Environmental and Molecular Mutagenesis 51:476^489 (2010)
Review Article
Targeting Mitochondria for Cancer Therapy
David M. Hockenbery*
Fred Hutchinson Cancer Research Center, Seattle, Washington
Several recent insights into the roles of mitochon-
dria in cancer have renewed efforts to develop
nongenotoxic therapies targeting mitochondrial
proteins and functions. Mitochondria are central
hubs for intrinsic apoptotic pathways that are
activated by cellular stress and injury, and as a
consequence, cancers often have defects in these
pathways. Bcl-2, the first identified regulator of
apoptotic cell deaths, was discovered as an
oncogene in human cancers. BCL-2 inhibits mito-
chondrial pathways of apoptosis through local
effects at mitochondrial and endoplasmic reticu-
lum membranes. Increased expression of BCL-2
and the related antiapoptotic proteins BCL-XL,
MCL-1, and BCL-W occurs in significant subsets
of common cancer types (Table I) and is gener-
ally correlated with poor response. Although
Key words: apoptosis; Bcl-2; BH3; mitochondria; PTP
INTRODUCTION
The mode of cell death known as apoptosis consists
of a choreographed disintegration of a cell into mem-
brane-enclosed fragments suitable for phagocytosis by
neighboring healthy cells. Cytoskeletal changes, leading
to membrane blebbing, and DNA fragmentation in com-
pacted chromatin are hallmarks of apoptosis, resulting
from activity of caspase proteases [Bellamy et al., 1995;
Nicholson, 1999]. Proximal to caspase activation are a
limited number of adaptor/receptor molecules, which are
activated in response to extracellular death receptor
ligands or intracellular markers of cell damage or infec-
tion [Barnhart et al., 2003; Fumarola and Guidotti,
2004]. However, most examples of cell stress or damage
elicit apoptotic responses via cytoplasmic translocation
of mitochondrial intermembrane space proteins, in par-
ticular, cytochrome c. Cytochrome c binds to the adaptor
protein, apoptotic protease activating factor-1 (APAF-1),
leading to oligomerization of APAF-1 in a heptameric
wheel and exposure of a caspase-9-binding caspase
recruitment domain [Shi, 2006]. Induced dimerization of
caspase-9 promotes an active-site conformation, setting
in motion a cascade of proteolytic activations of down-
stream caspases [Riedl and Salvesen, 2007].
V
C 2010 Wiley-Liss, Inc.
incomplete, the emerging understanding of BCL-2
functions through structural, biochemical, and or-
ganelle physiology studies has provided paths
for targeting BCL-2 with small molecules. Cancer
cells also exhibit metabolic differences with their
normal cell counterparts, including aerobic
glycolysis, known as the Warburg effect, mito-
chondrial membrane hyperpolarization, and
unusual dependence on nutrient substrates such
as glucose and glutamine. This knowledge has
prompted reexamination of the potential cancer
selectivity of previously identified mitochondrio-
toxic compounds, including approved drugs for
other indications, and screening programs to
identify new compounds with mitochondrial activ-
ities. Environ. Mol. Mutagen. 51:476–489,
2010. VC 2010 Wiley-Liss, Inc.
BCL-2 ANTIAPOPTOTIC PROTEINS
The BCL-2 family of proteins controls the release
of cytochrome c and other proapoptotic proteins from
mitochondria in response to a wide variety of stimuli
(e.g., cytotoxic drugs, nutrient deprivation, and cell
detachment). BCL-2 proteins are constitutively localized
at the mitochondria and endoplasmic reticulum as intrin-
sic membrane proteins or undergo translocation to these
sites following apoptotic stress. The founding member of
this family, Bcl-2, was discovered as the defining onco-
gene in human follicular B cell lymphomas, located at
one reciprocal breakpoint of the t(14;18) (q32;q21) chro-
Abbreviations: AA, antimycin A; BH3, (Bcl-2 homology 3); CLL,
chronic lymphocytic leukemia; FP, fluorescence polarization; NMR, nu-
clear magnetic resonance; PTP, permeability transition pore; SCLC,
small-cell lung cancer.
*Correspondence to: David M. Hockenbery, Fred Hutchinson Cancer
Research Center, 1100 Fairview Avenue North, D2-190, Seattle, WA
98019-1024, USA. E-mail: dhockenb@fhcrc.org
Received 18 November 2009; and in final form 7 January 2010
DOI 10.1002/em.20552
Published online 8 March 2010 in Wiley InterScience (www.interscience.
wiley.com).
 Environmental and Molecular Mutagenesis. DOI 10.1002/em
Targeting Mitochondria for Cancer Therapy 477

TABLE I. Association of Bcl-2 Antiapoptotic Family Members With Human Cancers

Bcl-2 Bcl-Xl Mcl-1 Bcl-w

Non-Hodgkin’s lymphoma Prostate adenocarcinoma Multiple myeloma Gastric cancer

Small-cell lung cancer
Acute and chronic
lymphocytic leukemia
Breast adenocarcinoma ER1
Lung adenocarcinoma Anaplastic large Colorectal
cell lymphoma adenocarcinoma
Hepatocellular carcinoma Melanoma
Breast adenocarcinoma ER2
Colorectal carcinoma

Fig. 1. (a) Ribbon diagram of BCL-XL with conserved domains. (b) Molecular surface representation of
BCL-XL and complex with BH3 peptide from Bak (magenta a-helix). The BH1 (red), BH2 (yellow), and
BH3 (blue) domains of BCL-XL line the hydrophobic groove. [Color figure can be viewed in the online
issue, which is available at www. interscience.wiley.com.]

