Enzyme-Instructed Intracellular Molecular Self-Assembly to Boost Activity of Cisplatin against Drug-Resistant Ovarian Cancer Cells

Article abstract of DOI:10.1002/anie.201507157

Anticancer drug resistance demands innovative approaches that boost the activity of drugs against drug-resistant cancers without increasing the systemic toxicity. Here we show the use of enzyme-instructed self-assembly (EISA) to generate intracellular supramolecular assemblies that drastically boost the activity of cisplatin against drug-resistant ovarian cancer cells. We design and synthesize small peptide precursors as the substrates of carboxylesterase (CES). CES cleaves the ester bond pre-installed on the precursors to form the peptides that self-assemble in water to form nanofibers. At the optimal concentrations, the precursors themselves are innocuous to cells, but they double or triple the activity of cisplatin against the drug-resistant ovarian cancer cells. This work illustrates a simple, yet fundamental, new way to introduce non-cytotoxic components into combination therapies with cisplatin without increasing the systemic burden or side effects. Cisplatin-boosting nanofibers: The design and synthesis is reported of small peptide precursors that can be cleaved by carboxylesterase (CES) to form peptides that self-assemble in water to form molecular nanofibers. The precursors themselves are innocuous to cells at optimal concentrations, but they double or triple the activity of cisplatin against drug-resistant ovarian cancer cells.

Full text of DOI:10.1002/anie.201507157

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201507157
German Edition: DOI: 10.1002/ange.201507157
Prodrug Design
Enzyme-Instructed Intracellular Molecular Self-Assembly to Boost
Activity of Cisplatin against Drug-Resistant Ovarian Cancer Cells
Jie Li, Yi Kuang, Junfeng Shi, Jie Zhou, Jamie E. Medina, Rong Zhou, Dan Yuan,
Cuihong Yang, Huaimin Wang, Zhimou Yang, Jianfeng Liu, Daniela M. Dinulescu,* and
Bing Xu*
Abstract: Anticancer drug resistance demands innovative
approaches that boost the activity of drugs against drug-
resistant cancers without increasing the systemic toxicity. Here
we show the use of enzyme-instructed self-assembly (EISA) to
generate intracellular supramolecular assemblies that drasti-
cally boost the activity of cisplatin against drug-resistant
ovarian cancer cells. We design and synthesize small peptide
precursors as the substrates of carboxylesterase (CES). CES
cleaves the ester bond pre-installed on the precursors to form
the peptides that self-assemble in water to form nanofibers. At
the optimal concentrations, the precursors themselves are
innocuous to cells, but they double or triple the activity of
cisplatin against the drug-resistant ovarian cancer cells. This
work illustrates a simple, yet fundamental, new way to
introduce non-cytotoxic components into combination thera-
pies with cisplatin without increasing the systemic burden or
side effects.
of treating drug-resistant ovarian cancers. One of the most
explored strategies is combination chemotherapy (such as the
combination of cisplatin with other therapeutics) because the
advantages of cisplatin promote the rapid translation from
preclinical to clinical settings. Despite the remarkable clinical
success of combination therapies,[3] the 5-year relative sur-
vival rate of ovarian cancer hardly improved over the past
decade (45% (2004–2010) vs. 45% (1996–2003)).[4e] Thus,
there is an urgent need for innovative approaches in cisplatin-
based combination therapies.
We have been exploring enzyme-instructed molecular
self-assembly[5] inside cells,[6] and we recently showed that
intracellular molecular nanofibers promiscuously interact
with cytoskeleton proteins[7] yet selectively inhibit cancer
cells.[8] Recently, Maruyama and co-workers,[9] Pires and
Ulijn,[10] Yang et al.,[11] and Wells[12a] also reported inhibition
of cancer cells by nanofibers formed by the self-assembly of
small molecules. The exceptional selectivity[8] and new
mechanisms[7,13] of the molecular nanofibers against cancer
cells encouraged us to explore the utilization of enzyme-
instructed intracellular molecular self-assembly for combina-
tion therapy with cisplatin. Unlike the previous approaches,
this work focuses on the use of d-peptides for intracellular
self-assembly, and is the first demonstration of combining
intracellular enzyme-instructed self-assembly with cisplatin.
