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Introduction
Epidermal growth factor (EGF), a small polypeptide
(6 045 Da), was first isolated from the submandibular glands
of mice[1]. It was found to have stimulating effect on
epidermal cell proliferation,
differentiation[2], and
migration[3,4]. EGF exerts its effects via EGF receptor (EGFR), which belongs to
the superfamily of receptor tyrosine
kinases[5,6]. After binding to ligands, this receptor dimerizes, leading to
autophosphorylation, initiation of intracellular signaling
cascade, and eventually receptor
internalization[7_9].
The main pathway linked to EGFR activation involves
Ras, a small GTP-binding protein, followed by the activation
of the mitogen-activated protein kinase pathway, which
eventually leads to the phosphorylation of extracellular signal
response kinases (ERK) 1 and 2. Phosphorylated ERK
enters the nucleus and activates the transcription of various
genes, including those required for cell proliferation,
differentiation, and migration[8]. The binding of EGF to its
receptor also causes the receptor's downregulation at the cell
surface due to receptor-mediated internalization. The
internalized ligand_receptor complex is eventually sent to
endosomes and lysosomes and degraded by enzymes present
in these compartments[10,11]. Therefore, internalization of EGFR
might be used as an indicator of its activation by agonists and
the activation of downstream signaling pathways.
EGFR is the receptor most often found to be upregulated
in a wide variety of human tumors[12]. The inhibition of EGFR
may present a promising method for attacking tumor
invasiveness as an adjunct therapy for antimitotic therapy. EGFR
has been the target of various anticancer drug development
strategies, including therapeutic antibodies, kinase inhibitors,
ligand-toxin conjugates, and antisense
nucleotides[13]. On the other hand, EGFR activation has tremendous effect in
promoting wound repair and limiting scar
formation[14,15]. Thus, modulators of EGFR signaling pathway might provide
important therapeutic opportunities in several areas,
including cancer, organ repair, and cell production.
To identify novel EGFR modulators, an accurate and
robust high-throughput assay is highly desirable to screen a
large collection of chemical entities. Traditional
high-throughput screening (HTS) methods usually involve
expression and purification of the receptor protein and
performance of kinase assay in vitro. The conditions of such
assays are very different from the natural cellular
environment and radioactive materials are normally used. In this
paper, we describe a new, quantitative, cell-based assay for
the screening of EGFR modulators based on receptor
internalization and high-content screening (HCS) technology.
HCS is a high-throughput, multi-parameter image analysis
technology recently developed based on fluorescent
microscopy. Fluorescent-tagged EGF was used to visualize
the internalized ligand_receptor complex. The fluorescent
intracellular spots were detected and measured with an
ArrayScan HCS reader. Compounds that competitively bind
to EGFR or interfere with EGFR internalization process will
result in a reduced number and intensity of the intracellular
fluorescent spots. This assay was validated, optimized,
and applied to a large-scale screening of a library
containing 48 000 synthetic compounds. Thirteen novel compounds
with a relatively high degree of interference with EGFR
internalization were identified. One of the compounds was proven
to be an agonist of EGFR since it induced phosphorylation
of the receptor and the downstream ERK protein.
Materials and methods
Reagents EGF and Hoechst 33342 were purchased from
Sigma (St Louis, MO, USA). Cell culture medium, EGF
biotinylated and complexed to Alexa Fluor 555 streptavidin
(Alexa Fluor 555 EGF) and wheat germ agglutinin
(WGA)_oregon green were bought from Invitrogen (Carlsbad, CA, USA).
Formaldehyde, DMSO, Tris base, SDS, glycerol, dithiothreitol,
and bromophenyl blue were purchased from Sinopharm Group
Chemical Reagent Co (Shanghai, China). The phototope
horseradish peroxidase (HRP) Western blot detection system,
antibodies to ERK1/2, phospho-ERK1/2, EGFR, phospho-EGFR and
goat anti-rabbit IgG antibody conjugated to HRP were
purchased from Cell Signal Technology (Beverly, MA, USA).
Cell culture HeLa cells obtained from the American Type
Culture Collection (Manassas, VA, USA) were maintained in
low-glucose Dulbecco's modified Eagle's medium (LG_DMEM)
supplemented with 10% fetal bovine serum, 100 mg/L penicillin,
and 100 mg/L streptomycin at 37 °C in a humidified atmosphere
of 5% CO2. Cells were trypsinized and seeded onto 96-well
plates at proper density for 18_24 h before the experiments.
