Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells

Article abstract of DOI:10.1016/j.bmc.2012.03.002

AMP-activated protein kinase (AMPK) is a critical monitor of cellular energy status and also controls processes related to tumor development, including cell cycle progression, protein synthesis, cell growth and survival. Therefore AMPK as an anti-cancer t

Full text of DOI:10.1016/j.bmc.2012.03.002

Bioorganic & Medicinal Chemistry 20 (2012) 3031–3037
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated
protein kinase pathway and target cancer stem cells
Di Chen a, Sreedhar Pamu b, Qiuzhi Cui a, Tak Hang Chan b, , Q. Ping Dou a,
⇑
⇑
a The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and Department of Oncology, School of Medicine, Wayne State University, 540.1 HWCRC,
4100 John R. Road, Detroit, MI 48201-2013, USA
b Department of Chemistry, McGill University, Montreal, Quebec, Canada H3A 2K6
a r t i c l e i n f o
a b s t r a c t
Article history:
AMP-activated protein kinase (AMPK) is a critical monitor of cellular energy status and also controls pro-
cesses related to tumor development, including cell cycle progression, protein synthesis, cell growth and
survival. Therefore AMPK as an anti-cancer target has received intensive attention recently. It has been
reported that the anti-diabetic drug metformin and some natural compounds, such as quercetin, geni-
stein, capsaicin and green tea polyphenol epigallocatechin gallate (EGCG), can activate AMPK and inhibit
cancer cell growth. Indeed, natural products have been the most productive source of leads for the devel-
opment of anti-cancer drugs but perceived disadvantages, such as low bioavailability and week potency,
have limited their development and use in the clinic. In this study we demonstrated that synthetic EGCG
analogs 4 and 6 were more potent AMPK activators than metformin and EGCG. Activation of AMPK by
these EGCG analogs resulted in inhibition of cell proliferation, up-regulation of the cyclin-dependent
kinase inhibitor p21, down-regulation of mTOR pathway, and suppression of stem cell population in
human breast cancer cells. Our findings suggest that novel potent and specific AMPK activators can be
discovered from natural and synthetic sources that have potential to be used for anti-cancer therapy
in the clinic.
Received 9 December 2011
Revised 28 February 2012
Accepted 1 March 2012
Available online 9 March 2012
Keywords:
EGCG analogs
AMP-activated protein kinase
Cancer stem cells
Breast cancer
Cell proliferation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
pathways, which consume ATP, including protein fatty acid and
cholesterol syntheses. Activation of AMPK also induces cell cycle
AMP-activated protein kinase (AMPK) is a serine/threonine pro-
tein kinase, which serves as a metabolic sensor and plays a key role
in cellular energy homeostasis in all eukaryotic cells.1 AMPK is a
heterotrimeric protein complex composed of three subunits: a cat-
arrest and inhibits cell proliferation in malignant cells through
multiple mechanisms including up-regulation of the p53–p21
axis11 and down-regulation of the mammalian target of rapamycin
(mTOR) pathway.12,13 Investigation into the regulation and bio-
functions of AMPK in cancer field has been intensively conducted.
Accumulating evidence suggests that targeting AMPK may be a
promising therapeutic option for cancer therapy.
Metformin, an anti-diabetic drug, could also activate AMPK,
raising a hypothesis that metformin may reduce the risk of cancer
in patients with type 2 diabetes through activation of AMPK path-
way.14 It has been reported that metformin not only inhibits the
tumor cell growth in both cell lines15,16 and mouse models,17 but
also selectively kills cancer stem cells.18 Besides metformin, green
tea polyphenol EGCG (Fig. 1A) demonstrated ability to induce
apoptosis in cancer cells, associated with activation of AMPK sig-
naling pathway.19,20 However, the caveat and limitation to the
use of EGCG in cancer prevention and therapy is its low bioavail-
ability due to its instability under neutral or alkaline conditions
(i.e., physiologic pH) and to biologically inactivating processes that
include methylation.21,22 An octa-acetate derivative of EGCG (pro-
EGCG, Fig. 1A) has been found to function as the pro-drug of EGCG
with improved bioavailability.23
alytic (
a
) subunit and two regulatory (b and
c
) subunits. AMPK is
subunit un-
activated through phosphorylation of Thr-172 on the
a
der multiple conditions including: (i) an energy-depletion stress,
such as ATP level declines and AMP level increases2,3; (ii) hypoxia4;
(iii) hormone and cytokines such as adiponectin and leptin5,6; (iv)
cellular kinases including liver kinase B1 (LKB1, a primary up-
stream kinase of AMPK),7,8 Calmodulin-dependent protein kinase
kinase (CaMKK)9 and proto-oncogene tyrosine-protein kinase Src
(c-Src)10; (v) the anti-diabetic medicine metformin (see below).
Once activated, AMPK switches on catabolic pathway and activates
ATP-generating processes including the uptake and oxidation of
glucose and fatty acids. Activated AMPK also switches off anabolic
⇑
Corresponding authors. Tel.: +1 313 576 8301 (Office); fax: +1 313 576 8307
(Q.P.D.).