mosomal translocation [Bakhshi et al., 1985; Cleary et al.,
1985; Tsujimoto et al., 1985]. Transgenic mice bearing a
BCL-2-Ig mini-gene, recapitulating the t(14:18) chromo-
somal translocation, develop B cell follicular hyperplasias
with progression over time to B cell lymphomas, the first
demonstration of an oncogene whose primary effect is to
maintain cell survival [Vaux et al., 1988; McDonnell and
Korsmeyer, 1991].
A second protein with BCL-2 homology, BAX, was
discovered as a coimmunoprecipitant with BCL-2 [Oltvai
et al., 1993]. Unlike BCL-2, BAX-transfected cells died
rapidly in the absence of growth factor, and BAX was
subsequently shown to be capable of directly triggering
apoptosis. Since the discovery of BCL-2 and BAX, the
mammalian BCL-2 family in mammals has expanded to
19 members, with six antiapoptotic and 13 proapoptotic
proteins. Conserved BH (BCL-2 homology) domains,
numbered BH1-4, define the BCL-2 protein family
(Fig. 1a). BH domains correspond to a-helical and con-
necting segments in the three-dimensional protein struc-
tures. All antiapoptotic members, and three proapoptotic
family members, BAX, BAK, and BOK, are ‘‘multi-
domain’’ proteins sharing sequence homology within 3-4
BH domains. The ‘‘BH3-only’’ subset of proapoptotic
molecules, including BAD, BID, BIM, NOXA, and
PUMA, show sequence homology only within a single
amphipathic a-helical segment, the BH3 domain [Wang
et al., 1996]. The BH3 domain is both necessary and suf-
ficient for the function of proapoptotic members. This
function correlates with binding of the isolated BH3 pep-
tide to a hydrophobic groove found in the three-dimen-
sional structure of the antiapoptotic BCL-2 proteins
[Muchmore et al., 1996; Sattler et al., 1997]. The interac-
tion of BH3-only proteins with BCL-2, BCL-XL, and
MCL-1 can be viewed as inhibiting their function, most
clearly by displacing proteins bound to these antiapoptotic
proteins. These bound proteins include the multidomain
proapoptotic proteins BAK and BAX as well as certain
BH3-only proteins capable of triggering oligomerization
of BAK and BAX.
Two of the multidomain proapoptotic members, BAX
and BAK, are required for apoptosis in responses to mul-
tiple types of stress and, furthermore, are necessary for
the proapoptotic function of BH3-only proteins [Wei
et al., 2001]. Monomeric BAX and BAK undergo a con-
formational change leading to dimerization and formation
of high-molecular-weight oligomers during apoptosis.
These changes occur at mitochondrial membranes and are
promoted by local availability of selected BH3-only pro-
teins (BIM, BID, and PUMA). Oligomerization of mem-
brane-bound BAX and BAK coincides with mitochondrial
outer membrane permeabilization that releases cyto-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
478 Hockenbery
chrome c to the cytoplasm [Scorrano and Korsmeyer,
2003]. Addition of individual BCL-2 family members,
including proapoptotic (BAX and BID) and antiapoptotic
(BCL-XL and BCL-2) proteins, to synthetic lipid bilayers
and vesicles demonstrated an intrinsic pore-forming
capacity [Antonsson et al., 1997; Minn et al., 1997; Schen-
del et al., 1997; Saito et al., 2000]. This is consistent with
the tertiary structures of multidomain BCL-2 family mem-
bers, which are structurally homologous to the known pore-
forming diphtheria toxin T domain and bacterial colicins
[Fesik, 2000], although other mechanisms of BAX/BAK-
dependent cytochrome c release have been proposed.
BAX and BAK oligomerization can be triggered directly
or indirectly by the BH3-only subset of proapoptotic BCL-
2 family members. BH3-only proteins are not expressed,
physically separated from mitochondrial-binding partners,
or kept in a nonbinding conformation in healthy cells. Spe-
cific cellular or environmental stress signals induce expres-
sion, posttranslational modification, or other events leading
to translocation of particular BH3-only proteins to mito-
chondria. NOXA and PUMA are transcriptionally regu-
lated by p53 in response to DNA damage [Oda et al.,
2000; Nakano et al., 2001; Yu et al., 2001]. BAD associ-
ates with 14-3-3 proteins following growth factor-depend-
ent phosphorylation, BIM binds to dynein light chain 1 at
the microtubule dynein motor complex and BMF binds
dynein light chain 2 in the myosin V-actin motor complex
[Huang and Strasser, 2000]. Cytosolic BID is activated
upon cleavage by caspase-8, leading to mitochondrial
translocation, BAX/BAK activation, and cytochrome c
release [Li et al., 1998; Luo et al., 1998].
The short BH3 domain forms an amphipathic a-helix
in binding to the hydrophobic groove found in the antia-
poptotic BCL-2, BCL-XL, MCL-1, and BCL-W structures.
Several BH3-only proteins appear to be intrinsically
unstructured (Bim, Bad, and Bmf), with localized folding
of the BH3 domain coupled to hydrophobic groove bind-
ing, whereas others (Bid) have tertiary folds highly simi-
lar to the multidomain proteins and presumably require
local unfolding to present the hydrophobic face of the
BH3 alpha helix ‘‘ligand’’ into the ‘‘receptor’’ hydropho-
bic groove [Hinds et al., 2007]. BH3 peptide assays dem-
onstrate some selectivity for binding partners among
BH3-only proteins. In particular, BH3 peptide from BAD
binds to BCL-2, BCL-XL, and BCL-W, but not MCL-1,
whereas NOXA BH3 peptide binds only to MCL-1. A
second level of specialization occurs at the level of acti-
vating proapoptotic BAX and BAK. Only BIM, BID, and
PUMA appear able to induce oligomerization of BAX
and BAK directly, whereas the other BH3-only proteins
counter the neutralizing effect of BCL-2 antiapoptotic
proteins by competitively displacing BIM, BID, or
PUMA. These studies support two classes of ‘‘activator’’
and ‘‘sensitizer’’ BH3-only proteins. BH3 domains from
activator BH3-only proteins can bind to BAX and BAK,
at a binding groove distinct from the BH3-binding site in
antiapoptotic proteins, and induce oligomerization of
BAX/BAK by an unknown mechanism [Letai et al., 2002;
Kuwana et al., 2005; Walensky et al., 2006; Gavathiotis
et al., 2008].
Current understanding of the cytotoxic effects of anti-
neoplastic agents involves the activation of endogenous
cell suicide programs (apoptosis) by one or more damage
or stress response pathways. The choice of apoptosis or
survival depends on the balance of pro- and antiapoptotic
BCL-2 proteins within the cell and, more particularly, at
mitochondrial membranes. Basal expression of Bcl-xL is a
strong negative predictor of sensitivity to diverse classes
of chemotherapeutics in the 60 cell lines of the National
Cancer Institute anticancer drug screen [Amundson et al.,
2000]. BCL-2 antiapoptotic proteins have been regarded
as ripe targets for anticancer drug development for at least
a decade [Minn et al., 1995], yet only recently have can-
didate small molecule inhibitors emerged.
TURNING KILLERS LOOSE VERSUS INTRINSIC
SURVIVAL FUNCTIONS
One of the most intriguing discoveries concerning
BCL-2 was the existence of both anti- and proapoptotic
branches of the BCL-2 protein family. Within the proa-
poptotic branch, there are three multidomain proteins
and 10 ‘‘BH3-only’’ proteins [Antsson and Martinou,
2000]. Recent models have focused on the direct lethal
activities of the proapoptotic BH proteins, with BCL-2
antiapoptotic proteins acting as restraints by sequestering
these proteins by direct physical association [Yin et al.,
1994]. In this regard, the sole antiapoptotic function of
BCL-2 proteins resides in their ability to bind to proapop-
totic proteins. However, BCL-2 antiapoptotic orthologs in
D. melanogaster and C. elegans function epistatically to
proapoptotic orthologs, consistent with a separate intrinsic
survival function [Kornbluth and White, 2005]. Although
this is a rather nebulous concept for mammalian cells at
the present time with few proposed molecular mecha-
nisms, it is notable that several of the small molecular
inhibitors of BCL-2 act as mitochondrial uncouplers,
respiratory inhibitors, and sensitizers to permeability tran-
sition [Milanesi et al., 2006]. Our understanding of the
relationships between BCL-2 and physiological mitochon-
drial functions, including membrane transport, redox equi-
librium, and network architecture, is still incomplete. The
very recent report that BCL-XL and the nematode ortho-
log CED-9 disassemble ceramide channels in defined
phospholipid membranes is a relevant example [Siskind
et al., 2008]. Improvements in knowledge of how BCL-2
functions are integrated with mitochondrial physiology
may provide additional, new routes for targeting BCL-2
with small molecules.

