Overcoming Drug Resistance by Targeting Cancer Bioenergetics with an Activatable Prodrug

Article abstract of DOI:10.1016/j.chempr.2018.08.002

Nearly without exception, all known cancer chemotherapeutics elicit a resistance response over time. The resulting resistance is correlated with poor clinical outcomes. Here, we report an approach to overcoming resistance through reprogramming oncogene-di

Full text of DOI:10.1016/j.chempr.2018.08.002

Article
Overcoming Drug Resistance by Targeting
Cancer Bioenergetics with an Activatable
Prodrug
Amit Sharma, Min-Goo Lee, Hu
Shi, ..., Jin Yong Lee, Sung-Gil
Chi, Jong Seung Kim
sessler@utexas.edu (J.L.S.)
jinylee@skku.edu (J.Y.L.)
chi6302@korea.ac.kr (S.-G.C.)
jongskim@korea.ac.kr (J.S.K.)
HIGHLIGHTS
A conjugate that targets cancer
bioenergetics through delayed
prodrug activation
Restoration of mitochondrial
oxidative phosphorylation in
cancer cells
Active doxorubicin translocation
from the mitochondria to the
nucleus
Excellent tumor growth inhibition
in both drug-sensitive and
-resistant tumor models
Sharma et al. report a cancer-selective multicomponent strategy to overcome drug
resistancel. The current strategy centers around a conjugate that triggers a
reprogramming of the mitochondrial metabolism. This results in delayed prodrug
activation and allows drug efflux mechanisms to be evaded. Compared with
various controls, the lead conjugate exhibits cancer-selective activation, restores
mitochondrial oxidative phosphorylation, and promotes active doxorubicin (Dox)
translocation from mitochondria to the nucleus. It is also significantly more
effective at inducing cancer cell apoptosis and tumor growth inhibition in both
Dox-sensitive and -resistant tumor models.
Sharma et al., Chem 4, 1–14
October 11, 2018 ª 2018 Elsevier Inc.
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Article
Overcoming Drug Resistance
by Targeting Cancer Bioenergetics
with an Activatable Prodrug
Amit Sharma,1,6 Min-Goo Lee,2,6 Hu Shi,3,4,6 Miae Won,1 Jonathan F. Arambula,5
3,
2,
1,
Jonathan L. Sessler,5,7, Jin Yong Lee, Sung-Gil Chi, and Jong Seung Kim
*
*
*
*
SUMMARY
The Bigger Picture
Drug resistance is a particularly
pernicious feature of modern
cancer chemotherapy. In many
instances, drug resistance is
correlated with aberrant
Nearly without exception, all known cancer chemotherapeutics elicit a resis-
tance response over time. The resulting resistance is correlated with poor clin-
ical outcomes. Here, we report an approach to overcoming resistance through
reprogramming oncogene-directed alterations in mitochondrial metabolism
before drug activation while simultaneously circumventing drug efflux pumps.
Conjugate C1 increases cancer cell apoptosis and inhibits regrowth of drug-
resistant tumors, as inferred from efficacy studies carried out in human cancer
cells and in Dox-resistant xenograft tumor models. It also displays minimal
whole-animal toxicity. These benefits are ascribed to an ability to evade chemo-
resistance by switching cancer cell metabolism back to normal mitochondrial
oxidative phosphorylation while helping target the active Dox to first the mito-
chondrion and then the nucleus.
metabolic adaptations that favor
aerobic glycolysis over oxidative
phosphorylation. To date, efforts
have been made to overcome
resistance by targeting the
metabolic changes associated
with resistance, albeit with limited
effect. The hypothesis underlying
the present work is that a
INTRODUCTION
multipronged strategy might be
more effective. As detailed in this
study, we have found that a
rationally designed conjugate that
both restores oxidative
Cancer chemotherapeutics play a critical role in the management of malignant dis-
ease. Unfortunately, drug resistance often presents a barrier to successful clinical
outcomes. Resistance is associated with lethal side effects and increased recurrence
rates.1 Initially, many tumors are responsive to chemotherapy; however, over time,
they become resistant owing to the rapid proliferation of small subpopulations of
resistant cells that survive during the initial treatment periods.2 These problems
are exacerbated when acquired resistance from one drug is transferred to other anti-
cancer treatment regimens despite mechanistic and structural differences in the
agents being used. This latter phenomenon is generally referred to as multidrug
resistance (MDR).3,4 Although other factors can contribute, it is estimated that innate
or induced drug resistance poses a serious risk in approximately 90% of metastatic
cancer patients and is correlated with most treatment failures.5 Typically, an increase
in drug efflux mediated by ATP-binding cassette (ABC) transmembrane proteins,
such as ABCG2 and MDR1 (p-glycoprotein), is the primary reason for chemoresist-
ance seen in a variety of cancers.6 This is true even though these proteins play pos-
itive roles in normal physiology.7 Despite continuous efforts to develop effective
modulators of drug transport proteins, fully satisfactory clinical benefits have yet
to be realized.8,9 As detailed below, we believe that progress can be made by ad-
dressing the aberrant metabolic adaptations typically associated with solid tumors.
phosphorylation and permits the
targeted delivery of doxorubicin
(a known anticancer agent) to the
nucleus overcomes resistance, as
judged from both in vitro and
in vivo studies. The present work
highlights the benefits of using a
multicomponent small-molecule
approach to overcome the
complexities associated with drug
resistance. It may represent the
vanguard of a new paradigm in
anticancer drug discovery.