We design and synthesize two enantiomeric peptidic precur-
sors (l-1 and d-1) that turn into the self-assembling molecules
(l-2 and d-2) upon the catalysis of carboxylesterases (CES;
Scheme 1).[14] Our study confirms that CES are able to
convert both l-1 and d-1 into the corresponding molecules of
S
ince its serendipitous discovery five decades ago,[1] cisplatin
has become the most successful therapeutic agent for
anticancer chemotherapy.[2] Particularly, cisplatin has drasti-
cally extended the progression-free survival (PFS) of patients
with ovarian cancers.[3] However, owing to the lack of early
detection of ovarian cancer and the almost inevitable relapse
in the patients with advanced ovarian cancer, drug resistance
remains a major obstacle in treating ovarian cancers.[4] Many
approaches have been investigated to address the urgent need
[*] J. Li, Y. Kuang, J. Shi, J. Zhou, R. Zhou, D. Yuan, Prof. Dr. B. Xu
Department of Chemistry, Brandeis University
415 South St, Waltham, MA 02454 (USA)
E-mail: bxu@brandeis.edu
J. E. Medina, Prof. Dr. D. M. Dinulescu
Department of Pathology, Brigham and Women’s Hospital,
Harvard Medical School
Boston, MA 02115 (USA)
E-mail: ddinulescu@rics.bwh.harvard.edu
H. Wang, Prof. Z. Yang
State Key Laboratory of Medicinal Chemical Biology and
College of Life Sciences, Nankai University
Tianjin 300071 (China)
C. Yang, Prof. J. F. Liu
Tianjin Key Laboratory of Radiation Medicine and
Molecular Nuclear Medicine,
Institute of Radiation Medicine,
Chinese Academy of Medical Science and
Peking Union Medical College
Tianjin 300192 (P.R. China)
Scheme 1. Enzymatic transformation of the precursor (1) as a substrate
of carboxylesterase (CES) to the corresponding hydrogelator (2) for
intracellular self-assembly.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507157.
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l-2 and d-2, respectively. l-2 or d-2 self-assembles in water to
form molecular nanofibers. At the optimal concentration, l-
1 or d-1 is innocuous to cells. The co-incubation of l-1 or d-
1 at the optimal concentration with cisplatin significantly
boosts the activity of cisplatin against SKOV3 and A2780cis,
two lines of drug resistant ovarian cancer cells. The efficacy of
this simple approach (inhibiting over 80% of SKOV3 by
20 mm of cisplatin and 15 mgmLÀ1 of d-1), in fact, is
comparable to that of the innovative approach based on the
co-delivery of siRNA and cisplatin nanoparticles (80%
inhibition of SKOV3 by 75 mm of cisplatin).[15] We chose to
work on the enantiomeric precursors l-1 and d-1 to assess the
influences of the cell uptake of the precursors and the
proteolytic stability of the intracellular nanofibers to the
efficacy of combination therapy. These results confirm that
enzyme-instructed self-assembly promises a new approach to
boost the activity of cisplatin against drug-resistant ovarian
cancers without increasing the systemic toxicity.
The synthesis of 1 and 2 is simple and straightforward. The
facile synthetic route (Supporting Information, Scheme S1)
combines liquid-phase synthesis and solid-phase peptide
synthesis (SPPS) for making the precursors. For example, by
loading N-Fmoc-protected phenylalanine (Fmoc-Phe-OH)
onto 2-chlorotrityl resin and carrying out SPPS, we obtain
Nap-FF[16] for coupling with ethanolamine to produce l-2.
