Time-dependent intracellular trafficking of FITC-conjugated epigallocatechin-3-O-gallate in L-929 cells

Article abstract of DOI:10.1016/j.bmc.2008.10.009

Many in vitro studies about green tea polyphenol, (-)-epigallocatechin-3-O-gallate (EGCG) focused on its pro-apoptotic and anti-proliferative effects on various types of cancer cells, while less attention has been paid to its incorporation into the cytoplasm and nuclear translocation. This study concentrated on the time-dependent intracellular trafficking of EGCG in L-929 cells. EGCG was conjugated with fluorescein-4-isothiocyanate (FITC) via the 3″-OH or 5″-OH group, as confirmed by NMR analysis, and then treated to either suspended or cultured cells. Confocal microscopic observations revealed that FITC-EGCG was clearly seen onto the membrane of suspended cells as well as into the cytoplasm and nucleus within 1 h. As an increase in treatment time, it concentrated on the nucleus and then was located at any places of the cells. The cellular uptake of FITC-EGCG in cultured cells was not observed until 1 h of culture, but started to be observed after at least 2 h. These results imply that although the cellular sensitivity and response to EGCG would be different from those of FITC-EGCG, it would be incorporated into the cytoplasm of cells and further be translocated into the nucleus in a time-dependent manner.

Full text of DOI:10.1016/j.bmc.2008.10.009

Bioorganic & Medicinal Chemistry 16 (2008) 9652–9659
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Time-dependent intracellular trafficking of FITC-conjugated
epigallocatechin-3-O-gallate in L-929 cells
Dong-Wook Han a,b, Kazuaki Matsumura a, Bongju Kim c, Suong-Hyu Hyon a,
*
a Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho,
Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
b Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology, Pusan National University, San 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, South Korea
c Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Many in vitro studies about green tea polyphenol, (–)-epigallocatechin-3-O-gallate (EGCG) focused on its
pro-apoptotic and anti-proliferative effects on various types of cancer cells, while less attention has been
paid to its incorporation into the cytoplasm and nuclear translocation. This study concentrated on the
time-dependent intracellular trafficking of EGCG in L-929 cells. EGCG was conjugated with fluorescein-
4-isothiocyanate (FITC) via the 300-OH or 500-OH group, as confirmed by NMR analysis, and then treated
to either suspended or cultured cells. Confocal microscopic observations revealed that FITC-EGCG was
clearly seen onto the membrane of suspended cells as well as into the cytoplasm and nucleus within
1 h. As an increase in treatment time, it concentrated on the nucleus and then was located at any places
of the cells. The cellular uptake of FITC-EGCG in cultured cells was not observed until 1 h of culture, but
started to be observed after at least 2 h. These results imply that although the cellular sensitivity and
