Real-time monitoring of caspase-3/8 activity by self-assembling nanofiber probes in living cells

Article abstract of DOI:10.1039/d0cc07821b

Caspase-3/8 are key members of the cysteine-aspartyl protease family with pivotal roles in apoptosis. We have designed and synthesized self-assembling probes, Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC, with fluorescence 'turn-on' properties for real-time mo

Full text of DOI:10.1039/d0cc07821b

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Real-time monitoring of caspase-3/8 activity by
self-assembling nanofiber probes in living cells†
Cite this:DOI: 10.1039/d0cc07821b
Li-Song Zhang,a Hong-Lei Xu,a Ying Xia,a Jian-Peng Bi,a Chuan-Zeng Zhang,a
a
Zhen Xi,b Lu-Yuan Lia and Zhi-Song Zhang
*
Received 1st December 2020,
Accepted 15th December 2020
DOI: 10.1039/d0cc07821b
rsc.li/chemcomm
Caspase-3/8 are key members of the cysteine–aspartyl protease family Caspase-8 and Caspase-3,14–16 respectively, to aid the detection
with pivotal roles in apoptosis. We have designed and synthesized self- of caspase activities. Fluorescent group such as 7-amino-4-trifluoro-
assembling probes, Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC, methylcoumarin (AFC), which has
a fluorescent property
with fluorescence ‘turn-on’ properties for real-time monitoring of depending on the electron donating ability of the 7-amino group,
Caspase-3/8 activity in living cells.
has been utilized to modify peptidyl substrates of caspases,17–19
such that the highly fluorescent group AFC is released when the
Apoptosis, the major type of programmed cell death, is a highly amide bond between the peptide and the coumarin is cleaved, as
regulated process involved in tissue and organ development, in the cases of Ac-DEVD-AFC and Ac-IETD-AFC that are used for
immune system functions, and many diseases including detecting Caspase-3 and Caspase-8 activities in cell lysates.20,21
cancer.1–4 Dysregulated apoptosis may lead to tumorigenesis and However, these probes cannot be readily used for real-time
cancer drug resistance.5,6 A number of factors may induce a detection of apoptosis in living cells because of their poor ability
cascade of apoptotic signals leading to pathological changes in to penetrate membrane of living cells.
cells such as DNA breakage and protein degradation.7,8 The
In this study, we designed and synthesized fluorescent probes
caspase family of proteins play an important role in this process. Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC for the detection
Caspases are usually synthesized as inactive zymogens which when of activated caspases in living cells. Results show that when
cells respond to cell death or inflammatory stimuli convert to their catalysed by alkaline phosphatase (ALP), which is widely distributed
active forms.9 At the onset of apoptosis the initiator caspase is on the cell surface,22 the Nap-GFFpY moiety promotes self-assembly
activated and, in turn, cleaves and activates the effector caspases to of the probes to form nanofibers, which enter the cell through
catalyze proteolysis of respective protein substrates.4 Caspase-8 and endocytosis at high efficiency.22 Activated Caspase-8/3 will then
Caspase-3 are a typical pair of initiator caspase and effector caspase recognize IETD/DEVD sequences, cleave the probes, and release
that are activated sequentially, and the activation status of these the AFC groups. The fluorescence intensity of the free AFC is
two enzymes is a quintessential mark for classic apoptosis.1,10–12 proportional to the activity of the corresponding caspase in the
Therefore, there is an urgent need for rapid and real-time monitor- cytoplasm (Scheme 1).
ing of this process in biomedical research as well as in clinical
applications.
The details of syntheses of Nap-GFFpYDEVD-AFC, Nap-
GFFpYIETD-AFC, Nap-GFFYDEVD-AFC and Nap-GFFYIETD-AFC
The detection of caspase activities, unlike analyses of DNA are given in the ESI,† (Fig. S1). Fmoc-Asp(OH)-AFC was synthesized
fragmentation or cell membrane phospholipids, not only reflects in two steps with a total yield of 73%19(Fig. S1, ESI†). Nap-
the intensity of the apoptotic process at an earlier stage, but also GFFpYDEVD-AFC, Nap-GFFpYIETD-AFC, Nap-GFFYDEVD-AFC and
quickly identifies apoptotic signalling pathways the cell Nap-GFFYIETD-AFC were prepared by standard Fmoc solid phase
utilizes.13 Short peptide sequences on the substrates of caspase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the
enzymes have been reported, such as IETD and DEVD for corresponding N-Fmoc protected amino acid as previously
described23 (Fig. 1A and Fig. S1, ESI†). The carboxyl group was
a State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and
Tianjin Key Laboratory of Molecular Drug Research, NanKai University,
Tianjin 300350, China. E-mail: zzs@nankai.edu.cn
b Key Laboratory of Elemento-Organic Chemistry and College of Chemistry,
Nankai University, 94 Weijin Road, Tianjin 300071, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc07821b
deprotected with 95% trifluoroacetic acid and cleaved from the
resin to produce Nap-GFFpYDEVD-AFC, Nap-GFFpYIETD-AFC, Nap-
GFFYDEVD-AFC and Nap-GFFYIETD-AFC as described,24 which
were further purified by high performance liquid chromatography
(HPLC). The chemical synthesis route of ALP inhibitor (DQB)25 is
shown in Fig. S2 (ESI†). The structures of the products were
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Scheme 1 Schematic of the mechanism of Nap-GFFpYIETD-AFC and
Nap-GFFpYDEVD-AFC imaging of Caspase-8 and Caspase-3 activity in
apoptotic tumour cells.
