Enzymatic Self-Assembly Confers Exceptionally Strong Synergism with NF-κB Targeting for Selective Necroptosis of Cancer Cells

Article abstract of DOI:10.1021/jacs.7b12368

As a promising molecular process for selectively inhibiting cancer cells without inducing acquired drug resistance, enzyme-instructed self-assembly (EISA) usually requires relatively high dosages. Despite its discovery 30 years ago, the translation of the

Full text of DOI:10.1021/jacs.7b12368

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Article
Enzymatic Self-Assembly Confers Exceptionally Strong Synergism
with NF-kB Targeting for Selective Necroptosis of Cancer Cells
Jie Zhou, Xuewen Du, xiaoyi chen, Jiaqing Wang, Ning Zhou, Difei Wu, and Bing Xu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12368 • Publication Date (Web): 29 Jan 2018
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Enzymatic Self-Assembly Confers Exceptionally Strong Syner-
gism with NF-κB Targeting for Selective Necroptosis of Cancer
Cells
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Jie Zhou‡, Xuewen Du‡,#, Xiaoyi Chen, Jiaqing Wang, Ning Zhou, Difei Wu, Bing Xu*
Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA 02453, USA
ABSTRACT: As a promising molecular process for selectively inhibiting cancer cells without inducing acquired drug re-
sistance, enzyme-instructed self-assembly (EISA) usually requires relatively high dosages. Despite its discovery 30 years
ago, the translation of the knowledge about NF-κB signaling into clinic remains complicated due to the broad roles of NF-
κB in cellular regulation. Here we show that integrating EISA and NF-κB targeting boosts the efficacy of EISA over an or-
der of magnitude without compromising selectivity against cancer cells. That is, in-situ enzymatic self-assembly of a
tetrapeptide results in nanofibers, which hardly affect cell viability, but lead to inductive expression of tumor necrosis
factor receptor 2 (TNFR2) and decreased expression of three key proteins at the up-stream of NF-κB pathway in the can-
cer cells. Adding the inhibitors targeting NF-κB further decreases the expressions of those up-stream proteins, which
turns the otherwise innocuous nanofibers to being lethal to the cancer cells, likely causing necroptosis. As the first case of
using supramolecular processes to enable synthetic lethality, this work illustrates a versatile approach to translate key
regulatory circuits into promising therapeutic targets.
effective against cancer cells. Being discovered 30 years
ago,18 NF-κB, as a family of related protein hetero- or ho-
modimers, exhibits remarkable capabilities for regulating
the transcription of hundreds of target genes.19 Because
activation of NF-κB is an essential feature of the survival
of cancer cells during treatment, which contributes to
cancer drug resistance, considerable efforts have focused
on targeting NF-κB for cancer therapy. In fact, one of
FDA-approved drugs for treating multiple myeloma,
bortezomib (BTZ), works by blocking NF-κB activation.20
This success inspired the development of NF-κB inhibi-
tors to sensitize cancer cells of solid tumors to anticancer
drugs.21 A notable example of such development is BAY 11-
7085 (BAY), which blocks proteasomal degradation of
IκBα,22 allowing it to sequester NF-κB in an inactivated
state.23 Therefore, we choose to integrate EISA with NF-
κB inhibitors, such as BAY and BTZ for boosting the activ-
ity of EISA. Two fundamental biological facts support this
choice: (i) Intracellular or pericellular peptide nanofibrils
cause cell stress,10a,24 which results in up-regulation of
transcription factors;25 (ii) NF-κB, as a rapidly inducible
transcription factor,18-19 is critical for the survival response
of cells to stress.26 Thus, blocking NF-κB signaling should
sensitize cancer cells to the nanofibers formed by EISA
(Scheme 1). In fact, our previous study has indicated the
synergism of NF-κB inhibitors and pericellular nanofibers
for apoptosis, however, the concentrations of the precur-
sors required are still high10a and the relevant mechanism
remains to be elucidated.
