has serious toxicity.
Inhibition of Complex I by utamide causes decreases in ATP and
glutathione depletion and oxidation leading to hepatitis.
Targeted drug delivery to cancer cells is a means by which untoward
effects could be avoided. Strategies and methods of rational drug
development using moieties such as triphenylphosphonium cations or
delocalized lipophilic cations could be employed to combat potentially
harmful off-target effects associated with systemic inhibition of
complex I by exploiting the higher mitochondrial membrane potentials
observed in tumors.
5.4.2. Succinate dehydrogenase links TCA cycle and OXPHOS
Succinate dehydrogenase (Complex II) is the only complex of the
ETC that is fully encoded in the nuclear DNA. It is not subject to
alterations imparted by mutations in mtDNA and is rarely altered in
non-germline cancers [101]. Complex II functions in both TCA cycle to
catalyze the oxidation of succinate to fumarate, and in OXPHOS by
transferring the liberated electrons stored as FADH2 into the electron
transport chain. Complex II is located on the IMM. Unlike the other
complexes of the ETC, Complex II does not pump protons across the IMM
and therefore does not contribute to mitochondrial membrane potential (Fig. 5). Post-translational modications such as phosphorylation,
1260
Fig. 6. Structures of Complex I inhibitors.
N-terminal acetylation and methionine oxidation have been observed.

The functional consequences of these modications are still uncertain
[102].
Inhibitors of complex II can be classied according to their mechanisms of action. Inhibitors such as antimycin, funiculosin and the
hydroxyquinoline-N-oxides block the QD site. Other inhibitors block
the transfer of electrons through the QP site. Examples include the
1,2 hydroxy-1-4, benzoquinone derivatives and monoamine oxidase
inhibitors.
Complex II dysfunction is correlated with pseudohypoxia, enhanced
glycolysis, and resistance to apoptosis in patients [103,104]. Inhibition of
Complex II with thenoyltriuoroacetone slows cell cycle progression
and increases ROS production and glutathione oxidation [105]. In
neuroblastoma cells inhibition of Complex II leads to increased ROS
production, p38 MAPK activation and BAX mediated apoptosis [106].
Compounds such as the benzoquinone derivatives, monoamine
oxidase inhibitors and Vitamin E analogs contain moieties that resemble
ubiquinone and function to inhibit Complex II via competitive binding to
the ubiquinone sites. Ether, ester and amide linked redox-silent Vitamin
E analogs such as -tocopheryl succinate (TOS) (Fig. 7) show potential
as anti-cancer agents. TOS has been shown to inhibit tumor cell growth
and induce apoptosis in animal xenograft models. TOS competes with

ubiquinone in binding to Q sites on Complex II [107]. Displacement of
ubiquinone from the distal and proximal Q sites allows the escape of
electrons. Reduction of oxygen by these free electrons promotes the
formation of ROS that are capable of activating BAX. Once activated BAX
dimerizes and initiates the intrinsic pathway of apoptosis. ROS production is required for apoptosis in response to TOS [108]. Treatment
of cells with the TPP conjugated antioxidant MitoQ abrogates ROS
production and prevents TOS-induced cell death [109].
5.4.3. Cytochrome c reductase
Cytochrome c reductase (Complex III) (Fig. 5) is embedded in the
mitochondrial inner membrane and functions as a dimer to facilitate
the transfer of electrons from reduced ubiquinone to cytochrome c in
a process known as the Q cycle. Complex III is a major site of ROS
production in the ETC. Under conditions of hypoxia, Complex III increases production of ROS. ROS generation by Complex III in response
to decreased oxygen levels acts to stabilize HIF1 leading to induction
of the glycolytic pathway and VEGF (vascular endothelial growth
factor) mediated angiogenic signaling [72,110]. In this way, Complex
III acts as an oxygen sensor. Complex III also acts to mediate MPT
1261
Fig. 7. Structures of Complex II and III and IV antagonists. Alpha-tocopheryl succinate is a redox-silent vitamin E analog that inhibits complex II. Benzoisothiocyanate and stigmatellin
both inhibit Complex III function. Complex IV is inhibited by ditercalinium.
(membrane permeability transition) via its role in the regulation of

Ca2+ ux [111].
Myxothiazol (Fig. 7) is a highly potent inhibitor of Complex III
belonging to a group of quinol antagonists that includes the
strobilurines and oudemansins. The methoxyacrylate group common
amongst these inhibitors resembles the structure of ubiquinone such
that their binding to the Qo site of Complex III blocks ubiquinol
oxidation [112]. The antibiotic antimycin inhibits Complex III function
by antagonizing the transfer of electrons at the Q1 center of Complex
III. Due to their structural similarities and the ability to mimic ubiquinone binding these compounds may not be selective for Complex III.
Stigmatellin (Fig. 7) inhibits Complex III in a different manner.
Stigmatellin binds Complex III preventing the transfer of electrons
from the Fe2S2 complex to cytochrome c1 in the bc1 complex of
Complex III. In this way it acts as a strong oxidant to generate reduced
Fe2S2 complex and oxygen radicals [113].
Benzylisothiocyanate (Fig. 7) is a dietary cancer preventive agent
that has been shown to inhibit complex III. In MCF7 and MDA-MB-231
cell lines, treatment with benzylisothiocyanate leads to JNK (c-Jun Nterminal kinase) and MAPK p38 mediated activation of BAX and
apoptosis. Benzylisothiocyanate showed selectivity for cancer cells as
breast epithelial cells were resistant to apoptosis in response to
treatment [114].
5.4.4. Cytochrome c oxidase is the nal electron acceptor
Electrons are transferred from cytochrome c to cytochrome c
oxidase (Complex IV) where they are used in the reduction of oxygen
to water. The protons generated in this reaction are pumped across
the IMM into the intra-membrane space to further increase the proton
motive force generated by the H+ gradient (Fig. 5). Complex IV
function is regulated endogenously by multiple mechanisms. Binding
of ATP at the matrix side of Complex IV provides allosteric control of
enzyme activity thereby regulating the rate of energy production to
match ATP demand. Nitric oxide (NO) at low concentrations reversibly inhibits the activity of Complex IV [115]. Multiple Zn2+ and Ca2+
binding sites have been identied in mammalian Complex IV. The
functional signicance of Ca2+ binding has yet to be determined. Zinc
binding leads to inhibition of proton pumping in particular although