E-mail addresses: tak-hang.chan@mcgill.ca (T.H. Chan), doup@karmanos.org
(Q.P. Dou).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.002

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D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
We have thus synthesized a focused library of EGCG analogs of
general structure A (Fig. 1A) with the substituents X, Y and Z being
H, OH, OAc, NH2, NHAc, alkyl and halogens (Fig. 1B). By screening
this library of about 58 different compounds (U.S. Patent Applica-
tion 2011/0152210 A1, June 23, 2011),30 we found that compared
with natural product EGCG and metformin, analogs 4 and 6 were
AMPK activators with greater potency. Activation of AMPK by
these EGCG analogs resulted in inhibition of cell proliferation, sup-
pression of tumorsphere formation and decrease of cancer stem
cell population in human breast cancer cells, which is associated
with down-regulation of mTOR pathway and up-regulation p21
protein. The results also showed that these EGCG analogs were
able to enhance efficacy of anti-cancer drugs in human breast can-
cer cells, associated with activation of AMPK pathway. Therefore, it
would be an attractive approach to develop novel AMPK activators
as anti-cancer agents from structure-modified natural compound,
which possess unique properties of higher efficacy, lower toxicity
and targeting cancer stem cells as well.
2. Chemistry
2.1. General
Compounds
2 and 3 were synthesized as previously de-
scribed.25 The library of compounds of general structure
A
(Fig. 1A) were synthesized as reported.30 Generally, the starting
materials and reagents, purchased from commercial suppliers,
were used without further purification. Anhydrous methylene
chloride was distilled under nitrogen from CaH2. Anhydrous DMF
was distilled under vacuum from CaH2. Reaction flasks were
flame-dried under a stream of N2. All moisture-sensitive reactions
were conducted under a nitrogen atmosphere. Flash chromatogra-
phy was carried out using silica-gel 60 (70–230 mesh). The melting
points were uncorrected. 1H NMR and 13C NMR (300 MHz) spectra
were measured with TMS as an internal standard when CDCl3,
CD3OD and acetone-d6 were used as solvent. High-resolution
(ESI) MS spectra were recorded using a QTOF-2 Micromass spec-
trometer to analyze the compounds.
Figure 1. Chemical structures of EGCG analogs, their chemical synthetic scheme
and their effect on AMPK activation. (A) Chemical structures of natural EGCG,
synthetic pro-EGCG and general structure A of EGCG analogs. (B) Brief summary of a
synthetic scheme of EGCG analogs 4, 5 and 6 including steps a–e. The step (a) 4-
bromo-3,5-dimethoxymethoxybenzoic acid/diisopropylethylamine/dicyclohexyl-
carbodiimide in DCM; (b) p-toluenesulfonic acid/MeOH; (c) N-boc-p-aminobenzoic
acid/diisopropylethylamine/dicyclohexylcarbodiimide in DCM; (d) TFA in DCM; (e)
Ac2O/pyridine. (C) EGCG analogs 4 and 6 are more potent AMPK activators when
compared with natural EGCG and metformin. MDA-MB-231 cells were treated with
2.2. Synthesis of EGCG analog 4
2.2.1. 4-Bromo-3,5-dimethoxymethoxybenzoic acid
A solution of 4-bromo-3,5-dihydroxylbenzoic acid (500 mg,
2.1 mmol) in dry DCM at 0 °C was added with diisopropylethyl-
amine (2.49 g, 18.9 mmol) dropwise, followed by methoxymethyl
chloride (1.5 g, 18.9 mmol) dropwise and stirred for 48 h at room
temperature. After completion of the reaction as indicated by
TLC, the reaction mixture was quenched with saturated NH4Cl
sol (10 ml) and extracted twice with DCM. Removal of the solvent
gave the intermediate ether ester. To this ether ester in MeOH
(80 ml) was added 15% NaOH in MeOH (80 ml) and the whole
was heated at 70 °C for 3 h. The pH of the reaction was adjusted
to 5–6 by addition of 6 N HCl at 0 °C followed by filtration. The
MOM protected benzoic acid was obtained as white solid, mp
172 °C, (400 mg, 60% yield): 1H NMR (400 MHz, CDCl3) d: 7.54 (s,
2H), 5.32 (s, 4H), 3.53 (s, 6H); 13C NMR (400 MHz, CH3OH-d4) d:
167.3, 154.8, 130.7, 109.6, 108.5, 94.8, 55.2; ESI MS m/z: 318(Mꢀ1).