Fig. 2 Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC are catalyzed by
recombinant human Caspase-3/8 protein to release AFC. (A) Concentration
gradient release culve: AFC fluorescence test results of Nap-GFFpYDEVD-
AFC and Nap-GFFpYIETD-AFC at different concentrations (5, 10, 20, 40,
80 mmol LÀ1) after catalysis by recombinant Caspase-3/8 protein for 120 min.
(B) Time gradient release culve: AFC fluorescence test results of
Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC (40 mmol LÀ1) after hydro-
lysed by recombined Caspase-3/8 protein for different period of time (0, 10,
20, 30, 40, 60, 80 min).
from 440 nm to 492 nm. The fluorescence intensity of free AFC
also increased significantly at the same concentration
(Fig. S10A–D, ESI†). The strong fluorescence of AFC can be
observed at the excitation wavelength of 440 nm, but the
fluorescence of the probe itself is not observed at all
(Fig. S10E, ESI†). Therefore, under the excitation wavelength
of 458 nm of the confocal microscope, the activity of Caspase-3
and Caspase-8 protein in living cells can be detected because of
the fluorescence intensity of the freed AFC.
Fig. 1 (A) Synthesis route of Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-
AFC. (B) TEM images of the hydrogels formed by
5
mmol LÀ1
Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC probes with addition of
ALP (2.0 U mLÀ1) at pH = 7.4. Inset: Optical images. Scale bar: 100 nm.
confirmed by NMR spectroscopy analysis and shown in Fig. S3A–
S8B (ESI†).
We determined the direct interaction between self-assembled
peptide probes Nap-GFFpYDEVD-AFC or Nap-GFFpYIETD-AFC
For the solubility of the probes, Nap-GFFpYDEVD-AFC and and the corresponding recombinant human Caspase-3 or
Nap-GFFpYDEVD-AFC at 10 mmol LÀ1 in PBS (pH 7.4) form Caspase-8 proteins. In 120 min, the curves in Fig. 2A exhibit
clear solutions due to the presence of the phosphate group in positive correlation between probe concentration and free AFC
the anionic form (Fig. S9, ESI†). However, the solubility of fluorescence intensity. When the concentration of probes was
probes without the phosphate group significantly decreases maintained at 40 mmol LÀ1, the fluorescence intensity of free
(Fig. S9, ESI†). Addition of recombinant ALP (2.0 U mLÀ1
,
AFC gradually increased with the prolonging of the action time
37 1C) lead to self-assembly of the probes to form nanofibers of corresponding Caspase protein (Fig. 2B). These in vitro enzy-
with a diameter of about 10 nm (Fig. 1B), measured with a matic assays demonstrate that Caspase-3 recognizes the DEVD
transmission electron microscopy (TEM). The self-assembled sequence in the probe Nap-GFFpYDEVD-AFC and Caspase-8
nanofiber hydrogel remains stable for more than 15 days at recognizes the IETD sequence in the probe Nap-GFFpYIETD-
37 1C.
AFC. Both self-assembled probes exhibit the breakage of the
We used a fluorescence spectrophotometer to test the exci- amide bond between aspartic acid and AFC, releasing the
tation and emission wavelengths of the free AFC and the self- fluorescent group AFC.
assembled probes. After AFC dissociated from the probes, the
We treated cervical cancer HeLa cells and breast cancer
maximum excitation wavelength is red-shifted from 384 nm to MCF-7 cells with the two probes at the concentrations up to
440 nm, and the maximum emission wavelength is red-shifted 200 mmol LÀ1 for 48 hours. Analysis of the treated cells by MTT
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and flow cytometry based Anexin-V assays indicated that the
treatment did not induce any significant cytotoxicity or apop-
tosis (Fig. S11 and S12, ESI†), suggesting good biocompatibility
of the probes.
