Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to Cancer Therapy

Article abstract of DOI:10.1016/j.chempr.2020.03.004

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most

Full text of DOI:10.1016/j.chempr.2020.03.004

Article
Mitochondrial Relocation of a Common
Synthetic Antibiotic: A Non-genotoxic
Approach to Cancer Therapy
Kyoung Sunwoo, Miae Won,
Kyung-Phil Ko, ..., Jonathan L.
Sessler, Peter Verwilst, Jong
Seung Kim
chi6302@korea.ac.kr (S.-G.C.)
sessler@cm.utexas.edu (J.L.S.)
peter.verwilst@kuleuven.be (P.V.)
jongskim@korea.ac.kr (J.S.K.)
HIGHLIGHTS
Mitochondrial re-localization of
ciprofloxacin reduces nuclear
genotoxicity
Mitochondrial localization and
spatiotemporal drug activation
were confirmed
qPCR analyses provide support
for the proposed mode of action
In vivo experiments support the
efficacy and target specificity
DATA AND CODE AVAILABILITY
GSE137679
Therapy-associated nuclear DNA damage is a key driver for secondary
malignancies. It represents a major problem in clinical practice that has yet to be
overcome. The mitochondria-targeted ciprofloxacin conjugate reported here
eliminates therapy-induced nuclear DNA mutagenicity and induces cancer cell
death as a result of oxidative damage. These findings could provide a new
framework for the development of non-genotoxic therapeutics that overcome the
conceptual limitations of the inherently mutagenic chemotherapeutics that define
the current standard of care in cancer chemotherapy.
Sunwoo et al., Chem 6, 1–12
June 11, 2020 ª 2020 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2020.03.004

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Article
Mitochondrial Relocation of a Common
Synthetic Antibiotic: A Non-genotoxic
Approach to Cancer Therapy
Kyoung Sunwoo,1,5 Miae Won,1,5 Kyung-Phil Ko,2 Miri Choi,3 Jonathan F. Arambula,4 Sung-Gil Chi,2,
*
1,6,
1,
Jonathan L. Sessler,4,7, Peter Verwilst,
and Jong Seung Kim
*
*
*
SUMMARY
The Bigger Picture
Cancer recurrence is a major
concern in clinical practice,
severely limiting the life
Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major
issue in cancer treatment. Currently, only a few examples of potentially non-
genotoxic drugs have been reported. Mitochondrial re-localization of ciproflox-
acin, one of the most commonly prescribed synthetic antibiotics, is reported
here as a new approach. Conjugation of ciprofloxacin to a triphenyl phospho-
nium group (giving lead Mt-CFX) is used to enhance the concentration of cipro-
floxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the
mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large
bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to
classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in
a xenograft mouse model and proved to be well tolerated. Mitochondrial re-
localization of antibiotics could emerge as a useful approach to generating anti-
cancer leads that promote cell death via the selective induction of mitochondri-
ally mediated oxidative damage.
expectancy of patients with
cancer. Recurrence is often the
result of therapy, which can also
lead to drug-induced secondary
tumorigenesis. In many cases, the
resulting secondary malignancies
are the result of nuclear DNA
lesions induced by traditional
genotoxic chemotherapeutics. In
this study, we show that by
rerouting a commonly used
microbiocidal, ciprofloxacin, to
the mitochondria, it is possible to
produce a highly selective and
well-tolerated anticancer agent.
Mitochondrial targeting not only
induces a high degree of cancer
specificity but also minimizes
nuclear DNA damage. On the
other hand, it promotes
INTRODUCTION
Tumor recurrence remains the greatest challenge in cancer therapy and is respon-
sible for up to 90% of cancer mortality.1 Paradoxically, recurrence is often elicited
by chemotherapeutics due to the accumulation of nuclear genomic mutations dur-
ing the treatment regime.2 Antineoplastic agents relying on direct nuclear DNA
damage, such as alkylating agents, are common chemical mutagens and have
been implicated in tumor recurrence, e.g., therapy-related acute myeloid leukemia
(t-AML).3–6 Additionally, drug-induced genomic alterations are carried over into the
next generation, detectable up to two generations in mice.7 Given the steady in-
crease in pediatric malignancy survival rates,8 prevention of these long-term effects
mitochondrial damage-
associated cancer cell death and
tumor growth reduction. Thus, the
present targeted ciprofloxacin
derivative illustrates what could
emerge as a new approach to
generating less genotoxic and
more effective
might warrant
a paradigm shift toward rationally designed non-genotoxic
chemotherapeutics.
Mitochondria lie at the nexus of cellular signaling events, including the induction of
apoptosis via the caspase cascade. As such, mitochondrial-damage-induced pro-
grammed cell death represents an appealing pathway for cancer chemotherapy.
Agents that target the mitochondria could bypass direct nuclear DNA damage
mechanisms, thus preventing the accumulation of nuclear DNA mutations. Mito-
chondria are widely believed to have a bacterial evolutionary origin and share
many similarities with the bacterial genome and biosynthetic machinery.9 While
operating via disparate mechanisms in bacteria and eukaryotic cells, common anti-
biotics (such as fluoroquinolones, aminopenicillins, aminoglycosides, and
chemotherapeutics.
Chem 6, 1–12, June 11, 2020 ª 2020 Elsevier Inc.
1