Alexa Fluor 555 EGF induced EGFR internalization
Cells were planted onto 96-well plates overnight at desired density.
Cells were then starved in serum-free LG_DMEM for 3 h.
Alexa Fluor 555 EGF was added at a final concentration
ranging from 0.2 to 1 mg/L and incubated at 37 °C for the
indicated times. (To show clear membrane binding of Alexa Fluor
555 EGF, the first 2 min of incubation were on ice.) To study
the competition between EGF and Alexa Fluor 555 EGF or
compound interference with EGFR internalization, cells were
pretreated with unlabeled EGF or compounds for 10 min
before the addition of Alexa Fluor 555 EGF. Cells were then
washed 3 times with prewarmed phosphate-buffered saline
(PBS) and fixed with 3.7% formaldehyde for 15 min. The cell
membrane was stained with 5 mg/L WGA_oregon green for
30 min and the cell nuclei were stained with 10 mg/L Hoechst
33342 for 15 min, respectively. The plates were either
observed with an Olympus IX51 inverted fluorescent
microscope or analyzed with an ArrayScan 4.0 HCS Reader
(Cellomics, Pittsburgh, PA, USA).
Imaging and data generation Images and data of the
cells were obtained with an ArrayScan HCS 4.0 Reader.
Appropriate filter sets were used for the detection of
Hoechst-labeled nuclei and the Alexa Fluor 555 EGF/EGFR complex. A
10 or 20× microscope objective was used for the imaging,
and the spot detector bioapplication was used to acquire
and analyze the images after optimization of the application
protocol. Around 500 cells from randomly-picked fields were
analyzed for each well.
HCS campaign The compound library used for the
screening was comprised of 48 000 different compounds. A
10 compound pool/well mix was applied to the primary
screening, with an average final concentration of 3 µmol/L
for each compound. This matrix system maximized the
advantage of HTS and allowed duplicate screening of each
compound[16]. In each 96-well plate, 8 wells were used as
positive controls (10 nmol/L EGF in 1% DMSO) and another
set of 8 wells as negative controls (1% DMSO). The
inhibition rate of 10 nmol/L EGF was normalized to 100% and that
of the negative control was 0. The inhibition rate of each
compound was calculated with the following equation:
Inhibition %=(Spot countcompound_Spot
count1%DMSO)/(Spot countEGF_Spot
count1%DMSO)×100%. The samples showing
more than 80% inhibition were considered "hits" in the
primary screening.
Western blotting HeLa cells were seeded onto 6-well
plates and cultured overnight. Then the cells were serum
starved for 16 h. Compounds were added directly into the
culture medium at desired concentration and incubated for
various time periods. After removal of the culture medium,
the cells were lysed with sample buffer (62.5 mmol/L
Tris-HCl, 2% SDS, 10% glycerol, 50 mmol/L dithiothreitol, and
0.01% bromophenyl blue [pH 6.8]). After immediate scraping,
the lysates were sonicated for 10_15 s and then boiled at
95_100 °C for 5 min. The protein concentration was determined
with the Bradford method and 10 μg of total protein was
loaded onto 12% SDS_PAGE gels and electrophoresed at 60
V with Tris-glycine running buffer (25 mmol/L Tris-HCl, 250
mmol/L glycine, and 0.1% SDS [pH 8.3]). The proteins were
then transferred onto a PVDF membrane (Millipore, MA, USA)
at 100 V for 60 min (for ERK) or 120 min (for EGFR). The
membrane was incubated with blocking buffer (20 mmol/L Tris,
137 mmol/L NaCl, 0.1% Tween-20, and 5% non-fat dry milk) for
2 h at room temperature. After washing, the membranes were
probed simultaneously with rabbit anti-ERK (1:1000) and rabbit
anti-p-ERK (1:1000) antibodies or rabbit anti-EGFR (1:1000) and
rabbit anti-p-EGFR (1:1000), respectively, for 8 h and then with
goat anti-rabbit IgG HRP (1:2000) antibody for 3 h. After the
final wash, the membrane was developed on Kodak X-Omat BT
film (Eastman Kodak, Rochester, NY, USA) using the phototope
HRP Western blot detection system (Cell Signal Technology,
MA, USA) according to manufacturer's instructions.
