Selective tracking of ovarian-cancer-specific γ-glutamyltranspeptidase using a ratiometric two-photon fluorescent probe

Article abstract of DOI:10.1039/c8tb01735b

Real-time tracking of GGT enzymatic activity in human ovarian cancer cells is a reliable method for accurate prediction of cancer diagnosis and management. Here, we report the two-photon ratiometric tracking of GGT activity in cancer cells based on a probe with switchable F?rster resonance energy transfer properties. In the absence of GGT, the designed probe showed two well-resolved emission bands at 461 and 610 nm, corresponding to the 7-hydroxycoumarin donor and BODIPY acceptor, respectively. In contrast, GGT catalyzed cascade reactions including cleavage of the γ-glutamyl group and subsequent aromatic hydrocarbon transfer from the S to N atom increased the distance between the two chromophores, thus decreasing the FRET efficiency, with the recovery of the donor fluorescence at 461 nm. By exploiting this enzyme-triggered ratiometric measurement, successful differentiation of ovarian cancer cells from normal cells with this probe was realized by two-photon fluorescence confocal microscopy.

Full text of DOI:10.1039/c8tb01735b

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Selective tracking of ovarian-cancer-specific
c-glutamyltranspeptidase using a ratiometric
two-photon fluorescent probe†
Cite this: J. Mater. Chem. B, 2018,
6, 7439
Ben Shi,‡a Zhenyu Zhang,‡a Qingqing Jin,‡b Zhiqiang Wang,c Jie Tang,a Ge Xu,a
c
a
Tianli Zhu,a Xueqing Gong,
Xinjing Tang *b and Chunchang Zhao
*
Real-time tracking of GGT enzymatic activity in human ovarian cancer cells is a reliable method for
accurate prediction of cancer diagnosis and management. Here, we report the two-photon ratiometric
tracking of GGT activity in cancer cells based on a probe with switchable Forster resonance energy
¨
transfer properties. In the absence of GGT, the designed probe showed two well-resolved emission
bands at 461 and 610 nm, corresponding to the 7-hydroxycoumarin donor and BODIPY acceptor,
respectively. In contrast, GGT catalyzed cascade reactions including cleavage of the g-glutamyl group
and subsequent aromatic hydrocarbon transfer from the S to N atom increased the distance between
the two chromophores, thus decreasing the FRET efficiency, with the recovery of the donor fluorescence at
461 nm. By exploiting this enzyme-triggered ratiometric measurement, successful differentiation of ovarian
cancer cells from normal cells with this probe was realized by two-photon fluorescence confocal microscopy.
Received 3rd July 2018,
Accepted 4th October 2018
DOI: 10.1039/c8tb01735b
rsc.li/materials-b
reliable method for accurate prediction of cancer diagnosis
and management.
Introduction
Ovarian cancer is one of the most common tumors in women
Indeed, a number of analytical methods have been explored
and accounts for a major cause of cancer death.1 Primary for GGT assays to better understand GGT-related pathological
cytoreductive surgery is vital to the treatment of women with sequences, including high-performance liquid chromatography
ovarian carcinoma.2 Since successful surgical management (HPLC), and colorimetric and fluorescent assays.8 Because of their
crucially relies on the precise localization of cancerous tissues, operational simplicity, spatiotemporal resolution and noninvasive
measurement of ovarian-cancer-related biomarkers assisted by features, fluorescent probes and sensors have attracted intense
molecular imaging probes will improve surgical prognosis.3 interest.9–14 Accordingly, several fluorescent probes for GGT detec-
Among many studied biomarkers, g-glutamyltranspeptidase tion have been developed based on the GGT-catalyzed cleavage of
(GGT) has been reported to express at high levels in human the g-glutamyl bond.15–25 However, many reported sensing systems
ovarian cancers.4–6 GGT, a cell-membrane-bound enzyme, are still subject to limitations concerning a fluorescence turn-on
catalyzes the metabolism of GSH, wherein a complementary response with a single detection signal. As is known, fluorescence
supply of cysteine is provided, thus conferring the advantage turn-on signals heavily depend on experimental conditions such
for tumorigenesis and invasion.7 Real-time tracking of GGT as probe concentration and excitation intensity.26–29 In sharp
enzymatic activity in human cancer cells should thus be a contrast, ratiometric probes can eliminate adverse effects and
provide quantitative measurements of biomolecules.30–34 Recently,
we and others have reported two such ratiometric probes for GGT
a Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of
activity assay.35,36 However, the shallow penetration depth of the
Chemistry and Molecular Engineering, East China University of Science &
short excitation wavelength restricts their practical applications.