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
tetracyclines) inhibit the mitochondrial electron transfer chain (ETC).10 The associ-
ated reduction in mitochondrial electron transfer efficiency leads to the production
of reactive oxygen species (ROS),10 resulting in mitochondrial damage. This damage
can trigger apoptosis. We thus thought that by targeting antibiotics to the mito-
chondria, it might be possible to discover new non-genotoxic drugs for cancer
chemotherapy. The present study was designed to test this hypothesis.
Efforts to apply antibiotics to cancer chemotherapy have a long history.11,12 For
instance, bacteria-derived bactericides, such as doxorubicin (DOX) and mitomycin
C, have found broad clinical applications in chemotherapy; however, their mode
of action is thought to involve direct nuclear DNA damage, which can lead to
drug-induced genomic alterations.13,14 In contrast, many commonly prescribed syn-
thetic bactericidal drugs were developed with a goal of maintaining low toxicity
levels in eukaryotic cells. This has made the standard repurposing of such Food
and Drug Administration (FDA)-approved drugs as potential anticancer agents
attractive. However, difficulties in achieving effective sub-cellular localization of
such antibiotics has, in part, prevented success. Although large doses of synthetic
antibiotics have been shown to reduce cancer cell viability, typically non-cancerous
cells have proved more susceptible to these antimicrobiocidal drugs than cancer
cells.15 Nonetheless, the potential role of antibiotics as chemotherapy adjuvants
has been noted.16 Here, we show that redirection of the antibiotic ciprofloxacin
(CFX) to mitochondria via chemical modification results in a new agent with can-
cer-cell-selective antiproliferative activity. A reduction in vivo is also seen, as inferred
from tumor regrowth experiments in murine models. The approach illustrated here
could overcome the difficulties encountered in the standard repurposing of antibi-
otics for cancer treatment.
RESULTS AND DISCUSSION
Mt-CFX Induces Cell Death in a Mitochondrial-Membrane-Potential-
Dependent Way
CFX is one of the most commonly prescribed synthetic antibiotics worldwide. It is
thus an attractive target for repurposing as a potential anticancer agent. However,
we hypothesized that in order to be effective in this regard, CFX would need to
be directed to the mitochondria. To test this hypothesis, CFX was linked to a
triphenyl phosphonium subunit, a group known to promote mitochondria localiza-
tion.17,18 The resulting conjugate, Mt-CFX (Figure 1A; for synthesis, see the Supple-
mental Information), was found to reduce cell viability in a dose-dependent manner
in MDA-MB-231 cancer cells (Figure 1B). An IC50 value of 31 mM was recorded for
1Department of Chemistry, Korea University,
Seoul 02841, Korea
Mt-CFX, whereas CFX or the phosphonium targeting group alone proved several
orders of magnitude less effective (Figure 1B). An associated BrdU incorporation
assay revealed a statistically significant reduction in cell proliferation in the presence
of Mt-CFX as compared with CFX or vehicle only (Figure S1). Relative to MDA-MB-
231, a reduced cytotoxicity profile was seen for Mt-CFX in the non-cancerous
MCF10A mammary gland epithelial cell line (Figure 1C), thus illustrating cancer
selectivity in vitro. The antiproliferative effect of Mt-CFX was found to be on the
same order of magnitude across several cancerous cell lines (e.g., MDA-MB-231,
SW620, DU145, A549, and PC3). The toxicity of Mt-CFX was at least an order of
magnitude lower in the normal human fibroblast cell line (BJ) as compared with
that in the cancer cell lines. Moreover, as noted above, virtually no toxicity was
observed in the MCF 10A cell line (Figure 1F).
2Department of Life Sciences, Korea University,
Seoul 02841, Korea
3Chuncheon Center, Korea Basic Science
Institute, Chuncheon 24341, Korea
4Department of Chemistry, University of Texas at
Austin, Austin, TX 78712-1224, USA
5These authors contributed equally
6Present address: KU Leuven, Rega Institute for
Medical Research, Medicinal Chemistry, B-3000
Leuven, Belgium
7Lead Contact
*Correspondence: chi6302@korea.ac.kr (S.-G.C.),
sessler@cm.utexas.edu (J.L.S.),
peter.verwilst@kuleuven.be (P.V.),
jongskim@korea.ac.kr (J.S.K.)
JC-1 monomer versus aggregate flow-cytometry assays were used in this study to
assess the mitochondrial membrane potentials (MMPs), with high proportions of
https://doi.org/10.1016/j.chempr.2020.03.004
2
Chem 6, 1–12, June 11, 2020