response to EGCG would be different from those of FITC-EGCG, it would be incorporated into the
cytoplasm of cells and further be translocated into the nucleus in a time-dependent manner.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 2 September 2008
Revised 2 October 2008
Accepted 3 October 2008
Available online 7 October 2008
Keywords:
EGCG
Intracellular trafficking
Nuclear translocation
L-929 cells
NMR analysis
FITC-EGCG
1. Introduction
dant activities, its time-dependent intracellular trafficking is still
unclear. In these previous studies, EGCG was added to cell culture
Conventional studies have shown that green tea catechins have
potent suppressive effects on cellular responses of various cancer
media, and the effects were examined 24, 48 or even 72 h later. It is
not known whether the action of EGCG was exerted in the first few
hours or throughout the experimental period. Our earlier study
also demonstrated the suppressive effects of EGCG on vascular
smooth muscle cells after 48 h.13 In the present study, the binding
of EGCG onto membranes, its incorporation into cytoplasm and
subsequent translocation into nucleus in suspended and cultured
L-929 cells for 4 or 8 h were demonstrated by FITC-conjugation,
which would be exploited to give a clue to identify the primary tar-
gets and action mechanisms of EGCG.
and references therein
cells and their intracellular signaling cascades.1–3
Most of this anti-cancer activity of green tea are believed to be
mediated by its major polyphenolic antioxidant constituent,
(ꢀ)-epigallocatechin-3-O-gallate (EGCG).4–6 Furthermore, some
previous reports have demonstrated the incorporation of EGCG
into the cytosol and/or even the nucleus of cells as well as its tis-
sue distribution and metabolism in animals by using radioiso-
tope-labeled EGCG, for example, [3H]EGCG.7–12 This cellular
internalization of EGCG is considered to be involved in the
mechanism of the anti-cancer activity of EGCG as a possible che-
mopreventive agent, since catechins exert pro-apoptotic and
anti-proliferative activities on cancer cells.
2. Results
2.1. Characterization of EGCG conjugated with FITC
Although the biological effects of EGCG have been extensively
investigated and believed to be mainly due to its potent antioxi-
Figure 1 shows representative HPLC profiles of free FITC, EGCG
and their conjugate, FITC-EGCG, submitted to HPLC analysis with
UV detection at 280 nm.14 One major peak and other minor were
shown on the chromatogram of FITC-EGCG, which were named
as peak 1 and 2, respectively (Fig. 1C). Peak 1 was identified as
EGCG by comparison with the chromatogram of EGCG and its
authentic standard.15 Since the column used in HPLC was not
Abbreviations: EGCG, (–)-epigallocatechin-3-O-gallate; FITC, fluorescein-4-iso-
thiocyanate; FITC-EGCG, FITC-conjugated EGCG; PBS, phosphate-buffered saline; PI,
propidium iodide; UV, ultraviolet.
* Corresponding author. Tel.: +81 75 751 4125; fax: +81 75 751 4141.
E-mail address: biogen@frontier.kyoto-u.ac.jp (S.-H. Hyon).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.009