Technology, Shanghai 200237, P. R. China. E-mail: zhaocchang@ecust.edu.cn
As an alternative and innovative strategy, designing two-photon
excitation (TPE) probes with ratiometric imaging should address
the above mentioned issues. TPE, utilizing two near-IR photons
as the excitation source, endows the advantages of deep tissue
penetration, minimum photodamage to biosamples, and low
background fluorescence.37,38 However, to the best of our
knowledge, no ratiometric probes with two-photon excitation
for GGT assay have been reported yet.
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing, 100191, China. E-mail: xinjingt@bjmu.edu.cn
c Key Laboratory for Advanced Materials, Centre for Computational Chemistry and
Research Institute of Industrial Catalysis, School of Chemistry and Molecular
Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai, 200237, P. R. China
† Electronic supplementary information (ESI) available: Procedures for synthesis,
characterization data, and supplementary figures. See DOI: 10.1039/c8tb01735b
‡ These authors contributed equally to this work.
This journal is ©The Royal Society of Chemistry 2018
J. Mater. Chem. B, 2018, 6, 7439--7443 | 7439

View Article Online
Paper
Journal of Materials Chemistry B
Scheme 1 GGT switchable FRET in the designed probe PZS1 and its
conversion to PPZS1.
In this contribution, we reported the two-photon ratiometric
detection of cancer cells by tracking GGT activity based on a
¨
probe with switchable Forster resonance energy transfer prop-
erties (Scheme 1). In our design, 7-hydroxycoumarin is expected
to act as an energy donor and a BDIPIY derivative as an energy
acceptor. In the free probe, the two fluorophores are installed in
close proximity to each other and FRET from the 7-hydroxycoumarin
to BODIPY chromophore occurs efficiently, affording two well-
resolved emission bands at 461 and 610 nm. Upon interaction
with GGT, the cleavage of the g-glutamyl group and subsequent
aromatic hydrocarbon transfer from the S to N atom lead to the
unfavorable arrangement of the two fluorophores (increasing
the distance between the two chromophores), thus decreasing
the FRET efficiency, with the recovery of the donor fluorescence
at 461 nm. By means of this enzyme-triggered ratiometric
measurement, this probe successfully demonstrated its capa-
bility for differentiation of ovarian cancer cells from normal
cells by two-photon fluorescence confocal microscopy.
Scheme 2 Synthesis of PZS1.
Results and discussion
The synthetic procedures for probe PZS1 are depicted in
Scheme 2. In brief, treatment of 5-(tert-butoxy)-4-((tert-butoxy-
carbonyl)amino)-5-oxopentanoic acid with N-hydroxysuccin-
imide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) in CH2Cl2 led to the production of an
activated intermediate, which was coupled to S-trityl-L-cysteine
to afford the Gly-Cys peptide. Subsequently, this peptide was
conjugated to 7-hydroxylcoumarin via a piperazine linkage by a
standard coupling reaction to provide compound 5, which was
then subjected to sequential deprotection with TFA (trifluoroacetic
acid) and TES (triethylsilane) to produce compound 6. Finally, the
probe PZS1 was obtained by the aromatic nucleophilic substitution
reaction between 6 and BODIPY via the formation of a thioether
bond. The chemical structures of the new compounds were fully
characterized by 1H NMR, 13C NMR and HRMS.
Fig. 1 Time-dependent absorption (a) and fluorescence (b) spectral
changes of PZS1 (10 mM) in the presence of GGT (600 U LÀ1). lex
=
350 nm. (c) The fluorescence intensity ratio (I461/I610) of PZS1 (10 mM) when
GGT was pretreated with various concentrations of GGsTOP before
treatment with the probe for 110 min. (d) Time dependent fluorescence
changes of the BODIPY unit in PZS1 after the addition of GGT (600 U LÀ1).
lex = 510 nm.
