An Ethacrynic Acid-Brominated BODIPY Photosensitizer (EA-BPS) Construct Enhances the Lethality of Reactive Oxygen Species in Hypoxic Tumor-Targeted Photodynamic Therapy

Article abstract of DOI:10.1002/anie.202012687

Despite being a clinically approved intervention for cancer, photodynamic therapy (PDT) still suffers from limitations. Prime among these is a therapeutic response that is mostly oxygen dependent. This limits the utility of PDT in treating hypoxic tumors

Full text of DOI:10.1002/anie.202012687

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: An Ethacrynic Acid-Brominated BODIPY Photosensitizer
(EA-BPS) Construct Enhances the Lethality of Reactive Oxygen
Species in Hypoxic Tumor-Targeted Photodynamic Therapy
Authors: Jonathan L. Sessler, Miae Won, Seyoung Koo, Hao Li, Jin
Yong Lee, Amit Sharma, and Jong Sung Kim
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202012687
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10.1002/anie.202012687
Angewandte Chemie International Edition
RESEARCH ARTICLE
An Ethacrynic Acid-Brominated BODIPY Photosensitizer
(EA-BPS) Construct Enhances the Lethality of Reactive Oxygen
Species in Hypoxic Tumor-Targeted Photodynamic Therapy
Miae Won,[a]† Seyoung Koo, [a]† Hao Li, [b]† Jonathan L. Sessler,*[c] Jin Yong Lee,*[b] Amit Sharma,*[d] and
Jong Seung Kim*[a]
Abstract: Despite being a clinically approved intervention for
cancer, photodynamic therapy (PDT) still suffers from limitations.
Prime among these is a therapeutic response that is mostly
oxygen dependent. This limits the utility of PDT in treating hypoxic
tumors since lower levels of cytotoxic reactive oxygen species
(ROS) are generated in regions of low oxygen tension.
Glutathione-pi (GST-pi) is a key enzyme that militates against
ROS-mediated apoptosis. We report here a new construct, EA-
BPS, that contains both a brominated BODIPY photosensitizer
(BPS) and an ethacrynic acid (EA) GST-pi inhibitor.
Photoirradiation of EA-BPS induces a synergistic antitumor effect
that results from the combination of ROS production and GST-pi
inhibition. Relative to BPS alone, an enhanced cell-killing effect is
seen under hypoxic conditions both in vitro and in vivo. We
conclude that by making better use of the available oxygen in
tumor environments, improved therapeutic PDT outcomes should
be achievable even under hypoxic conditions.
considerable body of effort has been devoted to improving the
performance of PDT photosensitizers, particularly in the context
of cancer therapy.[7] For example, to avoid the low penetration
depth problem associated with the excitation laser,[8] PS with
near-infrared excitation features[9-10] have been developed, as
have a variety of upconverting nanomaterials.[11-12] Moreover,
both chemiluminescence and bioluminescence have been used
as excitation light sources.[13-15] To improve further PDT efficacy
with minimal undesired side effects, the targeted delivery of PS to
tumor parenchymal cells and subcellular organelle has also been
explored.[16-18] Unfortunately, PDT is an oxygen-dependent
therapy and an adequate supply of oxygen at the tumor site is
necessary to generate sufficient ROS to induce a robust
therapeutic effect. This oxygen dependence limits the utility of
PDT in the hypoxic microenvironments of solid tumors.[19-20] To
avoid such limitations, hyperbaric oxygen (O2) has been used
clinically to augment endogenous O2 supplies in the blood and in
tumor tissues.[21-23] Such approaches are, however, not without
risk and can, for instance, lead to O2-derived toxicity effects in the
central nervous system and in the lungs.[24-25] Various O2 carriers
and in situ generators of O2 have also been explored in an effort
to enhance the effectiveness of PDT.[26-29] However, such
strategies can actually lead to the overproduction of ROS under
conditions of photoirradiation, thus engendering various side
effects.[30] We suggest that an attractive alternative would involve
making the ROS produced during PDT more fatal to malignant
cells. This can be accomplished by hampering the endogenous
antioxidant-defense systems inherent to cancer cells.
Cancer cells rely on several protective mechanisms to reduce
the cellular damage from extracellular oxidants. Inhibiting one or
more of these mechanisms could serve to enhance the effective
toxicity of the limited ROS produced under conditions of hypoxic
PDT. In this study we set out to test the viability of this hypothesis
by targeting glutathione S-transferase-pi (GST-pi). GSTs are
classified as phase II detoxifying enzymes that play a significant
role in homeostasis and intracellular signal transduction.[31-33]
Cytosolic GST-pi participates in various cellular processes,
including cellular proliferation, through its non-enzymatic
activity.[34-36] Several reports have highlighted a direct correlation
between elevated levels of GST-pi and resistance to anticancer
therapies.[37-38] The combination of PDT with GST-pi gene
silencing was used successfully in the case of hepatic carcinoma
cells.[39] Also, therole of GST-pi in modulating the response of
cancer cells to PDT has been studied.[40-42] However, these
studies are mostly confined to investigations at the cellular level.