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 1. Mt-CFX Induces Cell Death in a MMP-Dependent Manner
(A) Chemical structure of Mt-CFX.
(B) Concentration-dependent cell viability of MDA-MB-231 cells incubated with Mt-CFX, CFX, and TPP as determined by a Cyto-Tox96 Assay (72 h).
(C) Concentration-dependent cell viability of MCF10A cells incubated with Mt-CFX, CFX, and TPP as determined by a CytoTox96 Assay (72 h).
(D) Proportion of JC-1 monomer fluorescence in a flow-cytometry assay of MDA-MB-231 pretreated with 30 mM Mt-CFX as per the indicated incubation
times.
(E) Time-dependent Annexin V-FITC flow-cytometry analysis of MDA-MB-231 pretreated with 30 mM Mt-CFX as per the indicated incubation times.
(F) Concentration-dependent cell viability of different cell lines incubated with Mt-CFX as determined by a CytoTox96 Assay (72 h).
(G) Different proportions of JC-1 monomer fluorescence in a flow-cytometry assay of untreated cells.
(H) Summarized JC-1 monomer fractions from (G) and IC50 values of Mt-CFX-treated cell lines from (F).
All cytotoxicity experiments were carried out three times and in triplicate wells. Data are represented as mean G SEM. Statistical significance was
determined by a one-way ANOVA test with a post-hoc Bonferroni test. Different letters (e.g., a–d) signify data that are statistically different (p < 0.05).
monomer fluorescence corresponding to a decreased MMP. The addition of Mt-CFX
to MDA-MB-231 cells resulted in a time-dependent MMP depolarization (Figures 1D
and S2) with a concurrent increase in Annexin-V binding (Figures 1E and S3). Such
findings are consistent with an apoptosis-induced cell death mechanism. We thus
sought to investigate the relationship, if any, between cell viability and the MMP.
A population of each cell line was subjected to a JC-1 assay (Figures 1G and S4).
A strong general trend toward lower cell viability with stronger MMP hyperpolar-
ization was found (Figure 1H).
Localization of the Mt-CFX System to the Mitochondria as Evidenced by
Studies of a Fluorescent Probe, Bo-Mt-CFX
To obtain further support for the suggestion that Mt-CFX localizes to the mitochon-
dria, we synthesized a related conjugate incorporating the fluorophore resorufin.
This system, termed Bo-Mt-CFX, was designed to allow for an accurate spatiotem-
poral analysis of the drug action in vitro and in vivo (see the Supplemental Informa-
tion for synthetic details).19–22 A phenylboronate moiety was used to prepare Bo-Mt-
CFX because it is a known trigger that will undergo peroxide-induced cleavage, thus
allowing release of both the fluorophore and putative Mt-CFX drug under condi-
tions of ROS over-production (i.e., in cancerous tissues).23 As can be seen in
Figure 2A, peroxide-triggered drug release results in the liberation of the CFX
Chem 6, 1–12, June 11, 2020
3

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 2. Bo-Mt-CFX, a ROS-Triggered DDS System Incorporating Mt-CFX
(A) Peroxide-induced self-immolative activation mechanism of Bo-Mt-CFX.
(B) Localization of resorufin release from 10 mM Bo-Mt-CFX in MDA-MB-231 cells as compared with MitoTracker Green FM, LysoTracker Green DND-26,
and ER-Tracker Green.
(C) Western blotting of apoptosis-related proteins in MDA-MB-231 cells treated with 10 mM Bo-Mt-CFX or a control.
(D) Western blotting of cytochrome c release from mitochondria to the cytosol seen in MDA-MB-231 cells treated with 3 or 10 mM Bo-Mt-CFX, as well as a
control.
and the fluorophore resorufin. This self-immolative release relies on the formation
and subsequent nucleophilic attack of various quinone methide moieties, which
are produced throughout the release mechanism.
We validated the peroxide-triggered self-immolative release of the resorufin fluoro-
phore from Bo-Mt-CFX in aqueous solution (5 mM) by observing the characteristic
fluorescent band centered at 580 nm. We observed a clear time-dependent
response with a half-life of approximately 6,000 s in the presence of 50 molar equiv-
alents of H2O2 (Figure S5A). A concentration-dependent effect was also observed
(Figure S5B). Moreover, the identity of the fluorescent species was confirmed to
be resorufin via a time-dependent high-performance liquid chromatography
(HPLC) assay. On this basis, we conclude that under these test conditions, the self-
immolative reaction is complete within 8 h (Figure S6). Mt-CFX release was also
confirmed by HPLC-mass spectrometry (HPLC-MS) (Figure S7). The influence of
Bo-Mt-CFX on intracellular cellular ROS and its effects on cellular activity (i.e., cell
viability across a panel cell lines, glutathione homeostasis, and ROS dependence)
4
Chem 6, 1–12, June 11, 2020

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 3. Mt-CFX Induces Mitochondrial ROS Production with Minimal Nuclear DNA Damage
(A) Confocal microscopic fluorescence intensity of MDA-MB-231 cells treated with Mt-CFX (30 mM) or a control co-incubated with 400 mM Amplex Red
for 30 min (n = 6).
(B) Confocal microscopic fluorescence intensity of Mt-CFX (30 mM) or control-treated MDA-MB-231 cells incubated with 5 mM Mito-Sox for 15 min (n = 5).
(C) Confocal microscopic fluorescence intensity of Mt-CFX (30 mM) or control-treated MDA-MB-231 cells incubated with 10 mM CM-H2DCFDA for 30 min
(n = 15).
(D) ELISA DNA oxidation and protein carbonylation assay results of MDA-MB-231 cells treated with 30 mM Mt-CFX, CFX, and control samples.
Colorimetric malondialdehyde (MDA) assay results, as a measure of lipid peroxidation, of similarly treated MDA-MB-231 cells.
(E) qPCR analysis of DNA repair genes in MDA-MB-231 cells treated with 30 mM Mt-CFX, CFX, DOX, and control samples.
(F) qPCR analysis of housekeeping genes in MDA-MB-231 cells treated with 30 mM Mt-CFX, CFX, DOX, and control samples.
(G) Taq1 restriction-site mutation assay using MDA-MB-231 cells treated with 30 mM Mt-CFX, CFX, DOX, and control samples.
Chem 6, 1–12, June 11, 2020
5