During the early stage of carcinogenesis, aerobic glycolysis (the Warburg effect) usu-
ally dominates over oxidative phosphorylation (OXPHOS).10,11 This is ascribed to the
inherent need to meet the urgent energy demands and metabolic requirements
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associated with uncontrolled tumor growth.12,13 In mammalian cells, pyruvate serves
as an intermediary in both processes. Its fate is regulated by pyruvate dehydroge-
nase (PDH), a mitochondrial gatekeeper enzyme. PDH activity itself is modulated
by pyruvate dehydrogenase kinase (PDK). A direct correlation between PDH and
PDK in promoting metabolic alterations associated with chemoresistance has
been established.14–17 In addition, the glycolytic byproducts associated with aerobic
metabolism provide advantages to rapid cellular proliferation, including reduced
apoptosis, enhanced cell mobility, increased efflux rates, and augmented metastatic
potential, although a few positive outcomes in improving chemosensitivity have
been noted.18,19 Given these correlations, the ability to alter aberrant metabolic ad-
aptations in tumors and restore OXPHOS has appeal in the context of cancer ther-
apy. However, to date only a few reports involving efforts to effect MDR reversal
through tumor metabolic regulation have appeared.20–22 Presumably, this reflects
the poor pharmacokinetics and dose-limiting toxicity of the metabolic blockers
and regulators tested to date. We think a more targeted strategy could prove
beneficial.
So-called targeted drugs, wherein a putative chemotherapeutic is localized prefer-
entially to a tumor site, hold promise in terms of improving the survival rates and
quality of life for cancer patients. In addition, targeted therapeutics may be able
to bypass some of the known MDR mechanisms, thereby preventing chemoresist-
ance.23,24 To date, a number of localizing agents, including cancer-specific receptor
inhibitors, peptides, polymers, ionic liquids, dendrimers, metal organic frameworks,
and nanoparticles, have been explored in the context of cancer drug develop-
ment.25–27 Similar approaches have also been successfully utilized in the diagnosis
and treatment of pathogenic bacterial infections,28 neurologic disorders,29 inflam-
mation, and immune system diseases.30,31 However, to our knowledge a targeting
strategy that also overcomes unfavorable alternations in metabolism has yet to be
pursued in the context of cancer drug discovery. Such an approach has particular ap-
peal in terms of overcoming the effects of MDR.
In this study, we report a small-molecule targeted drug conjugate, compound C1,
that circumvents drug resistance in MDR cells even at low doses. We designed
this agent and various control compounds (e.g., C2–C6) to test whether metabolic
reprogramming, followed by drug activation, could be achieved in the context of
a single multifunctional chemical entity. Compound C1 contains a doxorubicin
(Dox) moiety reversibly connected to a dichloroacetic acid (DCA) subunit, as well
as a triphenylphosphonium (TPP) mitochondrial targeting group. The control com-
pounds contain some, but not all, of these presumably key subunits (Figure 1).
1Department of Chemistry, Korea University,
Seoul 02841 Korea
2Department of Life Sciences, Korea University,
Seoul 02841, Korea
Dox is an anthracycline antibiotic that is the standard of care for many cancer pa-
tients. However, it suffers from drug efflux mediated by various drug transporter pro-
teins and in many cases fails to produce curative clinical outcomes.32 DCA is a PDK
inhibitor33 that, when incorporated into a larger structure in the form of, e.g., an
amide derivative, offers the possibility of sustained drug release.33 In the context
of conjugate C1, the DCA moiety is attached in the form of an aniline amide that
was expected to be susceptible to enzymatic-induced release. The associated
bond cleavage was expected to fulfill a dual role. First, it would provide a metabolic
modulator (DCA) that would weaken drug resistance mechanisms. Second, on the
basis of literature precedent,34 it would induce a cascade of reactions to release
free Dox that would be finally translocated from mitochondria to the nucleus to
evade the various Dox-refractory mechanisms in the initial stages of the drug activa-
tion process. Usually, cancer cells have higher mitochondrial membrane potentials
3Department of Chemistry, Sungkyunkwan
University, Suwon 16419, Korea
4School of Chemistry and Chemical Engineering,
Shanxi University, Taiyuan, 030006, China
5Department of Chemistry, University of Texas at
Austin, Austin, TX 78712-1224, USA
6These authors contributed equally
7Lead Contact
*Correspondence: sessler@utexas.edu (J.L.S.),
jinylee@skku.edu (J.Y.L.),
chi6302@korea.ac.kr (S.-G.C.),
jongskim@korea.ac.kr (J.S.K.)