INSIGHTS FROM BCL-2 PROTEIN STRUCTURE AND
ISOLATED BH3 DOMAIN PEPTIDES
One approach to identify critical binding sites for drug
targeting is to first identify peptide ligands with desirable
activities, perturbing protein complex assembly, allosteric
regulation, or active-site function [Kay and Hamilton,
2001; Xu et al., 2001]. A structural element unique to the
BH pore-forming proteins is the hydrophobic groove on
the protein surface (Fig. 1b), the base, and sides created by
the BH1, 2, and 3 domains. The BH3 region of sequence
homology was first reported in the BAK proapoptotic pro-
tein as an amphipathic helical domain required both for
BAK function and physical association with BCL-XL
[Chittenden et al., 1995]. Once the BCL-XL structure was
solved, the hydrophobic groove was shown to be an inter-
face for BH3 peptide binding [Sattler et al., 1997]. Alpha-
helical peptides representing proapoptotic BH3 sequences
bind to hydrophobic grooves (Fig. 1b) in antiapoptotic BH
proteins with affinities in the low nanomolar range. BH3
peptides can displace bound ‘‘activator’’ BH3-only pro-
teins from a BCL-2 antiapoptotic protein or, in principle,
mimic the ‘‘activator’’ in promoting multidomain proapop-
totic protein oligomerization. Discovery and development
efforts for small molecular inhibitors of BCL-2 antiapop-
totic proteins have focused on BH3 peptidomimetics. All
but one of the small molecule inhibitors of the antiapop-
totic BH proteins reported to date bind to the BH3 peptide-
binding hydrophobic groove.
SPECIFIC SMALL MOLECULAR INHIBITORS
Gossypol
Gossypol is a dinaphthyl pigment derived from cotton
plants and initially linked to male infertility in cotton-pro-
ducing provinces of China [Dodou et al., 2005]. WHO-
sponsored research into gossypol as an antifertility drug in
China was terminated in 1986 because of two major side
effects, irreversible azoospermia and hypokalemia. Natural
gossypol has a chiral structure due to sterically hindered
rotation about the internaphthyl bond (Fig. 2). The (2) lev-
orotatory enantiomer is associated with higher activity in
most bioassays. In addition to antifertility effects, gossypol
has been reported to have antiviral, antimalarial, and anti-
cancer activities. Initial hypotheses for a cytotoxic mecha-
nism involved disruption of mitochondrial function.
Kitada et al. reported in 2003 that gossypol displaced
BAD BH3 peptide from BCL-2 with a Ki of 0.3 lM
[Kitada et al., 2003]. Binding affinities in the high nano-
molar to low micromolar range for BCL-XL, MCL-1, and
BCL-W have also been reported (Table II]. Chemical
shift perturbation by 2D [15N, 1H] nuclear magnetic reso-
nance (NMR) spectroscopy indicates that gossypol
binds to an extensive surface of BCL-XL surrounding the
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Targeting Mitochondria for Cancer Therapy 479
hydrophobic groove. Gossypol was also recovered in a
natural compound library screen for apoptotic activity in
bak2/2/bax2/2 cells [Lei et al., 2006]. Bax/Bak-inde-
pendent apoptosis is associated with a gossypol-dependent
conformational change in BCL-2 involving exposure of a
BH3 domain epitope, proposed to represent a proapoptotic
conformation of BCL-2. Additional activities of gossypol
are known, including inhibition of phosphoinositol kinases
[Mayr et al., 2005]. Recently, a modified gossypol com-
pound with removal of two reactive aldehyde groups
(apogossypol) was shown to retain BCL-XL-binding
activity and cytotoxicity [Becattini et al., 2004], although
minimal cytotoxicity was reported for apogossypol in
comparison to native gossypol with different cell lines
[Shelley et al., 2000].
Several Phase I human trials have been conducted with
racemic and more recently (2) gossypol. Treatment of 34
patients with advanced malignancies with 5–60 mg/day
racemic gossypol led to no objective responses in 20
assessable patients with emesis as a dose-limiting toxicity
[Stein et al., 1992], whereas trials in metastatic adrenal
carcinoma (3 PR, 1 SD in 18 patients), malignant glioma
(2 PR, 4 SD in 15 pts), and metastatic breast cancer
(1 MR, 2 SD in 20 pts) demonstrated low response rates
[Flack et al., 1993; Bushenow et al., 1999; Van Poznak
et al., 2001]. A Phase I/II trial in chemotherapy naı̈ve,
castrate-resistant prostate cancer was published this year,
with no objective responses by RECIST criteria [Liu
et al., 2009]. Dose-limiting gastrointestinal toxicity (diar-
rhea, nausea, anorexia, and small intestinal obstruction)
necessitated dose reductions.
Obatoclax (GX15-070)
The first recognized Bcl-2 small molecular inhibitor in
clinical trials was obatoclax (GX15-070, Gemin X), a
compound related to prodigiosin, a tripyrrole pigment
from Serratia marcescens. Obatoclax is reported to bind
BCL-XL, BCL-W, and MCL-1 with
0.5 lM Kd by
direct radioligand binding assay (Table II), although
because of the poor solubility of obatoclax, the reliability
of solution-phase binding assays has been questioned
[Nguyen et al., 2007]. Experimental validation of target
specificity was addressed in cytotoxicity assays with cells
dependent on BCL-2 or MCL-1 survival functions, includ-
ing yeast expressing proapoptotic Bak or Bax. Cytotoxic-
ity was not observed in bak2/2/bax2/2 cells.
Phase I studies in advanced solid tumors began in late
2004, followed by Phase Ib studies in chronic lymphocytic
leukemia (CLL) in early 2005. An intermittent dosing
scheme (intravenous q1–3 weeks) was selected for the
Phase I studies. The CLL trial compared 3.5–14 mg/m2 vs.
20–40 mg/m2 in 26 patients, with 1 partial response
[O’Brien et al., 2009]. Pharmacodynamic effects assessed
by plasma histone-oligodeoxynucleotide concentrations
Environmental and Molecular Mutagenesis. DOI 10.1002/em
480 Hockenbery