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 3. Continued
(H) Long-range qPCR DNA lesion assay using MDA-MB-231 cells treated with Mt-CFX (30 mM), TMZ (1 mM), CPT (250 nM), CDDP (3 mM), CLB (50 mM), and
5-FU (30 mM). Concentrations were selected on the basis of their respective IC50 values.
Data are represented as mean G SEM (n = 3, unless specified otherwise). Statistical significance was determined by a one-way ANOVA test with a post-
hoc Bonferroni test. Different letters (e.g., a–d) signify data that are statistically different (p < 0.05).
was further evaluated in vitro (Figures S8–S14), revealing the expected correlation
between cancer cell inhibition and cellular oxidative stress.
Confocal microscopy studies confirmed that in MDA-MB-231 cells, the release of re-
sorufin from Bo-Mt-CFX occurs in the mitochondria (Figure 2B). The Mt-CFX
released from Bo-Mt-CFX was found to be largely retained in the mitochondria for
at least 8 h (Figure S15). The addition of Bo-Mt-CFX to MDA-MB-231 cells also re-
sulted in the activation of the caspase pathway (Figures 2C and S16) while engen-
dering a dose-dependent cytoplasmic re-localization of cytochrome c (Figures 2D
and S16). An in-depth study of several pro-apoptotic (BAX, BAC, BAD, PUMA,
Noxa, and BID) and anti-apoptotic (Bcl-2, Bcl-w, Mcl-1, Bcl-xl, and A1) members
of the Bcl-2 family was conducted in the presence of Mt-CFX (Figure S17). A lowered
or constant protein expression level was found for the anti-apoptotic proteins,
whereas the pro-apoptotic BAX levels were increased. The PUMA and Noxa levels
were scarcely elevated, which is consistent with a lack of (nuclear) genotoxicity.24
Finally, a comparison between Mt- HCT116 WT and HCT116 Baxꢀ/ꢀ cells revealed
the requirement of BAX for apoptosis induction following Mt-CFX treatment (Fig-
ure S18), a finding that supports mitochondrial involvement in the cell death process.
Taken together, these results lead us to conclude that the activity of Mt-CFX is
strongly dependent on the mitochondrial accumulation of the drug, a phenomenon
that generally tracks with the MMP.
Mt-CFX Induces Mitochondrial ROS Production with Minimal Nuclear DNA
Damage
Several antibacterial drugs have been reported to cause mitochondrial damage as a
result of ROS production.10 Therefore, we investigated the influence of Mt-CFX on
ROS production in MDA-MB-231 cells. As can be seen in Figure 3A, the intracellular
Amplex Red fluorescence was elevated in cells treated with Mt-CFX and this indica-
tor. The fluorescence intensity ascribed to Mito-SOX, a mitochondria-selective su-
peroxide (O2_ꢀ) fluorescent dye, is likewise enhanced upon treatment with Mt-
CFX. Such findings are consistent with an increase in ROS production within the
mitochondria (Figures 3B and S19A). The use of the non-selective fluorescein-based
oxidative stress indicator, CM-H2DCFDA, provided further support for the conten-
tion that ROS are being generated in the presence of Mt-CFX (cf. Figures 3C and
S19B). Furthermore, cells pre-incubated with N-acetyl cysteine (NAC) (cf. Figures
S20 and S21) or other antioxidants (Figure S21) not only decreased the cellular
ROS burden but also rescued cells from Mt-CFX-induced cytotoxicity.
We next investigated whether damage to cellular components ascribable to ROS
was seen upon incubation of MDA-MB-231 cells with Mt-CFX or CFX (Figures 3D
and S22). We found elevated 8-hydroxy-20-deoxyguanosine (8-OHdG) levels, a
classic marker for radical-induced oxidative damage to nuclear and mitochondrial
DNA. Both protein carbonylation and elevated malondialdehyde (MDA) levels
were seen. These are indicators of protein oxidation and lipid peroxidation, respec-
tively. Thus, considered in concert, these results lead us to suggest that Mt-CFX in-
terferes with the mitochondrial ETC process, inducing a significantly enhanced level
of oxidative stress. Transmission electron microscopy (TEM) revealed noticeable
morphology changes, including the loss of cristae, deformation, and mitochondrial
6
Chem 6, 1–12, June 11, 2020