Introduction
Despite the significant advances in cancer biology and
cancer therapy, cancer remains a challenge in drug devel-
opment due to its great complexity.1 The elucidation of
the mechanisms2 of drug resistance to current chemo-
therapy reveals the inherent drawbacks of molecular tar-
geting therapy and the demands of new approaches for
cancer therapy.3 Contrasting to targeting only a specific
enzyme or protein, the integration of enzymatic reaction
and molecular self-assembly4 leads to a multiple-step mo-
lecular process (termed as enzyme-instructed self-
assembly (EISA))5 that is able to interact with multiple
cellular targets and selectively inhibit cancer cells5c,6
without inducing acquired drug resistance.7 In fact, EISA
is an inherent cellular process that affords spatiotemporal
control of higher order structures from nanoscales to mi-
cron scales.8 Moreover, EISA is general and applicable for
a
wide range of substrates (e.g., peptides,9
carbohydrates,5c and nanoparticles10) and different en-
zymes (e.g., phosphatases,5b proteases,11 esterases,6c and
glycosidases12), and is finding increased number of appli-
cations (e.g., efficient adjuvant for a vaccine,13 imaging of
enzyme activity,14 monitoring autophagy,15 inhibiting bac-
teria,16 and removing unwanted iPS cells17).
However, EISA usually requires relatively high concen-
tration for inhibiting cancer cells (e.g., about 1.0 mM5c) or
killing iPS cells (e.g., 300 ꢀM17). To address this limitation,
we combine EISA with targeting the transcription factor
nuclear factor-κB (NF-κB) and expect that their synergis-
tic action would make NF-κB targeting selective and EISA
Here, this study shows exceptionally strong synergism
between EISA and NF-κB targeting when EISA of a C-
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terminal methylated phosphotetrapeptide (pTP-Me) re- thyl group (Scheme 2, Figs. S1-S11). Being generated after
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sults in extra- and intracellular nano-assemblies (Scheme
1). Self-assembling to form nanoparticles to facilitate cell
uptake27 and intracellular EISA and being dephosphory-
lated in cellular milieu of cancer cells (e.g., Saos-2), pTP-
Me becomes TP-Me, which forms nanofibers and results
in inductive expression of tumor necrosis factor receptor
2 (TNFR2)28 and decreased the expression of three key
proteins (PI3K,29 Akt,30 and MEKK331) at the up-stream of
NF-κB signaling in the cancer cells. Though exhibiting
low cytotoxicity, the precursors drastically decrease can-
cer cell viability (about an order of magnitude) in the
presence of the inhibitors targeting NF-κB signaling. Pre-
liminary mechanistic study indicates that the combina-
tion of the EISA of pTP-Me and NF-κB inhibitors further
decreases the expressions of those up-stream proteins,
which eventually results in the death of Saos-2 via necrop-
tosis. As the first report to focus on combining supramo-
lecular chemistry with a chemical biology approach, this
work illustrates the use of molecular processes to trans-
late key regulatory circuits into promising therapeutic
targets.
enzymatic dephosphorylation of pTP-Me by ALP, TP-Me
forms a hydrogel with higher storage modulus (G’) than
that of the soft hydrogel formed by pTP at the same con-
dition (i.e., concentration (wt%), pH, and amount of ALP),
indicating that methylation of the peptide indeed signifi-
cantly increases self-assembling ability (Figs. S12, S13). In
certain cases,
a small portion of precursors being
dephosphorylated would result in gelation. Since pTP-Me
has much lower critical micelle concentration (CMC)
than that of pTP (vide infra), it is reasonable for the mix-
ture of TP-Me and pTP-Me to form a stronger gel than
that of TP and pTP even though more pTP has been
dephosphorylated than pTP-Me has. While it supports
that formation of nanofibers contributes to cell death,
this rheological observation also permits other factors to
contribute to cell death.
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Scheme 2. Molecular structures of pTP-Me, pTP, TP-Me,
and TP.