https://doi.org/10.1016/j.chempr.2018.08.002
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O
O
O
Cl
Cl
HN
N
HN
HN
N
Cl
Cl
OH
OH
O
O
O
O
O
O
O
OH
OH
O
N
H
O
O
O
O
OH
OH
O
N
H
O
HO
N
O
O
N
N
N
N
N
N
OH
OH
OH
OH
Br
Br
Br
P
P
P
C1
C3
C5
O
O
O
Cl
Cl
HN
HN
HN
Cl
Cl
OH
OH
O
O
O
O
O
O
OH
O
N
H
O
O
O
OH
OH
O
N
H
O
HO
O
O
OH
OH
O
OH
O
OH
OH
C2
C4
C6
Figure 1. Chemical Structure of Compound C1 and Controls (C2–C6)
(Djm = 220 mV) than normal cells (À140 mV). A tethered lipophilic TPP cation was
included in conjugate C1 to provide the predicative mitochondrial targeting.35
Because of its multicomponent nature, conjugate C1 was expected to provide a
greater therapeutic benefit than Dox alone, combinations of Dox + DCA, and
even alternative Dox-based targeting strategies. Experimental support for these
suggestions, involving studies of both human cancer cell lines and Dox-resistant mu-
rine xenograft tumor models, is provided below. The results presented here lead us
to suggest that the combination of metabolic alternation and subcellular targeting
could provide an attractive strategy for addressing chemoresistant tumors.
RESULTS
Design and Synthesis of Compounds
From a synthetic perspective, our challenge involved linking a recognized metabolic
mediator, DCA, with an active drug with a recognized resistance profile (Dox)
through a cleavable linker that would permit further tagging with a cancer targeting
entity. To be effective, we believed that the resulting molecular construct would
need to be stable yet capable of releasing both the DCA and Dox moieties in
response to a single triggering event. With these considerations in mind, we chose
to target for synthesis an intermediate, I-2 (Scheme S1), that could be used to link a
DCA subunit to a Dox-based construct while concurrently expressing an alkyne func-
tionality that would allow for further copper-assisted tagging.36 As prepared, I-2
contains a strongly donating aniline group (sp = À0.66) that could be elaborated
to produce amides and carbamates, which are weakly donating (Hammett constant,
sp = 0.00). The resulting changes in electronic features,37 we believed, could be ex-
ploited to effect both Dox and DCA release by taking advantage of the carboxyles-
terase-based triggering strategy previously used with success in several drug
delivery formulations.38 Aromatic amides are known to be hydrolyzed by carboxyles-
terase at the N-terminal position.39,40 However, at the onset of the present project, it
remained an open question whether the presence of the bulkier dichloromethyl
group in derivatives of I-2 (e.g., compounds C1 and C3; see below) would still allow
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Figure 2. Chemical Activation of C1
(A) Fluorescence intensity response of C1 (5 mM) seen upon incubation with carboxylesterase (1 U/
mL) in phosphate-buffered saline (PBS) (37ꢀC).
(B) Time-dependent fluorescence enhancement seen upon incubation of C1 with carboxylesterase
(1 U/mL) in PBS (37ꢀC). The change in fluorescence intensity is directly related to active Dox release
from C1.
(C) Reverse-phase high-performance liquid chromatography (RP-HPLC) curves of C1 treated with
esterase at 37ꢀC for 4 hr.
(D) Binding of C1 to human CE1.
the compound to fit into carboxylesterase enzymatic active site and undergo
cleavage.
To gain further insight into the preliminary stability and activation of conjugates con-
taining DCA, Dox, and a TPP cation targeting entity, we prepared both the target
conjugate C1 and the control system C3 (an analog of C1 lacking the chloromethyl
groups present in DCA; cf. Figure 1). These two conjugates were obtained in excel-
lent yield, as shown in Scheme S1. Upon incubation of C1 in phosphate-buffered sa-
line (PBS [pH 7.4]) at 37ꢀC for 24 hr, no decomposition was observed. This was taken
as an augury that C1 would likely prove stable under physiologic conditions (Fig-
ure S1). Conjugate C1 displayed a very weak fluorescence emission at 590 nm (Fig-
ure 2A). On the other hand, exposure of C1 to carboxylesterase (1 U/mL) resulted
in scission of the aniline-amide bond and release of Dox from the reactive intermedi-
ate, presumably via the 1,6-elimination route characteristic of these kinds of aniline
derivatives.34,40 This release was accompanied by a strong emission band at
560 nm corresponding to free Dox (Figures 2A and 2B). Further support for the
release of free Dox under these conditions came from time-dependent fluorescence
experiments, high-performance liquid chromatography (HPLC), and liquid chroma-
tography-mass spectrometry, carried out over a 2 hr analysis window (Figures 2B,
2C, and S2). Similar results were observed for conjugate C3 under the same experi-
mental conditions (Figures S4–S7). To test the reaction of C1 with various other bio-
analytes, we recorded the fluorescence response in the presence of various amino
acids, enzymes, and biomolecules. Distinct and large fluorescence enhancements
in the 590 nm emission feature were observed upon treatment with the esterase
but not in its absence. This finding leads us to suggest that C1 is an effective substrate
for this enzyme and that it may be activated in a selective manner (cf. Figure S3).