(Fig. 1b), with a FRET efficiency of 88.8% (Fig. S1, ESI†), and the
The fluorescence response of PZS1 to GGT was first evaluated in solution of probe PZS1 showed a pink fluorescence. Upon addition
aqueous buffer (0.01 mM PBS, pH 7.4). In the absence of GGT, of GGT (600 U LÀ1), the emission band at 461 nm exhibited a
PZS1 shows intense absorption at 358 nm and 538 nm (Fig. 1a), dramatic enhancement accompanied by a decrease of the emis-
derived from the 7-hydroxylcoumarin and BODIPY chromophore, sion at 610 nm, and the fluorescence color of the solution was
respectively. Upon excitation at 350 nm, two well-resolved emission bluish green (Fig. S2, ESI†). Notably, a 23.5-fold enhancement of
bands were noted at 461 nm and 610 nm for the free PZS1 the fluorescence intensity ratio (I461/I610) was observed (Fig. 1c),
7440 | J. Mater. Chem. B, 2018, 6, 7439--7443
This journal is ©The Royal Society of Chemistry 2018

View Article Online
Journal of Materials Chemistry B
Paper
which indicated that PZS1 can serve as a potent ratiometric
probe. In contrast, the ratiometric fluorescence change was
reduced when the GGT was pretreated with GGsTOP (Fig. S3,
ESI†), an inhibitor of GGT, before treatment with probe PZS1.
The reduction was GGsTOP dose-dependent. The intensity ratio
(I461/I610) decreased from 23.5 to 13.8, 8.5 and 1.8, respectively,
after pretreatment with 1 mM, 3 mM and 5 mM GGsTOP,
indicating that GGT is vital for triggering the FRET-based ratio-
metric fluorescence signal change. According to our previous
studies,35 GGT can catalyze the cleavage of the g-glutamyl bond,
which was followed by the aromatic hydrocarbon transfer reaction
between the sulfur and the nitrogen atom in BODIPY, leading to
obvious fluorescence variations.39,40 In the case of PZS1, such
GGT-induced translation was demonstrated by observation of
the optical changes of BODIPY. As expected, a dramatic decrease
in the emission of PZS1 at 610 nm was afforded in the presence of
GGT, along with a simultaneous buildup of an emission at 567 nm
corresponding to amino-substituted BODIPY (Fig. 1d). This result
confirmed that GGT triggered the transformation from PZS1
(sulfur-substituted BODIPY) to amino-substituted BODIPY. HRMS
analysis was also used to confirm this reaction process. The peak
of our proposed amino-substituted BODIPY (m/z = 719.2021,
[M + H]+) was clearly manifested (Fig. S4, ESI†).
Obviously, the GGT-triggered transformation reaction con-
tributed to the switchable FRET process and the recovery of the
donor fluorescence at 461 nm. To gain a deep understanding of
the FRET, theoretical calculations using density functional
theory (DFT) with G09 and B3LYP/6-311G were carried out.
Based on the optimized chemical structures of PZS1 and PPZS1,
the distance between the coumarin and BODIPY chromophore
was estimated to be 8.19 Å and 11.84 Å, respectively (Fig. 2). It is
known that the energy transfer efficiency is inversely propor-
tional to the sixth power of the distance between the donor and
acceptor, and increasing the distance will rapidly lead to a
decrease of energy-transfer efficiency. GGT-triggered conversion of
PZS1 to PPZS1 increases the distance between coumarin and
BODIPY, presumably contributing to the switchable FRET process.
The selective response of PZS1 to GGT activity was then
tested. The data in Fig. 3 indicated that PZS1 exhibited negligible
change in the fluorescence ratio (I461/I610) upon treatment with
potential interfering species, including glucoamylase, aprotinin,
collagen hydrolase, phosphatase, lipase, human plasma, FBS and
inorganic salts (KCl, MgCl2, CaCl2, ZnCl2, FeCl2, FeCl3, and MnCl2).