solid evidence as to the role of zinc in Complex IV regulation remains
elusive. Complex IV is a substrate of multiple Ser/Thr and Tyr kinases
involved in cell signaling. Phosphorylation of Complex IV has been
shown to have inhibitory or activating effects depending on the type
of kinase [65]. N-retinyl-N-retinylidene ethanolamine (A2E) is an
endogenously produced lipophilic cation implicated in age related
macular degeneration. N-retinyl-N-retinylidene ethanolamine or A2E,
is highly selective, inhibiting Complex IV activity by blocking its
interaction with cytochrome c in mammalian cell cultures [116].
Ditercalinium (Fig. 7) was designed as a DNA bis-intercalating agent
but its anti-tumor properties have been ascribed to its inhibition of
Complex IV [117].
5.4.5. Targeting the F0F1 ATPase prevents ATP synthesis
The F0F1 ATPase (Complex V) is located in the mitochondrial inner
membrane. The membrane bound F0 subunit transports protons
through inner membrane and the F1 unit acts as the ATP synthase.
Complex V produces the majority of ATP utilized by the cell. It does so
using the proton gradient established by complexes I, III and IV of the
ETC to drive the synthesis of ATP from ADP and inorganic phosphate
and therefore is a signicant target for disrupting energy metabolism
(Fig. 5).
The beta-F1 subunit of ATPase is signicantly reduced in breast
and gastric adenocarcinomas, and squamous esophageal and lung
carcinomas. A decrease in ATPase activity has also been noted in
hepatocellular carcinomas [118120]. Presumably, the decreased
activity of ATP synthase in the presence of a functional ETC would
increase mitochondrial membrane potential and could partially
account for the selectivity of delocalized lipophilic cations (DLCs)
observed in carcinoma cells. Point mutations in the mtDNA encoding
subunits of the F0F1 ATPase have been shown to sensitize cancer cells
to chemotherapeutic agents such as etoposide [121]. Considering this
observation, it is possible that therapeutic agents targeting F0F1
ATPase may synergize with known chemotherapeutic agents in vivo.
Numerous compounds have been identied for their ability to
inhibit ATP synthesis (Table 4). The Rhodamine dyes, MKT-077 and
1262
Rhodamine 123 (Fig. 8), have shown the most potential thus far
having completed clinical trials for the treatment of solid tumors.
Development of the rhodamine dyes prompted the observation
that some analogues sequestered into the mitochondria. It was later
found that the esteried carboxyl and delocalized positive charge
imparted the mitochondria-selective properties seen in some rhodamine analogues. This discovery subsequently led to the identication
of a series of mitochondriotropic rhodamines (rhodamine 123) and
rhodacyanin dyes (MKT-077). It was noted that the rhodamines
exhibited prolonged retention in tumor cells compared to normal
cells.
Rhodamine DLCs and rhodacyanin dyes were found to have potent
antitumor activity in mouse xenograft models and drugs belonging to
both classes advanced to and completed Phase I clinical trials [122].
MKT-077 was tested in treatment refractory solid tumors for its ability
to inhibit ATP synthesis by selectively targeting tumor mitochondria.
Treatment with MK-077 resulted in stable disease in one patient but
the number of cycles administered was limited by recurrent
nephrotoxicity. Evidence of mitochondrial accumulation was not
seen in the early stages of treatment but after prolonged treatment
toxicities that correlated with mitochondrial dysfunction were
observed. Phase II testing was rejected on the basis of prolonged
and recurrent nephrotoxicity seen in nearly all study participants
[123].
Rhodamine 123 completed phase I clinical trials for the treatment
of prostate cancer and the results were presented in 2005. Rhodamine
123 was well tolerated at 6 infusions over a 28 day treatment schedule
reaching a MTD of 96 mg/m2. Drug related toxicities were manifest as
hypertension, vomiting and rash all of which subsided 6 h post
administration. Preliminary evaluation of Rhodamine 123 efcacy
showed increased PSA (prostate specic antigen) doubling time in
80% of patients (8/10 patients). Increased tumor drug levels versus
serum levels validates the theoretical basis for the utility of DLCs as
anticancer agents and may explain the modest levels of drug related
toxicities and increased PSA doubling time observed in patients
treated with Rhodamine 123 [124]. Currently there are no reports of
Phase II clinical trials of Rhodamine 123 in any type of cancer.
6. The other side of the coin: biosynthetic drive

Tumor cells must increase their cellular biomass prior to cell
division. The metabolic changes are necessary to provide tumor cells
with sufcient substrates for the biosynthetic machinery. Moreover,
oncogene products, tumor suppressor gene products and HIF-1
regulate the enzymatic machinery, and availability of substrates for
the biosynthesis of cellular macromolecules. These biosynthetic
precursors are formed by the metabolism of glucose and glutamine
(Fig. 1) [11].
6.1. Biosynthesis of nucleotides
The synthesis of purines and pyrimidines requires ribose-5phosphate, which is derived from the oxidative and non-oxidative
branches of the pentose phosphate pathway. Some of the glycolytic
intermediates feed the pentose phosphate pathway leading to the
synthesis of ribose-5-phosphate. Moreover the non-essential amino
acids obtained from glucose and glutamine metabolism are also
required for the synthesis of nucleotides [125].
Oncogene and tumor suppressor gene products play an important
role in diverting glycolytic metabolites into the branches of the pentose
phosphate pathway. Several enzymes of the nucleotide biosynthetic
pathway are the target of c-Myc [43]. TIGAR suppresses glycolysis by
decreasing the levels of PFK-1 and PGM. TIGAR decreases the expression
of PFK1 activator (fructose-2,6-biphosphate) and thus leads to the
accumulation of fructose-6-phosphate, which is retained for ribose-5phosphate synthesis by the pentose phosphate pathway [125]. c-Myc
and Ras are also known to activate PFK1 [43]. In p53 negative tumors,
pyruvate kinase-M2 exists as a dimer (less active form of the enzyme),
leading to accumulation of upstream glycolytic intermediates that are
subsequently used by the pentose phosphate pathway [125].
Increased expression of HIF-1 regulates the entry of glycolytic
intermediates into the pentose phosphate pathway. HIF-1 potentiates the expression of transketolase and pyruvate kinase-M2, which
promote the synthesis of ribose-5-phosphate via the pentose phosphate pathway [43].
Fig. 8. Mitochondriotropic delocalized lipophilic cations under investigation for anti-tumor properties.
1263
6.2. Biosynthesis of fatty acids
7.1. Dicholoroacetate (DCA)
The biosynthesis of fatty acids is required for the formation of

lipids which are incorporated into the membranes of cells and organelles. Furthermore, these lipids can also function to modify proteins
destined to be associated with membranes. Lipogenic enzymes are
overexpressed in tumor cells. The expression of ATP citrate lyase, a
lipogenic enzyme favors the Warburg effect by preventing the citrate
build up in the cytosol (increased citrate can suppress glycolysis).
Oncogenic mutations play an important role in fatty acid synthesis
in tumor cells. Activation of the PI3K/Akt pathway promotes the
expression of lipogenic enzymes, while suppressing the -oxidation
of fatty acids. Additionally the PI3K/Akt pathway increases the
expression of glucose transporters providing substrate for the
reactions [125].
DCA is a pyruvate analog that binds the N-terminal region of PDK.

Phase I trials for clinical and pharmacological studies of DCA are being
carried out in patients with recurrent and/or metastatic solid tumors.
Studies of the safety and efcacy of DCA for the treatment of brain
cancer are currently in phase II clinical trials [127,130,131].
6.3. Biosynthesis of proteins