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
swelling, when treated with Mt-CFX alone, whereas NAC pre-treatment also pre-
vented discernable mitochondrial damage (Figure S23). Mitochondrial de-energiza-
tion was found to mirror these observations, as the cellular ATP content was reduced
in the case of Mt-CFX treated cells, but not for those pre-treated with NAC
(Figure S24)
The nature and extent of Mt-CFX-mediated DNA was then explored. Oxidative DNA
damage induces the expression of base-excision repair proteins in both the nucleus
and mitochondria, whereas nucleotide-excision repair (NER) expression is localized
to the nucleus.25 We thus employed quantitative PCR (qPCR) to probe the expres-
sion levels of organelle-specific repair enzymes upon Mt-CFX treatment. DOX was
also studied since this clinically approved drug is known to induce ROS production.8
POLG, encoding a mitochondria-selective base-excision repair protein, was found
to be significantly increased following incubation with Mt-CFX only (Figure 3E).
DOX, on the other hand, elicited a clear upregulation of NER, as observed for
both ERCC1 and DDB2, whereas Mt-CFX did not cause significantly increased
expression levels. Under these conditions, treatment with CFX alone did not lead
to an upregulation in any of these repair enzymes.
The impact of Mt-CFX on cellular protein metabolism was investigated by moni-
toring the expression levels of genes encoding nuclear (GAPDH, glyceraldehyde
3-phosphate dehydrogenase) and mitochondrial (ND1, NADH-ubiquinone oxidore-
ductase chain 1) housekeeping proteins. Although DOX treatment produced a
reduction in GAPDH expression levels, no statistically alterations in GAPDH were
seen upon exposure to either Mt-CFX or CFX. Conversely, Mt-CFX was found to
interfere with mitochondrial function (Figure 3F). Furthermore, Mt-CFX was found
to reduce the ND1/GAPDH expression-level ratio in both a dose- and time-depen-
dent manner. However, a much smaller reduction in the mitochondrial/nuclear
expression-level ratio was observed for CFX (Figure S25).
The extent of drug-induced mitochondrial DNA mutation was then measured using a
TaqI restriction-site mutation assay.26 This restriction enzyme cleaves the double
stranded 50-TCGA-30 DNA sequence leading to DNA digestion. In contrast, restric-
tion-enzyme-resistant sites undergo PCR amplification. Mt-CFX-treated cells
showed an approximately 4.5-fold increase in resistance to TaqI restriction, whereas
no significant difference in the amount of TaqI resistance was observed in the case of
DOX-treated cells (Figure 3G).
We also investigated the location of predominant DNA mutagenicity for Mt-CFX, as
well as that for several conventional chemotherapeutic drugs, including DOX, temo-
zolomide (TMZ), camptothecin (CPT), cis-platin (CDDP), chlorambucil (CLB), and
fluorouracil (5-FU). Long-range qPCR (3,129 and 3,723 bp for nuclear and mitochon-
drial targets, respectively)27 revealed that all the conventional drugs induced a
considerable amount of nuclear DNA mutations, while having relatively little effect
on the mitochondrial genome. In contrast, Mt-CFX was found to promote mitochon-
drial DNA damage while engendering little in the way of nuclear DNA damage (Fig-
ure 3H). This mirrors the lack of increase in the PUMA and Noxa protein levels (cf. Fig-
ure S17). Taken in aggregate, these results are consistent with our core hypothesis,
namely that Mt-CFX localizes to the mitochondria and induces levels of ROS that are
increased to the point where mitochondrial (but not nuclear) DNA damage is seen.
Chem 6, 1–12, June 11, 2020
7

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Identification of Cellular Processes Affected by Mt-CFX
A DNA microarray study, comprising 197 genes, was conducted to determine the
impact of Mt-CFX on protein expression and pathway activation in MDA-MB-231
cells (Figures 4A and 4B). Several pathways related to programmed cell death,
such as apoptosis, ferroptosis, and necroptosis, were altered in a statistically signif-
icant manner. Genes corresponding to the biosynthesis and metabolism of amino
acids, fatty acids, and pyruvate were also affected.
Selected examples of gene expression levels were assayed using qPCR (Figure 4C).
On this basis, it is inferred that cellular metabolism is strongly affected by Mt-CFX, as
reflected in an increased expression of the transaminase, pyruvate synthesis, and
folate metabolism pathways (GPT2, ME1, and MTHFD2). Importantly, we also found
that several ROS-induced cellular responses were upregulated in the presence of
Mt-CFX. A mitochondrial aldehyde dehydrogenase (ALDH2) gene was significantly
overexpressed. Presumably, this overexpression reflects a need to mitigate the ef-
fects of increased protein carbonylation, which is a recognized form of ROS damage
(Figure 4D). GADD45A, encoding a protein involved in stimulating DNA excision
repair and inducing cell-cycle arrest, was also overexpressed by ꢁ12-fold (Figure 4E).
An approximately 20-fold overexpression of NLRP1 was also seen, which is taken as
evidence of cellular structure damage and the induction of the NLRP1 inflammasome
(Figure 4E). DDIT4 and SESN2, involved in inhibiting mTORC1 and thus enabling
autophagy, were overexpressed by ꢁ40-fold and ꢁ23-fold, respectively, indicating
a need for the degradation of damaged cellular components. Overexpression of
MAP1LC3B was also detected; this result suggests that damaged mitochondria
are being degraded via mitophagy and may indicate onset of ferroptosis (Figure 4F).
In Vivo Tumor-Growth Reduction by Mt-CFX and Bo-Mt-CFX
Finally, we investigated the in vivo activity of Mt-CFX in MDA-MB-231 tumor xeno-
graft-bearing mice. All agents (e.g., vehicle, CFX, Mt-CFX, and Bo-Mt-CFX) were
administered intravenously to these mice via tail vein injection. To assess bio-
localization in vivo, Bo-Mt-CFX was administered to mice and its fluorescence was
monitored. Fluorescence was detected at the tumor site and was found to persist
>48 h (Figure 5A). We confirmed that the inferred selectivity was indeed cancer spe-
cific by ex vivo analyses of organs harvested from mice euthanized 48 h after a single
Bo-Mt-CFX treatment. Nearly all fluorescence was confined to the tumor site,
whereas other organs, such as the heart and reticuloendothelial system, remained
unaffected (Figure 5B).
Chemotherapeutic intervention was investigated using a 3-week regimen involving
1-dose per week treatment with 5 mmol kgꢀ1 CFX, Mt-CFX, Bo-Mt-CFX, or vehicle
alone. The study revealed a statistically significantly reduction in tumor growth in
mice treated with Bo-Mt-CFX or Mt-CFX from the sixth week onward (Figure 5C).
The experiment was terminated at the 12th week, when the tumors in the control
group reached 1,000 mm3. The reduction in tumor burden produced by Bo-Mt-
CFX or Mt-CFX was mirrored in the excised tumor weights (Figures 5E and 5F).
This treatment regime was well tolerated by the mice, and no significant differences
in animal body weights were observed during the course of treatment (Figure 5D).
Liver-function tests at the experimental endpoint revealed no significant differences
relative to the outset, and the overall aspartate transaminase (AST) and alanine trans-
aminase (ALT) ratios of all mice were within the normal range.28
Tissue samples from the four treatment groups were subjected to immunohisto-
chemistry. Cells with cleaved caspase were clearly more prevalent in the Mt-CFX
8
Chem 6, 1–12, June 11, 2020