20
followed by Western blot analysis. The numbers underneath the Western results of
p-AMPK indicate normalized phosphor-AMPK /b-actin ratios.
lM of natural EGCG or synthetic EGCG analogs or 10 mM of metformin for 3 h,
a
a
We have previously shown that (+)-EGCG, the synthetic enan-
tiomer of natural (ꢀ)-EGCG, is equally potent in the inhibition of
the chymotrypsin-like activity of proteasome.24 This suggested
that the b5 active site binds equally well to (+)- and (ꢀ)-EGCG
and is thus pseudo-symmetric. On this basis, we synthesized sim-
ple, symmetrical analogs 2 and 3 of general structure A (Fig. 1A)
and they were found to be inhibitors of proteasome as well.25 Pre-
sumably, they bind to the same b5 active site and the gallate func-
tion in 2 resembles the gallate ester of EGCG.26 Furthermore, the 40-
deoxy analog 3 can also mimic EGCG with the additional advantage
that 3 is not a substrate of human catechol-O-methyltransferase
(COMT) and can not be biomethylated as natural EGCG. The com-
pound 3 exhibits higher cytotoxicity than natural EGCG in human
breast cancer cells which harbor high catechol-O-methyltransfer-
ase activity.27 We also found that p-aminobenzoate28 and fluor-
obenzoate29 could replace the gallate group of EGCG to induce
cancer cell apoptosis.
2.2.2. 2,3-Naphthalene-1,2,3,4-tetrahydro-2,3-bis(4-bromo-3,5-
dimethoxymethoxybenzoate), (2R,3S)rel (8)
The acid (466 mg, 1.4 mmol) obtained above and dicyclohexyl-
carbodiimide (296 mg, 1.4 mmol) were taken in dry DCM and stirred
for 1 h at room temperature (rt). Then 4-dimethylaminopyridine
(14 mg, 0.08 mmol) and the cis-diol 7 (100 mg, 0.6 mmol), was
added and stirred at rt. After 24 h the formed ppt was filtered off,

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
3033
and purified by column chromatography (3:7, ethyl acetate/hexane)
to give the product as clear white solid, mp 138 °C, (400 mg, 85%
yield): 1H NMR (400 MHz, CDCl3) d: 7.41 (s, 4H), 7.22–7.16 (m,
4H), 5.71–5.65 (m, 2H), 5.2 (s, 8H), 3.42 (s, 12H), 3.38–3.22 (m,
4H); 13C NMR (400 MHz, CDCl3) d: 165.0, 154.8, 132.0, 130.1,
129.0, 126.5, 110.2, 109.8, 95.1, 70.6, 56.4, 32.0; ESI MS m/z: 793
(M+ Na); HRMS (ESI) m/z Calcd for (M+23) C32H34Br2O12Na,
791.0309, Found 791.0338.
6H); 13C NMR (300 MHz, DCM-d2) d: 169.7, 165.5, 142.9, 132.4,
130.6, 129.0, 126.3, 124.9, 118.7, 70.0, 31.9, 24.0; ESI MS m/z:
509 (M+Na); HRMS (ESI) m/z, Calcd for (M+23) C28H26N2O6Na,
509.1683, Found 509.1702.
3. Materials and methods for biological studies
3.1. Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), dimethyl sulfoxide (DMSO) and metformin were pur-
chased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies
2.2.3. 2,3-Naphthalene-1,2,3,4-tetrahydro-2,3-bis(4-bromo-3,5-
dihydroxybenzoate), (2R,3S)rel (4)
para-Toulenesulfonic acid (35 mg, 0.18 mmol) was added to a
solution of the diester 8 (350 mg, 0.45 mmol) obtained above in
MeOH and refluxed for 3 h. After completion of the reaction, the
reaction mixture was neutralized by solid NaHCO3, filtered and
dried over NaSO4. Purification by column chromatography gave
the product 4 as white solid, mp 164 °C, (180 mg, 65% yield): 1H
NMR (400 MHz, CDCl3) d: 9.13 (br s, 4H), 7.20 (s, 4H), 7.11 (s,
4H), 5.75–5.6 (m, 2H), 3.4–3.2 (m, 4H); 13C NMR (400 MHz,
CH3Cl-d3) d: 205.4, 164.8, 155.3, 132.4 130.2, 129.0, 126.4, 107.8,
70.2, 31.6; ESI MS m/z: 594 (M); HRMS (ESI) m/z, Calcd for
(M+23) C24 H18 Br2 O8 Na, 614.9260, Found 614.9280.
against phosphor-AMPKa (T172), AMPKa, phosphor-Raptor, were
purchased from Cell Signaling Technology, Inc. (Danvers, MA).
Antibodies against b-actin and p21 were obtained from Santa
Cruz Biotechnologies (Santa Cruz, CA, USA). Mouse monoclonal
antibody against human PARP was purchased from BIOMOL
International LP (Plymouth Meeting, PA, USA). Antibodies
CD44-FITC and CD24-PE were purchased from BD Biosciences
(San Jose, CA, USA).