The complexity of the in vivo environment makes it inherently
challenging to translate results from in vitro work into more
advanced animal studies. Issues of deliver, various protective
mechanisms, the presence of potential inhibitory agents, and a
variety of other factors can influence the spatial distribution and
pharmacokinetics of an administrated agent. These factors,
individually or collectively can limit the actual PDT performance.
The diuretic drug ethacrynic acid (EA) is widely employed for
high blood pressure and proscribed to patients in a severe
edematous state.[43] EA is known to form a covalent Michael
Photodynamic therapy (PDT) is a clinically established oncologic
intervention that relies on the light-mediated activation of
photosensitizers (PS) to produce reactive oxygen species (ROS).
Many, but not all, PS act to convert molecular oxygen from its
triplet ground state to its singlet excited state under conditions of
photoirradiation. Singlet oxygen is a recognized ROS that can
promote oxidation of key cellular macromolecules and trigger
tumor cell death.[1] PDT enjoys various merits, such as minimal
invasiveness, high regioselectivity, low systematic toxicity, and
reduced long-term morbidity in certain cancer types.[2-3] PDT is
also being used as an adjunctive treatment after surgical
resection where it serves to reduce residual tumor burden.[4-6]
A
[a] Dr M. Won, Dr S. Koo, Prof J. S. Kim
Department of Chemistry
Korea University
Seoul 02841, Korea
E-mail: jongskim@korea.ac.kr
[b] H. Li, Prof J. Y. Lee
Department of Chemistry
Sungkyunkwan University
Suwon 16419, Korea
E-mail: jinylee@skku.edu
[c] Prof J. L. Sessler
Department of Chemistry
University of Texas at Austin
Austin, Texas 78712-1224, United States
E-mail: sessler@cm.utexas.edu
[d] Dr. A. Sharma
CSIR - Central Scientific Instruments Organisation, Sector-30 C
Chandigarh 160030, India
E-mail: amitorg83@gmail.com
[†] These authors contributed equally to this work
Supporting information and the ORCID identification numbers for the
authors of this article can be found under:
https://doi.org/10.1002/anie.xxxxxxxxx.
1
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10.1002/anie.202012687
Angewandte Chemie International Edition
RESEARCH ARTICLE
structures proposed for all new compounds (Supporting
Information, SI, Figure S1-S17).
Figure 1. Structure of EA-BPS and schematic illustration of the synergistic
multi-functional benefit to PDT it is expected to provide including under
hypoxic conditions.
addition complex with reduced glutathione (GSH) through the
cysteine side chain. This serves to lower the GSH levels and
reduce resistance to oxidative stress in cancer cells.[44] EA has
been explored as an anticancer enhancing agent in combination
with several chemotherapeutic drugs to improve the treatment
outcomes in drug resistance tumors.[45-48] The EA-GSH conjugate
that results from reaction of EA with GSH is relatively non-toxic;
however, it has a strong inhibitory effect on the activity of GSTs.[49]
Because cellular damage by ROS is reduced under conditions of
elevated GST-pi activity,[50] we envisaged that GST-pi inhibition in
conjunction with PDT would produce a synergistic effect and
impart greater lethality to the ROS produced through PDT, even
under hypoxic conditions.
Here we show that EA, when covalently linked to a brominated
BODIPY-based photosensitizer (BPS) to give conjugate EA-BPS,
enhances the anticancer performance of PDT under hypoxic
conditions. EA-BPS was designed to deliver EA to scavenge GSH
and simultaneously downregulate GST-pi activity as shown
schematically in Figure 1. Our experimental results provide
support for the conclusion that EA conjugated to BPS exerts a
synergistic effect with PDT both in vitro in MDA-MB-231 cells and
in a corresponding animal tumor model. In vitro mechanistic
studies revealed that as compared to various controls, EA-BPS
significantly reduces the GST-pi activity through a presumed
lysosomal degradation pathway and enhances lipid peroxidation.
EA-BPS also improves PDT-based cytotoxicity under hypoxic
conditions and its use regulates the protein expression level of
related pro-apoptotic genes. In vivo studies, involving the use of
a MDA-MB-231 tumor xenograft mouse model, revealed a
superior therapeutic effect relative to BPS alone, thus supporting
our hypothesis that inhibition of GST-pi activity can lead to PDT-
based protocols that exploit relatively effectively the limited
oxygen available in tumor microenvironments under hypoxic
conditions.
Scheme 1. Synthesis of R-BPS and EA-BPS.