Both glucose and glutamine metabolism are involved in generating
amino acids, tRNAs and ribosomes required for protein synthesis. The
ribose-5-phosphate synthesized as a result of shunting of glycolytic
intermediates to the pentose phosphate pathway is used up in the
synthesis of nucleotides. These nucleotides are the constituents of the
protein synthesis machinery of the cells (tRNA, ribosomes). The
increased glutaminolysis also adds to the cellular pool of ribose-5phosphate and contributes to protein synthesis. Moreover, metabolism of glucose and glutamine is involved in increasing cellular supply
of amino acids for synthesis of proteins [125].
6.4. Anaplerosis and NADPH production: role of glutaminolysis
in biosynthesis
The increased demand for biosynthetic precursors depletes the
intermediates of glycolysis and the TCA cycle. Citrate produced by TCA
cycle is transferred out of the mitochondria to the cytosol, where it is
used in fatty acid synthesis. There is a need for replenishment of these
metabolic intermediates. Glutamine anaplerosis plays a key role in
compensating for the depletion of these metabolites. Glutaminolysis
also serves as a source of reducing power, providing NADPH required
for nucleotide and fatty acid biosynthesis [125]. The uptake of glutamine by tumor cells is regulated by c-Myc. It also induces the expression and activity of enzymes involved in the biosynthetic processes
(Fig. 4) [43].
7. Critical switch between glycolysis and TCA cycle:
pyruvate dehydrogenase complex/pyruvate dehydrogenase
kinase (PDC/PDK)
The PDC/PDK interaction regulates the entry of pyruvate into the
TCA cycle. PDC is a multienzyme complex. It is composed of pyruvate
decarboxylase (E1 subunit), dihydrolipoyl acetyltransferase (E2 subunit), and dihydrolipoyldehydrogenase (E3 subunit). PDC catalyzes the
oxidative decarboxylation of pyruvate to give rise to acetyl-CoA. PDC
activity is controlled by PDKs (14) and PDPs (pyruvate dehydrogenase
phosphatases 1,2). Phosphorylation and inactivation of PDC by PDK
favors glycolysis [126,127]. Recent studies have shown that induction
of PDK3 by HIF-1 promotes the metabolic switch and resistance to
conventional anticancer drugs like cisplatin and paclitaxel [128]. In vitro
assays have shown decreased invasiveness of human head and neck
squamous carcinoma (UM-22B) cells upon PDK inhibition. Additionally,
a decrease in tumor volume was seen for mouse xenograft models
expressing shPDK1. Therefore, inhibition of PDK can serve as an effective
anticancer strategy [129]. In addition to its role in cancer, the PDC/PDK
interaction has also been implicated in diabetes and some types of heart
disease (Table 1) [127]. Some known inhibitors of PDK are listed below
and are shown in Fig. 9.
7.2. Radicicol
Radicicol binds to the nucleotide (ATP) binding site of PDK3. It is
also known to inhibit heat shock protein Hsp90 and TopoVI [130].
7.3. Nov3r and AZD7545
AZD7545 and other related compounds inhibit PDK2 and PDK1 but
do not affect the activity of PDK4. AZD7545 binds to the lipoyl-binding
pocket in the N-terminal domain of PDK1 and inhibits the enzyme
[127,128,130].
Oximes of diterpenes and triterpenes, analogs of ATP, lactones, and
(R)-3,3,3-Triuro-2-hydroxy-2-ethyl-propionamides also inhibit
PDK. However, these are more useful in the treatment of diabetes
[132134].
8. Mitochondrial OXPHOS uncouplers and cancer
Under normal circumstances the process of electron transfer is
tightly coupled to the production of ATP. Mitochondrial OXPHOS
uncouplers cause the uncoupling of the phosphorylation from the
oxidation in the mitochondria (Fig. 2). DNP (2,4-dinitrophenol), FCCP
(p-triuoromethoxyphenylhydrazone) and CCCP (carbonylcyanide3-chlorophenylhydrazone) are classical examples of mitochondrial
OXPHOS uncouplers [135]. Studies have revealed that OXPHOS
uncouplers can inuence tumor cell growth. CCCP, a proton ionophore
and a mitochondrial OXPHOS uncoupler showed toxicity in human
bladder carcinoma cell line (MGH-U1) and in murine mammary
sarcoma cells (EMT-6) [136]. Another study demonstrated that
rottlerin synergistically potentiated imatinib induced apoptosis in
tumors expressing BCR/ABL. These effects were independent of
rottlerin's effect on PKC (protein kinase C delta). Furthermore similar
results were obtained with FCCP and DNP, two well known mitochondrial uncouplers [137]. Additionally rottlerin and FCCP decreased
the HIF-1 transcription activity in PC-3 and DU-145 prostate cancer
cells both under hypoxic and normoxic conditions. Moreover the
uncoupler decreased the expression of HIF targeted genes [138]. Thus
using mitochondrial uncouplers may prove to be a valid strategy for
anticancer therapy. Further studies are required to establish this eld.
9. Mitochondrial DNA mutations in cancer
The circular mitochondrial genome is composed of a circular
double stranded DNA molecule ~ 16,000 base pairs in length. MtDNA
encodes 12S and 16S rRNA, 22 tRNAs and 13 subunits of the OXPHOS
machinery [139]. The remaining OXPHOS proteins as well as ~ 1500
Fig. 9. Pyruvate dehydrogenase kinase inhibitors. Dichloroacetate and radicicol are PDK
inhibitors under investigation for their anticancer potential (radicicol is a known Hsp
90 and TOPO VI inhibitor).
1264
other gene products are encoded by the nuclear genome. MtDNA lacks
histones, has limited DNA repair mechanisms and is in proximity to
high levels of ROS produced in the course of normal energy metabolism taking place in the mitochondria. These factors contribute to the
high rate of mtDNA damage and mutation [140].
Damage and mutations, regardless of their source can result in
alterations in gene products that impair mitochondrial function.
Mutations that compromise OXPHOS components may initiate cycles
of ROS production and subsequent damage to mtDNA culminating in
dysregulation of energy metabolism [141]. Mutations in the mtDNA
promoter region, aka the D-loop, or in tRNA coding regions may have
deleterious effects on gene transcription of OXPHOS components
[121]. Mutations in mtDNA have been observed in cancers of the
breast, lung, thyroid and prostate. Mutations have also been reported
in renal cell carcinoma and hepatocellular carcinoma [142].
Point mutations deletions, insertions, tandem duplications and
copy number changes are among the types of alterations that have
been identied in human cancers. Both the type and extent of mutations observed have been correlated with increased invasive and
metastatic potential, drug resistance and poorer prognosis [143].
MtDNA mutations have also been observed in normal cells in the
urine and bodily uid of cancer patients and thus have been explored
as a means of early detection of cancers. Unfortunately, in the studies
performed the percentages of patients with detectable mutations in
bodily uid was below optimal for diagnostic testing parameters
[144].
The number and uniformity of mtDNA mutations has been observed
to change in the course of cancer progression [145]. Depending on the
type, mammalian cells generally contain 103104 copies of mtDNA that
randomly distribute to daughter cells upon division. With further cell
divisions, mutated DNA may become enriched in certain cells resulting
in a phenotype of metabolic dysfunction conducive to the initiation of
carcinogenesis. During the process of cancer progression the complement of mitochondria harboring mutations within a given cell progress
toward a state of homoplasmy. Studies demonstrate that homoplasmic,
mutated mtDNA imparts metastatic potential to cancer cells regardless
of the genomic DNA complement. Mitochondria from highly metastatic
cancer cells were transferred to cells with low metastatic potential.
These cells, when grafted into immune compromised mice, produced
tumors that were highly metastatic [146].
10. TCA cycle enzymes as tumor suppressors: succinate
dehydrogenase (SDH) and fumarate hydratase (FH)
TCA cycle is a metabolic process which consists of sequential
enzymatic reactions linked to OXPHOS. TCA cycle and OXPHOS are the
main source of energy in the cell. Recent ndings have ascribed
remarkable properties to two TCA cycle enzymes, succinate dehydrogenase and fumarate hydratase. Germline mutations in the SDH gene
were found to give rise to hereditary paragangliomas and phaeochromocytomas. FH gene germline mutations were observed in hereditary
leiomyomas and renal cell cancer [147].
The tumor suppressor activity of these two enzymes is evidenced
by emergence of a pseudohypoxic environment in SDH/FH decient
cells. Decreased activity of SDH or FH leads to pseudohypoxia which
stabilizes HIF-1. The PHDs responsible for degradation of HIF-1 use
molecular oxygen and -ketoglutarate as their substrates, to generate
succinate and prolyl hydroxylated HIF-1 (degradation of hydroxylated HIF-1 is discussed in the section for HIF-1 inhibitors). Impaired
SDH/FH function leads to the accumulation of succinate/fumarate in
cells, which causes inhibition of PHDs (details in HIF-1 section) [18].
Loss of SDH function causes increased ROS production. ROS
degrade PHDs and thus stabilize HIF-1. Furthermore, it is proposed
that fumarate accumulation leads to activation of some unknown
physiological signaling pathways. These pathways may be responsible
for development of uterine leiomyoma/renal cancer cell syndromes
[148]. Recently, it has been reported that inhibition of LDH-A can be