3.2. Cell culture, protein extraction, and Western blot assay
Human breast cancer MDA-MB-231 cells were grown in DMEM/
F12 (1:1) supplemented with 10% fetal bovine serum, 100 U/ml
2.3. Synthesis of compounds 5 and 6
penicillin, and 100 lg/ml streptomycin and maintained at 37 °C
2.3.1. 2,3-Naphthalene-1,2,3,4-tetrahydro-2,3-bis(N-t-
in a humidified incubator with an atmosphere of 5% CO2. A whole
cell extract was prepared as previously described.31,32 For Western
blot analysis, the cell extracts were separated by SDS–PAGE and
transferred to a nitrocellulose membrane, followed by visualiza-
tion using the enhanced chemiluminescence (ECL) reagent (Amer-
sham Biosciences, Piscataway, NJ, USA).
butoxycarbonyl-4-aminobenzoate), (2R,3S)rel (9)
The N-boc-p-aminobenzoic acid (45 mg, 0.21 mmol) and dicy-
clohexylcarbodiimide (42 mg, 0.21 mmol) were taken in dry DCM
and stirred for 2 h at rt. 4-Dimethylaminopyridine (3 mg,
0.03 mmol) and the cis-diol 7 (20 mg, 0.12 mmol) were added
and stirred at rt. After 24 h the formed precipitate was filtered
off, and purified by column chromatography (1.5:8.5, ethyl ace-
tate/hexane) to give compound 9 as white solid, mp 110 °C,
(45 mg, 60% yield): 1H NMR (400 MHz, CDCl3) d: 8.78 (br s, 2H),
7.90 (d, 4H), 7.63 (d, 4H), 7.2 (s, 4H), 5.8–5.65 (m, 2H), 3.4–3.35
(m, 4H), 1.47 (s, 18H); 13C NMR (400 MHz, acetone-d6) d: 205.3,
165.0, 152.4, 144.2, 132.7, 130.5, 129.0, 126.3, 123.7, 117.2,
117.1, 79.8, 69.8, 31.8, 27.5; ESI MS m/z: 625 (M+Na). HRMS (ESI)
m/z, Calcd for (M+23) C34H38N2O8Na, 625.2520, Found 625.2543.
3.3. Cell viability/proliferation assay
The MTT assay was used to determine the effects of various
compounds on proliferation of MDA-MB-231 breast cancer cells.
Cells were plated onto a 96-well plate and grown to 70–80% con-
fluency followed by adding indicated EGCG analogs or metformin
for 24 h incubation at 37 °C. Inhibition of cell proliferation was
measured as previously described.33 All samples were assayed in
triplicate in three independent experiments, and the mean value
for each experiment was calculated. The results are displayed as
mean ( standard deviation) and are expressed as percentage of
the control, which was considered to be 100%.
2.3.2. 2,3-Naphthalene-1,2,3,4-tetrahydro-2,3-bis(4-
aminobenzoate), (2R,3S)rel (6)
Compound 9 (130 mg, 0.2 mmol) was dissolved in DCM and a
little excess of trifluoroacetic acid (246 mg, 2 mmol) was added
and stirred at rt for overnight. The excess trifluoroacetic acid was
removed and the crude product was purified by column chroma-
tography to give compound 6 as white solid, mp 94 °C, (80 mg,
92% yield): 1H NMR (300 MHz, acetone-d6) d: 7.69 (d, 4H), 7.17
(m, 4H), 6.62 (d, 4H), 5.62 (t, 2H), 5.42 (br s, 4H), 3.29–3.36 (m,
4H); 13C NMR (400 MHz, acetone-d6) d: 205.3, 165.4, 132.9,
131.3, 128.9, 126.1, 117.6, 112.8, 69.2, 32.0; ESI MS m/z: 425
(M+Na); HRMS (ESI) m/z, Calcd for (M+23) C24 H22 N2 O4 Na,
425.1471, Found 425.1471.
3.4. Mammosphere formation assay
Mammosphere formation assay was performed to assess the
capacity of cancer stem cell self-renewal. Single cell suspensions
of MDA-MB-231 cells were thoroughly suspended and plated on
ultra low adherent wells of 6-well plates (Corning, Lowell, MA) at
1000 cells/well in 1.5 ml of sphere formation medium (1:1
DMEM/F12 medium supplemented with 50 units/ml penicillin,
50 mg/ml streptomycin, B-27, and N-2). One milliliter of sphere
formation medium was added every 3–4 days. After 7 days of incu-
bation with different concentrations of EGCG analogs 4, 6 or met-
formin, the formed spheres were collected by centrifugation at
300 g for 5 min and counted with an inverted phase-contrast Zeiss
Axiovert 25 microscope.