We next examined the absorption and emission properties of
R-BPS and EA-BPS in dimethyl sulfoxide (DMSO). An intense
absorbance band corresponding to the brominated BODIPY core
(BPS) was observed for both R-BPS (molar extinction coefficient,
ε = 6.45 × 104 M-1cm-1 at 667 nm) and EA-BPS (ε = 7.35 × 104 M-
1cm-1 at 667 nm) (Figure S18). The fluorescence spectra of R-
BPS and EA-BPS showed an emission peak centered at 698 nm
(Figure S18b). The fluorescence emission of both R-BPS and EA-
BPS exhibited hypsochromic shifts (up to 40 nm) with increasing
the solvent polarity (p) (tetrahydrofuran, p: 4.0 < acetone, p: 5.1 =
methanol, p: 5.1 < acetonitrile, p: 5.8) (Figure S19). We also
tested the absorbance and emission properties of R-BPS and EA-
BPS in phosphate buffered saline (PBS) (10 mM, pH 7.4, 1 %
Triton X100). Both compounds showed similar absorption (671
nm) and emission (693 nm) characteristics, as determined in
organic solvents (Figure S18). However, it is important to
appreciate that although emissive, these conjugates incorporate
heavy atoms (bromine) in the photosensitizer subunit; this serves
to lower the fluorescence quantum yield by promoting inter-
system crossing (ISC) to the excited triplet state.[51]
We next explored whether the enhancement in the ISC rates
expected for R-BPS and EA-BPS would translate into features
attractive for PDT. Toward this end 1,3-diphenylisobenzofuran
(DPBF) was used as a chemical probe for singlet oxygen (Type 2
ROS) under photosensitizing conditions.[52] Light irradiation of a
mixture of EA-BPS and DPBF with 660 nm light showed a sharp
decrease in the content of DPBF over 30 min (Figure 2A and 2C).
Similar results were obtained for R-BPS under otherwise identical
experiment conditions, while in the absence of these BPS
derivatives, the DPBF content remained unchanged (Figures 2C
and S20). The singlet oxygen quantum yields (ΦΔ) for R-BPS and
EA-BPS were determined using a protocol that relies on
methylene blue (MB) as the standard (MB: ΦΔ = 0.52 in DMSO)[16]
giving ΦΔ values of 0.14 and 0.13 for R-BPS and EA-BPS,
respectively (Figure S20).
The synthesis of molecular conjugates, EA-BPS and R-BPS
is shown in Scheme 1. Briefly, the diol protected acid intermediate
1 was coupled with 2-bromoethanol in the presence of 1-ethyl-
3(3-dimethylaminopropyl)carbodiimde hydrochloride (EDC·HCl)
and N,N’-dimethylaminopyridine (DMAP) in dimethylformamide
(DMF) to give compound 2. Subsequent diol deprotection and
coupling with ethacrynic acid (EA) in the presence of EDC·HCl,
DMAP, and DMF furnished compound 4. Compound 4 was then
treated with sodium azide in DMF to give compound 5. In parallel,
Separately, 2,7-dichlorodihydrofluorescein diacetate (DCFH-
DA) was used as a ROS indicator. DCFH has been reported to
behave as a broad ROS sensor (primarily for Type 1 ROS) that
reacts with hydroxyl radicals (HO·), peroxyl radicals (ROO·), and
compound 10 was obtained from compound
dimethylpyrrole by following
reported procedure.[16]
Intermediate R-BPS was then obtained by means of
8 and 2,4-
a
a
Knoevenagel condensation between 7 and 10. Finally, the target
compound EA-BPS was prepared in good yield via a Cu(I)-
mediated alkyne-azo click reaction between intermediates 5 and
R-BPS in methanol/dichloromethane (10:1). The combined
results from nuclear magnetic resonance (NMR) spectroscopy
and mass spectrometry (MS) proved consistent with the
·-
to a lesser extent superoxide radicals (O2 ), to give a highly
fluorescent daughter product, 2’,7’–dichlorofluorescein (DCF).[53]
Both R-BPS and EA-BPS produced
a
time-dependent
fluorescence enhancement at 523 nm ascribed to DCF upon
irradiation (660 nm) for an extended time (Figure 2B and 2D). In
contrast, when DCFH-DA was subject to photoirradiation in the
2
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10.1002/anie.202012687
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RESEARCH ARTICLE
absence of R-BPS or EA-BPS the fluorescence increases at 523
nm proved negligible (Figure S21). Moreover, R-BPS and EA-
BPS gave rise to fluorescence spectral changes of
dihydrorhodamine 123 (DHR123) and hydroxyl phenyl fluorescein
(HPF), which are specific indicators of O2·- and HO·, respectively,
upon light irradiation (Figure S22). Collectively, these results
provide support for the contention that under photosensitizing
conditions, R-BPS and EA-BPS are capable of promoting ROS
production (in both DMSO and aqueous media) (Figures S20, S21
and S22). We thus considered them worthy of further study.