Fig. 3 (a) The fluorescence intensity ratio (I461/I610
)
changes in the
presence of potential interfering species including collagen hydrolase,
phosphatase, aprotinin, glucoamylase, 10% FBS or 10% human plasma.
(b) The effects of some important metal ions on the responsive process of
PZS1 (10 mM) to GGT (600 U LÀ1).
However, a dramatic enhancement was noted when treated with
GGT, suggesting that PZS1 has sufficient selectivity for application.
Notably, fluorescence titration experiments showed that the fluores-
cence ratio (I461/I610) change of PZS1 is GGT dose-dependent within
a concentration range from 0 to 800 U LÀ1 (Fig. S5, ESI†). Utilizing
the Michaelis–Menten equation, the kinetic values of PZS1 against
GGT were then estimated. The Km and Vmax values were calculated
to be 0.75 mM minÀ1 and 18.62 mM, respectively (Fig. S6, ESI†).
Having established the specificity of PZS1 for monitoring
GGT activity in aqueous solutions, we then explored the capability
of PZS1 for differentiating ovarian cancer cells from normal cells
via living-cell tracking of GGT activity. The feasibility of this probe
Fig. 4 Differentiation of ovarian cancer cells from normal cells via living-
cell tracking of GGT activity with PZS1 (10 mM). For imaging of HUVEC cells
or OVCAR5 cells, these cells were incubated with PZS1 (10 mM) for
30 minutes. For assaying the inhibitory effect of GGsTOP, ovarian cancer
cells were pretreated with GGsTOP for 30 minutes and then loaded with
PZS1 (10 mM) for 30 minutes. The excitation wavelength was 700 nm and
the emission was collected at 400–500 nm (blue channel) and 600–
650 nm (red channel); the ratio image was generated from the blue to red
channel. The scale bar is 10 mm.
Fig. 2 The optimized chemical structures of PZS1 and PPZS1 by theoretical
calculations using density functional theory (DFT) with G09 and B3LYP/
6-311G.
This journal is ©The Royal Society of Chemistry 2018
J. Mater. Chem. B, 2018, 6, 7439--7443 | 7441

View Article Online
Paper
Journal of Materials Chemistry B
3 Y. Liu, J. Tan, Y. Zhang, J. Zhuang, M. Ge, B. Shi, J. Li, G. Xu,
S. Xu, C. Fan and C. Zhao, Biomaterials, 2018, 173, 1.
4 M. H. Hanigan, H. F. Frierson Jr., J. E. Brown, M. A. Lovell
and P. T. Taylor, Cancer Res., 1994, 54, 286.
5 A. Pompella, V. D. Tata, A. Paolicchi and F. Zunino, Biochem.
Pharmacol., 2006, 71, 231.
6 D. Yao, D. Z. Huang, J. Lu, Q. Tao, Z. Yu and X. Meng,
Cancer, 2000, 88, 761.
7 A. Pompella, V. De Tata, A. Paolicchi and F. Zunino, Biochem.
Pharmacol., 2006, 71, 231.
8 Y. Urano, M. Sakabe, N. Kosaka, M. Ogawa, M. Mitsunaga,
D. Asanuma, M. Kamiya, M. R. Young, T. Nagano, P. L.
Choyke and H. Kobayashi, Sci. Transl. Med., 2011, 3, 110.
9 T. Kowada, H. Maeda and K. Kikuchi, Chem. Soc. Rev., 2015,
44, 4953.
10 M. Vendrell, D. Zhai, J. C. Er and Y. T. Chang, Chem. Rev.,
2012, 112, 4391.
11 J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012,
4, 973.
12 D. Wu, A. C. Sedgwick, T. Gunnlaugsson, E. U. Akkaya,
J. Yoon and T. D. James, Chem. Soc. Rev., 2017, 46, 7105.
13 M. H. Lee, A. Sharma, M. J. Chang, J. J. Lee, S. Son, J. L.
Sessler, C. Kang and J. S. Kim, Chem. Soc. Rev., 2018, 47, 28.
14 L. Wu and X. G. Qu, Chem. Soc. Rev., 2015, 44, 2963.
15 L. H. Li, W. Shi, Z. Wang, Q. Y. Gong and H. M. Ma, Anal.
Chem., 2015, 87, 8353.