Data analysis Data were analyzed with GraphPad Prism
software (GraphPad, San Diego, CA, USA). Non-linear
regression analyses were performed to generate dose-response curves,
and linear regression was used to analyze data reproducibility.
Mean±SEM was calculated automatically with this software.
Results
Observation of EGFR internalization with Alexa Fluor
555 EGF HeLa cell is a human cervical cancer cell line with a
natural high expression of EGFR. To observe the EGFR
internalization process, a fluorescent-tagged EGF (Alexa Fluor
555 EGF) was used. HeLa cells were incubated with Alexa
Fluor 555 EGF for various time periods and conditions. An
initial 2 min incubation on ice revealed binding of Alexa Fluor
555 EGF with the EGFR on the plasma membrane, as
indicated by the colocalization of membrane marker WGA_oregon
green (Figure 1). Incubation at 37 °C for 10 min led to the
cointernalization of the receptor_ligand complex and
formation of bright red intracellular spots. Thirty minutes later,
the intracellular spots became larger and aggregated, which
might be automatically identified and analyzed with a
software algorithm. To visualize the competition between EGF
and Alexa Fluor 555 EGF, different concentrations of
unlabeled EGF were added to the cells before the addition of the
Alexa Fluor 555 EGF. Higher concentrations of unlabeled
EGF showed advantages in competitive binding to the EGFR
and resulted in a reduced number and intensity of
intracellular spots (Figure 2). Compounds that competitively bind to
the EGFR or interfere with the internalization process should
also cause reduced intracellular spots.
Imaging and analysis by the HCS reader An ArrayScan
4.0 HCS reader, equipped with a software module, the spot
detector bioapplication, was used for imaging and data
analysis. Two imaging channels with proper filters were used
to photograph nuclei (Figure 2, lane 1) and intracellular spots
(Figure 2, lane 3). After optimization of the bioapplication's
protocol settings, the software was able to automatically
detect nuclei (Figure 2, lane 2) and intracellular spots (Figure
2, lane 4). Relevant readouts from this software included cell
density, spot count/cell, spot total intensity/cell, and spot
total area/cell. The results also indicated that an increase in
the dose of unlabeled EGF significantly reduced the Alexa
Fluor 555 EGF-labeled intracellular spot number and intensity.
Assay optimization Various experimental conditions were
tested for assay optimization. The intracellular spot counts
increased with the elongation of the incubation time with
Alexa Fluor 555 EGF. After 30 min of incubation, the spot
counts reached a plateau (Figure 3A). The use of a
high-power microscope objective (20× vs
10×) resulted in increased spot counts due to better image resolution (Figure 3B).
Different cell densities were also tested. As shown in Figure
3B, the maximal spot count exceeded 20 with low cell density
(10 000 cells/well), but in high cell density wells (30 000
cells/well), the maximal spot count was only about 14. Higher
concentration (1 mg/L) of Alexa Fluor 555-EGF significantly
increased the signal (Figure 3B,3C), but due to the increased
cost, we eventually decided on the low concentration (200
μg/L), which proved to be adequate for screening as
discussed later.
Several readouts, including spot count/cell, spot total
intensity/cell and spot total area/cell compared in the
EGF/Alexa Fluor 555 EGF competition assay (Figure 3D) were
found to yield similar results. The IC50
values of unlabeled EGF calculated from different readouts were all approximately
0.2 nmol/L, which is in agreement with previously reported
values (0.2_0.4 nmol/L)[17].
The final assay conditions for HCS were determined as
follows: cell density was 10 000 cells/well, the concentration
of Alexa Fluor 555 EGF was 200 μg/L; the incubation time
with Alexa Fluor 555 EGF was 30 min, and the quantitative
readout was spot count/object.
Assay performance The Z' value is used to assess the
robustness of an assay for screening and is the normalized 3
SD window between the negative and positive
controls[18]. The Z' value for the assay was 0.75 and the
signal/background (S/B) ratio was 170, indicating that the system was
adequately optimized for HCS (Figure 4A). Furthermore, to
investigate reproducibility between duplicate plates, the
corresponding wells from 2 different 96-well plates were
treated with same concentration of EGF followed by Alexa
Fluor 555 EGF treatment. The data from corresponding wells
of different plates were analyzed with linear regression
analysis[19]. The correlation coefficient was 0.97, showing a high
degree of reproducibility between duplicate sample plates.