therapeutically benecial for hereditary leiomyomas and renal cell
cancer treatment. As already stated, FH deciency leads to stabilization of HIF-1, which in turn increases the expression of HIF-1 target
genes. LDH-A is a HIF-1 targeted gene and is overexpressed in
hereditary leiomyomas and renal cell cancer. Studies have revealed
that LDH-A inhibition leads to apoptosis of A549 (surrogate FH
knockdown cell line) cells via ROS production [149].
11. Targeting mitochondrial redox status
11.1. Inhibition of mitochondrial thioredoxin reductase disrupts
mitochondrial redox balance and induces tumor cell apoptosis
The formation of ROS occurs as a by-product of oxidative
phosphorylation and increased formation of ROS is a trait often
associated with pathological states, including cancer. ROS production
results in the modication of proteins via the oxidation of thiol groups
of cysteine and methoinine residues. Disulde linkages formed by ROS
may impinge on the functionality, reactivity, interactions and
structure of the modied proteins and therefore mechanisms to
restore the redox status of the protein and the cell exist within both
the cytosol and mitochondria. One mechanism by which the internal
environment of cells is maintained in the reduced state occurs
through thiol transferase reactions carried out by the thioredoxin/
thioredoxin reductase (Trx/TrxR) system. This system, via disulde
exchange, reverses inter- and intra-protein disulde linkages that
occur in thiols of oxidized cysteine residues [150]. Noteworthy
substrates of the Trx/TrxR system include DNA replication enzymes,
transcription factors and components of the electron transport chain
[151153]. Recent experimental evidence suggests NADPH-dependent reduction via the Trx/TrxR system may function as a regulatory
mechanism thereby inuencing the rate of cell division and protein
synthesis. Upregulation in the activity of the Trx/TrxR system seen in
many tumors is thought to be a result of increased ROS production
associated with oncogenic transformation and correlates with
resistance to apoptosis and tumor cell proliferation [154,155].
Increased expression of thioredoxin has been observed in numerous
cancers, and its increase has been correlated with decreased survival
in colorectal cancer and diffuse large B cell lymphoma [154,156].
Elevated levels of Trx are observed in drug resistant cancer cell lines
and have been implicated in steroid resistance in the treatment of
certain leukemias [157,158]. Disruption of the cellular redox state via
inhibition of TrxR, either in the cytosol or the mitochondria has been
shown to increase the ratio of oxidized/reduced Trx, increase ROS,
modulate cell signaling, sensitize cancer cell lines to chemotherapeutic agents, and induce cell cycle arrest, senescence and apoptosis of
cancer cells [159,160].
Maintenance of intracellular redox state is critical to cellular
homeostasis therefore tumor selectivity in targeting at the cellular
and enzymatic levels are of great interest to the rational design of TrxR
targeted compounds. The lipophilic and cationic Au(I) diphosphines
(Fig. 8) and the Au(I) N-heterocyclic carbene compounds are goldbased compounds rationally designed to show selectivity for both
TrxR and tumor cell mitochondria compared to earlier generation
gold (I) and gold (III) based compounds that targeted both gluthione
and thioredoxin reductases and were not tumor cell specic
[161,162].
12. Cancer therapy with mitochondrial potassium
channel modulators
Mitochondrial potassium uxes are important for controlling the
proton motive force in energized mitochondria [163]. It has been
reported that the mitochondria-NFAT-Kv (NFAT, nuclear factor of
activated T cells) channel axis plays a signicant role in the electrical
remodeling of mitochondria that characterizes human cancers. The