Fig. 2. Chemical structures of small molecule inhibitors of BCL-2/BCL-XL.

TABLE II. Selectivity of Small Molecule Bcl-2 Inhibitors cent subsites in the BCL-XL hydrophobic groove [Oltersdorf
Bcl-xL Bcl-2 Mcl-1 Bcl-w et al., 2005]. Site 1 and Site 2 have significant hydrophobic
character, separated by a narrowed channel between Phe 97
Gossypol 1 1 1 1
and Arg 139. Two fragments with low to submicromolar
Obatoclax 1 1 1 1
ABT-737 1 1 1 affinities were linked. The most active compound, ABT-
HA14-1 1 1 737, has Ki values for BCL-XL and BCL-2 below the lower
BH3I-1 1 detection limit of the assay (0.5 and 1 nM, respectively).
BH3I-2 1 ABT-737 binds with high affinity to BCL-2, BCL-XL,
2-Methoxy antimycin A 1 1
and BCL-W, but not to MCL-1 (Table II). Target-based ac-
Chelerythrine 1 1
tivity in cellular assays was validated using a chiral center
in ABT-737 [Oltersdorf et al., 2005; Wendt et al., 2006].
correlated to dose. A maximum tolerated dose of 28 mg/m2 The S enantiomer at this site reduces Ki for BCL-XL by
was reported, with dose-limiting toxicities of somnolence, 60- to 500-fold, with biologic activity reduced by 3- to
ataxia, and dysphoria. >100-fold. A comparison of BCL-XL and MCL-1 struc-
tures with their hydrophobic grooves occupied by BH3
ABT-737 peptides to the X-ray crystal structure of the BCL-
XL:ABT-737 complex identifies different subsite geome-
Oltersdorf et al. used ‘‘structure-activity relationship by tries in the MCL-1 binding groove that may account for the
NMR’’ screening to identify compounds that bind to adja- lack of ABT-737 binding to MCL-1 [Lee et al., 2007].