After l-2 reacts with succinic anhydride, another step of
amide bond formation allows the attachment of taurine to
form l-1. After the purification by HPLC, the overall yield of
l-1 is about 60%. The same synthetic approach also produces
d-1.
Figure 1. TEM images of the hydrogels (inset: optical images) formed
by the addition of CES (2 UmLÀ1) to the solution of A) l-1 or B) d-1 at
the concentration of 0.4 wt% in PBS buffer (Scale bar: 100 nm). The
signal intensity ratio of static light scattering (SLS) of the solution of
C) l-1 or D) d-1 at concentrations from 10 to 100 mm before (black
bar) and after (gray bar) being treated CES (2 UmLÀ1) for three hours.
example, nanofibers or nanoparticles) in the solution of l-
1 (or d-1) at concentrations lower than the mgc and after the
addition of CES (2 UmLÀ1). We chose the concentrations
from 10 mm to 100 mm to analyze whether there are differences
in self-assembly of the molecules before and after the addition
of CES. Before being treated with CES, the signal intensity
ratios of the solution of l-1 (or d-1) at concentrations from
10 mm to 50 mm were close to zero (Figure 1C,D), indicating
that there are hardly any assemblies of l-1 (or d-1) in the
solution. When the concentration of the solution of l-1 (or d-
1) increases to 100 mm, there was a slight increase of intensity
ratio, suggesting that small amounts assemblies of l-1 (or d-1)
exist in the solution. In contrast, the addition of CES to the
solution of l-1 (or d-1) at concentrations from 10 mm to
100 mm results in a significant increase of the signal intensity
ratios, especially when the concentration of l-1 (or d-1) is at
or above 50 mm. For example, the signal intensity ratio of the
solution of l-1 (or d-1) at 50 mm drastically increased from
about zero (before the addition of CES) to about 17 (after the
addition of CES), which revealed the formation of assemblies
of l-2 (or d-2). Moreover, the solution of 100 mm l-1 showed
a 9-fold increase of the signal intensity ratio after the addition
of CES, indicating the formation of a larger amount of
assemblies after enzymatically converting the precursors to
the hydrogelators (Supporting Information, Figure S6). Sim-
ilarly, the signal intensity ratio of the solution of 100 mm d-
1 increased significantly after the addition of CES, which
agrees with the observation that CES converts d-1 into d-2 to
form self-assembling nanoscale assemblies of d-2 in water
(Figure 1B).
Because enzyme-instructed self-assembly usually leads to
the formation of supramolecular hydrogels,[17] we evaluated
the hydrogelation resulting from the esterase catalyzed
conversion of l-1 and d-1 as a facile method to assay the
self-assembly. After obtaining the precursors, we tested the
use of CES to convert the precursors into the hydrogelators
that self-assembly in water to form molecular nanofibers. The
addition of l-1 (or d-1) in PBS buffer at pH 7.4 at a concen-
tration of 0.4 wt% (5.5 mm) afforded a transparent solution.
After the addition of CES (2 UmLÀ1) into the solution of l-
1 (or d-1), a translucent hydrogel formed after 24 h. We also
found the minimum gelation concentration (mgc) of l-2 or d-
2 is about 0.1 wt% (1.4 mm). While CES efficiently converts
both l-1 and d-1 into l-2 and d-2, respectively, the hydrogel
of l-2 is apparently weaker than the hydrogel of d-2
(Supporting Information, Figure S5). We speculate that this
subtle difference might originate from weaker interactions
between d-2 and CES than between l-2 and CES. The
transmission electron microscopy (TEM) images of the
resulting hydrogels reveal the formation of uniform nano-
fibers after the addition of CES (Figure 1A,B). The diame-
ters of the nanofibers of the hydrogel formed by l-2 or d-2
after the addition of CES in the solution of l-1 or d-1 are 10 Æ
2 nm or 8 Æ 2 nm, respectively.