4
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To obtain further insights into the structural and interaction features associated with
the putative interactions of C1 and C3 within the carboxylesterase active side, we
carried out docking and molecular dynamics (MD) simulations (Figures 2D and
S4D). After docking and further MD simulations (cf. Supplemental Information for
details), the binding free energy of C1 to Carboxylesterase1 (CE1) was calculated
to be À60.22 kcal/mol (Figure S8). In the binding site of CE1, there are five residues
(K92, A93, L96, L304, and M364) that play an important role. They contribute À1.59,
À2.16, À1.56, À2.20, and À3.55 kcal/mol to the binding energetics, respectively.
The binding free energy of C3 to CE1, À53.41 kcal/mol, was found to be slightly
lower than that of C1. However, the residues in the binding site of CE1 that interact
with C3 were found to be different from those used to bind C1 (Figure S8). The res-
idues A93, W357, M361, S365, Y366, P367, and M459 play a crucial role in the bind-
ing of C3 to CE1, each contributing to À1.44, À1.70, À3.87, À3.15, À3.24, À2.62,
and À1.54 kcal/mol, respectively. In addition, residues D90 and K257 had repulsive
interactions with C1, contributing +0.84 and +1.19 kcal/mol, respectively. Our re-
sults lead us to suggest that although both C1 and C3 can fit very well into the active
pocket of CE1 and are bound strongly, the specific interactions supporting enzyme-
substrate interactions differ in the case of these two conjugates.
Validation of Mitochondrial Targeting and Carboxylesterase-Dependent
Cytotoxicity of C1
To explore whether compounds C1 and C3 induce a cytotoxic response in cancer
cells, we first compared the tumor cell selectivity of the compounds, as well as their
ability to target mitochondria, in various cancer cell lines. Fluorescence image ana-
lyses revealed that C1 and C3, which contain the mitochondria-targeting lipophilic
TPP moiety as noted above,35 were found to be taken up specifically by cancer cells
(A549 and HepG2) but not by normal cells (NHDF and IMR90). In contrast, the con-
trol systems C2 and C4, which do not incorporate a TPP moiety, exhibited no cellular
selectivity (Figure S9). Further analyses using immunofluorescence in conjunction
with MitoTracker as a dye revealed that C1 and C3 were located predominantly
within the mitochondria for at least 24 hr after treatment, whereas C2 and C4 were
distributed throughout the cell with no detectable mitochondrial localization (Fig-
ure 3A). These findings provide support for the conclusion that the TPP moiety pro-
motes mitochondrial targeting in the case of conjugates C1 and C3.
Next, we evaluated the cytotoxicity of conjugates C1–C6 by using cell-survival as-
says. On the basis of these studies, we conclude that C1 and C2 induce greater
cell death in a dose-dependent manner than either C3 or C4 (which lack the DCA
and TPP moieties, respectively) or controls C5 and C6 (which lack the Dox subunit)
(Figure 3B). The half maximal inhibitory concentration values of C1 and C2 were
22.1 G 0.325 and 10.6 G 1.03 mM, respectively (Figure S10A). An immunoblot assay
of cleaved caspase-3 and PARP-1 revealed that C1 has greater apoptosis-inducing
activity than C2 but induces apoptosis at a later time than C2 (Figure S10B).
To understand the role of the carboxylesterase in the release of DCA and Dox, we
tested the effect of the known carboxylesterase inhibitor, bis-(4-nitrophenyl)phos-
phate (BNPP) on the cytotoxicity of C1.41 BNPP pretreatment strongly attenuated
the C1-driven decrease in cell viability, as well the dose-dependent induction of
apoptosis (Figure 3C). Moreover, C1 treatment led to up- and downregulation of
p21Waf1 and phosphorylated PDH levels, which are mediated by Dox and DCA,
respectively. Moreover, these effects of C1 were strongly inhibited by pretreatment
with BNPP (Figures 3C and 3D). Unlike C1, conjugate C3, which lacks a DCA subunit,
showed no effect on p21Waf1 or PDH phosphorylation (Figure 3E). Together, our
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Figure 3. Cell Localization and Viability Assays of Compounds C1–C6
(A) Confocal microscopy analysis of intracellular localization of compounds C1–C4. HepG2 cells
treated with each compound (5 mM, 24 hr) were co-stained with the mitochondrial marker
MitoTracker (green). Boxed regions show co-localization of the test compounds and probe within
the mitochondria.