To identify the active sites and obtain insights into the
presumed interactions between GST-pi and EA-BPS-(GSH)2 (a
molecule conjugate presumably formed between EA-BPS and
GSH), docking and molecular dynamics (MD) simulations were
performed (Figure 2E and 2F). The binding free energy between
GST-pi and EA-BPS-(GSH)2 was calculated to be -65.1 kcal/mol
from the MD simulation. Five residues (Chain A: F7, W37 and
Y107; Chain B: Y107 and A120) were found to play an important
role in the active site, as reflected in the associated deconstructed
free energy values of -2.9, -2.4, -4.2, -1.9 and -2.3 kcal/mol,
respectively. It is worth noting that Y107 residues are present in
the active site, a finding that leads us to infer that EA-BPS-(GSH)2
is interacting with the GST-pi enzyme pocket through both
ethacrynic subunits rather than just one (Figure S23).
The stability of agents in biological milieus is an important
issue that must be considered in the context of their therapeutic
development. Given the fact that EA-BPS has an ester functional
group, which could lead to low stability in biological media (e.g.,
be reactive towards esterases), we monitored the time-dependent
change in composition of EA-BPS, under various solution phase
conditions using high performance liquid chromatography (HPLC).
As indicated in Figure S24, a peak with retention time of 24.6 min,
corresponding to EA-BPS, remained unchanged after incubation
for 6 h in 10 mM PBS, RPMI 1640, and RPMI 1640 containing 10%
FBS. Furthermore, EA-BPS was not found to be appreciably
hydrolyzed in the presence of esterase while EA-ester, the
carboxylic ethyl ester form of EA, was quickly hydrolyzed under
these same conditions (Scheme S1 and Figure S25). On this
basis, we conclude that EA-BPS was sufficiently stable so as to
allow for further biological studies.
To test whether GST-pi inhibition leads to an improvement in
PDT efficacy, the cellular uptake behavior and cytotoxicity of R-
BPS and EA-BPS were investigated. First, the endogenous
expression of GST-pi in the human breast cancer cell lines the
MDA-MB-231 (GST-pi positive) and MCF7 (GST-pi negative)
were confirmed using Western blot analyses. As expected, the
GST-pi expression in MDA-MB-231 proved significantly higher
than in the MCF7 cell line (cf. Figure S26). Because of their
differential expression behavior, these two cell lines were
selected for further experiments.
Figure 2. Photodynamic and GST-pi binding properties of EA-BPS.
Time-dependent (a) absorption spectral changes of DPBF (1O2
detection) and (b) fluorescence spectral changes of DCFH (ROS
detection) upon 660 nm light irradiation (Xe-lamp, slit width 15/1.5 nm)
of EA-BPS. (c) absorption intensity change of DPBF at 412 nm and (d)
fluorescence intensity change of DCFH at 535 nm in the presence of
light irradiation (LED lamp, 100 mW/cm2) with EA-BPS, R-BPS or
without BPSs. Binding study of GST-pi and GSH-EA-BPS. (e)
Calculated GST-pi binding sites. (f) The free energy of binding of each
residue of GST-pi with GSH-EA-BPS. Bar graph data are presented as
the mean, while the error bars indicate the standard deviation from the
mean (n = 3). **p<0.001.
Next, we performed flow cytometry analyses to evaluate the
cellular uptake of compounds R-BPS and EA-BPS in the MDA-
MB-231 and MCF7 cell lines. As shown in Figures 3A and S27,
EA-BPS was taken up more effectively than R-BPS. The uptake
proved time dependent in both cell lines, with the uptake being
greater for the MDA-MB-231 cell line. Considering the structural
features of these two compounds, the greater uptake of EA-BPS
may be due to its more lipophilic character as compared to R-
BPS. It is known that the lipid bilayer of the cell membrane in
cancer cells facilitates the transport of lipophilic agents.[54] The
higher retention EA-BPS in the MDA-MB-231 cell line as
compared to the MCF7 cell line may also reflect a possible
covalent interaction between GSH and the EA subunit and further
interactions of the resulting adduct with GST-pi.[55]
To assess the therapeutic potential of R-BPS and EA-BPS,
cell viability assays were performed without irradiation (test of
dark toxicity) using the MCF7 and MDA-MB-231 cancer cell lines.
While R-BPS was not appreciably cytotoxic up to 50 µM in both
cell lines, the same concentration of EA-BPS produced 21% and
42% reduction in the cell viabilities in the MCF7 and MDA-MB-
231 cell lines, respectively (Figure S28). Upon photoirradiation
(660 nm, LED lamp, 100 mW/cm2) for 10 min, the toxicity
difference (R-BPS vs.EA-BPS or MCF7 vs. MDA-MB-231) was
enhanced even when 5 µM concentrations of R-BPS and EA-
BPS were used (Figure S29). EA-BPS led to a significant
reduction of cell viability in MCF7 cells (31%) and MDA-MB-231
(70%), respectively; however, R-BPS produced only a slight
decrease in the cell viability in both tested cell lines under similar
conditions. These results support our core hypothesis, namely
that EA-based blocking of GST-pi activity under conditions of
photoirradiation should translate into increased cytotoxicity for
cells that are particularly reliant on the GST-pi antioxidant system.
