A highly selective fluorogenic substrate for imaging glutathione S-transferase P1: Development and cellular applicability in epigenetic studies

Article abstract of DOI:10.1039/c9cc03064f

Pi-class glutathione S-transferase (GSTP1) is a molecular marker enzyme whose expression level is altered in various malignant tumour tissues. Herein, we report the first highly selective fluorogenic GSTP1 substrate, Ps-TG, and its membrane-permeable deri

Full text of DOI:10.1039/c9cc03064f

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A highly selective fluorogenic substrate for imaging glutathione S-
transferase P1: development, and cellular applicability to
epigenetic studies†
Received 00th January 20xx,
Accepted 00th January 20xx
Masay Mori, Yuuta Fujikawa,* Manami Kikkawa, Moeho Shino, Mei Sawane, Shiho Sato, and
Hideshi Inoue
DOI: 10.1039/x0xx00000x
Pi-class glutathione S-transferase (GSTP1) is a molecular marker reports have shown that GSTP1 is a crucial factor for
enzyme whose expression level is altered in various malignant tumorigenesis and tumour growth, based on the fact that GSTP1
tumour tissues. Herein, we report the first highly selective knockout or knockdown has a negative effect on the viability or
fluorogenic GSTP1 substrate, Ps-TG, and its membrane-permeable proliferation rate of cancer cells9,14. On the other hand, loss or
derivative Ps-TAc, for specific visualization of intracellular GSTP1 reduction of GSTP1 expression compared to that of the
activity in cancer cells or epigentically regulated GSTP1 expression. surrounding normal cells has often been observed in several
types of cancers, including prostate cancer. The loss of GSTP1
Enzymes play essential roles as biological catalysts in most
physiological and pathological contexts. Therefore, methods for
detecting or visualising the activity of a specific enzyme at the
single-cell level are valuable1. For example, the visualization of
cancer-associated enzyme activity using fluorogenic substrates
with higher sensitivity and specificity than existing substrates
would facilitate the accurate detection of small tumours. Various
“smart” (activatable) fluorogenic substrates have been reported
to enable the early diagnosis of tumours on a molecular basis and
to allow determination of the need for subsequent therapeutic
intervention2–5 and monitoring of the effects of therapeutic
intervention. However, it is still difficult to target or visualise the
activity of a single specific enzyme from others with similar
activity.
expression results from the aberrant hypermethylation of CpG
islands in the promoter region of the GSTP1 gene15,16. Therefore,
the hypermethylation of CpG islands in the GSTP1 promoter,
and the resulting disappearance of GSTP1 expression, can serve
as a molecular marker for the detection and diagnosis of prostate
and several other cancers16. In addition, restored expression of
the GSTP1 protein by treatment with epigenetic drugs has been
suggested to be a marker of the efficacy of drug treatment in
some types of tumours17,18
.
The detection and visualisation of altered GSTP1 activity in cancer
cells would therefore be useful for cancer diagnosis and assessment of
the efficacy of drug treatment. However, fluorogenic substrates
reported to date19-24 are not suitable for the selective visualisation of
GSTP1 activity in living cells due to their lack of GSTP1 selectivity
Glutathione S-transferases (GSTs) are
a
group of or rapid non-enzymatic background reaction with GSH. Thus, to our
multifunctional enzymes that catalyse the nucleophilic attack of knowledge, currently no optimal fluorogenic substrate is available for
reduced glutathione (GSH) on electrophiles such as real-time and selective visualization of intracellular GSTP1 activity.
halonitroaromatics and α,β-unsaturated carbonyls, thereby
detoxifying endogenously generated or exogenous reactive
electrophiles to protect cells from the formation of undesirable
protein or nucleotide adducts6. The human soluble GSTs are
categorized into eight classes (Alpha, Mu, Pi, Theta, Omega,
Sigma, Kappa, and Zeta) based on their amino acid sequence
similarity. It is known that the Pi-class GST GSTP1 is frequently
and highly expressed in a variety of solid tumours7–12 as well as
in benign or pre-neoplastic lesions of colon cancer13. Several
Fig.