2.3.3. 2,3-Naphthalene-1,2,3,4-tetrahydro-2,3-bis(4-
acetamidobenzoate), (2R,3S)rel (5)
Compound 6 (40 mg, 0.09 mmol), acetic anhydride (0.5 ml) and
pyridine (0.5 ml) were stirred at rt for 24 h. After completion of the
reaction, ethyl acetate was added and stirred for 5 min then 1 N
HCL (1 ml) was added and stirred for another 5 min. The solution
was washed with CuSO4 solution (2 ꢁ 10 ml), water (2 ꢁ 10 ml),
brine (2 ꢁ 10 ml), dried over Na2SO4 and purified by column chro-
matography to give compound 5 as white solid, mp 128 °C, (40 mg,
82% yield): 1H NMR (300 MHz, DCM-d2) d: 7.90 (d, 6H). 7.55 (d,
4H), 7.18 (s, 4H), 5.5.72–5.62 (m, 2H), 3.34–3.24 (m, 4H), 2.14 (s,
3.5. Flow cytometry
MDA-MB-231 cells were cultured on low adherent plates in
sphere formation medium for 5 days and the formed colonies were
washed with PBS, and incubated with accutase solution (Sigma) at

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D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
37 °C for 5 min. The cells were then seeded on p100 dishes and
treated with different concentrations of compounds 4 or 6 for
48 h. The treated cells were detached by using 0.02% EDTA in
phosphate-buffered saline (PBS), counted and washed in 0.1%
BSA in PBS, and followed by staining with anti-CD24-PE and anti-
CD44-FITC (BD Pharmingen, San Diego, CA, USA), or respective iso-
type controls for 30 min in the dark at 4 °C. After washing steps,
the labeled cells were analyzed by flow cytometry using a FACS
FACscan (Becton Dickinson, CA, USA).
4. Results
4.1. EGCG analogs 4 and 6 significantly activate AMPK in human
breast cancer cells
In order to discover AMPK activators from natural resource, we
screened a series of EGCG analogs (see Fig. 1A) in breast cancer
cells. Treatment of human breast cancer MDA-MB-231 cells with
20 lM of the EGCG analogs was performed. Metformin and natural
product EGCG were used as controls. After treatment for 3 h, the
cell lysates of the treated cells were analyzed by Western blot to
measure levels of phosphor-AMPKa (T172), an active form of
AMPK protein (Fig. 1C). As reported, metformin and EGCG can acti-
vate AMPK signaling pathway.17–19,34 Our results (Fig. 1C) showed
that pro-EGCG, the pro-drug of EGCG, was as effective as EGCG in
activating the AMPK signaling pathway. Among our synthetic
EGCG analogs, 4 and 6 not only significantly activated AMPK but
also with greater potency compared with metformin, even at lower
concentrations (20 lM vs 10 mM) (Fig. 1C). The compounds 4 and
6 were also more potent AMPK activator than natural product
EGCG (Fig. 1C).
Figure 2. EGCG analogs 4 and 6 inhibited breast cancer cell proliferation through
activation of AMPK and upregulation of p21. (A) EGCG analogs 4 and 6 inhibited
breast cancer cell proliferation. MDA-MB-231 cells were treated with indicated
concentrations of 4, 6 or metformin for 24 h, followed by a MTT assay. ⁄, P <0.1; ⁄⁄, P
<0.01. Columns, mean of three experiments; bars, SD. (B) EGCG analogs 4 and 6
activated AMPK at
a dose-dependent manner, measured by elevated level of
phosphor-AMPK and its downstream proteins of phosphor-Raptor. Treatment
a
with 4 and 6 also showed increased level of p21. MDA-MB-231 cells were treated
with indicated concentrations of 4 or 6 for 3 h, followed by Western blot analysis
with the indicated antibodies. The numbers underneath the Western results of
4.2. EGCG analogs 4 and 6 inhibit proliferation of breast cancer
cells in a dose-dependent manner associated with activation of
AMPK and induction of p21 protein
p-AMPK
underneath the Western results of p-AMPK
AMPK /b-actin ratios.
a
indicated normalized phosphor-AMPK
a/b-actin ratios. The numbers
indicate normalized phosphor-
a
a
It has been reported that AMPK activation by an authentic
AMPK activator AMP-mimetic 5-aminoimidazole-4-carboxamide
ribonucleoside (AICAR) results in cell cycle arrest and inhibition
of cell proliferation in hepatoma HepG2 cells.35 In order to deter-
mine whether EGCG analogs 4 and 6 could play a similar role as AI-
CAR in suppression of cell proliferation, we treated human breast
cancer MDA-MB-231 cells with different concentrations of com-
pounds 4 and 6, followed by Western-blot analysis and cell prolif-
eration assay. The cells treated with metformin and natural
product of EGCG were served as controls. The results showed that
both EGCG analogs 4 and 6 could inhibit cell proliferation in a
dose-dependent manner and their inhibitory effects were more po-
tent than EGCG and metformin even when analogs 4 and 6 were
used at much lower concentrations compared with metformin
treatment (Fig. 2A). Results in Western blot showed that com-
pounds 4 and 6 activated AMPK in a dose-dependent manner as
4.3. Compounds 4 and 6 significantly inhibit mammosphere
formation
Tumor stem cells have the characteristic of forming tumor
sphere. An experiment of mammosphere formation is a useful tool
to identify a human mammary stem/progenitor-cell population
and measure stem cell-like behavior. To examine whether com-
pounds 4 and 6 can target cancer stem or stem-like cells and inhi-
bit mammosphere formation, we conducted a mammosphere
formation assay. Metformin and EGCG were used as controls. The
results showed that treatment of MDA-MB-231 cells with 10 or
20
formation by 45.1% and 66.7% or 52.2% and 73.3%, respectively
(Fig. 3). As comparisons, treatment with 10 or 20 M of EGCG only
lM of 4 or 6 for 7 days resulted in inhibition of mammosphere
l
inhibited mammosphere formation by 20.1% and 51.3%, and treat-
ment with 5 and 10 mM of metformin inhibited mammosphere
formation by 41.4% and 67.6%, respectively (Fig. 3). Therefore,
EGCG analogs 4 or 6 are much more potent than EGCG and metfor-
min in terms of inhibition of mammosphere formation.