O
O
O
O
P
P
OH
OH
HO
HO
O
O
O
O
H
H
N
H
H
N
O
N
N
OH
N
H
N
H
N
H
N
H
Me
O
O
O
O
O
O
Scheme 1. EISA in cellular milieu causes cell stress to
activate NF-κB signaling for cell survival and the combi-
nation of EISA and NF-κB targeting effectively inhibit
pTP-Me
pTP
OH
OH
O
O
O
O
H
H
H
N
H
N
N
N
OH
O
cancer
cells.
N
H
N
H
N
H
N
H
Me
O
O
O
O
O
O
TP
TP-Me
Transmission electron microscopy (TEM) reveals that,
at 100 µM (i.e., a working concentration in cellular envi-
ronment), pTP-Me itself already self-assembles to form
nanoparticle in water, and undergoes EISA to form nano-
fibers after the addition of ALP (Figure 1A). While pTP
hardly aggregates itself at 100 ꢀM, it forms nanofibers
after EISA (Fig. S14). Being concentration-dependent,
static light scattering (SLS) signals of the solutions of
pTP-Me increase considerably after adding ALP (Figure
1B), suggesting significant aggregation. Notably, pTP-Me
solutions exhibit considerable SLS signals around 7.5-15
µM before ALP treatment and around 1.9-3.7 µM after
EISA, agreeing with the CMC of pTP-Me before (i.e., 14.8
µM) and after (i.e., 2.5 µM) the addition of ALP (Figure 1C,
D). pTP exhibits similar SLS signal trend (Figure 1E), but
with higher CMCs (128.66 µM and 8.69 µM before and
after adding ALP, respectively) (Figure 1F, G). These re-
sults validate the enhanced self-assembly ability con-
ferred by the methyl group at the C-terminal of DFDFDFDY.
Because dephosphorylation plays an essential role in the
multiple-step process of EISA, we monitored the
dephosphorylation of pTP-Me and pTP treated by ALP at
physiological conditions. As shown in Figure 1H, ALP
dephosphorylates pTP-Me at a much slower relative rate
than dephosphorylating pTP, likely due to the structural
hinder of methyl group and the aggregation of pTP-Me at
the concentration higher than its CMC.
Results and Discussion
Molecular design and enzymatic self-assembly.
Scheme 2 shows the structure of the precursor, pTP-Me,
which consists of a tetrapeptide (D-Phe-D-Phe-D-Phe-D-
Tyr (DFDFDFDY)) as the self-assembling motif, a phosphate
on D-Tyr as the enzymatic trigger, a 2-acetylnaphthyl
group at the N-terminal and a methyl group at the C-
terminal of the peptide as the additional motifs for pro-
moting self-assembly in water. To examine the roles of
EISA and methylation, we synthesized three controls:
pTP, without C-terminal methylation; TP-Me, lacking
enzymatic trigger; TP, without both phosphate and me-
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Figure 2. 48-hour relative cell viability of (A) Saos-2, (B)
HeLa, (C) SK-OV-3, and (D) A2780cis cells treated by pTP-
Me and pTP at different concentrations (20, 40, 60, 80, 100,
200, 300, 400, and 500 µM).
Table 1. IC50 summary of pTP-Me and pTP against differ-
ent cells lines shown in Figure 2.
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Figure 1. Before and after adding ALP (1 U/mL), (A) TEM
IC50 (ꢀM)
Cell lines
images of the nanoparticles and nanofibers in the solutions
of precursor pTP-Me (100 µM, pH 7.4), (B) SLS signals (at 30o)
of the solution of pTP-Me and (C, D) CMCs of pTP-Me. (E)
SLS signals (at 30o) of the solution of pTP and (F, G) CMCs of
pTP before and after adding ALP (1 U/mL). (H) Time-
dependent course of dephosphorylation of pTP-Me and pTP
(500 µM) treated by ALP (0.01 U/mL).