(B) WST cell viability assay of compound cytotoxicity. Experiments were carried out in triplicate and
the data shown represent means G SD. **p < 0.01.
(C) Attenuation of C1 cytotoxic effect by BNPP. HepG2 cells were incubated with BNPP (100 mM) for
2 hr prior to C1 treatment. **p < 0.01.
(D) Immunoblot assay showing the effect of C1 on p21Waf1 and phosphorylated PDH (p-PDH) levels
and its blocking by BNPP pretreatment. Cleaved caspase-3 and PARP-1 levels were examined to
assess apoptosis induction. C, cleaved.
(E) Immunoblot assay for comparison of the effect of C1 and C3 on p21Waf1 and p-PDH levels.
results provide support for the suggestion that the rationally designed nature of C1
and the concurrent presence of the three design elements it embodies (DCA, Dox,
and TPP) allows for synergistic PDK inhibition in combination with Dox drug release
in human cancer cells.
Cytotoxic Effect of C1 on Drug-Resistant Tumor Cells
Previous studies have shown that the drug resistance of many tumor cells can be
traced in large measure to the drug efflux activity of these cells.42 To assess whether
our strategy of targeting bioenergetics (via DCA-based PDK inhibition) combined
with triggered Dox release overcomes the MDR property of tumor cells, we
compared the effect of our conjugates on the breast cancer cell line MCF7 and its
Dox-resistant subline MCF7/Dox. The latter cell line is characterized by an enhanced
drug efflux capability. A fluorescence assay revealed that C2 (an analog of C1 that
lacks the TPP targeting subunit) was accumulated at a substantially lower level in
MCF7/Dox than in MCF7 (Figure S11A). In contrast, C1 accumulated at a compara-
ble level in both MCF7 and MCF7/Dox cells for up to 12 hr after treatment (Fig-
ure 4A). Immunoblotting studies revealed that C1, but not C2, exhibited mitochon-
drial localization in both MCF7 and MCF7/Dox cells (Figure S11B). Similar results
were obtained in another MDR tumor cell line, NCI/Dox (Figure S12).
6
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A
B
E
C
D
Figure 4. C1-Induced Apoptosis in Multidrug-Resistant Tumor Cells
(A) Comparison of intracellular levels of C1 and C2 in MCF7 and MCF7/Dox cells. Fluorescence
intensities were measured with SpectraMAX I3. Data represent means G SD. **p < 0.01.
(B) Cell fractionation assay showing the mitochondria-to-nucleus trafficking of C1. Data represent
means G SD. **p < 0.05.
(C) Dose-associated effect of C1 on intracellular lactate accumulation. Data represent the mean G
SD (n = 3 experimental replicates). *p < 0.05, **p < 0.01.
(D) Immunoblot assay showing the C1-induced release of cytochrome c from the mitochondria.
MCF7/Dox cells were treated with C1 or C3 as indicated, and the cytosolic (C) and mitochondrial
(M) fractions were subjected to immunoblot assay. COX-IV and tubulin were used as markers for the
mitochondria and cytosol fractions, respectively.
(E) Suppression of colony-forming ability of MCF7/Dox cells induced by C1 but not C2.
In order to evaluate more precisely the subcellular localization of C1, we performed a
cell fractionation assay. On this basis, we concluded that after treatment, C1 local-
izes initially to the mitochondria. After enzymatic cleavage of DCA and release of
free Dox, the Dox then translocates to the nucleus over the course of approximately
2 hr (Figure 4B). This ability to localize to the nucleus is considered particularly bene-
ficial for an agent, such as Dox, which is thought to mediate its antitumor action
within the nucleus. Mitochondrial targeting of Dox and other nuclei-dependent
drugs has been reported.43,44 However, to our knowledge, the subsequent time-
dependent nuclear translocation behavior seen after administration of C1 to MDR
cancer cells is without precedent.
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To address whether carboxylesterase-mediated DCA release blocks aerobic glycol-
ysis and thus drives apoptosis in the case of the drug-resistant cells, we tested the
effect of C1 on lactate accumulation. Treatment of MCF7/Dox cells with C1 led to
a dose-dependent reduction of intracellular lactate levels (Figure 4C). This was
accompanied by decreased glucose uptake and lowered intracellular ATP levels
(Figure S13). This finding is consistent with our design hypothesis, namely that con-
jugate C1 would trigger mitochondrial dysfunction by releasing DCA and modu-
lating the pyruvate metabolic pathway in these cancer cells.
The crucial role of carboxylesterase-mediated drug release was further supported by
the observation that C1 induces cytochrome c release from the mitochondria, cleav-
age of caspase-3 and PARP-1, and a decrease in cell viability; in contrast, C3, which
does not provide for PDK inhibition, fails to evoke these effects (Figures 4D, S14A,
and S14B). Moreover, C1 showed a strong inhibitory effect on the colony-forming
ability of both MCF7 and MCF7/Dox cells, whereas C2 showed this effect only in
the case of the MCF7 cells but not the resistant MCF7/Dox cells (Figure 4E).