We next investigated the ability of R-BPS and EA-BPS to
produce ROS inside MDA-MB-231 cancer cells under conditions
of photoirradiation. The cells were incubated separately with R-
BPS, EA-BPS, and 1% DMSO as a control in the presence of an
oxidant-sensitive ROS probe (DCFH-DA, green). Upon
photoirradiation (660 nm, 100 mW/cm2, 10 min), an intense green
3
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10.1002/anie.202012687
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RESEARCH ARTICLE
Figure 3. PDT sensitizer potential of EA-BPS. (A) Time-dependent uptake of EA-BPS (5 µM) into MDA-MB-231 cells. (B) The effect of NAC on
the ROS burden in R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h) and control (1% DMSO for 12 h) challenged cells in the absence and presence
of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (C) Singlet oxygen sensor green (SOSG) fluorescence (5 μM for 30 min) in MDA-
MB-231 cells incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h) and the absence of a BPS (control, 1% DMSO) in the absence and
presence of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (D) Dihydroethidium (DHE) fluorescence (30 μM for 30 min) in MDA-
MB-231 cells incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h) and the absence of a BPS (control, 1% DMSO) in the absence and
presence of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (E) Hydroxy phenyl fluorescein (HPF) fluorescence (10 μM for 30 min)
in MDA-MB-231 cells incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h) and the absence of a BPS (control, 1% DMSO) in the absence
and presence of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (F) Intracellular thiol-tracker fluorescence (10 μM for 30 min) in
MDA-MB-231 cells incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h) and the absence of a BPS (control, 1% DMSO) in the absence
and presence of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. Data are presented as the mean, while the error bars indicate the
standard deviation from the mean (n = 9). Statistical significance was determined using a one-way ANOVA test with a post-hoc Bonferroni test.
Different letters signify data that are statistically distinct (p < 0.05). Scale bar = 20 μm.
fluorescent signal corresponding to the formation of DCF (the
oxidized, fluorescent form of the DCFH-DA probe) was observed
from cells incubated with R-BPS and EA-BPS. Quantitative
analyses revealed that the fluorescence signal intensity in the
cells treated with EA-BPS was greater than that in
the corresponding R-BPS group (Figure 3B). In contrast, no
fluorescence signal was observed in the control group (1%
DMSO). Pre-incubation of the cells with N-acetylcysteine (NAC,
an effective ROS quencher)[56] resulted in a diminished green
fluorescence when the experiments were carried out under
otherwise identical experimental conditions. Based on these
results, we conclude that R-BPS and EA-BPS trigger intracellular
ROS production upon light activation and that the extent of ROS
generation was greater in the case of EA-BPS than R-BPS by
~1.4-fold (Figure 3B).
molecular oxygen (3O2) to reactive singlet oxygen (1O2))
mechanisms.[57-58] In an effort to ascertain which of these limiting
mechanisms might be dominant in the case of EA-BPS, we first
determined whether 1O2 was being produced in cells under
conditions of photoirradiation. For this analysis, a well-known
probe, Singlet Oxygen Sensor Green (SOSG), was used as the
indicator.[59] Illumination of cells incubated with R-BPS and EA-
BPS as above were characterized by a high level of green
fluorescence ascribed to the SOSG probe (Figure 3C). This
finding provides support for the contention that these two PS are
able to promote singlet oxygen formation.
To test whether R-BPS and EA-BPS are also able to generate
ROS through a Type I mechanism, dihydroethidium (DHE), a
widely used fluorescence assay for the detection of intracellular
O2 ,
˙− [60] was used as indicator. As can be seen from an inspection
Typically PDT photosensitizers are classified by whether they
promote light-mediated production of ROS through Type I (radical
ions formed by the abstraction of an electron/hydrogen atom from
a substrate by a photoexcited PS) or Type II (photoexcited PS
serving to promote the conversion of triplet ground state
of Figure 3D the fluorescence intensities ascribed to oxidized
DHE proved similar for the R-BPS or EA-BPS treated cells,
˙−
indicating O2 production upon irradiation. Analogous studies
were then carried out with hydroxyl phenyl fluorescein (HPF), a
chemosensor used for the detection of hydroxyl radical and
4
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10.1002/anie.202012687
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RESEARCH ARTICLE
To obtain further insights into the potential benefits of the EA
moiety present in EA-BPS, we monitored the levels of GSH
expression with and without photoirradiation using a thiol-tracker,
Thiol-trackerTM Violet.[65] In the absence of photoirradiation, a
modest (ca. 15%) decrease in the GSH levels in the EA-BPS
treated cells was observed, while the R-BPS treated cells showed
nearly the same levels as control (1% DMSO). A very different
situation was seen under conditions of photoirradiation. Here, a
7.3-fold reduction in the GSH levels was seen for the EA-BPS
treated cells compared to the R-BPS treated cells (Figure 3F). We
conclude that per our design expectations the EA moiety in EA-
BPS when combined with light serves to reduce the endogenous
GSH levels.