We then determined the ability of the two probes to detect
apoptotic signals in HeLa and MCF-7 cell cultures. The cells
were treated with either Nap-GFFpYDEVD-AFC or Nap-
GFFpYIETD-AFC (50 mmol LÀ1, 4 hours), using Ac-DEVD-AFC
or Ac-IETD-AFC, respectively, in control experiments. Confocal
laser scanning microscopic analysis of the cells showed only
marginal fluorescent background. Upon the addition of H2O2
(1 mmol LÀ1), which induces apoptosis,14,26 the blue-green
fluorescence from free AFC emerged and the fluorescence
intensity became stronger with time, clearly indicating the
occurrence of apoptosis in the cells (Fig. 3 and Fig. S14, ESI†).
Cells treated with either Ac-DEVD-AFC or Ac-IETD-AFC showed
no fluorescence after 60 minutes of H2O2 treatment (Fig. S13,
ESI†). These findings indicate that self-assembly into nanofi-
bers is a prerequisite for efficient cellular uptake of the probes.
We designed a siRNA that was capable of knock down ALP
protein on the surface of tumor cells. Western Blotting analysis
results show that ALP siRNA can significantly reduce the
expression of ALP protein on the cell membrane surface in
MCF-7 and HeLa cells (Fig. S15, ESI†). During the process of
apoptosis of MCF-7 cells and HeLa cells induced by hydrogen
peroxide, using a confocal microscope, we show that the
fluorescence brightness of AFC in the two living cells decreased
significantly after siRNA-treatment, similar to the results from
alkaline phosphatase inhibitor treatments (Fig. S16 and S17,
ESI†). These findings indicate that inhibiting the activity of ALP
of tumor cells significantly reduces the efficiency of self-
assembled probes entering into the cells.
Fig. 4 Confocal images of HeLa cells incubated with Nap-GFFpYDEVD-
AFC or Nap-GFFpYIETD-AFC (50 mmol LÀ1) and Caspase-8/3 inhibitors for
2 h with or without further treatment by H2O2 (1.0 mmol LÀ1). AFC:
lex = 458 nm, lem = 492 nm, RedDot 2: lex = 561 nm, lem = 700 nm.
Scale bar: 20 mm.
the cells were treated with H2O2. These findings indicate that our
probes are highly specific toward the intended caspases, thus
appropriate for the detection of the apoptotic signals in
living cells.
One apparent application of these probes is for high
throughput screening of drug candidates that induce apoptosis
in cancer cells. We treated HeLa cells with either of the two
caspase-responsive fluorescent probes in the presence or
absence of one of the clinically used anti-cancer drugs, namely,
taxol (5 mmol LÀ1), doxorubicin (5 mmol LÀ1), etoposide
(20 mmol LÀ1) and cisplatin (10 mmol LÀ1) (Fig. 5). After 3 hours
of incubation, the fluorescence intensity of the cell cultures was
measured by using a confocal laser scanning microscope. AFC
fluorescence was detected in all of the drug-treated cells,
indicating the activation of the apoptosis process. We repeated
the experiments with MCF-7 cells and obtained similar results
(Fig. S19, ESI†). These data demonstrate the utility of the two
probes in rapid evaluation of the activities of cancer drugs.
We determined the specificity of the two probes for the
caspase cascade by using selective inhibitors of Caspase-8 and
Caspase-3, Z-IETD-FMK and Z-DEVD-FMK, respectively
(Fig. 4 and Fig. S18, ESI†). Results showed that Z-DEVD-FMK
nearly completely prevented Caspase-3-catalyzed proteolysis of
Nap-GFFpYDEVD-AFC, but that of Nap-GFFpYIETD-AFC. On the
other hand, Z-IETD-FMK inhibited Caspase-8-catalyzed proteo-
lysis of Nap-GFFpYIETD-AFC and Nap-GFFpYDEVD-AFC, when
Fig. 5 HeLa cells were co-incubated with Nap-GFFpYDEVD-AFC or
Fig. 3 Confocal images of HeLa cells incubated with 50 mmol LÀ1
Nap-GFFpYDEVD-AFC or Nap-GFFpYIETD-AFC for 4 h and further treated
with H2O2 (1.0 mmol LÀ1). AFC: lex = 458 nm, lem = 492 nm, RedDot 2:
lex = 561 nm, lem = 700 nm. Scale bar: 20 mm.
Nap-GFFpYIETD-AFC and
4 commonly used anticancer drugs, taxol
(5 mmol LÀ1), doxorubicin (5 mmol LÀ1), etoposide (20 mmol LÀ1) and cisplatin
(10 mmol LÀ1), the fluorescence of AFC were observed. AFC: lex = 458 nm,
lem = 492 nm, RedDot 2: lex = 561 nm, lem = 700 nm. Scale bar: 20 mm.
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In summary, we have developed fluorescence-generating,
self-assembling nanofiber probes Nap-GFFpYDEVD-AFC and
Nap-GFFpYIETD-AFC for the analysis of apoptosis in living cells.
The formation of the nanofibers facilitate the uptake of the
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This work was financial supported by National Natural
Science Foundation of China (NSFC) projects (grant no.
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Conflicts of interest
There are no conflicts to declare.
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