1 Fluorescence activation of Ps-TG by GSTP1-selective
glutathionylation. Before the reaction, the fluorescence of Ps-TG is
quenched by photo-induced electron transfer. After replacement of
the nitro group with GSH by GSTP1 catalysis, quenching is cancelled,
resulting in enhanced fluorescence.
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1
Horinouchi, Hachioji, Tokyo, 192-0392 (Japan) E-mail: yfuji@toyaku.ac.jp
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Herein, we report the development and biological application of the 104 M-1s-1 based on the linear relationship betweenViethweArtsiculbe sOtnrlaintee
DOI: 10.1039/C9CC03064F
first highly selective fluorogenic GSTP1 substrate, Ps-TG. Among concentration and the initial rate of product formation at 0.13-1.0 μM
the previously reported GST substrates, 3,4-dinitrobenzanilide was Ps-TG and 1 mM GSH (Fig. 2B, Table S3). The app. kcat/KM value
used as a fluorescence-modulating moiety that enables photo-induced for Ps-TG is comparable to that determined for the previously
electron transfer (d-PeT) quenching (Fig. S1)22,24,25. However, both characterized GSTP1-1 substrate FOMe-Ac23 [(9.5 ± 0.3) × 104 M-1s-
the lack of subtype selectivity and the non-enzymatic background 1], but seventy-fold smaller than that determined for the non-selective
reaction with intracellular GSH (e.g., 0.5-10 mM26) hampered fluorogenic substrate 3,4-DNADCF24 [(690 ± 100) × 104 M-1s-1]. The
selective imaging of GSTP1 activity under physiological conditions. app. kcat/KM value for Ps-TG of GSTM1-1 was (0.94 ± 0.08) × 104 M-
To overcome these drawbacks, we examined a series of nitroaromatics 1s-1, which is ten-times smaller than that of GSTP1-1. Thus, Ps-TG is
and identified 5-mesyl-2-nitrobenzalinide as a GSTP1-selective a preferred substrate for GSTP1-1 compared to GSTM1-1 (Fig. 2C).
substrate (Fig. 1). The kinetic and electrochemical properties of 5-
mesyl-2-nitrobenzalinide were characterized, as shown in Figs. S2-S3
and Table S1 (see Electronic Supplementary Information). Based on
these results, Ps-TG was designed by coupling the 5-mesyl-2-
nitrobenzanilide fluorescence-modulating moiety with the
TokyoGreen (TG) fluorophore moiety. The synthetic route for Ps-TG
Incubation of Ps-TG with various reductants showed only
negligible increases in fluorescence (Fig. 2D, Fig. S9),
suggesting essentially no false activation of Ps-TG by these
molecules. Taken together, these results suggested the
effectiveness of Ps-TG as a selective and specific fluorogenic
substrate for the detection of GSTP1 catalytic activity in vitro.
Although Ps-TG was loaded onto cells expressing GSTP1, no
fluorescence activation was observed, presumably due to the
1
and characterisation of each molecule by H-NMR, 13C-NMR, and
HRMS are detailed in the Supporting Information. Ps-TG showed an
absorption and emission maximum at 493 nm and 510 nm,
respectively, and a very weak fluorescence signal with a quantum
efficiency (QE) of 0.008 due to d-PeT quenching (Fig. 2A, Table S1).