D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
9653
Figure 1. HPLC chromatograms of free FITC (A), EGCG (B) and FITC-EGCG (C). The HPLC conditions were described under Section 4.
suitable to differentiate free FITC from FITC-EGCG, it was not clear
that peak 2 corresponded to the FITC-conjugate. From these re-
sults, it was concluded that the conjugation of EGCG with FITC
could not be confirmed by HPLC analysis.
relative cell viability with the IC50 value of 700
l
M (Fig. 3A). In
the cells treated with increasing concentrations of EGCG, the value
of IC50 was found to be approximately 690 M (Fig. 3B). However,
FITC-EGCG was more cytotoxic to the cells and its IC50 value was
about 325 M (Fig. 3C). This result implies that the conjugation
l
1H NMR analysis was carried out to obtain structural informa-
tion and confirm the conjugation of EGCG with FITC. Figure 2
shows the NMR results obtained for free FITC, EGCG and their con-
jugate, FITC-EGCG. A tentative assignment of free EGCG was car-
ried out by comparison with literature data.16,17 There was no
significant variation in the chemical shift values of the protons in
free FITC (Fig. 2A). A comparison between NMR spectrum of free
EGCG (Fig. 2B) with that of its FITC-conjugate (Fig. 2C) allowed
the assignment of the proton signals of the conjugate and showed
the existence of newly formed signals upon going from the free
compound to the conjugated form. New proton signals were lo-
cated at 7.330 and 7.220 ppm in FITC-EGCG, suggesting that the
conjugation to FITC might occur via the 300-OH or 500-OH groups, in-
stead of the usual conjugation mode via the 30-OH or 50-OH posi-
tions.18,19 On the other hand, the conjugation of EGCG with FITC
leaded to split of C-200,600 signal as well as slight shift in the d
(ppm) values ranging from 0.05 to 0.15 ppm, although significant
variations in the chemical shift values were not observed. Addi-
tionally, the signal of C-20,60 shifted from 6.557 ppm in free EGCG
to 6.628 ppm in the conjugate. C-2, C-3 and C-4 signals in free
EGCG disappeared after conjugation, but there were no significant
changes in the spectral profile of EGCG. Therefore, when comparing
Figure 2A–C, the formation of new proton signals in C-300 or C-500 of
FITC-EGCG is evident and strongly suggests that the conjugation
might occur via 300-OH or 500-OH group in the gallate ring of EGCG.
l
of EGCG with FITC induced a conformational change in EGCG and
resulted in quite different property from EGCG, rather than a syn-
ergistic effect in cytotoxicity.
2.3. Intracellular trafficking of FITC-EGCG in suspended cells
Employing L-929 cells in suspensions (Fig. 4) treated with
slightly toxic concentration (65 lM) of FITC-EGCG, the intracellular
trafficking of FITC-EGCG, namely the incorporation of FITC-EGCG
into cytoplasm and its subsequent nuclear translocation were ob-
served by confocal microscopy. Compared with the non-treated
controls (Fig. 4A), FITC-EGCG was seen mainly onto the membrane
of the cells and into the nucleus after 0.5 h of treatment (Fig. 4B).
After 1 h, it further concentrated on the nucleus while being
slightly distributed into the cytoplasm (Fig. 4C). Then, it was lo-
cated at any places of the cells including the cytosol after 4 h
(Fig. 4D). This result could be more confirmed by Supplemental
movies of suspended L-929 cells after 1 and 4 h of treatment with
FITC-EGCG. It was found that FITC (50
lM) alone could not pene-
trate into the suspended cells (Fig. 4E).
2.4. Internalization of FITC-EGCG into cultured cells
The internalization of FITC-EGCG could be also observed in the
mono-layered culture of L-929 cells treated with 130 lM FITC-
2.2. Cytotoxicity profiles
EGCG (Fig. 5). The cellular uptake of FITC-EGCG was not observed
until around 1 h of culture, but started to be observed between 2
and 4 h (Fig. 5A). Some cells showed severe alterations in the size
and morphology due to cytotoxicity of FITC-EGCG, but others did
not (Fig. 5B). As the progress in the incubation time, it was clearly
Cytotoxicity profiles of free FITC, EGCG and FITC-EGCG were
determined in L-929 cells by WST-8 assay (Fig. 3). The cells ex-
posed to free FITC for 24 h showed a dose-dependent decrease in