Next, to investigate the inhibitory effect of EA-BPS on GST-pi,
the enzymatic activity and protein levels of GST-pi were
measured (Figure 4A and 4B). To evaluate the effect of EA-BPS
conjugate, control experiments involving co-treatment of R-BPS
with EA were carried out. Given the presence of a carboxylic acid
functional group in EA, which might serve to limit permeability
across cellular membrane,[66-67] the carboxylic ethyl ester form of
EA (EA-ester) was prepared and tested (Scheme S1). As shown
in Figure 4A, photoirradiation of the EA-BPS treated group
resulted in a remarkable decrease in the GST-pi protein level. In
line with these results, EA-BPS when subject to photoirradiation,
served to reduce the GST-pi activity by 58% (Figure 4B). In
contrast, R-BPS and mixture of R-BPS and EA or EA-ester only
reduced the GST-pi activity by 5-20% under similar conditions
(Figure 4B).
Figure 4. Effect of EA-BPS with and without photoirradiation on
lysosomal degradation-dependent way. (A) Western blot analysis of
GST-pi expression in MDA-MB-231 cells incubated with R-BPS (5 µM
for 12 h), EA-BPS (5 µM for 12 h), EA (2 equiv.), EA-ester (2 equiv.)
and mixture of R-BPS (5 µM for 12 h) and EA or EA-ester (2 equiv.
for 12 h) in the absence and presence of photoirradiation (660 nm LED
lamp, 100 mW/cm2) for 10 min. (B) GST activity in MDA-MB-231 cells
incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h), EA (2
equiv.), EA-ester (2 equiv.) and mixture of R-BPS (5 µM for 12 h) and
EA or EA-ester (2 equiv. for 12 h) in the absence and presence of
photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (C) MDA
levels in MDA-MB-231 cells incubated with R-BPS (5 µM for 12 h),
EA-BPS (5 µM for 12 h), EA (2 equiv.), EA-ester (2 equiv.) and mixture
of R-BPS (5 µM for 12 h) and EA or EA-ester (2 equiv. for 12 h) in the
absence and presence of photoirradiation (660 nm LED lamp, 100
mW/cm2) for 10 min. (D) S-glutathionylation levels in MDA-MB-231
cells incubated with R-BPS (5 µM for 12 h), EA-BPS (5 µM for 12 h),
EA (2 equiv.), EA-ester (2 equiv.) and mixture of R-BPS (5 µM for 12
h) and EA or EA-ester (2 equiv. for 12 h) in the absence and presence
of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10 min. (E)
Western blot analysis of GST-pi expression in MDA-MB-231 cells
incubated with R-BPS and EA-BPS in the absence or upon treatment
of 10 µM chloroquine in the absence and presence of photoirradiation
(660 nm LED lamp, 100 mW/cm2) for 10 min. The plotted values are
the means, while error bars indicate the standard deviation from the
mean (n = 6). Statistical significance was determined using a one-way
ANOVA test with a post-hoc Bonferroni test. Different letters signify
data that are statistically distinct (p < 0.05).
Figure 5. Hypoxia induced apoptotic cell death by EA-BPS. (A) Cell
viability of MDA-MB-231 cells incubated with R-BPS, EA-BPS, mixture
of R-BPS and EA or EA under normoxia (21% O2) and hypoxia (2%
O2) in the absence and presence of photoirradiation (660 nm LED
lamp, 100 mW/cm2) for 10 min. (B) Cell population graph of MDA-MB-
231 treated with R-BPS and EA-BPS for 12 h in the absence and
presence of photoirradiation (660 nm LED lamp, 100 mW/cm2) for 10
min by flow cytometry. (C) Apoptotic cell death assay of R-BPS and
EA-BPS under 2% O2 (hypoxia) as determined by western blot
analysis. Values are the mean, while error bars indicate the standard
deviation from the mean (n
= 6). Statistical significance was
determined using a one-way ANOVA test with a post-hoc Bonferroni
test. Different letters signify data that are statistically distinct (p < 0.05).
peroxynitrite anions.[61] It was found that the oxidized HPF signal
from the EA-BPS treated cells was 3.6-fold higher than in the
case of R-BPS treated cells (Figure 3E), leading us to conclude
that hydroxyl (or peroxynitrite) radicals are being formed.