pTP-Me
pTP
120
Saos-2
HeLa
>500
>500
>500
>500
100
500
300
SK-OV-3
A2780cis
Cytotoxicity of the designed molecules. Contrary to
our observation of the cytotoxicity of a methylated phos-
photripeptide,32 pTP-Me, though having higher self-
assembling ability than pTP, exhibits less cytotoxicity
than pTP does against several cancer cell lines (e.g., HeLa,
Saos-2, SK-OV-3, and A2780cis) (Figure 2, Table 1). While
pTP has an IC50 of 100, 120, 300, and 500 µM against HeLa,
Saos-2, A2780cis, and SK-OV-3 cells, respectively, which
largely agree with the ALP activity from these cells,33 the
IC50 of pTP-Me against these two cells lines are higher
than 500 µM (Table 1). Since the self-assembly ability and
enzymatic dephosphorylation together dictate the biolog-
ical activities of these precursors, the weaker cytotoxicity
is likely due to the slower dephosphorylation rate of pTP-
Me (Figure 1H). For example, after being incubated with
ALP for 24 h, 87 % and 55 % of pTP and pTP-Me turn
into TP and TP-Me, respectively. The unexpected low
cytotoxicity pTP-Me makes it an ideal candidate to test
the synergistic effect of pTP-Me and BAY for killing can-
cers.
Figure 3. (A) 48-hour cell viability of Saos-2 cells incubated
with pTP-Me (20, 40, 60, 80, 100, 200, 300, 400, and 500 µM)
in combination with BAY (0, 8, 12, 16, and 20 µM). (B) 48-
hour cell viability of Saos-2 cells incubated with BAY at dif-
ferent concentrations (4, 8, 12, 16, and 20 µM). (C) ALP inhib-
itor (DQB, 10 µM) significantly rescues Saos-2 cells treated by
the combination of pTP-Me (100 µM) and BAY (10 µM). ***:
p<0.001. (D) Normalized Isobologram,34 (E) Combination
Index (CI)34 plot and (F) Dose-Reduction Index (DRI)34 plots
for the combination of pTP-Me and BAY. All the analysis
shown in (C-E) was based on the combination: pTP-Me 20-
500 µM and BAY 12, 16, 20 µM, 27 data points in total.
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data points on the left side of the DRI diagram (Figure
Synergism between EISA and NF-κB targeting. The
combination of pTP-Me (20-500 ꢀM) and a NF-κB inhibi-
tor (BAY, 8-20 µM) potently inhibit Saos-2 cells (Figure
3A) while neither of them alone significantly inhibits the
Saos-2 cells (Figure 2A, Figure 3B) at the concentrations
used for the combination. Notably, the combination of 20
µM pTP-Me (i.e., 17.7 µg/ml) and 16 µM BAY (i.e., 4.0
µg/ml) inhibits more than 95% Saos-2 cells, suggesting an
exceptionally strong synergism. An ALP inhibitor (e.g.,
DQB35) is able to rescue 60% of Saos-2 cells treated by the
combination of pTP-Me and BAY (Figure 3C), indicating
the involvement of EISA for the synergistic inhibition of
the cancer cells.
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3E), there is a 10-20 fold and 2-fold dose-reductions for
pTP-Me and for BAY, respectively, further confirming the
exceptionally strong synergism between EISA (of pTP-
Me) and NF-ĸB targeting.
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Table 2. Determination of synergism and antagonism
using CI analysis
CI
Description
Very strong synergism
Strong synergism
Synergism
< 0.1
0.1 - 0.3
0.3 – 0.7
0.7 - 0.85
0.85 – 0.9
0.9 – 1.1
1.1 – 1.2
1.2 – 1.45
1.45 – 3.3
3.3 – 10
> 10
Moderate synergism
Slight synergism
Nearly additive
Figure 4. (A) 48-hour cell viability of HeLa cells incubated
with pTP-Me (20, 40, 60, 80, 100, 200, 300, 400, and 500 µM)
in combination with BAY (0, 8, 12, 16, and 20 µM). (B) Nor-
malized Isobologram,34 (D) Combination Index (CI)34 plot
and (D) Dose-Reduction Index (DRI)34 plots for the combina-
tion of pTP-Me and BAY. All the analysis shown in (C-D)
was based on the combination: pTP-Me 20-500 µM and BAY
16, 20 µM, 18 data points in total.