Fluorescence was dramatically enhanced up to a QE of 0.76 (about a
95-fold enhancement) upon GSH conjugation catalysed by GSTP1
(Table S2). The glutathionylated product was confirmed by HPLC
and LC-MS analyses (Fig. S4). The detection limit of GSTP1-1 was
determined to be 0.115 ng/ml (Fig. S5). Non-enzymatic reaction with
GSH was minimal even at high concentrations of GSH up to 20 mM
at pH 7.4 (Fig. S6). Based on the pH dependence of the spectra, the
pKa values of the phenolic group were 6.2 for Ps-TG and 6.3 for the
fluorescent product (Figs. S7 and S8), suggesting that fluorescence
intensity is stable around physiological pH. The apparent specificity
constant (app. kcat/KM) for Ps-TG was determined to be (11.0 ± 0.3) ×
lack of membrane permeability by Ps-TG (Fig. S10). Therefore,
the phenol group of Ps-TG was acylated to obtain Ps-TAc with
sufficiently increased hydrophobicity to freely pass through the
plasma membrane (Fig. 3A). Absorbance spectrum changes
confirmed that Ps-TAc was deacetylated by incubation with
porcine liver esterase (PLE) or with GSH at room temperature
(
Fig. S11), suggesting that Ps-TAc is converted to Ps-TG after
entering the cells. The applicability of Ps-TAc as an imaging
probe was demonstrated in MCF7 cells transiently transfected
with a GSTP1-encoding fusion gene. When the cells transfected
with pIRES2-DsRed/3×FLAG-GSTP1 were loaded with 2.5 µM
Ps-TAc, an intense green fluorescence signal was observed
exclusively in the DsRed-positive cells (Fig. 3B). This suggests
that Ps-TAc efficiently enters the cells and becomes a substrate
for GSTP1. To further investigate subtype-selectivity in living
cells, we tested human GST enzymes, including eighteen
subtypes from the eight classes. First, MCF7 cells forcibly
Fig. 2 (A) Fluorescence spectra of 2 µM Ps-TG in sodium phosphate buffer
(pH 7.4) before (cyan) and after (magenta) reaction. Ex 490 nm. (B)
Relationship between the substrate concentration and the specific
activity of GSTP1-1 toward Ps-TG in the presence of 1 mM GSH in sodium
phosphate buffer (100 mM, pH 7.4) at 25°C. (C) Time course of the
change in fluorescence intensity of Ps-TG (2 µM) by GST catalysis (1
µg/ml) in the presence of 1 mM GSH. Ex 490 nm, Em 510 nm. Data are
shown as dots and lines as follows: red, GSTA1-1; green, GSTM1-1;
purple, GSTP1-1; cyan, no GST. (D) Reactivity of Ps-TG with various redox
active species (1 mM). 1. GSH, 2. DTT, 3. L-cysteine, 4. 2-
mercaptoethanol, 5. Na2S3, 6. NADH, 7. NAD+, 8. NADPH, 9. NADP+, 10.
ascorbic acid, 11. GSH/GSTP1-1, 12. none.
Fig. 3 (A) Molecular structure of Ps-TAc. (B) Representative fluorescence
images and their merged image of MCF7 cells transfected with pIRES2
DsRed-Express2/3×FLAG-GSTP1 and loaded with 2.5 µM Ps-TAc in HBSS
at room temperature for 5 min. Scale bars: 40 µm. (B) Box-whisker plot
representation of green fluorescence intensity in cells expressing DsRed.
n = 60 cells.
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Fig. 4 Various cancer cell lines stained with Ps-TAc (upper panels). The Ps-TAc fluorescence images (green) were merged with the corresponding images of
nuclei (blue) and bright-field images (grey) in various cancer cells (lower panels). Each cell line was incubated with 2.5 µM Ps-TAc in HBSS at room
temperature for 15 min, and fluorescence and bright-field images were acquired without removing excess fluorogenic substrate. Scale bars: 40 µm.
GSTP1 activity at the single-cell level and to visualise subcellular
localization of the glutathionylated product.
expressing GST were prepared for each GST subtype (Fig.