Although, in vivo the half-life of the hydroxyl radical is very short
(10-9 s),[62] due to its high reactivity, it is able to mediate the
oxidation of many macromolecules including carbohydrates, lipids,
and even amino acids.[63] Moreover, unlike superoxide, which can
be neutralized by superoxide dismutase, hydroxyl radicals are not
known to be eliminated via an enzymatic process.[64] Therefore,
the ability to produce hydroxyl (or peroxynitrite) radicals in
conjunction with other ROS could explain the greater cytotoxicity
seen for EA-BPS relative to R-BPS.
The effect of this reduction on downstream events was then
studied. It was previously reported that GST-pi facilitates the
detoxification and removal of lipid peroxides by activation of
peroxiredoxin VI (Prdx6), which has peroxidase activity.[68]
Appreciating this, malondialdehyde (MDA), one of the final
products of lipid peroxidation[69] was measured in the presence of
R-BPS and EA-BPS with and without photoirradiation using a
commercially available lipid peroxidation MDA assay kit (Figure
4C). Consistent with its effect on GST-pi activity, EA-BPS was
found to promote an increase in the MDA levels as compared to
R-BPS and mixtures of R-BPS and EA or EA-ester.
5
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10.1002/anie.202012687
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RESEARCH ARTICLE
GST-pi has also been reported to promote the S-
glutathionylation of damaged proteins.[70] The extent of protein S-
glutathionylated was thus also monitored in the presence of R-
BPS, EA-BPS and mixtures of R-BPS and EA or EA-ester under
conditions of photoirradiation. In accord with what would be
expected for a system that reduces GST-pi activity, EA-BPS was
found to lower the S-glutathionylated protein levels by roughly
50%, whereas the reduction seen in the case of R-BPS and
mixture of R-BPS and EA or EA-ester proved far more modest
(Figure 4D). These findings are consistent with a mode of action
for EA-BPS under conditions of photoirradiation that benefits from
the inhibition of innate detoxification processes normally exploited
by cancer cells.
To explore the mechanisms of GST-pi degradation mediated
by EA-BPS under conditions of photoirradiation, we pre-treated
MDA-MB-231 cells with 3 µM of chloroquine (CQ) (a lysosomal
inhibitor).[71] It was observed that LC3 (an autophagy marker)
protein expression was diminished in a statistically significant
manner under conditions of photoirradiation in the presence of
EA-BPS (Figure 4E). CQ treatment also induced GST-pi
expression. Collectively, these results provide support for the
notion that EA-BPS promotes a lysosomal degradation pathway
and inhibits GST-pi activity (Figure 4E). Overlap between the red
fluorescence ascribed to the BPS subunit and the green
fluorescence due to the LysoTracker® and MitoTracker® probes
revealed that after photoirradiation EA-BPS is not only localized
in lysosomes but also in the mitochondria (Figure S30).
To assess further the photo-induced toxicity of EA-BPS in
tumor micro-environments, MDA-MB-231 cells were pre-
incubated under normoxic and hypoxic conditions. The cells were
subsequently treated with R-BPS, EA-BPS, EA only, and EA in
conjunction with R-BPS with or without photoirradiation. As can
be seen from an inspection of Figure 5A, little appreciable toxicity
was seen when MDA-MB-231 cells treated with R-BPS, EA only,
or EA in combination with R-BPS were subject to photoirradiation
under hypoxic conditions. In contrast, remarkable cytotoxicity was
seen in the case of the cells treated with EA-BPS and then subject
to photoirradiation under both normoxic (21% O2) and hypoxic
conditions (2% O2).
Cell death signaling mechanisms for the cells treated with EA-
BPS under conditions of photoirradiation were investigated by
means of annexin V and western blot assays (Figure 5B and 5C).
An annexin-V/propidium iodide (PI) staining assay revealed that
EA-BPS under conditions of photoirradiation increased the
population of annexin V-positive apoptotic cells (to 50%)
compared to the other tested groups (Figures 5B and S31: ≤8%).
An increase in cleaved caspase-3 levels was also seen upon
photoirradiation under hypoxic conditions. This latter finding
supports the conclusion that treatment of MDA-MB-231 cells with
EA-BPS leads to reduced expression of the GST-pi protein
(Figure 5C) and that the observed improvements in therapeutic
efficacy can be ascribed to a hampering of the GST-pi mediated
endogenous antioxidant-defense system.
Figure 6. In vivo photodynamic effects and tumor regression seen in
MDA-MB-231 xenograft mouse models. (A) In vivo bodipy
fluorescence images of an MDA-MB-231 xenograft mouse 1, 24, and
48 h after intravenous tail-vein injection of a single dose of 5mg/ kg
EA-BPS (excitation at 660 nm; emission at 700 nm). (B) Ex vivo
images of excised tumors and organs recorded 48 h after injecting
single dose of 5mg/kg EA-BPS or vehicle alone (PBS containing 5%
DMSO). (C) In vivo tumor volumes (1/2 × length × width2) determined
in MDA-MB-231 xenograft mice treated 6 h after injecting with 5 mg/kg
R-BPS, EA-BPS or vehicle alone for 4 weeks (once a week) in the
absence and presence of photoirradiation (660 nm, 2 W/cm2, 10 min).