well, as measured by increased levels of phosphor-AMPK
a and
phosphor-Raptor, one of the direct downstream substrate proteins
of AMPK (Fig. 2B). Our data also showed that activation of AMPK by
4 and 6 could suppress mTOR pathway measured by decreased
phosphor-p70-S6K (Fig. 2B), demonstrating the functionality of
these EGCG analogs as AMPK activators.
4.4. Both EGCG analogs 4 and 6 significantly decreased the
CD44+ high/CD24ꢀ low population in breast cancer cells
The cell proliferation inhibitory results in the Figure 2 were sup-
ported by others’ reports, in which AMPK activation was able to in-
hibit cell proliferation and induce cell cycle arrest in various cancer
cells in vitro and in vivo through increasing p53–p21 axis.11,35 Inhi-
bition of breast cancer cell proliferation by treatment with com-
pounds 4 and 6 was indeed associated with increased levels of
p21 protein (Fig. 2B).
Metformin could selectively target cancer stem cells and reduce
CD44high/CD24low cell population in breast cancer cells through
activation of AMPK signaling.18 To determine whether EGCG ana-
logs 4 and 6 possess the similar property, we treated MDA-MB-

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
3035
5. Discussion
Breast cancer remains a leading cause of morbidity and mortal-
ity in women living in Western countries. The major reason of
breast cancer mortality is metastasis of the primary tumor. Exper-
imental evidence from preclinical and clinical studies suggests that
cancer stem cells may contribute to tumor progression and metas-
tasis spread. However, most chemotherapeutical drugs can not tar-
get cancer stem cells. Furthermore, even though chemotherapy
plays an important role as single modality for cancer treatment,
limited effectiveness of anti-cancer drugs are often observed be-
cause induction of drug-resistance and severe side-effects.36,37 In
an attempt to improve conventional chemotherapies, development
of more effective agents as an adjuvant therapy is critically impor-
tant, especially agents that can target cancer stem cells.
AMPK signaling pathway recently becomes a ‘hot’ field in can-
cer prevention and therapy. Hirsch et al. reported that metformin
selectively targets breast cancer stem cells and inhibited tumor
growth in mouse models mediated by activation of AMPK path-
way.18 It is becoming an attractive approach to discover more
AMPK activators with greater potent and more specific properties.
It has been reported that natural compounds genistein (rich in soy
been), EGCG (abundant in green tea), and capsaicin (from hot pep-
per) are able to activate AMPK pathway34 and anti-cancer effects of
these compounds have been also reported previously.38–40 Disad-
vantages of natural compounds as anti-cancer agents include poor
bioavailability, short half-life and weak potency.41 In order to in-
crease bioavailability and stability, we synthesized a pro-drug of
EGCG (termed pro-EGCG, Fig. 1A), in which all –OH group were
protected with acetate groups which can be cleaved by cellular
esterases to release EGCG in vivo. Through testing pro-EGCG
in vitro and in vivo, we reported that compared with natural EGCG,
Pro-EGCG increased the bioavailability, stability, and proteasome
inhibitory and anticancer activities in human breast cancer cells
and breast cancer xenografts.23
Figure 3. EGCG analogs 4 and 6 inhibited mammosphere formation. MDA-MB-231
cells were seeded in low attached 6-well plates (1000 cells/well) and treated with
indicated concentrations of 4 and 6 for 7 days, followed by calculating numbers of
mammosphere (A) and taking photos of mammosphere morphology (B). Metformin
and EGCG were served as controls. ⁄, P < 0.1; ⁄⁄, P < 0.01. Columns, mean of three
experiments; bars, SD.
231 cells with different concentrations of 4 and 6 for 48 h. The
treated cells were stained with specific antibodies against human
CD44 (FITC), CD24 (PE) or their respective isotype controls, fol-
lowed by washing, fixing and analysis by flow cytometry. The re-
sults showed that both EGCG analogs 4 and 6 could reduce
CD44high/CD24low cell population. Treatment of MDA-MB-231 cells
with 10 or 20 l /
M of 6 resulted in reduction of the CD44high
CD24low population by 43.3% and 71.7%, respectively (Fig. 4). The
same results were observed in the cells treated with compound
4, which was slightly less potent than 6 (Fig. 4).