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 4. Identification of Cellular Processes Affected by Mt-CFX
(A) Heatmap representing relative expression levels of the differentially expressed genes (DEGs) to control versus 10 mM CFX and Mt-CFX.
(B) KEGG pathway enrichment analysis of the 197 DEGs.
(C–F) qPCR gene expression levels of selected genes in cells MDA-MB-231 cells treated with 10 mM Mt-CFX, CFX, and a control sample. (C) Cellular
metabolism. (D) Mitochondrial detoxification. (E) Cellular repair and inflammasome induction. (F) Autophagy and mitophagy.
Data are represented as mean G SEM (n = 3 in C–F). Statistical significance in (C)–(F) was determined by a one-way ANOVA test with a post-hoc
Bonferroni test. Different letters (e.g., a–d) signify data that are statistically different (p < 0.05).
Chem 6, 1–12, June 11, 2020
9

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
Figure 5. In Vivo Tumor Growth Reduction by Mt-CFX and Bo-Mt-CFX
(A) In vivo images of an MDA-MB-231 xenograft mouse 1, 24, and 48 h after intravenous tail-vein injection of a single dose of 0.5 mmol kgꢀ1 Bo-Mt-CFX
(excitation at 550 nm; emission at 650 nm).
(B) Ex vivo imaging of excised tumors and organs 48 h after injection of a single dose of 0.5 mmol kgꢀ1 Bo-Mt-CFX or vehicle only (excitation at 550 nm;
emission at 650 nm).
(C) In vivo tumor volume determination (1/2 3 length 3 width2) of mice treated with 0.5 mmol kgꢀ1 CFX, Mt-CFX, Bo-Mt-CFX, or vehicle alone once a
week for 3 weeks.
(D) Body weight of mice during the treatment regime.
(E) Excised tumors at the treatment endpoint.
(F) Excised tumor weight per treatment group.
(G and H) Blood-serum AST (G) and ALT (H) activity levels as determined by a colorimetric assay.
(I) Immunohistochemistry (cleaved caspase 3 and Ki-67) of representative tumor tissue slices of the different treatment groups.
(J) H&E staining of representative tissue slices of the different treatment groups.
Data are represented as mean G SEM (n = 4 mice and n = 8 tumors per group in C, D, and F; n = 4 mice in G and H). Statistical significance was determined
by a one-way ANOVA test with a post-hoc Bonferroni test. Different letters (e.g., a–d) signify datasets that are statistically different (p < 0.05).
and Bo-Mt-CFX groups, whereas a Ki-67 cell proliferation also revealed a dramatic
reduction in cell proliferation in these two treatment groups (Figure 5E). Finally, tis-
sue samples from tumor, liver, heart, spleen, and kidneys were subject to
10
Chem 6, 1–12, June 11, 2020