(D) Body weight of mice recorded during the treatment regime. (E)
H&E staining of representative tissue slices of the different treatment
groups at the study endpoint. (F) Immunohistochemistry (C. cas 3 and
Ki-67) of representative tumor tissue slices taken from the different
treatment groups. (G) Blood serum AST, ALT and creatinine activity
levels, as determined using a colorimetric assay. The values are the
means, while the error bars indicate s.e.m. (standard error of the
mean). Panels c, d, g: n = 5 mice per group, Statistical significance
was determined using a one-way ANOVA test with a post-hoc
Bonferroni test. Different letters signify data that are statistically
different (p < 0.05). The symbols + and - are used to denote the
presence and absence of photoirradiation.
treatment; this was true for both the EA-BPS test groups and the
various controls (Figure 6D).
MDA-MB-231 tumor xenograft-bearing mice were used to
test the efficacy of EA-BPS as a PDT photosensitizer in a model
hypoxic tumor microenvironment. In these studies EA-BPS (5
mg/kg in PBS containing 5% DMSO) was administrated
The in vivo phototherapeutic effects were further confirmed
by examining tumor and tissue sections from animals in the test
and control groups through hematoxylin & eosin (H&E) staining
and immunohistochemistry (IHC) assays. The H&E-stained tumor
sections taken from the xenograft mice treated with EA-BPS and
subject to photoirradiation showed characteristic apoptotic cells,
while no such effect was seen in other groups (Figure 6E).
Cleaved caspase-3 (red) levels were greater in the mice treated
with EA-BPS and subject to photoirradiation. Also, the Ki-67
expression levels (green) were downregulated, indicating a
dramatic reduction in cell proliferation in the EA-BPS treated mice
(Figures 6F and S34). No such effects were observed in the tumor
sections taken from animals treated with R-BPS or in the control
group (5% DMSO in PBS). Further, no apparent clinical side-
effects were seen in the test and control groups during the
experiments or at their endpoint. Finally, the AST (aspartate
intravenously via tail vein injection.
A readily discernible
fluorescence signal was observed at the tumor site up to 48 h post
administration (Figures 6A and S32). Ex vivo imaging of dissected
organs collected from the mice treated with EA-BPS (48 h post-
administration) served to confirm that the observed fluorescence
signal corresponded to the tumor site. Little fluorescence was
seen for the other organs (liver, heart, kidney, spleen, testis, and
lung) (Figures 6B and S33). Mice treated with EA-BPS (5 mg/kg
in PBS containing 5% DMSO) and subject to photoirradiation (2
W/cm2, 10 min, once a week for 4 weeks) at the tumor regions
showed a statistically significant reduction in tumor growth and
volumes (Figure 6C). Furthermore, no significant changes in the
animal body weights was observed during the course of
6
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10.1002/anie.202012687
Angewandte Chemie International Edition
RESEARCH ARTICLE
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activity levels were within the normal range (Figure 6G).[72]
Collectively, these results were taken as evidence that under the
conditions of study, EA-BPS is biocompatible and is likely to
benefit from by an acceptable safety profile.
In conclusion, one of the major limitations of PDT, and one
with severe clinical repercussions, is the fact that the endogenous
antioxidant system of mammalian cells serves to mitigate the
effects of ROS generation. In this study we have shown that an
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Acknowledgements
This work was supported by the National Research Foundation of
Korea (NRF) funded by the Ministry of Science and ICT (CRI
project
2019M3E5D1A01068998, J.S.K.) and funded by the Ministry of
Education (Basic Science Research Program
no.
2018R1A3B1052702
and
NRF-
2020R1A6A3A01100551, M.W. and 2020R1A6A3A01100558,
S.K.). A.S. thanks the Department of Biotechnology, New Delhi,
for a prestigious Ramalingaswami Fellowship 2019 (Grant No.
BT/RLF/Re-entry/59/2018). The Robert A. Welch Foundation (F-
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Conflict of interest
The authors declare no conflict of interest.
Keywords: Ethacrynic acid • Photodynamic therapy •
Glutathione S-transferase -pi • Reactive oxygen species •
BODIPY photosensitizer • Hypoxia
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RESEARCH ARTICLE
Entry for the Table of Contents
A molecular construct (EA-BPS) has been prepared that overcomes hypoxia-mediated
resistance in pre-clinical PDT therapy models.
M. Won, S. Koo, H. Li, J. L. Sessler,* J. Y. Lee,* A. Sharma,* J. S. Kim*
Page No. – Page No.
Enhancing the Lethality of Reactive Oxygen Species in Photodynamic Therapy Targeting Hypoxic Tumors
9
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