downregulation of Kv channels contributes to apoptotic resistance in
cancer cells. Normalization of the NFAT-Kv channel by dichloroacetate
results in apoptosis [164]. Several agents are being developed that act
on mitochondrial potassium channels. The potassium channel openers such as diazoxide, and cromakalim and its derivatives affect the
mitochondrial as well as the plasma membrane KATP channels [165].
In vitro assays have revealed the antitumor potential of cromakalim
against human neuroblastoma (SK-N-MC) and human astrocytoma
(U-373 MG) cells [166]. However certain potassium channel openers
such as minoxidil have been shown to stimulate the growth of MCF-7
breast cancer cells, while potassium channel blockers like dequalinium and amioradone inhibit it [167]. Recent studies have shown that
glibenclamide, a KATP channel blocker acts as an antitumor agent for
the human gastric cell line MGC-803 [168]. These contradictory
observations make it difcult to assign a specic characteristic to
potassium channel openers/blockers with regard to their effects on
the tumor growth. Their lack of specicity for mitochondrial/plasma
membrane potassium channels further complicates the scenario.
Diazoxide is more specic for mitochondrial channels, but also has
cardio protective effects [169]. Additionally it has been reported that
pretreatment of cells with diazoxide exerts a protective effect against
rotenone induced cell death [170]. Preconditioning with pinacidil and
diazoxide exerts protective action against UV induced skin damage
[171]. Moreover levosimendan (potassium channel opener) has antiischemic effects [172]. Certain potassium channel openers (NS004
and NS1619) have been shown to inhibit the activity of mitochondria
in glioma cells. However these mitochondrial alterations do not lead
to any change in the survival of glioma cells [173]. The benzothiazine
diazoxide is reported to decrease the division of leukemic cells by
causing mitochondrial membrane depolarization [174].
In summary most of the potassium channel modulators lack
specicity for the mitochondrial channels, and exert varying effects
on the tumor tissue. This necessitates further studies for elucidating
the role of mitochondrial potassium channel modulators in cancer
therapy.
13. Targeting mitochondrial apoptotic machinery
13.1. Targeting membrane permeability transition (MPT) to
induce apoptosis
Signaling transduction cascades along the pathway of intrinsic
apoptosis descend upon the mitochondria to induce the membrane
permeability transition (MPT), the sentinel, irreversible event leading
to apoptosis. Membrane permeability transition is a sudden permeation of the mitochondrial inner membrane to solutes of greater than
1500 Da. The sudden permeation is thought to occur upon the
opening of a channel called the mitochondrial permeability transition
pore (PTP). Membrane permeability transition is characterized by a
rapid loss of m, accompanied by mitochondrial swelling and outer
membrane rupture. Upon rupturing, ATP and the apoptogenic
contents of the mitochondrial intermembrane space such as cytochrome c, Smac/DIABLO (second mitochondria derived activator of
caspases/Direct Inhibitor of Apoptosis Binding protein with a Low pI),
AIF (apoptosis inducing factor) and DNAseG are released into the
cytoplasm. Once in the cytoplasm, in the presence of ATP, cytochrome
c and AIF associate to form oligomers that recruit caspase 9. Caspase 9
in turn, auto-activates and together with cytochrome c and AIF
comprises the apoptosome to execute programmed cell death [175
177]. Thus, agents capable of modulation and induction of the MPT are
of great utility for the treatment of neoplasms.
The exact composition of the mitochondrial PTP and the mechanisms governing its response to pro-apoptotic signals are largely
unknown although is believed the Bcl-2 family member proteins are
intricately involved in the regulation of the PTP [178]. It is generally
1265
believed that the PTP spans the outer and inner membranes via
complexation of mitochondrial VDAC on the outer membrane, adenine
nucleotide transporter (ANT) on the inner membrane and cyclophilin
D [179]. Cyclophilin D is normally found in the matrix but is believed to
associate with ANT upon stimulation of MPT. The composition of the
PTP is a topic of ongoing debate. VDAC and ANT co-purify with
cyclophilin D by afnity chromatography of mitochondrial extracts
[180]. Inhibition of cyclophilin D with cyclosporine A blocks MPT [181].
The ANT ligands bongkrekic acid and atractyloside modulate MPT and
induce mitochondrial swelling [182]. In contrast to the evidence
obtained by pharmacological inhibition, cell culture based studies on
tissues derived from VDAC and ANT decient mice show that both are
dispensable for MPT [183,184]. Despite the conicting evidence
obtained in pharmacological and genetic studies, targeting antagonists
to VDAC and ANT have shown potential as anti-cancer therapeutics.
Three isoforms of VDAC have been identied thus far. In humans,
VDAC1 is ubiquitously expressed, and VDAC2 has been isolated from
heart mitochondria [185]. VDACs regulate the movement of metabolites, including ATP, to and from the mitochondria and cytosol [186].
Pro-apoptotic Bcl-2 family members BAX and BAK associate with, and
have been shown to cause opening of VDAC [187]. Association of
VDAC with Bcl-XL favors a closed conformation that suppresses MPT
[188]. In rats VDAC mRNA levels are increased in hepatomas as
compared to normal matched tissues. VDAC1 expression in human
solid tumor cell lines is increased compared to normal human broblasts. Cancer cells harboring mutations in mtDNA express higher
levels of VDAC and ANT [189]. The increased levels of VDAC seen in
cancer may be explained in part by the Crabtree effect. Under conditions of aerobic glycolysis, hexokinase II is bound to the outer mitochondrial membrane by association with VDAC. ATP produced by ATP
synthase in the inner membrane is transported through the channel
formed by VDAC and ANT. Hexokinase II, due to its proximity, co-opts
the newly formed ATP thereby sequestering it for use in the phosphorylation of glucose to glucose-6-phosphate. The Crabtree effect
favors aerobic glycolysis and prevents optimal energy metabolism, i.e.
state 3 respiration, for lack of adequate energy supply. In addition, the
association of hexokinase II with VDAC limits the amount of calcium
that enters the mitochondria further suppressing apoptosis [23].
The VDAC contribution to both the Crabtree effect and MPT suggest that it would be a signicant target for anti-cancer therapies.
Treatment of isolated mitochondria derived from head and neck
carcinomas with cisplatin showed preferential binding to VDAC
suggesting it as one potential target for cisplatin cytotoxicity [190].
Furanonapthoquinone (FNQs) (Fig. 10) derivatives have been shown
to target VDAC1. FNQs, isolated from the inner bark of tropical trees,
exhibit a 1014 fold higher toxicity in cancer cells versus normal cells.
FNQs induce apoptosis via NADH dependent ROS production at the
mitochondrial outer membrane causing collapse of the membrane
potential, cytochrome c release and caspase 9 activation [191]. Erastin
(Fig. 10) binds VDAC 2 and 3 to induce NADH dependent oxidative cell
death via the Ras-Raf-MEK pathway. Erastin shows greater activity in
cancer cells harboring mutations in HRas, KRas and BRaf [192].
In addition to its proposed role in MPT, ANT is responsible for the
exchange of ADP and ATP across the mitochondrial inner membrane
[193]. The rate of respiration is tightly linked to the ratio of ATP/ADP
within the mitochondria and modulation of ANT activity may provide
a means for controlling the rate of ATP synthesis, albeit indirectly.
Beutlinic acid, lonidamine and arsenic trioxide have been described as
ANT effectors based on their ability to induce MPT, but are not specic
for ANT [194]. The ANT2 gene has been shown to be upregulated in a
number of hormone dependent cancers. Consistent with previous
genetic studies, siRNA did not have deleterious effects on cell
functions but was able to potentiate lonidamine induced MPT [195].
The role of ANT in ATP and ADP exchange, its role in MPT and its
association with pro-apoptotic molecules such as BID and BAX suggest
it would be a good therapeutic target.
1266
domains. The pro-apoptotic proteins are either multidomain proteins (BH1BH3) or BH3-only proteins. All three classes play a
crucial role in regulation of apoptosis. Upon apoptotic stimulation, the
pro-apoptotic BAX and BAK undergo oligomerization causing mitochondrial membrane permeabilization and the release of cell death
promoting factors. The anti-apoptotic Bcl-2 proteins are known to
prevent this oligomerization. BH3-only proteins can act as sensitizers
(BAD, BIK, and NOXA) or activators (BIM, BID). Activators bind and
cause oligomerization of BAX and BAK leading to cytochrome c
release. The sensitizers act indirectly by inhibiting the interaction of
pro-survival Bcl-2 with activators or BAX/BAK [202,203].
Several strategies have been designed for targeting the Bcl-2
family of proteins. Antisense (G3139), anti-Bcl-2 antibody, anti-Bcl-2
ribozyme, BAK BH3 peptide, and SAHBs (stabilized alpha-helix of Bcl2 domains) of the BH3 domain from Bid have been developed as antiBcl2 therapies. Numerous small molecule inhibitors that bind the BH3
binding site of Bcl-2 have been designed to block the interaction of
Bcl-2 with pro-apoptotic proteins and thus promote apoptosis of the
cancer cells. ABT-263 and ABT-737 are examples of small molecule
inhibitors of the Bcl-2 proteins [203,204]. (For a detailed overview of
Bcl-2 inhibitors consult reference [205]).
Fig. 10. Structures of MPT modulators.
Cyclophilin D is a peptidyl prolyl-cis trans isomerase involved in