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
hematoxylin and eosin (H&E) staining. These studies revealed statistically significant
differences in the histology for the tumor samples obtained from the mice treated
with Mt-CFX or Bo-Mt-CFX relative to controls. Importantly no obvious signs of tis-
sue damage were found in any of the other samples subject to analysis (Figure 5E).
Conclusion
On the basis of the findings presented here, we propose that Mt-CFX acts as a mi-
tochondrially targeted chemotherapeutic drug. Mt-CFX was found to have inherent
selectivity toward cancer cells. The use of the fluorescent conjugate Bo-Mt-CFX re-
vealed mitochondrial localization, whereas an oxidative stress response was seen
when MDA-MB-231 cells were treated with either Mt-CFX system. Mt-CFX was
found to interfere with the mitochondrial ETC, inducing significantly enhanced levels
of ROS. This was reflected in increased protein carbonylation, lipid peroxidation,
and oxidative DNA damage in vitro. Moreover, Mt-CFX was found to cause a dra-
matic decrease in nuclear DNA damage as compared with DOX, TMZ, CPT,
CDDP, CLB, and 5-FU. Bo-Mt-CFX was retained in tumor xenografts as inferred
from fluorescent imaging, whereas treatment of xenograft-bearing mice with well-
supported doses of Mt-CFX and Bo-Mt-CFX resulted in reduced cancer burden.
We thus suggest that rerouting CFX and potentially other approved antibiotics to
the mitochondria could provide a new approach to generating small molecule che-
motherapeutics. Support for this notion comes from the finding that the present sys-
tem appears to be free of many of the genotypic-based side effects that plague the
majority of chemotherapeutics in current use.
EXPERIMENTAL PROCEDURES
The full experimental procedures are provided in the Supplemental Information.
DATA AND CODE AVAILABILITY
The accession number for the data of the microarray gene expression profiling re-
ported in this paper is GEO: GSE137679.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2020.03.004.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of Korea (NRF)
(2018R1A2A1A05020236 to S.-G.C. and CRI project no. 2018R1A3B1052702 to
J.S.K.) and funded by the Basic Science Research Program of the NRF
(2017R1D1A1B03032561 to P.V. and 2017R1D1A1B03030062 to M.W.) and the
Ministry of Education and the Korea Research Fellowship Program of the NRF
(2016H1D3A1938052 to P.V.). K.S. is a recipient of the Global PhD Fellowship
(GPF) program, funded by the NRF (2014H1A2A1020978). Funding from
the National Cancer Institute of the United States (RO1 CA68682 to J.L.S. and R15
CA232765 to J.F.A.) for the support in Austin is acknowledged.
AUTHOR CONTRIBUTIONS
K.S. contributed to synthesis, characterization, and solution experiments; M.W. and
K.-P.K. conducted biological experiments; M.W. performed statistical analyses; P.V.,
K.S., M.W., J.F.A., and J.L.S. contributed to writing, review, and editing; K.S., M.W.,
S.-G.C., P.V., J.F.A., J.L.S., and J.S.K. received study-enabling funding; K.S., P.V.,
and J.S.K. contributed to initial project conception; S.-G.C., J.L.S., P.V., J.F.A.,
Chem 6, 1–12, June 11, 2020
11