A mechanism-based assay with isolated mitochondria
was also developed, in which ABT-737 reversed BCL-2
inhibition of cytochrome c release induced by the BID
BH3-only protein. ABT-737 is inactive as a single
agent for most cancer cell lines, but exhibits synergistic
cytotoxicity with conventional chemotherapy and X-radia-
tion. However, ABT-737 is highly active against follicular
lymphoma (EC50 0.13–0.85 lM), CLL (<10 nM), and
small-cell lung cancer (SCLC) cells (<1 lM). Examina-
tion of cell line panels has demonstrated a correlation
between MCL-1 expression and resistance to ABT-737
[Konopleva et al., 2006; Van Delft et al., 2006; Chen
et al., 2007; Lin et al., 2007; Tahir et al., 2007]. Further-
more, CLL cells highly sensitive to ABT-737 exhibit con-
stitutive binding of proapoptotic BIM to mitochondrial
BCL-2, a ‘‘primed-to-die’’ state consistent with the mech-
anism of disrupting protein–protein interactions [Del
Gaizo Moore et al., 2007]. Follicular lymphomas harbor
t(14;18) translocations resulting in high levels of BCL-2
expression, whereas SCLCs exhibit chromosomal amplifi-
cations encompassing Bcl-2 and the Mcl-1-selective BH3-
only gene, NOXA, as markers of in vitro sensitivity to
ABT-737 in SCLC cell lines [Olejniczak et al., 2007].
Side effects of thrombocytopenia and lymphopenia have
been observed in mice treated with ABT-737 [Oltersdorf
et al., 2005]. ABT-737-induced thrombocytopenia is rapid
and reversible, a result of increased platelet clearance by
apoptosis and a process normally regulated by BCL-XL
[Mason et al., 2007; Zhang et al., 2007].
An orally available analog, ABT-263, entered clinical
trials for lymphoma, and more recently CLL and SCLC,
beginning in fall of 2006 [Lock et al., 2007].
HA14-1
The first published small molecule inhibitor of BCL-2,
HA14-1, was discovered in a virtual screening strategy
[Wang et al., 2000]. A total of 193,833 compounds in the
MDL Available Chemicals Directory database were tested
by simulated docking to the BCL-2 hydrophobic groove.
HA-14-1, a synthetic chromene molecule, achieved a high
docking score and, in direct binding assays, displaced
BAK-BH3 peptide binding to BCL-2 in a competitive flu-
orescence polarization (FP) assay with a Ki of 9 lM.
HA14-1 appears to act on BCL-2 and not BCL-XL
based on siRNA knockdown experiments [Manero et al.,
2006]. Milanesi et al. demonstrated that HA14-1 and two
other BCL-XL/BCL-2 inhibitors (BH3I-20 and chelerythr-
ine) produce mitochondrial uncoupling in rat liver mito-
chondria, with inhibition of respiration at higher concen-
trations [Milanesi et al., 2006]. At lower concentrations
without effects on respiratory rate, all three compounds
sensitized mitochondria to activation of the permeability
transition pore (PTP). These authors tested a series of
HA14-1 analogs and identified one candidate BCL-2
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Targeting Mitochondria for Cancer Therapy 481
inhibitor (EM20-25) without direct effects on rat liver mi-
tochondria respiration, although it retained PTP sensitiz-
ing activity.
No drug development efforts around HA14-1 are
known at this time.
2-Methoxy Antimycin A
An association of Bcl-2 expression and sensitivity to
antimycin A (AA), an inhibitor of mitochondrial electron
transport at the inner membrane ubiquinone-cytochrome c
oxido-reductase, was originally reported in fibroblasts
[Hennet et al., 1993]. Comparisons of isogenic TAMH
liver cells with graded expression of BCL-XL also demon-
strated markedly increased sensitivity to AA for cells
with the highest BCL-XL levels [Tzung et al., 2001]. A
2-methoxy derivative lacking inhibitory effects on elec-
tron transport retained selective cytotoxicity for BCL-XLhi
cells, indicating this activity was separable from respira-
tory chain inhibition.
Like BH3 peptides, the antimycins inhibit BCL-XL-
mediated pore formation in synthetic liposomes [Tzung
et al., 2001], but in contrast to BH3 peptides, do not in-
hibit membrane insertion of BCL-XL. Direct BCL-XL
binding studies based on the intrinsic fluorescence of AA
and isothermal titration calorimetry assays indicated a
stoichiometric interaction with Kd 1–2.4 lM. Competitive
binding studies with BAK BH3 peptide are consistent
with binding at the hydrophobic groove. This conclusion
is further supported by analysis of several point mutants
in the BCL-XL hydrophobic groove with reduced affinity
for AA and diminished sensitivity to AA in cell transfec-
tions [Manion et al., 2004]. Similar activities are observed
for the homologous BCL-2 protein [Kim et al., 2001]
(Table II).
The selective ‘‘gain-of-function’’ cytotoxicity of
2-methoxy AA for cells with high BCL-XL protein
expression is unusual among published BCL-2 inhibitors
[Mitsiades et al., 2007], as is the ability to inhibit mem-
brane pore formation with purified protein. A screening
strategy based on selective killing of BCL-XL-overex-
pressing cells in isogenic cell line pairs has led to the
identification of structurally distinct lead compounds with
2-MeAA-like activity [Schwartz et al., 2007]. An analysis
of bioenergetic metabolism in TAMH cells expressing
BCL-XL identified a glycolytic shift compared with con-
trol cells, with increased glucose consumption and lactate
production, reduced respiratory rates, and a reduced ratio
of mitochondrial to cytosolic NAD(P)H [Schwartz et al.,
2007] A similar phenotype has been observed in pancre-
atic islet beta cells expressing transgenic Bcl-xL [Zhou
et al., 2000], suggesting a ‘‘housekeeping’’ function of