protein folding [196]. It normally resides in the mitochondrial matrix.
In the event of MPT, cyclophilin D translocates to the inner membrane
where it associates with ANT where it is believed to induce a conformational change that augments channel opening [175]. Unlike VDAC
and ANT, genetic ablation of cyclophilin D in mice completely prevents MPT suggesting a critical role for cyclophilin D [181,197]. The
immunosuppressive drug cyclosporine A inhibits some forms of MPT
[198]. Despite this critical role in MPT, overexpression of cyclophilin D
is observed in many tumors. Overexpression of cyclophilin D in cell
lines imparts resistance to apoptosis [199]. These seemingly opposing
roles may be explained by the recent nding that cyclophilin D interacts with Bcl-2 in a pro-survival role [200]. Further investigation of the
pro and anti-apoptotic roles of cyclophilin D in apoptosis is needed to
fully understand role of cyclophilin D in cell fate determination.
The evidence for the composition of the MPT and the relative role
each component plays in the execution of apoptosis remains unclear
and is a source of on-going debate due to the inherent limitations of
current scientic methods of investigation. The inhibition of VDAC and
ANT has been shown to modulate MPT. Regardless of whether this
modulation is direct or indirect, these promising results warrant continued investigation for the development of anti-cancer therapeutics.
13.2. Bcl-2 inhibitors
Apoptosis is the major process that maintains tissue homeostasis.
Apoptosis is carried out by an extrinsic pathway (involving death
receptors), or an intrinsic pathway which is mediated by mitochondria. Disruption in the physiological balance between cell survival and
apoptosis is observed in most cancers. Pro-apoptotic signaling is
downregulated in tumors [201]. The Bcl-2 family of proteins is a key
player in apoptosis. The Bcl-2 family consists of pro-apoptotic (BAX,
BAK) and anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w, Mcl1,Bcl2-A1) members. The communication between the anti and pro-apoptotic Bcl-2
family members governs the destiny of the cell. There is higher expression of anti-apoptotic Bcl-2 family proteins in cancer. The higher
occurrence in cancers is responsible for chemo-resistance of some
tumors.
The Bcl-2 family of proteins is classied into three categories
depending on structure and function. All contain the Bcl-2 homology
(BH) domain. The anti-apoptotic Bcl-2 proteins consist of four BH
13.3. Smac/DIABLO
Apoptotic stimuli lead to permeabilization of the outer membrane
of mitochondria with a concomitant release of pro-apoptotic factors
into the cytosol. The mitochondrial apoptosis promoting factors include cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2 [206]. The XIAP
(X-linked inhibitor of apoptosis) and cIAPs (inhibitor of apoptosis)
consist of three tandem BIR (baculovirus IAP repeat) domains and a Cterminal RING domain. The RING domain is characterized by E3
ubiquitin ligase activity. cIAP1 and cIAP2 also contain a CARD domain
(CAspase Recruitment Domain). cIAP1/2 have been found to be associated with the TNFR2/TRAF (TNFR, tumor necrosis factor receptor;
TRAF, TNF receptor associated family of proteins) signaling complex,
whereas the XIAP is implicated in causing inactivation of caspases
[207,208]. XIAP interacts with the initiator caspase 9 through its BIR3
domain, and with the effector caspases 3 and 7 through its BIR2
domain [208210]. Smac/DIABLO is an endogenous inhibitor of IAPs.
Smac/DIABLO antagonizes IAPs and positively regulates caspase
activity [211,212]. Several types of cancers have upregulated IAPs
and hence are able to evade apoptosis [213]. Changes in the expression
pattern of Smac/DIABLO have also been observed in numerous tumors.
A lower Smac/Diablo expression is reported in renal cell carcinoma
and lung cancer [214,215]. However there is de novo expression of
Smac in some forms of cervical tumor [216]. Moreover recent ndings
have shown a higher expression of Smac/DIABLO in recurrent cervical
squamous cell carcinoma [217].
Agents that mimic the IAPsSmac interaction are under development for cancer therapy. Smac mimetics antagonize IAPs and promote
apoptosis of tumor cells expressing higher levels of IAPs and lower
Smac/DIABLO levels. Furthermore combined treatment with Smac
mimetics and other chemotherapeutics is also being studied. For
instance, Smac mimetics have shown to potentiate the apoptosis
mediated by tamoxifen, paclitaxel, doxorubicin, and irradiation. They
also sensitize the TRAIL-resistant tumor cells for apoptosis [218,219].
Smac/DIABLO has also been found to play an important role in
promoting pthalocyanine-PDT (photodynamic therapy) induced
apoptosis in a BAX dependent manner [220]. Studies have reported
that besides activating caspases, Smac mimetics can also stimulate
autoubiquitination and degradation of cIAPs, activate NFB, and lead
to TNF- dependent cell death [221223].
Several Smac mimics have been designed and are being tested for
their potential in cancer therapy. For example, AT-406 is in the late
stage of preclinical studies carried out by Ascenta Therapeutics. It has
shown promising results in xenograft models of human cancer [224].
Smac mimetics have been designed based on the interaction of

Smac/DIABLO with the IAPs. The N-terminal AVPI (alanine valine
proline isoleucine) sequence of Smac is involved in the interaction
and serves as the foundation for the designing of Smac mimics [225].
These are either peptidomimetics or small molecules mimicking
Smac. Some of the inhibitors possess only one AVPI mimic domain
(monovalent), whereas others are divalent.
Earlier studies showing the Structure Activity Relationship (SAR)
of the modied peptides derived from AVPI tetrapeptide, emphasized
the feasibility of designing Smac based XIAP inhibitors [226]. Further
modications of the Smac mimetic peptides led to the improvement
in their proteolytic stability and potency. These were synthesized by
incorporating capped peptides with unnatural amino acids. Such
peptides showed cytotoxic effects in various cell lines and slowed the
growth of breast cancer in mouse xenograft models [227]. Structure
based designing and synthesis have led to development of potent
Smac peptidomimetics showing activity in cell based assays (MDA
MB231 and PC-3 cells), and enhancing the apoptosis induced by
chemotherapeutic drugs [228]. Several Smac mimetics have been
designed by developing peptide isosteres for AVPI tetrapeptide [229].
Small molecule mimics of Smac were developed with the aim of
generating better drug entities. Heterocycles were used to replace the
amino acid moieties and develop non-peptidic Smac mimetics [230].
Advances in structure based drug design led to the development of
conformationally constrained bicyclic Smac mimetics. The nonpeptidic Smac mimetics showed promising results when tested in
various cell lines [231,232]. SM-122, SM-130, SM-131 and SM-230 are
some examples of Smac mimics that inhibited cell growth in MDAMB-231 breast cancer cells [233,234].
The eld of Smac mimics advanced with the development of divalent
Smac mimetics. Divalent Smac mimics such as SM-164 showed higher
potency in binding, functional and cellular assays as compared to the
monovalent mimics. The higher potency of divalent mimics can be
attributed to the fact that they target both the BIR2 and BIR3 domains of
the XIAP. SM-164 also induces the degradation of cIAPs [235237].
14. Small molecule delivery to mitochondria
14.1. Delocalized lipophilic cations selectively target the mitochondria of
tumor cells
DLCs, due to large hydrophobic surface areas and a delocalized
positive charge are readily absorbed through plasma and mitochondrial
membranes [238]. The lipophilic nature of DLCs allows for rapid
passage through membrane bilayers while the permanent cationic
charge, driven by the negative membrane potential, causes selective
1001000 fold accrual within the negatively charged cytosol and
mitochondrial compartments. Studies comparing the uptake of charged
molecules such as the anthracyclines and rhodamines show that
parameters such as size, lipophilicity and binding properties are a factor
in compartmental accumulation, but delocalization of charge is the key
element that determines selective mitochondrial accumulation [122].
DLCs are currently in various stages of preclinical and clinical
investigation for potential utility in targeting drugs to the mitochondria
of tumor cells. DLCs accumulate in cancerous cells to a greater degree
than in normal cells due to the more negative plasma and mitochondrial
membrane potentials typically observed in tumor cells and offer a
unique means by which to deliver drugs preferentially to tumors and
spare normal healthy cells in hopes of reducing drug related toxicities.
14.2. Lipophilic triphenylphosphonium cations rapidly accumulate
within mitochondria and have been utilized to target a wide
variety of molecules into the mitochondria of tumor cells
Lipophilic triphenylphosphonium cations (TPP) are the most
widely studied and reported on mitochondrial targeting DLC. TPP
1267
compounds were rst utilized as probes for assaying mitochondrial