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D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
Figure 2. 1H NMR spectra of free FITC (A), EGCG (B) and FITC-EGCG (C). The NMR analysis was performed under Section 4.
observed into the cytoplasm or even the nucleus, particularly with
intensive bright green and red fluorescence (Fig. 5C). Among the
cells incubated with FITC-EGCG for more than 8 h, some cells
highly sensitive to FITC-EGCG showed typical apoptotic appear-
ances, including blebbing, nucleus swelling and changes to the cell
membrane such as loss of membrane asymmetry and attachment
(Fig. 5D). After 24 h of treatment, FITC-EGCG was still incorporated
into the cytoplasm of the detached cells, but the nucleus was not
could not only enter the cultured cells, but also penetrate into
EGCG-pretreated cells as shown in the below fluorescence micro-
graphs (Supplementary Fig. S1).
3. Discussion
As a great deal of attention has been focused on various benefi-
cial activities of EGCG, exact information about the time-depen-
dent intracellular trafficking of this compound is required in
able to be delineated due to apoptosis (Fig. 5E). FITC (50 lM) alone

D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
9655
Fig. 2 (continued)
order to elucidate its primary targets of action and precise mecha-
nisms of its actions. For this purpose, EGCG was conjugated with
FITC via 300-OH or 500-OH group in the gallate ring of EGCG, which
was confirmed by NMR analysis (Fig. 2).
modulation of the exogenous signals directed to genes required
for the survival or apoptosis of cells.
Although the exact mechanism of the internalization of EGCG
into cells had not been elucidated yet, some evidence has been re-
ported using various cancer cell lines treated with [3H]EGCG. Oka-
be et al. demonstrated by microautoradiography that [3H]EGCG
was bound to the membrane as well as incorporated into the cyto-
plasm and the nucleus of PC-9 cells and that the radioactivity of the
cells was increased time-dependently.7 Furthermore, it was shown
that the uptake of [3H]EGCG by HT-29 cells displayed a concentra-
tion-dependent increase and did not plateau, suggesting that EGCG
uptake mainly occurs by a passive diffusion process.8 Those quan-
titative results using [3H]EGCG well agreed with our qualitative re-
sults showing that the intracellular trafficking of EGCG in L-929
cells took place time-dependently.
Consistently, recent study has demonstrated that expression of
the metastasis-associated 67 kDa laminin receptor confers EGCG
responsiveness to cancer cells at physiologically relevant concen-
trations, suggesting that the gallate moiety of EGCG may be critical
for receptor binding and subsequent activity.3,25 Moreover, the
observation that nucleic acids extracted from catechin-treated
cells were colored implied that because both galloyl and catechol
groups of EGCG were essential for DNA binding, both groups
seemed to hold strands of DNA via their branching structure.26
Nevertheless, it would remain to be determined whether conjuga-
tion with FITC via –OH group in the gallate ring of EGCG adversely
affects the receptor binding of EGCG and its subsequent activity or
not.
The binding of FITC-EGCG onto membranes, its incorporation
into cytoplasm and subsequent translocation into nucleus were
clearly observed in L-929 cells. In suspended cells (Fig. 4), FITC-
EGCG was incorporated into the cytoplasm via binding of the
membrane and further translocated into the nucleus of cells in a
time-dependent manner. Interestingly, a colored cell surface was
observed and possibly attributable to the binding of EGCG to mem-
branes. The cellular uptake of FITC-EGCG in the cultured cells
started to be observed after at least 2 h (Fig. 5). As an increase in
treatment time, internalized FITC-EGCG triggered apoptosis of
the cells. However, the incorporation of FITC-EGCG into cells was
appreciably decreased by pretreatment of free EGCG (data not
shown), suggesting that the conjugate will less accumulate within
cells by virtue of competition with unlabeled EGCG for membrane
binding sites. Moreover, it has been shown that the cell membrane
is not permeable for FITC, a frequently used derivatizing reagent
for laser-induced fluorescence detection and not able to penetrate
into viable cells by itself.20,21 These reports coincide with our re-
sults showing that FITC alone could enter neither the suspended
nor cultured cells (Fig. 4E and Supplementary Fig. S1). These results
imply that although the cellular response to EGCG and its binding
pattern to membrane receptors would be different from those of
FITC-EGCG, it might penetrate into the cytoplasm of cells thru
binding to specific receptors onto the membrane and forming com-
plexes with the receptors, and further be translocated into the nu-
cleus in a time-dependent manner. These phenomena might be
related to the intrinsic characteristics of polyphenolic compound,
EGCG. It has already been shown that this compound, due to its
amphipathic properties, readily binds to extracellular matrices, li-
pid membranes and any types of intracellular proteins.22–24 There-
fore, this compound would be allowed to penetrate into cell
cytoplasm and further to be translocated into nucleus, leading to
Collectively, the findings provide support to a scenario in which
the intracellular trafficking of EGCG takes place according to such
cascades as follows. First, EGCG freely binds to membrane recep-
tors on cells, resulting in the formation of EGCG-receptor com-
plexes. Next, it becomes to be internalized into cytoplasm thru
unknown mechanism, which would affect various signal factors
responsible for regulating transcription. Evidence that EGCG causes
internalization of the epidermal growth factor receptor in human