Collectively, the above results provide support for the conclusion that the presence
of the TPP subunit leads to initial targeting of C1 within the mitochondria and that
this helps overcome drug efflux processes that would normally serve to reduce the
effective concentration of Dox within a cancer cell. These results are also consistent
with the conclusion that DCA released from C1 modulates what is otherwise an aber-
rant mitochondrial metabolism in the case of resistant MCF7/Dox cells. A particular
benefit of C1 is that it allows the further translocation of released Dox to the nucleus.
The net result is synergistic apoptotic cell death.
C1-Induced Regression of Drug-Resistant Tumors
We also compared the cytotoxic effects of C1 with those produced by an equivalent
concentration of Dox and DCA (Dox + DCA). Whereas both C1 and Dox + DCA
induced a comparable reduction in cell viability in the case of the MCF7 cell line,
only C1 produced an appreciable cytotoxic effect in the resistant MCF7/Dox cells
(Figure 5A). Moreover, the effect of C1 was comparable in both cell lines. Treatment
with C1 also led to greater levels of p21Waf1, reduction of PDH phosphorylation, and
cleavage of caspase-3 and PARP-1 than did treatment with Dox + DCA. In the case of
C1, both time- and dose-dependent behavior was seen (Figures 5B and S14C).
Confocal laser scanning microscope imaging (CLSM) of the mitochondrial mem-
brane potential (Djm) revealed that treatment with C1 induces a drastic decrease
in Djm (by ca. 96%) and did so more effectively than either C3, Dox, or Dox +
DCA (Figures 5C and S15). Flow cytometric analysis of Annexin V also revealed
that C1 promotes both early and late apoptosis and does so more effectively than
either C3, free Dox, or Dox + DCA (Figure S16).
In light of the above results, we made an effort to test the potential of C1 to promote
an in vivo therapeutic effect. Toward this end, conjugate C1 and formulations con-
sisting of Dox + DCA (i.e., containing a mixture of Dox and DCA) were injected
into mice bearing MCF and MCF7/Dox xenograft tumors (Figure S17A). As can be
seen from an inspection of Figure 5D, conjugate C1 produced a greater growth in-
hibition than did the combined treatment. Specifically, C1 injection led to reductions
in the tumor volume of approximately 78% (MCF7) and 74% (MCF7/Dox), whereas
the combined (Dox + DCA) treatment resulted in only a 53% and 18% reduction,
respectively (Figures 5E and S17B). The difference was statistically significant. Like-
wise, C1 induced a statistically greater level of tumor inhibition than C2–C4 when
8
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Figure 5. Anticancer Activity of C1 versus Combination of DCA and Doxorubicin In Vitro and
In Vivo
(A) Effect of C1 or doxorubicin combined with DCA on cell viability in MCF7 and MCF7/dox cells.
Data represent the mean G SD. *p < 0.05.
(B) Comparison of the efficacy of C1 versus doxorubicin combined with cleaved PARP in the MCF/
Dox cells.
(C) Confocal laser scanning microscope (CLSM) images showing qualitatively the mitochondrial
membrane potential in MCF7/Dox cells observed upon incubation with C1, Dox + DCA, and Dox,
respectively, for 48 hr with MitoView 633 (Biotium) staining for the mitochondrial (red) channel and
Hoechst 33342 staining for the nucleus (blue) channel.
(D) Representative images of MCF7 and MCF7/Dox xenograft tumors treated with DMSO control,
Dox + DCA, and C1.
(E) C1-induced regression of MCF7 and MCF7/Dox tumors (mean G SD, n = 8 per group, **p <
0.01).
(F) Representative fluorescence images of mice bearing MCF7 and MCF7/Dox tumors and ex vivo
images of dissected organs.
retested in an independent comparative study using the same MCF/Dox animal
model (Figure S18). No appreciable weight loss was observed for any groups over
the course of treatment (Figures S17C and S18D). Measurement of alanine amino-
transferase (ALT) and aspartate aminotransferase (AST) levels in blood serum sam-
ples revealed no significant toxicity induced by either C1 or the control conjugates
C2–C4 (Figure S18E). Fluorescence image analysis of the mice and representative
dissected organs revealed a highly tumor-specific accumulation of Dox in the case
C1 (Figure 5F). This finding is consistent with the suggestion that conjugate C1 over-
comes efflux mechanisms associated with Dox-resistant tumor cells and can induce
regression of drug-resistant tumors in vivo. In purely operational terms, the key
finding is that C1 is active in promoting tumor growth inhibition for the resistant
cell line xenograft (MCF7/Dox), whereas Dox + DCA and other tested controls
C2–C4 are not appreciably effective.