S12A). Expression was exclusively observed in the DsRed-
positive cells (Fig. S12B). Then, the transfected MCF7 cells
were loaded with Ps-TAc and subjected to fluorescence imaging
using confocal microscopy. DsRed-positive cells were randomly
analysed. Notably, an intense green fluorescence signal was
observed only in the DsRed-positive cells (Fig. 3C). Thus, it was
demonstrated that Ps-TAc shows unprecedented selectivity as an
The applicability of Ps-TAc to the analysis of epigenetic regulation
of GSTP1 expression was also examined. Several cancer cell lines
have been reported to lack GSTP1 expression due to aberrant
hypermethylation of CpG islands in the GSTP1 promoter region16.
Treatment with decitabine (DAC), an inhibitor of DNA methylation,
is expected to lead to GSTP1 expression in cells where the expression
of GSTP1 is repressed due to hypermethylation in the promoter
region27. Previous reports suggested that restored GSTP1 expression
can be used as a marker of the efficacy of epigenetic drugs in vitro17,28
and in vivo18. In assessing the efficacy of DNA demethylating agents,
direct (e.g., bisulphate sequencing, methylation-sensitive restriction
imaging probe for GSTP1 activity.
Next, to demonstrate its applicability to the visualisation of
endogenous GSTP1 activity in tumour cells, Ps-TAc was loaded onto
various types of cancer cells with or without GSTP1 expression.
Remarkable fluorescence was observed in the GSTP1-positive cells
HT29, HCT116, HuCCT1, DU145 and HT-1080, but not in the GST-
fingerprinting,
chromatin
immunoprecipitation
on
DNA
microarray)29 and indirect methods (e.g., detection of re-expression of
target genes by qPCR, immunoblotting) have been used to evaluate
changes in the methylation levels of target genes. None of these
methods, however, are applicable to live cells and give information on
re-expression of the target gene with single cell resolution. To
investigate whether Ps-TAc is applicable to assessing the effect of
epigenetic drugs based on the GSTP1 re-expression level, MCF7 cells
were treated with the epigenetic drug DAC for 2-6 days. At each day
of treatment, cells were stained with Ps-TAc and subjected to
fluorescence confocal microscopic imaging (Fig. 5). The fluorescence
activation of Ps-TAc increased daily in cells treated with 1.25 µM
DAC whereas the fluorescence signal was only background level in
non-treated cells, even on day 6 (Fig. 5B). Propidium iodide (PI)
staining showed no significant increase in dead cells following DAC
treatment. Increased levels of GSTP1 protein upon DAC treatment
were confirmed by immunoblot analysis and immunofluorescence
staining (Fig. S14). GSTP1 knockdown with siRNA resulted in a
significant decrease in fluorescence activation of Ps-TAc in the DAC-
treated cells. These results suggest that fluorescence activation of Ps-
TAc in the DAC-treated cells is due to de-repression of GSTP1
expression by the demethylation of CpG islands (Fig. S15).
Collectively, these results demonstrated the utility of Ps-TAc as an
imaging agent to visualise and assess the effect of an epigenetic drug
at the single cell level based on the GSTP1 expression level. Sorting
cells with strong from very low fluorescence using a fluorescence
activated cell sorter (FACS) based on probe fluorescence would allow
further analysis of the action of epigenetic drugs at the single cell level
or subsequent investigation of tumorigenicity30. Therefore, probes
with characteristics more suitable for FACS analysis are currently
under development and will be reported elsewhere.
negative cells MCF7 and LNCaP (Fig. 4). Note that the staining
patterns (e.g., fluorescence intensity, subcellular localization of the
fluorescent product) of GSTP1-positive cells were very
heterogeneous within a single cell line; there was a large difference in
fluorescence intensity among individual cells. Treatment with short
interfering RNA (siRNA) against GSTP1 markedly decreased the
fluorescence signal concomitantly with decreased GSTP1 expression
in DU145 and HT-1080 cells (Fig. S13), consistent with the inference
that fluorescence should reflect intracellular GSTP1 activity. Taken
together, these results establish that Ps-TAc can be used to visualise
Fig. 5 Visualization of GSTP1 expression in MCF7 cells treated with DAC.