BCL-XL may be involved in partitioning of carbon and
electron flow between cytosolic and mitochondrial metab-
olism. Intriguingly, treatment with 2-methoxy AA resulted
Environmental and Molecular Mutagenesis. DOI 10.1002/em
482 Hockenbery
in reversal of the mitochondrial NAD(P)H deficit with a
substantial overshoot and mitochondrial hyperpolarization
[Schwartz et al., 2007]. These changes were noticeable
within 20 min of adding 2-methoxy AA to BCL-XL-over-
expressing cells, but were absent in similarly treated
control cells.
BH3I Compounds
Degterev et al. screened a Chembridge library of
16,320 compounds for small molecular inhibitors of BH3
peptide binding to BCL-XL [Degterev et al., 2001] Two
of the compounds identified were closely related benzyl-
substituted thiazolidines (BH3I-1s), and the third com-
pound was a dichlorobenzyl-substituted benzamide
(BH3I-2). Direct binding of each compound to the BCL-
XL hydrophobic cleft was confirmed using 2D 1H-15N
NMR spectroscopy. Ki values for inhibition of BH3 bind-
ing ranged from 3.3 to 15.6 lM, with higher affinities in
general for a series of BH3I-2 compounds. Subsequent
reports have demonstrated mitochondrial uncoupling ac-
tivity for BH3I-20 at low concentrations, together with
sensitization to PTP opening and respiratory inhibition
[Feng et al., 2003; Hao et al., 2004; Milanesi et al.,
2006]. Although these effects were not always correlated
with apoptosis, several were inhibited by increasing BCL-
2/BCL-XL expression. A dimerized BH3I-1 compound
has enhanced activity in BH3 displacement and cyto-
chrome c release assays [Wang et al., 2008].
Chelerythrine and Sanguinarine
Chan et al. reported results of a high-throughput screen-
ing of a 107,243 compound natural product library using
a competitive FP binding assay with BCL-XL and BAK
BH3 peptide [Chan et al., 2003]. Chelerythrine, a benzo-
phenanthridine alkaloid previously identified as a protein
kinase C inhibitor, demonstrated binding activity for both
BCL-XL (Kd 10.5 lM) and BCL-2 [Zhang et al., 2006]
(Table II). This compound induces apoptosis in mamma-
lian cells at 1–2 lM, with higher doses required for cells
transfected with BCL-XL. Previous studies demonstrated
in vivo activity of chelerythrine against head and neck
squamous cell carcinomas. A related natural product, san-
guinarine, has similar activity with an improved binding
affinity for BCL-XL. Sanguinarine is a major alkaloid
component in extracts from the bloodroot plant, Sangui-
naria canadensis, and an ingredient in mouthwash and
toothpaste products [Grenby, 1995].
Zhang et al. used 1H-15N and 1H-13C heteronuclear sin-
gle quantum correlation and 1H-saturation transfer differ-
ence NMR spectroscopy, site-directed mutagenesis, and
competitive binding studies to investigate the binding site
on BCL-XL and docking orientation of chelerythine and
sanguinarine [Zhang et al., 2006]. Their results indicate
that chelerythrine binds at a site termed the BH groove,
distinct from the major hydrophobic groove, and may also
interact with a large flexible loop joining alpha helices
1 and 2, while sanguarine binds within the BH3 binding
cleft. Chelerythrine also induces apoptosis efficiently in
bax2/2bak2/2 fibroblasts, with mitochondrial swelling,
cytochrome c release, and depolarization all inhibited by
cyclosporine A, an inhibitor of mitochondrial PTP open-
ing [Wan et al., 2008].
MCL-1AS A TARGET
As mentioned earlier, the high degree of selectivity of
ABT-737 for BCL-XL and BCL-2, with negligible bind-
ing to MCL-1, suggests significant differences in the bind-
ing pocket topology. This inference is supported by the
differential binding affinities for BH3 peptides, with
BCL-2 and BCL-XL exhibiting specificity for Bad BH3
peptide, whereas MCL-1 has high affinity for Noxa-
derived peptides. The hydrophobic BH3-binding pocket in
MCL-1 is notable for a more open conformation com-
pared with BCL-2 and BCL-XL, with a wider interhelical
angle between the alpha-3 and alpha-4 helices forming
the sides of the groove [Day et al., 2005]. Progress in
understanding the structural basis for peptide ligand speci-
ficity may aid in the rational design of pharmacophores
for the development of MCL-1-selective small molecule
inhibitors.
CANCER CELL METABOLISM
In 1924, Otto Warburg, a German chemist who subse-
quently won a Nobel Prize in Medicine and Physiology
for his studies of cellular respiration, began reporting dif-
ferences in the metabolism of cancer cells and normal
cells [Warburg, 1956]. He described a reduction in cellu-
lar respiration together with a marked increase in lactate
production, such that glycolysis accounted for 50% of cel-
lular ATP production in ascites cancer cells, whereas nor-
mal liver and kidney had very low rates of lactate produc-
tion. The aerobic glycolysis of cancer cells is now known
as the Warburg effect. Warburg interpreted these studies
as the result of defective mitochondrial oxidative phos-
phorylation with compensatory upregulation of glycolysis.
In hindsight, aerobic glycolysis is a characteristic of
proliferating cells and provides a high flux of central
carbon metabolism to supply metabolic intermediates for
lipid, nucleotide, and protein synthesis [Bental and
Deutsch, 1993; Houghton et al., 1996; Vander Heiden
et al., 2009]. One difference between cancer cells and
normal cells is how bioenergetic metabolism is regulated.
Nutrient uptake and metabolism in normal cell is licensed
by the availability of growth factors, via signaling path-
ways downstream of growth factor receptors, in particular