Our preliminary test of the cytotoxicity of l-1 and d-
1 indicates that l-1 and d-1 show significant cytotoxicity to
SKOV3 cells at concentrations below the mgc (Supporting
Information, Figure S4). Thus, we used static light scattering
(SLS) to help verify the existence of nanoscale assemblies (for
After confirming that CES converts the precursor l-1 (or
d-1) into the hydrogelator l-2 (or d-2), we determined the
stability of the precursors (l-1 or d-1) when incubated with
the ovarian cancer cells. After culturing the precursors with
SKOV3 or A2780cis cells at 378C for 4 h, we collected the cell
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lysates and culture medium for liquid chromatography–mass
spectrometry (LC-MS) analysis and determined the intra-
cellular concentrations of the precursors, the hydrogelators,
and the relevant proteolyzed products. After incubation with
SKOV3 or A2780cis cells for 4 h, more than 85% of the
precursors (l-1 or d-1) turned into the corresponding hydro-
gelators (l-2 or d-2; Table 1). Moreover, the intracellular
Table 1: The intracellular concentrations of the precursors and hydro-
gelators in SKOV3 and A2780cis cells.
Compd.
Precursor (1) [mm]
Hydrogelator (2) [mm]
Ratio[a]
Figure 2. Cell viability of ovarian cancer cells incubated with the
l-1[b]
d-1[b]
d-1[c]
62
16
69
431
108
582
6.95
6.75
8.43
precursors with and without cisplatin (CDDP). A) The cell viability of
SKOV3 cells incubated with the precursors d-1 or l-1 alone, or in
combination with CDDP for 72 h. B) The cell viability of A2780 cells
and A2780cis cells incubated with the precursors d-1 alone, or in
combination with CDDP for 72 h (***=pꢀ0.001, ****=pꢀ0.0001).
[a] ratio of hydrogelator to precursor after 4 h. [b] The cell lysates of
SKOV3 cells were collected after 4 h incubation with 20 mm (15 mgmLÀ1
)
of d-1 or with 50 mm (37 mgmLÀ1) of l-1 at 378C. [c] The cell lysates of
A2780cis cells were collected after 4 h incubation with 100 mm
(73 mgmLÀ1) of d-1 at 378C.
the inhibition of SKOV3 is about 80% or 86%, respectively.
The higher efficacy exhibited by l-1 agrees with the higher
uptake and incubation concentration of l-1.
concentrations of the hydrogelators were all above 100 mm,
which indicates the intracellular self-assembly of the hydro-
gelators. The cumulative intracellular concentration of l-
1 and l-2 was also about 10-fold higher than the incubation
concentration of l-1, and the cumulative intracellular con-
centration of d-1 and d-2 was about 5-fold higher than the
incubation concentration of d-1. These results not only
indicate that the cellular uptake of l-1 is more efficient than
that of d-1, but also confirm that the selective retention of
hydrogelators inside the cells originates from ester bond
cleavage catalyzed by CES. A fluorescent esterase substrate,
6-CFDA,[18] also confirmed high esterase activity in SKOV3
cells (Supporting Information, Figure S7). We also analyzed
the culture medium containing l-1 (or d-1), which was
incubated with SKOV3 cells or A2780cis cells. After 4 h
incubation with SKOV3 cells, about 19% of l-1 in the
medium turned into l-2 (Supporting Information, Table S2),
and the concentration of l-1 in the medium decreased from
50 mm to 39 mm; about 15% of d-1 converted d-2, and the
concentration of d-1 in the medium decreased from 20 mm to
16 mm. A similar trend was also observed in A2780cis cells.
These results further validate the hypothesis that intracellular
enzymatic conversion of the precursors catalyzed by CES
results in the intracellular self-assembly of the hydrogelators.
To evaluate the effect of intracellular self-assembly of l-2
or d-2 for cisplatin-based combination therapy, we test the
cell viability of three ovarian cancer cell lines by incubating
them with the mixture of precursors and cisplatin (CDDP).