Please cite this article in press as: Sunwoo et al., Mitochondrial Relocation of a Common Synthetic Antibiotic: A Non-genotoxic Approach to
Cancer Therapy, Chem (2020), https://doi.org/10.1016/j.chempr.2020.03.004
and J.S.K. contributed to supervision and project development. All authors proof-
read, commented on, and approved the final version of the manuscript.
DECLARATION OF INTERESTS
Mt-CFX and analogs as non-genotoxic anticancer agents are the subject of a
pending patent application, filed by Korea University, with J.S.K, K.S., P.V., and
M.W. named as inventors. J.L.S. holds a part-time summer position at Shanghai
University.
Received: September 22, 2019
Revised: December 2, 2019
Accepted: March 2, 2020
Published: March 25, 2020
REFERENCES
1. Mehlen, P., and Puisieux, A. (2006). Metastasis:
a question of life or death. Nat. Rev. Cancer 6,
449–458.
10. Kalghatgi, S., Spina, C.S., Costello, J.C., Liesa,
M., Morones-Ramirez, J.R., Slomovic, S.,
Molina, A., Shirihai, O.S., and Collins, J.J.
(2013). Bactericidal antibiotics induce
mitochondrial dysfunction and oxidative
damage in mammalian cells. Sci. Transl. Med.
5, 192ra85.
conjugates as targeted cancer theranostics.
Chem. Soc. Rev. 47, 28–52.
20. Zhang, R., Xu, Y., Zhang, Y., Kim, H.S., Sharma,
A., Gao, J., Yang, G., Kim, J.S., and Sun, Y.
(2019). Rational design of a multifunctional
molecular dye for dual-modal NIR-II/
2. Grosse, Y., Baan, R., Straif, K., Secretan, B., El
Ghissassi, F., Bouvard, V., Benbrahim-Tallaa, L.,
Guha, N., Galichet, L., and Cogliano, V.; WHO
International Agency for Research on Cancer
Monograph Working Group (2009). A review of
human carcinogens—part A: pharmaceuticals.
Lancet Oncol. 10, 13–14.
photoacoustic imaging and photothermal
therapy. Chem. Sci. 10, 8348–8353.
11. DeVita, V.T., Jr., and Chu, E. (2008). A history of
cancer chemotherapy. Cancer Res. 68, 8643–
8653.
21. Sun, Y., Ding, F., Zhou, Z., Li, C., Pu, M., Xu, Y.,
Zhan, Y., Lu, X., Li, H., Yang, G., et al. (2019).
Rhomboidal Pt(II) metallacycle-based NIR-II
theranostic nanoprobe for tumor diagnosis
and image-guided therapy. Proc. Natl. Acad.
Sci. USA 116, 1968–1973.
3. Eichenauer, D.A., Thielen, I., Haverkamp, H.,
Franklin, J., Behringer, K., Halbsguth, T.,
Klimm, B., Diehl, V., Sasse, S., Rothe, A., et al.
(2014). Therapy-related acute myeloid
leukemia and myelodysplastic syndromes in
patients with Hodgkin lymphoma: a report
from the German Hodgkin study group. Blood
123, 1658–1664.
12. Galm, U., Hager, M.H., Van Lanen, S.G., Ju, J.,
Thorson, J.S., and Shen, B. (2005). Antitumor
antibiotics: bleomycin, enediynes, and
mitomycin. Chem. Rev. 105, 739–758.
13. Tacar, O., Sriamornsak, P., and Dass, C.R.
(2013). Doxorubicin: an update on anticancer
molecular action, toxicity and novel drug
delivery systems. J. Pharm. Pharmacol. 65,
157–170.
22. Sun, Y., Ding, F., Chen, Z., Zhang, R., Li, C., Xu,
Y., Zhang, Y., Ni, R., Li, X., Yang, G., et al. (2019).
Melanin-dot–mediated delivery of
4. Pui, C.H., Ribeiro, R.C., Hancock, M.L., Rivera,
G.K., Evans, W.E., Raimondi, S.C., Head, D.R.,
Behm, F.G., Mahmoud, M.H., and Sandlund, J.T.
(1991). Acute myeloidleukemia in children treated
with epipodophyllotoxins for acute lymphoblastic
leukemia. N. Engl. J. Med. 325, 1682–1687.
metallacycle for NIR-II/photoacoustic dual-
modal imaging-guided chemo-photothermal
synergistic therapy. Proc. Natl. Acad. Sci. USA
116, 16729–16735.
14. Fu, D., Calvo, J.A., and Samson, L.D. (2012).
Balancing repair and tolerance of DNA
damage caused by alkylating agents. Nat. Rev.
Cancer 12, 104–120.
23. Liou, G.Y., and Storz, P. (2010). Reactive oxygen
species in cancer. Free Radical Res. 44,
479–496.
5. Travis, L.B., Holowaty, E.J., Bergfeldt, K., Lynch,
C.F., Kohler, B.A., Wiklund, T., Curtis, R.E., Hall,
P., Andersson, M., Pukkala, E., et al. (1999). Risk
of leukemia after platinum-based
chemotherapy for ovarian cancer. N. Engl. J.
Med. 340, 351–357.
15. Esner, M., Graifer, D., Lleonart, M.E., and
Lyakhovich, A. (2017). Targeting cancer cells
through antibiotics-induced mitochondrial
dysfunction requires autophagy inhibition.
Cancer Lett. 384, 60–69.
24. Yu, J., and Zhang, L. (2008). PUMA, a potent
killer with or without p53. Oncogene 27,
S71–S83.
16. Alibek, K., Bekmurzayeva, A., Mussabekova, A.,
and Sultankulov, B. (2012). Using antimicrobial
adjuvant therapy in cancer treatment: a review.
Infect. Agent Cancer 7, 33.
25. Dianov, G.L., Souza-Pinto, N., Nyaga, S.G.,
Thybo, T., Stevnsner, T., and Bohr, V.A. (2001).
Base excision repair in nuclear and
6. Bhatia, S., Robison, L.L., Oberlin, O.,
Greenberg, M., Bunin, G., Fossati-Bellani, F.,
and Meadows, A.T. (1996). Breast cancer and
other second neoplasms after childhood
Hodgkin’s disease. N. Engl. J. Med. 334,
745–751.
mitochondrial DNA. Prog. Nucleic Acid Res.
Mol. Biol. 68, 285–297.
17. Zielonka, J., Joseph, J., Sikora, A., Hardy, M.,
Ouari, O., Vasquez-Vivar, J., Cheng, G., Lopez,
M., and Kalyanaraman, B. (2017). Mitochondria-
targeted triphenylphosphonium-based
26. Vermulst, M., Bielas, J.H., and Loeb, L.A. (2008).
Quantification of random mutations in the
mitochondrial genome. Methods 46, 263–268.
7. Glen, C.D., and Dubrova, Y.E. (2012). Exposure
to anticancer drugs can result in
transgenerational genomic instability in mice.
Proc. Natl. Acad. Sci. USA 109, 2984–2988.
compounds: syntheses, mechanisms of action,
and therapeutic and diagnostic applications.
Chem. Rev. 117, 10043–10120.
27. Evans, S.O., Jameson, M.B., Cursons, R.T.,
Peters, L.M., Bird, S., and Jacobson, G.M.
(2016). Development of a qPCR method to
measure mitochondrial and genomic DNA
damage with application to chemotherapy-
induced DNA damage and cryopreserved
cells. Biology (Basel) 5, 39–49.
8. Smith, M.A., Seibel, N.L., Altekruse, S.F., Ries,
L.A.G., Melbert, D.L., O’Leary, M., Smith, F.O.,
and Reaman, G.H. (2010). Outcomes for
children and adolescents with cancer:
challenges for the twenty-first century. J. Clin.
Oncol. 28, 2625–2634.
18. Samanta, S., He, Y., Sharma, A., Kim, J., Pan,
W.H., Yang, Z.G., Li, J., Yan, W., Liu, L.W., Qu,
J., and Kim, J.S. (2019). Fluorescent probes for
nanoscopic imaging of mitochondria. Chem 5,
1697–1726.
28. Sher, Y., and Hung, M. (2013). Blood AST, ALT
and urea/BUN level analysis. Bio-protocol 3,
e931.
9. Dyall, S.D., Brown, M.T., and Johnson, P.J.
(2004). Ancient invasions: from endosymbionts
to organelles. Science 304, 253–257.
19. Lee, M.H., Sharma, A., Chang, M.J., Lee, J.,
Son, S., Sessler, J.L., Kang, C., and Kim, J.S.
(2018). Fluorogenic reaction-based prodrug
12
Chem 6, 1–12, June 11, 2020