phosphoinositide 3-kinase and Akt kinase [Schneider
et al., 1978; Vander Heiden et al., 2001]. In cancer
cells, nutrient uptake and metabolism is deregulated and
constitutively active—perhaps a universal characteristic of
cancer cells—by direct connections between metabolic
pathways and oncogenes and tumor suppressor genes
[Dang, 2009; Morrish et al., 2009; Vousden and Ryan,
2009]. This situation can lead to addiction to specific sub-
strates [Shim et al., 1998; Wise et al., 2008] or metabolic
inflexibility [Bonnet et al., 2007; Buzzai et al., 2007]. A
possibly related phenomenon in cancer cells is mitochon-
drial membrane hyperpolarization, due to reduced dissipa-
tion of membrane potential in coupled mitochondria due
to glycolytic ATP production [Davis et al., 1995; Bonnet
et al., 2007]. These features of bioenergetic metabolism in
cancer cells provide opportunities for selectively cytotoxic
drugs targeting cancer mitochondria.
SPECIFIC EXAMPLES OF SMALL MOLECULES WITH
MITOCHONDRIAL ACTIVITY
Metformin
Metformin is a biguanide oral hypoglycemic agent cur-
rently used in treatment of type 2 diabetes (Fig. 3). Origi-
nally described in 1957 [Ungar et al., 1957], metformin is
an inhibitor of mitochondrial electron transfer chain Com-
plex I [Owen MR, Doran E and Halestrap AP. 2000] and
activator of AMP-activated protein kinase [Hawley et al.,
2002] involving mitochondria-generated reactive nitrogen
species [Zou et al., 2004]. Metformin is selectively cyto-
toxic to p53-deficient tumors in mice [Buzzai et al., 2007]
and suppresses intestinal polyp growth in ApcMin/1 mice
[Tomimoto et al., 2008]. Cancer stem cells in breast can-
cer cell lines are selectively killed by metformin in vivo
and in vitro [Hirsch et al., 2009]. Epidemiologic studies
have shown reduced cancer incidence and cancer-related
mortality in patients with Type 2 diabetes treated with
metformin [Evans et al., 2005; Bowker et al., 2006; Libby
et al., 2009]. Diabetic patients on metformin who received
neoadjuvant therapy for breast cancer had higher patho-
logic complete response rates than a control group not
taking metformin (odds ratio 2.95) [Jiralerspong et al.,
2009]. A Phase I clinical trial of neoadjuvant metformin
in breast cancer is in progress (www.cancer.gov).
Arsenic Trioxide
Arsenic trioxide was approved by the FDA in 2000 for
treatment of acute promyelocytic leukemia (APL) refrac-
tory to all-trans retinoic acid. Trivalent arsenic binds to
reactive cysteines in proteins [He and Ma, 2010], and a
critical cysteine residue in the inner mitochondrial mem-
brane adenine nucleotide translocator (ANT) is suscepti-
ble to oxidation, leading to opening of the mitochondrial
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Targeting Mitochondria for Cancer Therapy 483
PTP [Costantini et al., 2000]. A similar effect on the outer
membrane voltage-dependent anion channel (VDAC) has
been proposed [Zheng et al., 2004]. The selectivity of
arsenicals for APL has been attributed to higher levels of
reactive oxygen species produced by activated NADPH
oxidases in cells expressing the PML/RARa fusion pro-
tein [Li et al., 2008].
Bisphosphonates
Bisphosphonates are analogs of naturally occurring
pyrophosphate compounds and are widely used in cancer
to prevent bone loss associated with antineoplastic thera-
pies. Some adjuvant therapy trials in breast cancer have
indicated a broader effect on nonbony metastases and
overall survival, now being tested in larger clinical trials
[Diel et al., 1998; Powles et al., 2002]. Bisphosphonates
are metabolized to nonhydrolyzable ATP analogs that in-
hibit mitochondrial ATP/ADP exchange via ANT [Lehen-
kari et al., 2002; Mönkkönen et al., 2006].
Atypical Retinoids
CD437 is a retinoid-related molecule that induces
stress-activated MAP kinases by inhibiting the phospha-
tase MKP-1 [Pfahl and Piedrafita, 2003]. Jun N-terminal
kinase phosphorylates the orphan nuclear receptor Nur77,
resulting in its translocation to the mitochondria [Holmes
et al., 2003; Han et al., 2006]. Nur77 binds to mitochon-
drial Bcl-2, inducing a conformational change that con-
verts Bcl-2 to a proapoptotic protein [Lin et al., 2004]. A
related molecule, ST1926, is in Phase I clinical trials for
advanced ovarian cancer.
Dichloroacetate
Dichloroacetate (DCA), a byproduct of water chlorina-
tion, inhibits pyruvate dehydrogenase kinase, a negative
regulator of pyruvate dehydrogenase, resulting in
increased entry of glycolytic carbons to the citric acid
cycle as acetyl-CoA [Whitehouse et al., 1974]. Clinical
studies of DCA have shown reduced lactate levels in
patients with congenital lactic acidosis and sepsis and
increased cardiac performance in patients with congestive
heart failure [Coude et al., 1978; Stacpoole et al., 1992;
Bersin et al., 1994]. Bonnet et al. reported a cancer-selec-
tive cytotoxicity, associated with a shift in metabolism
from glycolysis to oxidative phosphorylation, reduced mi-
tochondrial membrane potential, and expression of the
plasma membrane K1 channel Kv1.5 [Bonnet et al.,
2007]. A Phase I clinical trial of DCA is underway for
advanced solid tumors.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
484 Hockenbery