After 72 h, the mixture of CDDP (6 mgmLÀ1) with d-
1 (15 mgmLÀ1) or l-1 (37 mgmLÀ1) inhibits about 74% or
87%, respectively, of SKOV3 cells (Figure 2A). In contrast,
d-1 (15 mgmLÀ1) or l-1 (37 mgmLÀ1) alone is almost innoc-
uous to the cells, and CDDP (6 mgmLÀ1) alone inhibits only
48% SKOV3 cells. We also used another method to treat the
SKOV3 cells, in which the d-1 (or l-1) were added 12 h after
the addition of CDDP to SKOV3 cells. As shown in Fig-
ure 2A, 72 h after the addition of d-1 (15 mgmLÀ1) or l-
1 (37 mgmLÀ1) following the addition of CDDP (6 mgmLÀ1),
We also tested the combination of CDDP and d-1 for
treating A2780cis (cisplatin-resistant) and A2780 (cisplatin-
sensitive) cells. d-1 (15 mgmLÀ1) alone hardly exhibited any
cytotoxicity to A2780cis cells (Figure 2B). The combination
of d-1 and CDDP inhibited 70% of A2780cis cells, which is
double the activity of CDDP. The combination of d-1 and
CDDP significantly inhibits A2780 cell viability and decreases
the viability of A2780 from about 38% (without adding d-1)
to only 9%. Since SKOV3 and A2780cis are two drug-
resistant ovarian cell lines, CDDP shows lower inhibition
ability against these two cell lines compared with A2780 cells.
These results confirm that the addition of the precursors of
self-assembling small molecules in combination with cisplatin
drastically boosts the activity of cisplatin against drug-
resistant ovarian cancer cells. The IC50 values of l-1 and d-
1 against the ovarian cancer cells are 62–94 mm and 48–69 mm,
respectively (Supporting Information, Table S2), but their
concentrations for the combination therapy can be lower than
IC50 values because EISA accumulates the hydrogelators
intracellularly. Furthermore, the intracellularly formed nano-
fibers (of d-1) are about seven times more effective against
HeLa cells than the nanofibers of the dipeptides reported
previously (Nap-FF[8]; Supporting Information, Table S3).
To verify the critical role of enzyme-instructed self-
assembly, we synthesize a control compound (3), which
replaces the ester bond in d-1 by an amide bond (Supporting
À
À
À
À
Information, Scheme S2). This change ( COO to CONH
) renders 3 resistant to CES. Control compound 3 (500 mm)
alone hardly inhibited SKOV3 cells after 72 h incubation
(Supporting Information, Figures S8A,S9A). After 72 h
incubation with SKOV3 cells, while CDDP (6 mgmLÀ1
)
alone causes about 40% cell death, the mixture of 3
(15 mgmLÀ1) and CDDP (6 mgmLÀ1) inhibited only about
32% of SKOV3 cells. The innocuous effects of 3 also exclude
the possibility that l-1 or d-1 act as a surfactant to inhibit cell
survival. A similar trend is observed in A2780cis cells
(Supporting Information, Figures S8B, S9B). These results
further confirm that enzyme-instructed self-assembly inside
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cells is the main cause for boosting the efficacy of CDDP in
the combination therapy of CDDP with the precursors (d-
1 and l-1). Some of the cell viabilities slightly exceed 100%
(for example, Figure 2B) because MTT assay measures the
activity of mitochondrial reductase and it is not unusual for
treated groups to have higher enzyme activity than the control
group.