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D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
zerland).29 In order to visualize the intracellular trafficking of
EGCG, it was conjugated with fluorescein-4-isothiocyanate (FITC,
Dojindo Lab., Kumamoto, Japan) by using a modified labeling
method as reported previously.30 In brief, EGCG (1 g) was dissolved
in 100 ml of 0.2 M K2HPO4 (pH 9.2) and incubated for 72 h at 37 °C
with FITC (60 mg). After incubation, the conjugate was extracted
with 100 ml of ethyl acetate and then evaporated at 50 °C. It was
then dissolved in excess double distilled water and recovered by
freeze-drying. The resultant product was regarded as FITC-conju-
gated EGCG (FITC-EGCG) and quite stable, confirming that it did
not exchange with water or phosphate-buffered saline (PBS, pH
7.4) over a period of 24 h. The purity of the product was found to
be more than 85% by analytical HPLC and LC/MS (Supplementary
Fig. S2). The analytical HPLC was performed in a JASCO liquid chro-
matograph apparatus with a JASCO 870 ultraviolet (UV) detector.
Following are the conditions: column, CAPCELL PAK C18 UG120
(Shiseido Co., Ltd., 250 mm ꢁ 4.6 mm i.d., 5
lm, 110 Å); mobile
phase, 50% aqueous acetonitrile containing 0.01% acetic acid; flow
rate, 1.0 ml/min; injection volume, 20
detection, UV 280 nm.
ll; temperature, 35 °C;
4.2. HPLC analysis
FITC-EGCG was detected by HPLC analysis. The analysis was
performed on a Shimadzu (Kyoto, Japan) LC-10ADvp equipped
with an SPD-10Avp UV detector, a DGU-12A degasser, an SCL-
10Avp system controller, a C-R&A chromatopac data recorder and
a
CTO-20A column oven.
A
Shim-pack VP-ODS column
(150 mm ꢁ 4.6 mm i.d., 5 m, 110 Å) protected by a pre-column
l
GVP-ODS (10 mm ꢁ 4.6 mm i.d.) both from Shimadzu, was used
at 40 °C throughout this study. The mobile phase was composed
of 10 mM phosphate buffer: acetonitrile (85:15 v/v), and the aque-
ous phase was adjusted to pH 2.5 with phosphoric acid. A flow rate
was 1.0 ml/min, an injection volume was 20
tected at 280 nm.
ll, and peaks were de-
4.3. NMR analysis
Conjugation of EGCG with FITC was further confirmed by NMR
analysis. 1H NMR spectra were obtained at 300 MHz with a JEOL
JNM-LA400 spectrometer (Tokyo, Japan) at 25 °C in DMSO-d6.
Chemical shift values are reported relative to tetramethyl silane
(TMS, d = 0.00 ppm) in carbon tetrachloride as an external standard
inserted into an NMR tube (/ = 5 mm) with a coaxial cell. The dig-
ital resolution of the 1H NMR spectral data was 0.1 Hz.
Figure 3. Cytotoxicity profiles of free FITC (A), EGCG (B) and FITC-EGCG (C) to L-929
cells. The relative cell viability (%) was measured by WST-8 assay, as described in
Section 4.
colon cancer cells may explain, at least in part, that mechanism.27
Afterwards, its translocation into nucleus occurs via unknown
pathways, which might be partly explained by the fact that a phy-
toestrogen molecule, structurally related with catechin, binds to an
estrogen receptor and then moves to the nucleus thru nuclear
pores.28 Finally, it regulates various genes related to cellular re-
sponses including proliferation and cell cycle progression. Further
study about what signal transduction pathways are involved in
regulating cell cycle-related genes is required, and the exact mech-
anism needs to be elucidated. Since this is the first observation to
visualize the intracellular trafficking of EGCG, it is believed that the
results may be exploited to understand the mechanism of anti-can-
cer effects of EGCG as well as to craft strategies for chemopreven-
tion and/or therapy against cancer by EGCG.
4.4. Cell culture and conditions
A murine fibroblast cell line (L-929 cells from subcutaneous
connective tissue) was obtained from American Type Culture Col-
lection (CCL-1, Rockville, MD). The cells were routinely maintained
in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Co., St.
Louis, MO) supplemented with 10% fetal bovine serum (Sigma-Al-
drich Co.) and 1% antibiotic antimycotic solution (including
10,000 U penicillin, 10 mg streptomycin and 25 lg amphotericin
B per ml, Sigma-Aldrich Co.) at 37 °C in 95% humidity and 5%
CO2. Studies were performed with the cells at 30–50 passages.
4.5. Cytotoxicity assay
4. Materials and methods
The number of viable cells was quantified indirectly using a
highly water soluble tetrazolium salt [WST-8, 2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazo-
lium, mono-sodium salt] (Dojindo Lab., Kumamoto, Japan), re-
duced to formazan dye by mitochondrial dehydrogenases. Cell
viability was found to be directly proportional to the metabolic
4.1. Conjugation of EGCG with FITC
EGCG, the major polyphenolic component of green tea, was pur-
chased from DSM Nutritional Products Ltd. (TeavigoTM, Basel, Swit-