DISCUSSION
Altered cellular metabolism is a recognized hallmark of cancer. Metabolic flexibility
plays a crucial role in the acquisition and maintenance of various malignant features,
and this understanding has recently been parlayed into the development of effective
cancer therapeutics.45 Mitochondrial membrane potential plays a vital role in the
Chem 4, 1–14, October 11, 2018
9

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Chem (2018), https://doi.org/10.1016/j.chempr.2018.08.002
efflux of various pro-apoptotic signals through the mitochondrial transition pores
and also has an impact on the state of apoptosis resistance in cancer cells. The meta-
bolic switching induced by DCA, namely from aerobic glycolysis to oxidative phos-
phorylation, is correlated with promising anticancer effects across a number of can-
cer cell lines and rodent models.46 However, the clinical potential of DCA has yet to
be fully realized. Parallel with the efforts to develop metabolic modulators, a number
of approaches to overcoming drug resistance have been pursued in recent
years;47,48 however, translating these approaches to human use remains a largely
unmet challenge.
The approach detailed in this study is based upon the concept of switching the can-
cer cell metabolism back to normal mitochondrial OXPHOS. Effecting such a switch-
ing would reduce the effectiveness of several proliferation-enabling cancer-associ-
ated pathways. This putative benefit could, in principle, be further enhanced by
the targeted release of an active cancer chemotherapeutic. To the extent it could
be achieved, an organelle-specific drug ‘‘stopover’’ en route to trafficking an agent
to its ultimate site of action could help bypass various MDR-associated processes
and might provide yet additional anticancer benefits. To test these appealing pos-
tulates, we developed conjugate C1, a small molecule that embodies several sub-
units designed to allow for organelle targeting and triggered release of both a meta-
bolism modulator (DCA) and an active chemotherapeutic whose clinical use is
plagued by treatment-induced resistance (Dox). In conjugate C1, the DCA subunit
is incorporated in the form of an aniline amide. It thus not only serves as a meta-
bolism modulator but also functions as a trigger to release Dox in its activate
form. This lead also contains a tethered TPP moiety that, on the basis of prior
studies,35 was expected to direct the whole ensemble to the mitochondria of cancer
cells. In terms of details, esterase-mediated hydrolysis of the DCA aniline amide was
expected to release the DCA, which would inhibit PDK, thus promoting an increase
in intracellular pyruvate concentrations within the mitochondrion. This, in turn,
would act to reverse the metabolic disturbances that normally favor cancer cell pro-
liferation while reducing hyperpolarization and improving mitochondrial functions
(Figures 1 and 2).
Live-cell imaging experiments revealed that both conjugates C1 and C3 (an analog
of C1 lacking the chloromethyl groups present in DCA) accumulate preferentially in
the mitochondria (Figure 3A). The improved therapeutic efficacy of C1 over C3 was
confirmed via western blotting (Figures 3D, 3E, and S10B). Although compared with
C1, C2 (an analog containing the DCA subunit but not the targeting TPP moiety) eli-
cited higher apoptosis in non-malignant HepG2 cells in vitro, conjugate C1 was
found to provide enhanced drug retention and improved efficacy in tumor cells.
To evaluate further the therapeutic potential in MDR cell lines, we chose the human
breast cell lines MCF7 and MCF7/Dox (a Dox-resistant cell line), as well as the resis-
tant ovarian cell line NCI/Dox as models. Treatment with C1 gave rise to a time-
dependent fluorescent enhancement and accumulation in both the MCF7/Dox
and NCI/Dox cell lines, supporting the conclusion that the TPP present in this con-
jugate allows for better targeting and increased efficacy (Figures 4, S11, and S12).
The improved mitochondrial glycolysis expected to be induced by DCA release
was confirmed in the case of C1 by the observation of low cytosolic lactate,
decreased glucose uptake, and low ATP levels, in a concentration-dependent
manner (Figures 4C and S13). Further, western blot analysis revealed time-depen-
dent caspase activation, PARP cleavage, and cytosolic cytochrome c fractions that
were higher after treatment with C1 than with C3 (Figures 4D and S14A). After
10
Chem 4, 1–14, October 11, 2018

Please cite this article in press as: Sharma et al., Overcoming Drug Resistance by Targeting Cancer Bioenergetics with an Activatable Prodrug,
Chem (2018), https://doi.org/10.1016/j.chempr.2018.08.002
Scheme 1. Proposed Mode of Action of C1 in MCF7/Dox-Resistant Cells
After accumulation in cancer cell mitochondria, C1 becomes activated by carboxylesterase
enzymes. The released DCA (PDK inhibitor) shifts the aerobic glycolysis characteristic of cancerous
environments to OXPHOS. The free Dox produced via self-immolation in the mitochondria then
translocates over time to the nucleus where it mediates its function. DCA, dichloroacetic acid; PDK,
pyruvate dehydrogenase kinase; PDH, pyruvate dehydrogenase complex; TCA, tricarboxylic acid
cycle; Dox, doxorubicin; ATP, adenosine triphosphate; j, mitochondrial membrane potential.