16 X. F. Hou, F. Zeng and S. Z. Wu, Biosens. Bioelectron., 2016,
85, 317.
17 T. Nishihara, H. A. I. Yoshihara, H. Nonaka, Y. Takakusagi,
F. Hyodo, K. Ichikawa, E. Can, J. A. M. Bastiaansen,
Y. Takado, A. Comment and S. Sando, Angew. Chem., Int. Ed.,
2016, 55, 10626.
18 M. Chiba, Y. Ichikawa, M. Kamiya, T. Komatsu, T. Ueno,
K. Hanaoka, T. Nagano, N. Lange and Y. Urano, Angew.
Chem., Int. Ed., 2017, 56, 10418.
19 H. J. Tong, Y. J. Zheng, L. Zhou, X. M. Li, R. Qian, R. Wang,
J. H. Zhao, K. Y. Lou and W. Wang, Anal. Chem., 2016, 88, 10816.
20 S. Park, S. Y. Lim, S. M. Bae, S. Y. Kim, S. J. Myung and
H. J. Kim, ACS Sens., 2016, 1, 579.
for differentiation was investigated using two-photon microscopy
(TPM) with TP excitation at 700 nm. As shown in Fig. 4, OVCAR5
cells labeled with PZS1 under excitation for 30 min showed strong
fluorescence in the blue channel but negligible fluorescence in the
red channel with an average emission ratio (Iblue/Ired) of 4.52
(Fig. S7, ESI†). However, the signal ratio decreased when the cells
were pretreated with GGsTOP before incubation with probe PZS1.
The ratio decreased from 4.52 to 1.34 and 0.81, respectively, after
addition of 100 mM and 1 mM GGsTOP. Notably, Iblue/Ired was time-
dependent (Fig. S8, ESI†); for example, OVCAR5 cells incubated
with PZS1 for 15 min, 30 min, 45 min and 1 h led to ratiometric
ratios of 3.5, 4.6, 4.5 and 4.4, respectively, indicative of the enzyme-
mediated transformation from PZS1 to amino-substituted BODIPY
in transmembrane domains.35 These results clearly demonstrate
that PZS1 is capable of tracking GGT levels in living cells using
TPM. As is known, human umbilical vein endothelial cells (HUVECs)
express negligible GGT; thus HUVECs stained with PZS1 should
exhibit a decreased Iblue/Ired ratio when compared to ovarian cancer
cells. As expected, the emission ratio in HUVECs was reduced to
around 1.22. In addition, prolonging the treatment of HUVECs with
PZS1 showed no obvious Iblue/Ired change. These results firmly
evidenced the capability of PZS1 for differentiation of ovarian cancer
cells from normal cells by tracking GGT activity.
Conclusions
In conclusion, we have developed a switchable FRET probe for
assaying GGT activity that induces aromatic hydrocarbon transfer
from the S to N atom in the BODIPY unit of this probe, increasing
the distance between the donor and acceptor and thus modulating
the FRET. Utilizing GGT-triggered ratiometric responsiveness, this
probe was successfully applied to monitor GGT activity in living
cells by two-photon fluorescence confocal microscopy, which
illustrated that the probe is capable of differentiating ovarian
cancer cells from normal cells by tracking GGT activity.
Conflicts of interest
21 S. Li, R. Hu, C. L. Yang, X. Zhang, Y. Zeng, S. Q. Wang,
X. D. Guo, Y. Li, X. P. Cai, S. R. Li, C. W. Han and G. Q. Yang,
Biosens. Bioelectron., 2017, 98, 325.
22 Z. J. Hai, J. J. Wu, L. Wang, J. C. Xu, H. F. Zhang and
G. L. Liang, Anal. Chem., 2017, 89, 7017.
23 J. Tian, Q. L. Yan, Y. Zhu, J. C. Zhang, J. Li, B. Shi, G. Xu,
C. H. Fan and C. C. Zhao, Chin. J. Chem., 2017, 35, 1711.
24 X. F. Hou, Q. X. Yu, F. Zeng, C. M. Yu and S. Z. Wu, Chem.
Commun., 2014, 50, 3417.