HCS campaign Of the 48 000 samples initially screened,
184 hits (0.38%) showing more than 80% inhibition on Alexa
Fluor 555 EGF-induced EGFR internalization were
discovered (Figure 5A). Secondary screening (single
compound/well) was carried out to further confirm the hits (Figure 5B).
Finally, 13 compounds displaying consistent inhibitory
effects from secondary screening were picked out and
analyzed in a duplicate setup. Due to compound availability,
one of the hits with a novel structure (WJ-12) was further
analyzed with traditional Western blot assay.
EGFR and ERK activation by compound
WJ-12 We used Western blot analysis to distinguish the agonist or
antagonist nature of compound WJ-12. This compound was found
to induce EGFR and downstream ERK phosphorylation in a
dose- and time-dependent manner (Figure 6A_6D). The
activation of ERK by WJ-12 could be significantly blocked
by 1 µmol/L EGFR tyrosine kinase inhibitor, tyrphostin
AG1478[20] (Figure 6E), which indicated the phosphorylation
of ERK by WJ-12 was via direct activation of EGFR.
Discussion
In 1997, with the debut of first ArrayScan reader, a
dramatically new approach to early drug discovery was
introduced as HCS technology. With 3 key research components,
automatic fluorescence microscopy, fluorescence-based
reagents, and image analyzing software, HCS is a
technology platform designed to define the temporal and spatial
activities of genes, proteins, and other cellular constituents
in intact cells. Early research papers demonstrated that
cellular functions, such as transcription factor
activation[21],
cytotoxicity[22,23], and G-protein coupled receptor
internalization[24] could be quantified by HCS in a screening setup.
Ligand-induced EGFR internalization is an important
event in EGFR signal transduction and has been observed
with many previous studies[10,11]. With the development of
fluorescent-conjugated EGF and HCS technology, the
internalization of EGF/EGFR might be used as an indicator for
receptor activation and to search for new compounds that
interfere with this process.
In this article, we demonstrated a robust, cell-based HCS
assay for the identification of EGFR modulators. In this
assay, the Hoechst 33342 was used to label the cell nuclei
and fluorescent-conjugated EGF was used to monitor
EGF/EGFR complex internalization. Therefore, compounds
competitively binding to EGFR or interfering with the
internalization process would result in a reduced amount and intensity
of intracellular fluorescent spots.
In the optimization procedure of assay development,
many factors, such as cell density, incubation time, the
concentration of Alexa Fluor 555 EGF, image magnification, and
other parameters, have to be considered in order to maximize
the S/B ratio and assay throughput. For example, the amount
and intensity of the fluorescent spots would increase with
incubation time, but prolonged treatment would reduce the
assay speed and lead to fluorescent degradation, which
would eventually cause signal reduction. While testing the
concentration of Alexa Fluor 555 EGF, we found that an
increase in the concentration would increase the signal, but
considering the cost of large scale screenings, we chose the
low concentration of 200 μg/L without affecting the assay
performance. The Z' factor and S/B ratio were used to
evaluate the assay. The Z' factor was widely used for the
evaluation of HTS/HCS assay
qualities[18]. Generally speaking, if the Z' factor is above 0.5, the system could be considered
suitable for the HTS/HCS. The assay described here had a
Z' factor of 0.75 and an S/B ratio of 170, which indicated that
the assay was of high quality for HCS.
This assay was applied to a large-scale screening of a
compound library consisting of 48 000 synthetic compounds.
Thirteen compounds displaying consistent inhibitory effects
were found. One of the compounds with a novel structure
(WJ-12) was further analyzed with Western blotting to
identify its agonist or antagonist nature. The results revealed
that WJ-12 induced EGFR and downstream ERK
phosphorylation in a dose- and time-dependent way. The activation of
ERK was significantly blocked by EGFR tyrosine kinase
inhibitor, tyrphostin AG1478, which indicated the
phosphorylation of ERK by WJ-12 via direct activation of EGFR.
In summary, a quantitative, cell-based, high-content
screening assay was developed and validated for the
identification of compounds with specificity and functionality for
EGFR. Compared with the conventional radioactive kinase
assays, this assay was performed in a more physiological
environment, and due to its imaging nature, provided richer
information. It may be applicable to other membrane
receptors with the availability of fluorescent-tagged ligands.
Acknowledgements
The authors thank Ms Meilin SUN and Ms Hong YU for
their technical assistance and suggestions.
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