Slight antagonism
Moderate antagonism
Antagonism
Strong antagonism
Very strong antagonism
Table 3. Determination of synergism and antagonism
using isobologram.
Illustrated Diagno-
Data Points
sis
On the lower-left of the hypotenuse
On the upper-right of the hypote-
nuse
Synergism
Antagonism
On the hypotenuse
Addictive effect
Figure 5. (A) 48-hour cell viability of Saos-2 cells incubated
with different concentrations of pTP-Me (20, 40, 60, 80, 100,
200, 300, 400, and 500ꢀM) in combination with BTZ (100
nM). (B) CI-Fa34 plotting for the combination of pTP-Me and
BTZ. All the analysis shown in (B) was based on the combi-
nation: pTP-Me 20-500 µM and BTZ 100 nM, 9 data points in
total.
To quantitatively evaluate the therapeutic effect of the
combination of pTP-Me and BAY, we calculated the
Combination Index (CI) (Table 2), a term quantitatively
depicts synergism (CI < 1), additive effect (CI = 1), and
antagonism (CI > 1), and plotted Dose-Reduction Index
(DRI)-Fa diagram and normalized isobologram, according
to Chou-Talalay method.34 As shown in the normalized
isobologrm (Figure 3C), all these 27 combinations locate
on the lower-left of the hypotenuse, confirming the syn-
ergistic effect (Table 3). Moreover, most of the data points
in CI-Fa plot fall into the range of CI < 0.3 (Figure 3D),
indicating strong synergism (Table 2). Since a major aim
for achieving synergism in drug combination is dose re-
duction for reducing toxicity to host, DRI denotes how
many fold of dose-reduction is allowed for each drug due
to synergism when compared with each drug alone.
When inhibition (Fa) is very close to 1 (100% inhibition),
the calculated DRI value will become extremely large,
which in most case is meaningless. Thus, according to the
We also examined synergistic effect on the combination
of pTP-Me and BAY in the case of HeLa cells, which is
less significant than that on Saos-2 cells. As shown in Fig-
ure 4A, pTP-Me itself exhibits low cell inhibitory activity,
but kills most of the HeLa cells when being combined
with 20 µM BAY. However, the combination becomes
almost ineffective when BAY concentration decreases to
16 µM. According to Chou-Talalay analysis (Figure 4B, C,
D), most of the data points still locate in the area of syn-
ergism, but some are very close to or even on or above the
hypotenuse representing additive effects in isobologram graph
and have a calculated CI value equals to 1 or slightly below 1,
indicating weaker synergism than that on Saos-2 cells. In
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undergo dephosphorylation inside cells. The precursor
addition, the DRI values are also lower than that on Saos-
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2 cell (Figure 3E). This result originates from the different
ALP expressions between these two cell lines,36 implying
the cell selectivity of this combination, which is reasona-
ble since different cancer cells likely depend on different
survival mechanisms.
without C-terminal methylation, f-pTP, forms much
more extra- and intracellular fluorescent nanofibers than
f-pTP-Me does under the same condition (Fig. S20),
which explains the higher cytotoxicity of pTP than that of
pTP-Me.
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The combination of pTP and BAY only exhibits weak
synergism or additive effect on Saos-2 cells (Fig. S15),
while only additive effect or even antagonism on HeLa
cells (Fig. S16), further validating that the C-terminal
methylation is critical for the observed high synergism. In
addition, the synergism between pTP-Me and BAY occurs
on other cancer cell line (e.g., A2780cis, a drug resistant
ovarian cancer cell (Fig. S17)). Moreover, the combination
of pTP-Me and BTZ, a different NF-κB inhibitor, also ex-
hibits strong synergism although BTZ and BAY inhibit
NF-κB signaling via different mechanisms (Figure 5).37 As
shown in Figures 5A, pTP-Me, combined with only 100
nM BTZ, potently inhibits Saos-2 cells, while neither of
them shows strong cytotoxicity alone (Fig. S18). CI-Fa
plotting confirms very strong synergism between pTP-Me
and BTZ (Figure 5B, Table 2). These observations suggest
pTP-Me to be a promising therapeutic agent in the com-
bination therapy that employs various NF-κB targeting
molecules. There is little synergism observed between TP
or TP-Me and BAY (Fig. S19), further indicating the im-
portance of EISA and C-terminal methylation for the ob-
served synergism.