To gain insight into the action of intracellular self-
assembly within cells, we examined the change of the actin
filaments inside cells. SKOV3 cells treated by d-1 (20 mm
(15 mgmLÀ1), 20 h) exhibit far fewer well-defined, long actin
filaments than that in the control SKOV3 cells (without the
treatment of d-1; Figure 3). This trend becomes more
exchanging the media, the morphology of actin filaments was
restored to normal. These results suggest that intracellular
nanofibers formed by enzyme-instructed self-assembly
exhibit transient cytotoxicity that should help minimize
long-term systemic burden in combination therapy. Dissoci-
ation likely reduces the long-term cytotoxicity after the
apoptosis of cells so that the precursors and nanofibers cause
minimal systemic toxicity.
In conclusion, we demonstrated that enzyme-instructed
intracellular self-assembly of small molecules is a new
approach to boost the activity of CDDP against two drug-
resistant ovarian cancer cell lines. Moreover, at the optimal
concentrations, 20 mm (d-1) and 50 mm (l-1) used for boosting
the activities of the cisplatin, l-1 and d-1 hardly inhibit HS-5
and PC-12 cells (Supporting Information, Figures S15,S17),
despite cisplatin significantly inhibiting HS-5 and PC-12 cells
(Supporting Information, Figure S16).[19] Intravenous injec-
tion of l-1 or d-1 hardly affected the weight and organ index
of mice (Supporting Information, Figure S18), confirming the
low systemic toxicity of the precursors. The genome analysis
according to The Cancer Genome Atlas (TCGA) indicates
the amplification of CES in certain tumors (for example,
breast and ovarian cancer; Supporting Information, Fig-
ure S23), which not only supports the observations in this
work, but also provides useful guidance for treating other
cancers based on the self-assembly of intracellular nanofibers.
This work, together with other emerging evidence,[6–10,12]
indicates that enzyme-instructed self-assembly promises
a new way for developing combination therapy for cancer
treatment. Other than cisplatin, carboplatin has been used as
the preferred platinum-based drug,[20] and we will use
carboplatin for our future work.
Acknowledgements
Figure 3. The fluorescence images of SKOV3 cells stained with Alexa
Fluor 633 Phalloidin (F-actin) and Hoechst (nuclei) (upper) after
treatment of d-1 at concentration of 20 mm for 20 h or (bottom)
without the treatment of d-1. Scale bars: left=20 mm, right=10 mm.
This work was partially supported by the NIH (R01A142746),
DOD OCRP (W81XWH-10-1-0263, W81XWH-14-1-0092,
and W81XWH-14-1-0205), the International S&T Coopera-
tion Program of China (ISTCP, 2015DFA50310) and the
NSFC (81471727).
pronounced after increasing the concentration from 20 to
100 mm (Supporting Information, Figure S11), as evidenced
by the number of the actin filaments in the cells (Supporting
Information, Figure S19). This observation agrees with
hypothesis that the intracellular nanofibers of small peptides
interact with actin.[7] To verify the reversible assembly of d-2
inside cells, we treated the SKOV3 cells with d-1 at the
concentrations of 20, 50, and 100 mm respectively, for 20 h,
then replaced the media with fresh media and incubated the
cells for an additional 20 h. Actin filaments recovered after
being treated with fresh medium for 20 h when the concen-
trations of d-1 were 20 and 50 mm (Supporting Information,
Figure S12). The incomplete recovery of actin filaments, when
[d-1] = 100 mm, supports the hypothesis that d-1 starts to self-
assemble at 100 mm. After being incubated with l-1, SKOV3
cells exhibited similar behavior (Supporting Information,
Figures S13,S14): after 20 h, cells incubated with l-1 (50 mm)
exhibited fewer well-defined actin filaments compared with
the cells without the treatment of l-1. However, 20 h after
Keywords: cisplatin · combination therapy · drug-resistance ·
enzymes · self-assembly
How to cite: Angew. Chem. Int. Ed. 2015, 54, 13307–13311
Angew. Chem. 2015, 127, 13505–13509
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Received: July 31, 2015
Published online: September 14, 2015
Angew. Chem. Int. Ed. 2015, 54, 13307 –13311
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