D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
9657
Figure 4. Confocal microscopic photographs of suspended L-929 cells 0 h (A), 0.5 h (B), 1 h (A) and 4 h (D) after treatment with 65
l
M FITC-EGCG or over 4 h after 50
lM FITC
treatment (E). All the photographs shown in this figure are representative of four independent experiments, showing similar results (original magnification, 1200ꢁ).
reaction products obtained in WST-8. Briefly, WST-8 assays were
conducted as follows. L-929 cells were incubated with WST-8 for
the last 4 h of the culture period (24 h) at 37 °C in the dark. In order
to avoid a direct reaction between antioxidant, FITC-EGCG and
WST-8 to be reduced, the excess antioxidant was completely re-
moved and the medium was exchanged before adding WST-8.
For comparison, free FITC and EGCG were also assayed for their
cytotoxicity. Parallel sets of wells containing freshly cultured
non-treated cells were regarded as the controls. Absorbance was
determined at 450 nm using an ELISA reader (Spectra Max 340,
Molecular Device Co., Sunnyvale, CA). The relative cell viability
was determined as percentage of the optical densities in the med-
ium containing serially diluted concentrations of FITC-EGCG to the
optical densities in the fresh control medium. The LD50, the con-
centration (%) inhibiting growth of cells by 50%, was estimated
from the relative cell viability.
4.6. Confocal laser scanning microscopy
Preparation of L-929 cells was the same as described above.
After 20–30 min of preincubation in PBS, suspended cells were
treated with FITC-EGCG (65 lM) in 0.5 ml of PBS for 0.5–4 h. At dif-
ferent time points, the cell suspension was washed three times
with PBS and then centrifuged at 250g for 5 min at 4 °C. After cen-
trifugation, the cell pellets were re-suspended in PBS and fixed
with 3.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7) for
5 min at room temperature. The incorporation of FITC-EGCG into
the cytoplasm of the cells and its subsequent nuclear translocation

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D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
Figure 5. Confocal microscopic photographs of cultured L-929 cells 2–4 h (A and B), 8 h (C and D) and 24 h (E) after treatment with 130 lM FITC-EGCG. All the photographs
shown in this figure are representative of four independent experiments, showing similar results (original magnification, 400ꢁ in (A) and (C); 800ꢁ in (B) and (D); 1500ꢁ in
(E)).
were observed under a confocal laser scanning microscope (LSM
510, Carl Zeiss Advanced Imaging Microscopy, Jena, Germany).
For comparison, the suspended cells were treated with free FITC
PI as described above. The concentration of FITC-EGCG treated to
either suspended or cultured cells was predetermined from its
cytotoxicity profile.
(50 lM) and then observed. Cell nuclei were counterstained with
5 lM propidium iodide (PI, Sigma-Aldrich Co.) directly before
3–5 min of observation. For studies in cultured cells, ꢂ2 ꢁ 105 L-
929 cells in growth medium were plated into each well of a
24-well plate. When the cells reached 80–85% confluency, they
Acknowledgments
This work was supported by the Japan Society for the Promotion
of Science Grant funded by the Japanese Government (FY2006 JSPS
Postdoctoral Fellowship for Foreign researchers, P 06451). We
appreciate Dr. Naoki Nakajima and Mr. Hajime Sugai (Institute
for Frontier Medical Sciences, Kyoto University) for technical
assistance.
were treated with 0.5 ml of FITC-EGCG (130 lM in PBS), incubated
for 2–24 h and fixed with 3.5% paraformaldehyde as described
above. At different time points, the cell cultures were examined
for the time-dependent intracellular trafficking of FITC-EGCG by
confocal microscopy. Cell nuclei were counterstained with 5 lM

D.-W. Han et al. / Bioorg. Med. Chem. 16 (2008) 9652–9659
9659
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