Recently we synthesized a series of EGCG analogs based on the
general chemical structure A (Fig. 1A) to mimic natural EGCG in
their proteasome inhibition activity. By testing these synthetic
Figure 4. Treatment with EGCG analogs 4 and 6 effectively decreased cancer stem cell population in breast cancer cells. The MDA-MB-231cells were treated with indicated
concentrations of 4 and 6 for 48 h, followed by flow cytometry analysis of the CD44-FITC and CD24-PE labeled cells. ⁄, P < 0.1; ⁄⁄, P < 0.01. Columns, mean of three
experiments; bars, SD.

3036
D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
compounds we found that EGCG analogs 4 and 6 could inhibit
breast cancer proliferation, suppress mammosphere formation
and decrease cancer stem cell population. These effects of EGCG
analogs 4 and 6 appeared to be associated with their AMPK activat-
ing property. Furthermore these two synthetic compounds showed
greater potency when compared with EGCG and metformin in
terms of activation of AMPK, inhibition of cell proliferation and
mammosphere formation (Figs. 2 and 3).
tor, a mTOR binding partner and inhibitor, and decreased levels of
phosphor-p70S6K, a down-stream protein of mTOR (Fig. 2). These
data demonstrate that the compounds 4 and 6 are able to down-
regulate mTOR pathway mediated by activation of AMPK.
Breast cancer tissue contains a rare population of multi-potent
cells with the capacity to self-renew, termed cancer stem cells.
These breast cancer stem cells could be enriched and measured
by a colony formation assay in three-dimensional cultures, referred
to as mammosphere formations. Klopp et al. reported that cancer
stem cells can promote mammosphere formation.49 In the current
study, we examined whether exposure of breast cancer cells to
EGCG analogs 4 and 6 could block their mammosphere formation.
The results showed that mammosphere formation was decreased
in a dose-dependent manner in MDA-MB-231 cells treated with
the compounds 4 and 6. The inhibitory effect of these synthetic
compounds was more potent than the treatment with metformin
control (Fig. 3).
To further verify whether EGCG analogs 4 and 6 can target can-
cer stem cells, we selected cell surface protein CD44+ high/CD24ꢀ low
as breast cancer stem cell-related biomarkers. The CD44 antigen is
a cell-surface glycoprotein and a receptor for hyaluronic acid. The
CD44 protein belongs to cell surface adhesion molecules involved
in a wide variety of cellular functions including cell–cell and
cell–matrix interactions.50 CD44 expression is positively associated
with stem cell-like characteristics and CD24 is related to differen-
tiation of epithelial cells.51 Breast cancer stem cells, which are rich
in CD44+ high/CD24ꢀ low cells, were first identified by Al-Hajj et al.52
This pioneer study showed that this distinct population of cells had
the exclusive ability to form tumors in mice. Later, these human
The AMPK signaling pathway is composed of a series of tumor
suppressor genes including LKB1, p53, p21, Forkhead box O-class
(FoxO), Raptor and tuberous sclerosis complex 1/2 (TSC1/2). Accu-
mulating evidence indicates that AMPK activation strongly sup-
presses cell proliferation. These actions of AMPK activators
appear to be mediated through multiple mechanisms including
regulation of the cell cycle, inhibition of protein synthesis and
down-regulation of PI3K-Akt pathway. Cell cycle regulation by
AMPK activators is mediated by up-regulation of the p53–p21 axis
as well as down-regulation of TSC2–mTOR.42 The cell cycle
machinery is tightly controlled by multiple events that are posi-
tively regulated by cyclin-dependent kinases (CDKs) and nega-
tively regulated by CDK inhibitors (CDKIs),43 such as p21CIP and
p27KIP, and tumor suppressor proteins, such as p5344 and FoxO
transcription factors.45,46 Most of AMPK activating stimuli, such
as glucose deprivation and cellular stresses, can activate p53
through phosphorylation of p53 at the serine (Ser) 15.47 The phos-
phorylated p53 induces cell-growth arrest via the transcriptional
regulation of p53 response genes such as p21CIP and p53-regulated
apoptosis-inducing protein 1 (p53AIP1).48 In order to determine
mechanisms of inhibitory cell proliferation by EGCG analogs 4
and 6, we measured the protein level of p21 in MDA-MB-231 cells
treated with increasing doses of EGCG analogs 4 and 6. The results
revealed that treatment with the compounds 4 and 6 resulted in
breast cancer cells were further demonstrated to contain a defined
low
subpopulation of CD44+ high/CD24ꢀ
cells, which possess the
property of tumorigenesis.53 As we expected, EGCG analogs 4 and
6 indeed targeted cancer stem cells and significantly decreased
the CD44+ high/CD24ꢀ low cell population in MDA-MB-231 cells in
a dose-dependent manner (Fig. 4).
elevated protein level of p21, as well as phosphor-AMPK
172) (Figs. 2 and 3).
a (Thr
AMPK activation in breast cancer cells treated with EGCG ana-
logs 4 and 6 resulted in increased protein levels of phosphor-Rap-
Another candidate marker of cancer stem cells is aldehyde
dehydrogenase 1 (ALDH1),54 which is responsible for the oxidation
of intracellular aldehydes to carboxylic acids.55 Increased ALDH1
activity has been found in stem cell populations in both normal
and malignant cells.51,56,57 The ALDH1+ cell population has a small
overlap with the CD44+ high/CD24ꢀ
cells. Ginestier et al. re-
low
ported that the overlap of both cell populations represented
approximately 1% or less of the total cancer cell population. How-
ever, these small populations of cells appeared to harbor highly
tumorigenic capability and could generate tumors from as few as
20 cells.54 It will be worthwhile for us to further determine effects
of the EGCG analogs on both populations of ALDH1+ and CD44+ high
/
CD24ꢀ low cells and verify the finding in animal tumor models in
the near future.