membrane potential as it was observed that the lipophilic nature and
cationic charge facilitated the mitochondrial membrane potentialdependent accumulation of TPP within the negatively charged mitochondrial matrix [239]. By exploiting this property, TPP compounds
were used for the visualization and evaluation of mitochondrial
attributes. For these purposes, the TPP became a targeting motif via
which relevant molecules were conjugated to the TPP with the goal of
targeting their uptake into the mitochondrial inner membrane. Later,
routine screening of synthetic intermediates revealed a signicant
anti-leukemic activity in isoindolylalkyl phosphonium salts prompting the development of SAR to determine which substituents
imparted activity. These SAR studies showed the TPP moiety to be
essential for anti-tumor activity.
These same SARs showed that variations in the length of the carbon
side chains of the isoindolylalkyphopsphonium salts modied the antimitotic properties of the TPP halide moiety. The changes in potency
were likely related to changes in hydrophobicity [240]. Mathematical
based models predicting the selective uptake of DLCs such as TPPs into
tumor cells have been experimentally validated and show the
importance of logP values in the range of 2 to 2 [241]. Below this
range, absorption of compounds are predicted to be kinetically limited
and compounds having higher logP values are predicted to absorb to
cytosolic as well as mitochondrial lipids and therefore will lack compartmental selectivity [241]. The rate of mitochondrial TPP uptake in
cells can be greatly inuenced by hydrophobicity as shown by subsequent studies performed using TPP compounds conjugated to alkyl
chains of varying length. Addition of hydrophobic moieties to TPPs
results in increased rate of uptake in cellular mitochondria compared to
more hydrophilic TPPs, due to more rapid passage through the lipid
portion of the plasma membrane bilayer [242]. Once in the cytosol, entry
into the mitochondria occurs via adsorption of the TPP containing
molecule to the outer surface of the mitochondrial inner membrane,
permeation of the hydrophobic region of the bilayer, binding to the
inner surface of the inner membrane and nally desorption into the
mitochondrial matrix [239]. Increases in TPP uptake through the plasma
membrane imparted by alkyl groups or other hydrophobic motifs are
tempered by increased adsorption to mitochondrial inner membrane.
The increased hydrophobicity may ultimately result in impaired
function of therapeutic molecules via sequestration leading to decreased
potency, as well as a reduction in drug like properties such as solubility
and bioavailability.
14.3. Lipophilic cations as tumor selective radiotracers
Due to their selective accumulation in energized mitochondria,
numerous DLCs have been investigated as potential contrast agents
for the detection, diagnosis and therapeutic management of cancers
using PET (positron emission tomography), SPECT (single photon
emission computed tomography), CT (computed tomography) and
scintigraphy imaging modalities. In addition to accumulation in the
mitochondria of tumor cells, DLCs have been demonstrated to rapidly
accumulate in other mitochondria enriched tissues such as the
heart, liver and kidneys [243247]. For diagnostic purposes, a radiotracer must be able to detect small lesions with high delity.
Accumulation in mitochondria-rich, non-cancerous tissue, therefore
presents a signicant consideration for the development of tumor
specic radiotracers.
Radiolabelled DLCs such as quaternary ammonium, arsonium and
phosphonium complexes were developed to overcome the limitations
associated with Thallium 201 based imaging for the differentiation of
ischemia and myocardial damage [248]. In the process, biodistribution
studies of structural analogs of radioiodinated iodopentenyl-trisubstituted phosphonium, arsonium and ammonium cations provided
early insight for the development of tumor specic tracers. Elemental
substitutions, i.e. phosphorus in place of arsenic, had little effect on
1268
heart uptake nor did replacement of the phenyl rings system with a
cyclohexyl ring. Ultimately tissue distribution of these radioiodinated
compounds was inuenced by lipophilicity as alkyl substitution of the
ring system produced hydrophilic compounds that favored biliary
excretion over cardiac uptake [248].
Additional lipophilic cations such as 3H-tetraphenylphosphonium
(3H-TPP) and 4-(18F-benzyl) triphenylphosphonium (18FBzTPP) are
described in the literature and have been assessed for their utility in
tumor imaging. 3H-TPP administered in mice showed early uptake
and rapid clearance from the blood as expected for a radiotracer.
The tumor uptake of 3H-TPP was not signicantly different from that
of 18F-FDG (18F-deoxygluxose), the most common clinically used
tumor imaging radiotracer. 3H-TPP did exhibit some advantages over
18
F-FDG. 3H-TPP uptake was not inuenced by blood glucose levels, as
uptake in cell lines remained unchanged in the presence of increasing
concentrations of glucose in culture media. 3H-TPP also showed
minimal uptake in brown adipose tissue and at sites of inammation
in tumor and inammation bearing mice respectively, two common
means by which 18F-FDG contributes to false positive readings. The
high accumulation in heart and kidney and lower accumulation in
brain tissue may limit the diagnostic potential of 3H-TPP to detect
small lesions in the brain and abdominal regions [243].
18
F-FBzTPP uptake was correlated with membrane potential but
intracellular accumulation was driven mainly by m in accordance
with the lipophilic, cationic properties of the TPP substituent.
Biodistribution studies in rats and dogs revealed low tumor specicity
as high uptake was noted in liver, heart, kidney and spleen. Due to
these characteristics 18F-FBzTPP would not be suitable for diagnostic
purposes but could be useful for the detection of apoptosis in tumor
masses thereby indicating the level of response to treatment, if in fact
m does decrease in response to treatment [244].
99
m-Tc-Sestamibi (99m-Tc-MIBI) and 99m-TC-tetrofosmin are FDA
(Food and Drug Administration) approved contrast agents for cardiac
PET and SPECT imaging and have since been utilized for multiple
purposes related to cancer diagnosis and treatment. The biodistribution prole of 99m-Tc-MIBI shows rapid uptake and clearance from the
blood with optimal cardiac contrast in the timeframe of 6090 min
[247]. 99m-Tc-MIBI uptake ratios in heart versus lung and heart
versus liver were calculated to be 2:1 and 1:1 respectively and the
primary target organ was found to be the thyroid [249]. Accumulation
of 99m-Tc-MIBI in abdominal organs and the thyroid limit the
diagnostic potential on the whole body scale but localized imaging
modalities have been successfully applied. Scintigraphy using 99m-TcMIBI has been shown to be as effective as traditional mammography
for the detection of small breast lesions and may been even more
sensitive than conventional methods for the detection of locoregional recurrence and residual breast cancers [250252]. Decreased
99
m-Tc-MIBI and 99m-TC-tetrofosmin retention time in tumors has
been correlated with increased expression of MDR (multidrug
resistance protein) and Pgp (p-glycoprotein) expression and are currently under clinical investigation for their ability to serve as a noninvasive marker for MDR and Pgp expression in breast malignancies to
predict response to chemotherapies [253255]. Over-expression of
Bcl-2 inhibited the uptake of 99m-TC-MIBI in breast cancer cell lines
[256]. In breast cancer patients, absence of 99m-TC-MIBI uptake correlated highly with overexpression of Bcl-2 in tumors [257]. Inhibition
of Bcl-2 to induce MPT and tumor cell apoptosis is currently the
subject of intense investigation for the development of therapeutic
agents. The development of non-invasive imaging for the detection of
Bcl-2 expression in solid tumors would certainly aid drug development and complement patient stratication in the event that Bcl-2
inhibitors demonstrate clinical application.
Copper-labeled TPP cations are currently under investigation for
their potential as tumor selective PET imaging agents. The short halflife, low -emission and coordination chemistry of copper are favorable properties that have been exploited for the development of small
molecule radiotracers. First generation triphenylphosphonium and