Fig. 3. Chemical structures of small molecules with mitochondrial activity. [Color figure can be viewed in
the online issue, which is available at www.interscience.wiley.com.]

3-Bromopyruvate Phenethyl Isothiocyanate

3-Bromopyruvate (3-BP) is an alkylating agent with mul-
tiple enzyme targets, including hexokinase, glyceraldehyde
3-phosphate dehydrogenase, and succinate dehydrogenase
[Sanborn et al., 1971; Ko et al., 2001; Pereira da Silva
et al., 2009]. Local administration of 3-BP by intraperito-
neal or direct intratumoral injection [Ko et al., 2004] or as
an intraarterial infusion [Vali et al., 2008] resulted in long-
term control of transplantable epidermoid tumors. Increased
expression and mitochondrial localization of hexokinase II
in cancers has been proposed to selectively sensitize tumors
to 3-BP [Mathupala et al., 2006].
Isothiocyanates are phytochemicals that act as electro-
philes and inhibit carcinogen-induced tumors in preclini-
cal models by inhibiting activating cytochrome P450
enzymes [Zhang et al., 1994]. Proapoptotic activity of
phenethyl isothiocyanate (PEITC) in cancer cells is
associated with rapid and preferential oxidation of mito-
chondrial peroxiredoxin 3, disrupting mitochondrial redox
homeostasis [Brown et al., 2008]. Recently, PEITC has
been reported to inhibit glutathionylation of the antiapo-
potic Bcl-2 family member MCL1, promoting its degrada-
tion [Trachootham et al., 2008].

PK11195
PK11195 is an agonist-type isoquinoline carboximide
ligand of the peripheral benzodiazepine receptor, now
known as 18 kDa translocator protein (TSPO), present on
the outer mitochondrial membrane. TSPO binds choles-
terol and porphyrins with high affinity and transfers these
ligands from the outer to the inner mitochondrial mem-
brane for steroid and heme synthesis, respectively [Take-
tani et al., 1994; Rone et al., 2009]. TSPO ligands induce
cholesterol transport into mitochondria [Lacapere and
Papadopoulos, 2003]. TSPO is also associated with
VDAC and ANT [McEnery et al., 1992], and TSPO
ligands regulate the mitochondrial PTP [Park et al.,
2005].
F16
F16 is an example of a delocalized lipophilic cationic
dye, which like rhodamine 123, and the rhodacyanine
MKT077, selectively accumulates in cancer cell mito-
chondria with high mitochondrial membrane potentials
[Fantin et al., 2002; Modica-Napolitano and Aprille,
2001]. Eph4 immortalized mammalian epithelial cells
transformed with v-Ha-Ras or neu exhibited increased mi-
tochondrial membrane potential and underwent selective
mitochondrial damage and apoptosis with F16 treatment
[Fantin et al., 2002]. F16 acts as a weak protonophore,
with the resultant mitochondrial depolarization opening
the PTP.
Ditercalinium
Ditercalinium is a bifunctional symmetrical intercalat-
ing agent that causes selective loss of mitochondrial
DNA, while its monomeric analog produces nuclear DNA
strand breaks [Segal-Bendirdjian et al., 1988]. Antitumor
activity against leukemia, mammary carcinoma, and
SCLC has been reported [Pelaprat et al., 1980; Benard
et al., 1988; Le Moyec et al., 1988].
CONCLUSIONS
Mitochondria are increasingly recognized as important
targets in cancer because of their central roles in apoptotic
pathways and cellular metabolism. Disabled apoptotic
programs and altered central bioenergetic metabolism in
cancer cells present opportunities for selective targeting
of cancer cells with mitochondrially directed small mole-
cules. Antiapoptotic BCL-2 proteins have generated great
interest as drug targets for anticancer therapies. A path to
the development of small molecule inhibitors emerged
from structure–function studies of BCL-XL and the BH3
domain critical for the function of proapoptotic binding
partners. Several strategies appear to be feasible for dis-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Targeting Mitochondria for Cancer Therapy 485
rupting mitochondrial homeostasis in cancer cells, includ-
ing reestablishing oxidative metabolism in cells adapted
to the ‘‘Warburg effect’’ and using the increased mito-
chondrial membrane potential for selective accumulation
of cationic drugs in cancer cells. As many small mole-
cules with mitochondrial activity were discovered more
than a decade ago, newer targeted screening strategies
may uncover many additional compounds with desirable
anticancer activities.
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Accepted by—
P.J. Brooks