Our previous studies have indicated that EGCG is able to induce
tumor cell apoptosis and growth arrest in G1 phase of the cell cycle
via inhibition of the chymotrypsin-like activity of the protea-
some.40 However, the relationship between proteasome inhibition
and AMPK activation mediated by EGCG analog remain unclear. At
the present time, there is only limited information on the structure
activity relationship among these synthetic compounds.
6. Conclusions
The present study demonstrates that two EGCG analogs 4 and 6
of our library exhibit inhibitory properties in MDA-MB-231 human
breast cancer cells through inhibition of cell proliferation and
mammosphere formation, suppression of CD44+ high/CD24ꢀ low cell
populations and activation of AMPK signaling pathway. The activa-
tion of AMPK appears to be a key element in the various molecular
Figure 5. Schematic model for inhibition of cell proliferation by EGCG analogs.

D. Chen et al. / Bioorg. Med. Chem. 20 (2012) 3031–3037
3037
20. Hwang, J. T.; Ha, J.; Park, I. J.; Lee, S. K.; Baik, H. W.; Kim, Y. M.; Park, O. J. Cancer
Lett. 2007, 247, 115.
21. Chen, Z.; Zhu, Q. Y.; Tsang, D.; Huang, Y. J. Agric. Food Chem. 2001, 49, 477.
22. Lu, H.; Meng, X.; Yang, C. S. Drug Metab. Dispos. 2003, 31, 572.
23. Landis-Piwowar, K. R.; Huo, C.; Chen, D.; Milacic, V.; Shi, G.; Chan, T. H.; Dou, Q.
P. Cancer Res. 2007, 67, 4303.
24. Smith, D. M.; Daniel, K. G.; Wang, Z.; Guida, W. C.; Chan, T. H.; Dou, Q. P.
Proteins: Struct., Funct., Bioinf. 2004, 54, 58.
25. Huo, C.; Yang, H.; Cui, Q. C.; Dou, Q. P.; Chan, T. H. Bioorg. Med. Chem. 2010, 18,
1252.
events presented, and the involved mechanisms were summarized
in Figure 5. It is currently unclear how these EGCG analogs activate
AMPK, whether any other responsible molecular targets are in-
volved, and what the relationship among these potential targets
would be, since besides AMPK pathway, the synthetic EGCG ana-
logs might have multiple molecular targets in cancer cells. Future
investigation on this matter including in vivo study may provide
new insight into the questions and related mechanisms.
26. Kazi, A.; Wang, Z.; Kumar, N.; Falsetti, S. C.; Chan, T. H.; Dou, Q. P. Anticancer
Res. 2004, 24, 943.
Acknowledgments
27. Huo, C.; Yang, H.; Cui, Q. C.; Dou, Q. P.; Chan, T. H. Bioorg. Med. Chem. 2010, 18,
1252.
28. Osanai, K.; Huo, C.; Landis-Piwowar, K. R.; Dou, Q. P.; Chan, T. H. Tetrahedron.
2007, 63, 7565.
29. Osanai, K.; Landis-Piwowar, K. R.; Dou, Q. P.; Chan, T. H. Bioorg. Med. Chem.
2007, 15, 5076.
30. Chan, TH; Dou, QP. US patent application 0152210 A1, June 23, 2011.
31. Yang, H.; Sun, D. K.; Chen, D.; Cui, Q. C.; Gu, Y. Y.; Jiang, T.; Chen, W.; Wan, S. B.;
Dou, Q. P. Cancer Lett. 2010, 292, 48.
This work was partially supported by a pilot fund from Karm-
anos Cancer Institute and grants from the National Cancer Institute
(1R01CA120009, 3R01CA120009-04S1 and 5R01CA127258-05, to
Q.P.D.).
32. Chen, D.; Cui, Q. C.; Yang, H.; Dou, Q. P. Cancer Res. 2006, 66, 10425.
33. Chen, D.; Landis-Piwowar, K. R.; Chen, M. S.; Dou, Q. P. Breast Cancer Res. 2007,
9, R80.
34. Hwang, J. T.; Park, I. J.; Shin, J. I.; Lee, Y. K.; Lee, S. K.; Baik, H. W.; Ha, J.; Park, O.
J. Biochem. Biophys. Res. Commun. 2005, 338, 694.
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