(A) Fluorescence (upper panels) and merged (lower panels) with bright-
field (BF) images (middle panels) of MCF7 cells treated with 1.25 µM DAC
or vehicle for 6 days. Cells were stained with 2.5 µM Ps-TAc and 10 µg/ml
Hoechst 33258 for 20 min. Images were taken in HBSS containing 10
µg/ml propidium iodide (PI). Scale bars: 40 μm. (B) Dot plot
representation of the green fluorescence intensity in MCF7 cells treated
with DAC (n = 200 cells/each lane).
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Chem. Commun., 2013, 00, 1-3 | 3
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In this study, Ps-TAc, a plasma membrane-permeable Ps-TG 11 M. K. Kelley, A. Engqvist-Goldstein, J. A. Montali, J. B.
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derivative, was developed and demonstrated to be applicable to the
selective imaging of GSTP1 activity in living cells. The markedly
high selectivity of Ps-TAc for GSTP1 allows Ps-TAc to visualise
endogenously expressed GSTP1 at the single cell level. Consequently,
Ps-TAc holds promise for detecting GSTP1-positive cancer cells and
assessing the effect of an epigenetic drug based on its de-repressing
activity toward epigenetically silenced GSTP1 expression. Thus, this
study presents a new fluorogenic substrate as a powerful chemical tool
for investigation of the biological significance of intracellular GSTP1
activity, for the detection of cancer cells, and for assessing epigenetic
drugs.
15 D. S. Millar, K. Ow, C. L. Paul, P. J. Russell, P. L. Molloy, S.
J. Clark, Oncogene 1999, 18, 1313.
We thank Dr. Toru Komatsu (The University of Tokyo, Japan) for
critical discussions; Professor Hisanaka Ito (Tokyo University of
Pharmacy and Life Sciences) for support in organic synthesis and
spectroscopic measurements; and Dr. Tasuku Ueno (The University
of Tokyo, Japan) for cyclic voltammetry measurements and critical
discussions. This research was supported by the Platform Project for
Supporting Drug Discovery and Life Science Research (Platform for
Drug Discovery, Informatics, and Structural Life Science) from the
16 M. Schnekenburger, T. Karius, M. Diederich, Front.
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Japan Agency for Medical Research and Development (AMED); by 20 A. Shibata, Y. Nakano, M. Ito, M. Araki, J. Zhang, Y. Yoshida,
S. Shuto, B. Mannervik, R Mogenstern, Y. Ito, H. Abe, Analyst
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W. Liu, T. Deng, S. Yao, L. Feng, Anal. Chem. 2017, 89, 8097.
22 Y. Fujikawa, Y. Urano, T. Komatsu, K. Hanaoka, H. Kojima,
the Private University Research Branding Project of the Japanese
Ministry of Education, Culture, Sports, Science and Technology; by
MEXT/JSPS KAKENHI (18K05362); and by the Science Research
Promotion Fund from The Promotion and Mutual Aid Corporation for
Private Schools of Japan (to Y.F.).
T. Terai, H. Inoue, T. Nagano, J. Am. Chem. Soc. 2008, 130
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14533.
23 Y. Fujikawa, T. Nampo, M. Mori, M. Kikkawa, H. Inoue,
Talanta 2018, 179, 845.
24 Y. Fujikawa, F. Morisaki, A. Ogura, K. Morohashi, S. Enya,
R. Niwa, S. Goto, H. Kojima, T. Okabe, T. Nagano, H. Inoue,
Chem. Commun. 2015, 51, 11459.
Conflicts of interest
There are no conflicts to declare.
25 T. Ueno, Y. Urano, K. Setsukinai, H. Takakusa, H. Kojima, K.
Kikuchi, K. Ohkubo, S. Fukuzumi, T. Nagano, J. Am. Chem.
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Highly selective fluorogenic substrate was desined for the specific visualization of intracellular GSTP1
activity in cancer cells.
1