Examining the changes of actin filaments and microtu-
bules in Saos-2 cells treated by pTP-Me suggests that the
formation of nanofibers dramatically disrupts cytoskele-
tons (Figures 6C, 6D, S21, and S22). That is, in the cells
treated by pTP-Me, only few actin filaments and micro-
tubules attach to the cell membrane while there are nu-
merous well-defined actin filaments and microtubule
stretching through the control cells. The addition of BAY
barely influences the polymerization of either actin or
tubulin and hardly aggravates the disruption of cytoskele-
tons in cells treated by pTP-Me (Figs. S21 and S22). These
results suggest that the resulting extra- and intracellular
nanofibers from EISA of pTP-Me cause considerable cell
stress (Scheme 1).
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Figure 7. (A) Western blot shows the protein variations in
Saos-2 cells treated by pTP-Me, BAY, and the combination of
pTP-Me and BAY 11-7085 for different time (e.g., 4, 8, 12, 24,
36 hours). (B) Quantification of protein variation over time
in Saos-2 cells treated by pTP-Me or the combination of
pTP-Me and BAY (Table S2, S3).
Figure 6. (A) Molecular structure of f-pTP-Me. (B) Fluores-
cent image of Saos-2 cells treated by f-pTP-Me (400 µM) for
8 h. (C) (D) The distribution of (C) actin and (D) tubulin in
Saos-2 cells treated by pTP-Me (100 µM) for 4 hours.
Expression of signaling molecules. Using western blot
(WB), we investigated the possible mechanisms underly-
ing the anticancer activity of the combination of pTP-Me
and BAY against Saos-2 cells by focusing on NF-ĸB signal-
ing, an important factor and potential target in cancer
therapy.39 The stresses conferred by EISA of pTP-Me
alone lead to an inductive expression of TNFR2 and de-
creased expression of three key proteins—PI3K, Akt,3030
and MEKK3—at the up-stream of NF-κB pathway in Saos-
2 cells as a response to the cellular stress. Moreover, the
expressions of IKKα, IKKβ, and IĸBα, at downꢀstream of
MEKK3, decline (Figure 7A, B (left panel)). These results
indicate that the action of the nanofibers formed by EISA of
pTP-Me results in the down regulation of
Akt/PI3K/MEKK3 signaling, thus modulating constitutive
and inducible NF-ĸB activation. The activation of NF-ĸB
Intracellular EISA. pTP-Me forms nanoparticles, which
would be up-taken by the cells. To understand cellular
distribution of pTP-Me, we synthesized f-pTP-Me as a
structural analog of pTP-Me (Figure 6A, Fig. S6-S9). The
imaging contrast conferred by the assemblies consisting
of NBD-conjugated self-assembling peptides allows us to
evaluate the location of EISA in cellular environment.38 As
shown in Figure 6B, f-pTP-Me undergoes EISA on and
inside Saos-2 cells to form extra- and intracellular nano-
fibers. This result implies that, besides acting as the sub-
strate of extracellular EISA, pTP-Me allows intracellular
EISA, likely due to that the assemblies/nanoparticles of
pTP-Me, forming prior to EISA, favor cell uptake and
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signaling, as the survival response to stress, keeps the
contribute to the moderate synergistic effect of pTP-Me
and BAY on HeLa cells.
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cells alive (Scheme 1). Despite that BAY alone only in-
creases the expression of TNFR1 and hardly influences the
expressions of other proteins mentioned above (Figure
7A), the combination BAY with EISA, however, blocks NF-
ĸB activation, thus further decreases the expressions of
those up-stream proteins (i.e., PI3K, Akt,30 30 and MEKK3)
and the down-stream proteins (i.e., IKKα, IKKβ, and IĸBα),
which eventually results in cell death (Scheme 1 and Figure
7). The down regulation of Akt, MEKK3, IKKα, IKKβ, and
IĸBα depends on the concentration of pTP-Me (Figure 8).