triphenylarsonium (TPA) copper chelates displayed higher selectivity
for tumor mitochondria than 99m-Tc-MIBI and 99m-TC-tetrofosmin in
mouse xenograft imaging studies but were still retained in liver and
heart to a high degree [245]. Further structural renements of the TPA
and TPP chelates showed that moderate lipophilicity provided the
best selectivity presumably due to slightly lower uptake kinetics
compared to molecules having more positive logP values. The increase
in hydrophilicity ensured that uptake was dictated by charge
therefore imparting greater tumor selectivity [246]. These experiments were performed in glioma cell lines expressing low levels of
PgP and MDR. Further studies will be needed to determine what
effect, if any of these modulators will play in uptake and whether
copper labeled radiotracers will show additional utility for the prediction of response to treatment.
14.4. Lipophilic triphenylphosphonium cations as mitochondrial drug
carriers
Triphenylphosphonium conjugated antioxidant compounds,
owing to their ability to sequester selectively into tumor mitochondria, have been shown to act as ROS scavengers, induce cell
differentiation, inhibit cell proliferation, and suppress tumor growth
in animal xenograft models [258260]. Triphenylphosphonium
conjugated antioxidant compounds are currently the focus of intense
evaluation for their therapeutic potential in the treatment of
numerous disease states dened by mitochondrial dysfunction such
as cancer, Parkinson's disease and hepatitis C viral infection.
15. Mitochondrial targeted anti-cancer photodynamic therapy
Photodynamic therapy (PDT) harnesses the energy produced by
excitation of a photosensitizable molecule to generate ROS via energy
transfer. In chemotherapeutic PDT, the drug, a photosensitizer (PS) is
administered systemically. A locally applied external light will excite
the PS to a higher energy state caused by the absorption of a photon
of light. Upon returning to the ground state, the PS will transfer the
energy of absorbed light to proximal oxygen molecules producing
singlet state oxygen. It is the singlet oxygen that is believed to mediate
the primary effects of PDT [261].
The mechanisms of action of PDT are either direct or indirect and
vary according to the PS used and the host response. The ROS
generated by PDT causes lethal oxidative damage leading to rapid cell
death. In addition the photosensitization may also have an antiangiogenic effect, and the resulting loss of oxygen and nutrient supply
will indirectly kill the cells. PDT can also stimulate host immune
responses that impart anti-tumor activity [262]. Photodynamic
therapy using the photosensitizing agent pormer sodium is FDA
approved for the treatment of obstructing esophageal and endobronchial non small cell lung cancer.
Many photosensitizing drugs (Fig. 11) are known to target the
mitochondria. Depending on the photosensitizer used, the mechanism of action may involve direct or indirect targeting of mitochondria [263]. Cationic photosensitizers vary in mitochondrial afnity
based on the extent of their charge and lipophilicity [264]. Further,
interactions within the organelle are molecule specic. Cationic
photosensitizing dyes directly kill tumor cells as a result of mitochondrial photodamage. Indirect killing of proximal neovascular may
also occur as a result of the accumulation of the cation in the mitochondria of proximal endothelial cells [261]. Agents such as pormer
sodium, hemotoporphyrin and the pro-drug ALA, selectively target
specic components of the mitochondria [265]. Light interrogation in
the presence of these agents causes extensive photodamage. The
effects include mitochondrial swelling, morphological perturbations
and inhibition of mitochondrial function [266]. Indirect effects may
Fig. 11. Structures of PDT agents.
occur as the result of redistribution upon photosensitization resulting

in mitochondrial depolarization.
16. Conclusion and future directions
We provide an overview of the anticancer strategies targeting
cancer cell bioenergetics or mitochondria. The use of glycolytic inhibitors is a rational approach for targeting cancer's sweet tooth. Several
glycolytic inhibitors have also shown synergy with anticancer drugs.
The discovery of methyl jasmonate has provided a new gateway in the
cancer bioenergetics targeting. Targeting of glycolysis also decreases
the abundance of metabolic intermediates which serve as precursors
for macromolecule biosynthesis.
Targeting the mitochondrial apoptotic regulatory machinery is
also a promising anticancer approach. Recently, interest has developed in exploiting mitochondrial uncouplers and potassium channel
modulators as anticancer agents. However more research is required
in this eld to establish their importance.
We suggest using a combination of these agents to target different
aspects of cancer bioenergetics and mitochondrial functions to provide
synergistic therapies for cancer. Moreover, directing these compounds
specically to their targets would reduce the off target effects and
increase the potency. The physiological characteristics of cancer cells,
such as higher glucose uptake, and specic mitochondrial features
should be exploited for designing cancer cell specic treatments.
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Advanced Drug Delivery Reviews 61 (2009) 12501275

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Opportunities in discovery and delivery of anticancer drugs targeting mitochondria

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

intermembrane space (the space between the outer and inner

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

TCA cycle (tricarboxylic acid cycle/Krebs cycle/citric acid cycle) reside

cause of the Warburg effect. Several tumor types have functional

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

molecules of ATP. Overexpression of glycolytic genes has been seen

Some glycolytic enzymes have functions in addition to their enzymatic

Hexokinase catalyzes the rst step of glycolysis, phosphorylating

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

Clinical status of inhibitor

Colorectal cancer, hepatocellular

Clinical trials terminated

Showed efcacy in animal studies

Effective against breast

HLRCCb uterine tumor

HLRCC uterine tumor,

Colorectal cancer, HLRCC uterine

Colorectal cancer, HLRCCb

HLRCCb, B cell non-Hodgkin lymphoma

Transketolase like enzyme

Uterine, cervix cancer, non small

Benzene hexacarboxylic acid,

Completed Phase II clinical trials

competitively inhibits the enzyme. 2-DG decreases the mitochondrial

adriamycin and paclitaxel [27]. 2-DG exhibits synergistic effects with

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

3.3. Phosphofructokinase (PFK)

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

HIF-1 target gene product

Glucose transporters Glut1 and Glut3

Glucose uptake in cells

Decrease rate of TCA cycle and

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

Inhibit HIF-1 binding to DNA

Echinomycin/NSC-13502, synthetic polyamides

Inhibit HIF-1 activity

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

implicated in the regulation of mitochondrial respiration such as the

needs of transformed cells. Evidence in Saccharomyces cerevisiae

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

Cytotoxic in mammary adenocarcinoma, ovarian and hepatocellular

High logP, high number

Lacks HBD or HBA

Reasonable drug properties

I function was deciphered based on the inhibitory action of rotenone,

The prodrug TEMPOL, a piperidine nitroxide, is reduced by ubiquinol

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

Fig. 6. Structures of Complex I inhibitors.

N-terminal acetylation and methionine oxidation have been observed.

and induce apoptosis in animal xenograft models. TOS competes with

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

(membrane permeability transition) via its role in the regulation of

binding leads to inhibition of proton pumping in particular although

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

6. The other side of the coin: biosynthetic drive

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 12501275

6.2. Biosynthesis of fatty acids

7.1. Dicholoroacetate (DCA)