treatment with C1, the time-dependent movement of free Dox from the mitochon-
dria to the nucleus was observed (Figure 4B). We propose that conjugate C1 is taken
up first by the mitochondria and that this ‘‘stopover’’ following administration and
enzymatic release helps to evade the drug efflux mechanisms that contribute to
drug resistance. After activation (i.e., enzyme-mediated and chemically mediated
DCA and Dox release), enhanced production of reactive oxygen species results in
mitochondrial depolarization. These perturbations, we propose, allow the free
Dox to translocate to the nucleus, which gives rise to the observed improvements
in overall cytotoxicity (Scheme 1). It is important to stress that with our design, it is
free Dox present in the nucleus that is expected to mediate the proposed cytotoxic
effect. Conjugate C1 with its mitochondria targeting, activated release, and translo-
cation capability is expected to enhance this delivery of Dox while overcoming the
effects of MDR.
Finally, we tested the efficacy of C1 in an MCF7- and Dox-resistant MCF7/Dox-
derived xenograft mouse model. We found that C1 displayed greater tumor speci-
ficity and enhanced retention in the tumors than DCA and Dox combined, or the
various control systems prepared for this study. On this basis, we suggest that
DCA-induced metabolic switching concurrent with triggered release of Dox leads
to the enhancements in Dox retention in resistant tumor cells. Organelle targeting
by the appended TPP moiety allows these benefits to be enhanced. The net result
Chem 4, 1–14, October 11, 2018
11

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Chem (2018), https://doi.org/10.1016/j.chempr.2018.08.002
is a remarkable reduction in tumor burden (by 74% under conditions where com-
bined Dox + DCA treatment results in only an 18% reduction) in Dox-resistant
MCF7/Dox models (Figures 5 and S17). In addition, in an independent comparative
study conducted by using the same MCF/Dox xenograft animal model, C1 was
found to induce a statistically greater level of tumor inhibition than the various con-
trol systems, namely C2–C4 (Figure S18). Further, no appreciable hepatotoxicity was
seen in any of the treated animals (Figure S18E). To the best of our knowledge, there
are no other experimental agents that have demonstrated this kind of promise in
resistant tumor models. On this basis, we propose that further studies of C1 are war-
ranted as are efforts to develop conjugates that combine the favorable features of
individual subunits (i.e., DCA, Dox, and TPP in the present instance) within one
chemical entity. Ultimately, this could result in a next generation of small-mole-
cule-based therapeutics capable of producing improved clinical outcomes.
Conclusion
Resistance to chemotherapeutics and molecularly targeted therapies is a major
concern in the clinic and a challenge in cancer research. Although still in the early
stages of development, targeting cancer metabolism could provide an approach
to overcoming resistance. We have now provided experimental support for the sug-
gestion that the reversal of cancer metabolism prior to drug activation by a molec-
ular targeted agent that allows for initial stopover in the mitochondria can be used
to overcome resistance in cell cultures and in tumor xenograft animal models based
on studies involving both Dox-sensitive and Dox-resistant cell lines. The synthetic
ease, cancer selectivity, and effectiveness of this approach, coupled with the lack
of appreciable whole-animal toxicity during treatment, make this strategy one that
could have broad appeal as a paradigm for overcoming drug resistance.
EXPERIMENTAL PROCEDURES
The full experimental procedures are provided in the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, 37 fig-
ures, and 1 scheme and can be found with this article online at https://doi.org/10.
1016/j.chempr.2018.08.002.
ACKNOWLEDGMENTS
This research was supported in part by a Creative Research Initiatives project (no.
2018R1A3B1052702 to J.S.K.) and a grant from the National Research Foundation
of Korea (NRF-20158A2A1A01005389 to S.-G.C.). Funding from the National Can-
cer Institute (CA68682 to J.L.S.) and the Robert A. Welch Foundation (F-0018) is
also acknowledged.
AUTHOR CONTRIBUTIONS
A.S., M.-G.L., and H.S. contributed equally to this work (co-first authors). Synthesis
and Characterization, A.S.; Biological Experiments, M.-G.L. and M.W.; Docking
and Molecular Dynamics Simulation Calculations, H.S.; Supervision, J.S.K.,
S.-G.C., J.Y.L., and J.L.S.; Writing – Original Draft, A.S., J.S.K., S.-G.C., J.Y.L., and
J.L.S.; Writing – Review & Editing, A.S., J.S.K., J.L.A., and J.L.S.; Funding Acquisi-
tion, J.S.K., S.-G.C., and J.L.S. All authors proofread, commented on, and approved
the final submitted version of the manuscript.
12
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DECLARATION OF INTERESTS
A.S., M.-G.L., S.-G.C., J.Y.L., J.S.K., and J.L.S. are co-inventors of a patent applica-
tion related to this work.
Received: January 25, 2018
Revised: June 15, 2018
Accepted: July 30, 2018
Published: August 23, 2018
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