Such a dose-dependent response supporting that the EISA
of pTP-Me is able to modulate NF-ĸB signaling.
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Figure 9. 48-hour cell viability of Saos-2 cells incubated with
(A) pTP-Me (100, 200, 300, 400, and 500 µM) or (B) the
combination of pTP-Me and BAY (20 µM) with and without
the presence of pan-caspase inhibitor (Z-VAD-FMK, 40 μM)
or necroptosis inhibitor (Nec-1, 40 μM).
The modality of cell death. To examine the cell death
mode induced by the combination of pTP-Me and BAY,
we investigated the effects of zVAD-fmk, a pan-caspase
inhibitor,41 and Nec-1, a necroptosis inhibitor,42 on cell
death caused by pTP-Me. zVAD-fmk aggravates the Saos-
2 cell death caused by pTP-Me, but hardly affects the
cytotoxicity of the combination of pTP-Me and BAY; Nec-
1 barely changes the cytotoxicity of pTP-Me, but rescues
the cells treated by the combination of pTP-Me and BAY
(Figure 9, Fig. S25). These results indicate that the com-
bination of pTP-Me and BAY results in necroptosis of
Saos-2 cells.
Figure 8. (A) Western blot shows the concentration de-
pendent protein variations in Saos-2 cells treated by pTP-
Me for 24 hours. (B) Quantification of protein variation in
Saos-2 cells treated by pTP-Me at different concentra-
tions for 24 hours (Table S4).
In a control experiment, treating Saos-2 cells by pTP
also leads to an inductive expression of TNFR2, but barely
influences the expression levels of Akt, PI3K, or MEKK3
(Fig. S23), as well as the expressions of those down-stream
proteins (e.g., IKKα, IKKβ, and IĸBα). This observation
indicates a different cell killing mechanism, which corre-
sponds with different cytotoxicity of pTP-Me and pTP
against Saos-2 cells and underscores the notion that self-
assembly properties of small molecules is a critical factor
to affect how their assemblies to modulate cell signaling.
Meanwhile, since BAY increases the expression of TNFR1
(Figure 7A), the combination of pTP and BAY results an
increase in both TNFR1 and TNFR2, but hardly causes any
difference in the expression of other aforementioned sig-
naling proteins over time. Although the lower CMC of
pTP-Me likely is the key reason that pTP-Me has much
better outcome compared with pTP in the combination
with NF-κB inhibitors, other factors remain to be deter-
mined. We speculate that the aggregates of pTP-Me like-
ly lead to more endoplasmic reticulum (ER) stress than
that caused by pTP, which would decrease PI3K and
Akt.40 In addition, except the increase in TNFR2, the vari-
ation of the protein expression (Fig. S24) of the HeLa cells
treated by pTP-Me differ from those of Saos-2, confirm-
ing the selectivity of the combination of EISA and NF-kB
targeting. Although the concept of the work is straight-
forward, the details of enzyme actions are rather sophisti-
cated because both cell surface and intracellular phospha-
tases contribute to EISA, which may lead to the different
activities of the combination against different cell lines.
Moreover, the expression of esterases in HeLa cells may
Figure 10. Relative cell viability (48-hour) of wide type Saos-
2 cells (blue) or drugged Saos-2 cells (red) treated by (A) BAY
11-7085, (B) pTP-Me, and (C) the combination of pTP-Me
and BAY 11-7085. Drugged Saos-2 cells were constantly main-
tained in complete growth medium with (A) 4 µM BAY 11-
7085, (B) 100 µL pTP-Me and (C) the combination of 4 µM
BAY 11-7085 and 100 µL pTP-Me for 4 weeks.