25 P. S. Zhang, X. F. Jiang, X. Z. Nie, Y. Huang, F. Zeng, X. T. Xia
and S. Z. Wu, Biomaterials, 2016, 80, 46.
The authors declare no competing financial interest.
Acknowledgements
We gratefully acknowledge the financial support from the National
Science Foundation of China (grant number 21672062).
Notes and references
´
1 I. Vergote, C. G. Trope, F. Amant, G. B. Kristensen, T. Ehlen,
N. Johnson, R. H. M. Verheijen, M. E. L. van der Burg, A. J. 26 M. G. Ren, K. Zhou, L. W. He and W. Y. Lin, J. Mater. Chem.
Lacave, P. B. Panici, G. G. Kenter, A. Casado, C. Mendiola, B, 2018, 6, 1716.
C. Coens, L. Verleye, G. C. E. Stuart, S. Pecorelli and N. S. 27 L. Y. Niu, Y. Z. Chen, H. R. Zheng, L. Z. Wu, C. H. Tung and
Reed, N. Engl. J. Med., 2010, 363, 943. Q. Z. Yang, Chem. Soc. Rev., 2015, 44, 6143.
2 C. T. Griffiths and A. F. Fuller, Surg. Clin. North Am., 1978, 28 Z. G. Song, Y. N. Hong, R. T. K. Kwok, J. W. Y. Lam, B. Liu
58, 131.
and B. Z. Tang, J. Mater. Chem. B, 2014, 2, 1717.
7442 | J. Mater. Chem. B, 2018, 6, 7439--7443
This journal is ©The Royal Society of Chemistry 2018

View Article Online
Journal of Materials Chemistry B
Paper
29 Z. Ye, C. Duan, Q. Hu, Y. Zhang, C. Q. Qin and L. T. Zeng, 35 F. Y. Wang, Y. Zhu, L. Zhou, L. Pan, Z. F. Cui,
J. Mater. Chem. B, 2017, 5, 3600.
30 B. Shi, X. F. Gu, Z. J. Wang, G. Xu, Q. Fei, J. Tang and
C. C. Zhao, ACS Appl. Mater. Interfaces, 2017, 9, 35588.
Q. Fei, S. L. Luo, D. Pan, Q. Huang, R. Wang, C. C. Zhao,
H. Tian and C. H. Fan, Angew. Chem., Int. Ed., 2015,
54, 7349.
31 C. Zhao, X. Zhang, K. Li, S. Zhu, Z. Guo, L. Zhang, F. Wang, 36 S. Park, D. J. Bae, Y. M. Ryu, S. J. Myung and S. J. Kim, Chem.
Q. Fei, S. Luo, P. Shi, H. Tian and W.-H. Zhu, J. Am. Chem.
Soc., 2015, 137, 8490.
Commun., 2016, 52, 10400.
37 F. Helmchen and W. Denk, Nat. Methods, 2005, 2, 932.
32 G. Xu, Q. Yan, X. Lv, Y. Zhu, K. Xin, B. Shi, R. Wang, J. Chen, 38 W. R. Zipfel, R. M. Williams and W. W. Webb, Nat. Biotechnol.,
W. Gao, P. Shi, C. Fan, C. Zhao and H. Tian, Angew. Chem.,
Int. Ed., 2018, 57, 3626.
33 F. Wang, G. Xu, X. Gu, Z. Wang, Z. Wang, B. Shi, C. Lu,
X. Gong and C. Zhao, Biomaterials, 2018, 159, 82.
34 Z. S. Wu and X. J. Tang, Anal. Chem., 2015, 87, 8613.
2003, 21, 1369.
39 L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and
Q. Z. Yang, J. Am. Chem. Soc., 2012, 134, 18928.
40 F. Wang, L. Zhou, C. Zhao, R. Wang, Q. Fei, S. Luo, Z. Guo,
H. Tian and W.-H. Zhu, Chem. Sci., 2015, 6, 2584.
This journal is ©The Royal Society of Chemistry 2018
J. Mater. Chem. B, 2018, 6, 7439--7443 | 7443