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general process to disrupt the homeostasis of cancer cells
Drug resistance test. Combinational therapy is particu-
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larly attractive as it may result in a decrease in the onset
of drug resistance. We designed an easy experiment to
test whether the combination of EISA and NF-ĸB target-
ing will lead to acquired drug resistance. We maintained
the Saos-2 cells in complete growth medium with BAY (4
µM), pTP-Me (100 µM), or the combination of BAY (4 µM)
and pTP-Me (100 µM) in three culture dishes for 4 weeks.
Four weeks later, we tested the cytotoxicity of BAY, pTP-
Me and the combination of pTP-Me and BAY on both
wild type cells and the cells pretreated by BAY and pTP-
Me at suboptimal dosages. As shown in Figure 10A and
10B, constant treatment of BAY or pTP-Me alone both
evokes resistance in Saos-2 cells. For example, the IC50 of
BAY increases from 27 µM to 38 µM and 500 µM pTP-Me
hardly show any cytotoxicity after 4 weeks. Meanwhile,
the constant exposure of Saos-2 cells to the combination
of BAY and pTP-Me hardly causes any drug resistance
(Figure 10 C). This result indicates that the integration of
EISA with NF-ĸB targeting promises a fundamentally new
approach for developing anticancer therapeutics that may
counter drug resistance.
selectively, EISA is able to combine with the inhibitors
targeting other essential node proteins in other pathways
for anticancer therapy. In addition, this work provides
useful insights for understanding the cytotoxicity of aber-
rant protein or peptide aggregates in the context of NF-ĸB
modulation and the insights into gene function and drug
action.
Scheme 3. Cancer cells that have lost key functions (e.g.,
cytoskeleton protein dynamics) become reliant on backup
pathways (e.g., NF-κB signaling) so that “synthetic lethali-
ty” can be exploited to kill cancers.
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Conclusion
In conclusion, we demonstrate the exceptionally strong
synergism between the nanofibers formed by EISA and
NF-κB targeting, which turns the innocuous nanofibers
into the cell death signals to cause necroptosis in cancer
cells. The ability of EISA involved with pTP-Me for
modulating the expression of TNFR2, PI3K, Akt, MEKK3,
IKKs, and IĸB is significant because these proteins are the
key proteins in cell signaling circuitry. On the other hand,
NF-ĸB activation is essential for cell survival under stress.
By synergistically modulating NF-ĸB activation (e.g.,
regulating the key proteins: Akt/PI3K/MEKK3 and
IKKα/β), this combination possibly disrupts all the sup-
porting pathways for cancer cell survival, thus achieves
strong synergistic effects for dosage and toxicity reduc-
tion (Figure 3), as well as for decrease of the emergence of
drug resistance (Figure 10).
ASSOCIATED CONTENT
Supporting Information
See supporting information for synthetic procedures, NMR
and LC-MS characterization, and other supplementary re-
sults. This material is available free of charge via the Internet
at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
bxu@brandeis.edu
Author Contributions
‡These authors contributed equally.
Notes
#Deceased 26 December 2017.
In essence, EISA generated nanofibers inside cells dis-
rupt the cell homeostasis, which likely upregulate the
cellular processes that restore the cellular homeostasis.
Such disruption in homeostasis by the nanofibers, which
yields a non-lethal growth impairment, combines with
other types of perturbations (e.g., NF-ĸB blocking) to re-
sult in a form of “synthetic lethality” (Scheme 3).43 Besides
NF-ĸB inhibitors, the perturbation can also be the over-
expression of genes, the action of a chemical compound,
or environmental change. The concept demonstrated in
this work promises a new class of anticancer therapies
and future anticancer strategies that will increasingly
consist of combination therapies based on personalized
tumor vulnerabilities. Meanwhile, the use of alkaline
phosphatase-instructed self-assembly is particular im-
portant because of (i) the selectivity conferred by the
overexpression of certain ALPs on cancer cells,6b (ii) the
easy control of local formation of nanofibers at targeting
sites, and (iii) the reduced probability of resistance. As a
ACKNOWLEDGMENT
This work is partially supported by NIH (R01CA142746), the
W.M. Keck Foundation, NSF (DMR-1420382). J.Z. is an HHMI
International Research Fellow.
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