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Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 486568, 12 pages
doi:10.1155/2012/486568
Research Article
The Potential Utility of Curcumin in the Treatment of
HER-2-Overexpressed Breast Cancer: An
In Vitro
and
In Vivo
Comparison Study with Herceptin
Hung-Wen Lai,1, 2 Su-Yu Chien,3, 4, 5 Shou-Jen Kuo,1, 4, 5 Ling-Ming Tseng,6, 7 Hui-Yi Lin,8
Chin-Wen Chi,2and Dar-Ren Chen1, 4
1Comprehensive Breast Cancer Center, Changhua Christian Hospital, Changhua 50006, Taiwan
2Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan
3Department of Pharmacology, Changhua Christian Hospital, Changhua 50006, Taiwan
4School of Medicine, College of Health Care and Management, Chung Shan Medical University, Taichung 40201, Taiwan
5School of Nutrition, College of Health Care and Management, Chung Shan Medical University, Taichung 40201, Taiwan
6Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei 11217, Taiwan
7School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan
8Department of Pharmacology, School of Pharmacology, China Medical University, Taichung 40402, Taiwan
Correspondence should be addressed to Dar-Ren Chen, darren chen@cch.org.tw
Received 11 November 2010; Revised 21 March 2011; Accepted 2 May 2011
Academic Editor: Jae Youl Cho
Copyright © 2012 Hung-Wen Lai et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HER-2 is an important oncoprotein overexpressed in about 15–25% of breast cancers. We hypothesized that the ability of curcumin
to downregulate HER-2 oncoprotein and inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-κBactivationmay
be important in the treatment of HER-2-overexpressed breast cancer. To examine the effect of curcumin on breast cancer cells,
MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr (a herceptin resistant strain from SK-BR-3) cells were used for in vitro
analysis. The in vivo effect of curcumin on HER-2-overexpressed breast cancer was investigated with the HER-2-overexpressed BT-
474 xenograft model. Cell growth, cell cycle change, the antimobility effect, signal transduction, and xenograft volume analysis
between groups treated with herceptin and/or curcumin were tested. Curcumin decreased the cell growth of various breast cancer
cell lines (MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr). In Western blot analysis, the phosphorylation of Akt,
MAPK, and expression of NF-κB were reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. When
treated with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were decreased in both
BT-474 and SK-BR-3-hr cells. In the BT-474 xenograft model, though not as much as herceptin, curcumin did effectively decrease
the tumor size. The combination of curcumin with herceptin was not better than herceptin alone; however, the combination
of taxol and curcumin had an antitumor effect comparable with taxol and herceptin. The results suggested that curcumin has
potential as a treatment for HER-2-overexpressed breast cancer.
1. Introduction
Around 15–25% of breast cancers are noted to overexpress
the human epithelial growth factor receptor 2 (HER-2) [1–
3], patients with HER-2 overexpression were associated with
a poor prognosis, more disease relapse, and distant metas-
tasis [3–6]. Herceptin (generic name: Trastuzumab), which
effectively inhibits the HER-2-related PI3k/Akt and MAPK
pathways, is the first targeted therapeutic agent developed
and approved for the treatment of patients with HER-2-
overexpressed breast cancer [7–12]. Despite the success of
herceptin, a significant proportion of HER-2-positive breast
cancer patients responded poorly to the treatment [13,14].
In addition, some patients that initially responded to the
therapy became resistant within one year [14,15]. The
refractory state of these HER-2-positive breast carcinomas
2 Evidence-Based Complementary and Alternative Medicine
illustrates the need to examine the mechanisms underlying
tumor resistance and the necessity to seek novel treatment
strategies.
Curcumin (diferuloylmethane) is a yellow pigment de-
rived from the rhizome of the plant Curcuma longa L.
The powdered rhizome of this plant, called turmeric, is
commonly used in the preparation of curries. Curcumin,
as a polyphenol with a diarylheptanoid structure containing
two α,β-unsaturated ketones, is considered to be the major
active constituent of turmeric [16,17]. In addition to its
wide range of pharmacological activities, the anticancer
properties of curcumin have attracted great interest [18–20].
The ability of curcumin to downregulate EGFR [19,21,22]
and HER-2 [23] oncoproteins and affect the PI3K/Akt [24]
and MAPK [25] pathways, with which herceptin interfered,
raised interest in the potential utility of curcumin in the
treatment of HER-2-positive breast cancer.
Taxol (generic name: Paclitaxel) combined with her-
ceptin is one of the current preferred regimens for the
treatment of HER-2-overexpressed breast cancer [11,26].
NF-κB is an important factor related to proliferation and
antiapoptosis [27,28], and the activation of NF-κBplays
an important role in the chemoresistance of taxol [29,30].
Curcumin has the well-known ability to downregulate NF-
κB[31,32]. The combination of curcumin with taxol could
suppress taxol-related NF-κB activation and enhance the
antitumor effect of taxol [31,33]. The evolving concept
of combining a monoclonal antibody (herceptin or per-
tuzumab) for the extracellular domain of HER-2 and small
molecule tyrosine kinase inhibitors (TKI) for EGFR (gefi-
tinib, erlotinib) [34–36] or EGFR/HER-2 (lapatinib) [37]has
shown benefit in some preclinical studies and patient trials.
The multifunctions of curcumin in downregulating EGFR
and HER-2 oncoproteins, reducing the phosphorylation of
Akt and MAPK and suppressing NF-κB activation, led to
interest in using curcumin in the treatment of HER-2-
overexpressed breast cancer, along with herceptin and/or
taxol.
The aim of this preclinical study was to explore the
potential application of curcumin in the treatment of HER-
2-overexpressed breast cancer and examine the interaction of
curcumin and herceptin, which has rarely been reported.
2. Materials and Methods
2.1. Cell Culture and Reagents. To examine the effect of
herceptin and curcumin on breast cancer cell lines with
various estrogen receptors (ER) and HER-2 receptors, MCF-
7 [ER(+), HER-2(−)], SK-BR-3-hr (a herceptin-resistant
strain from SK-BR-3 breast cancer cells) [ER(−), HER-
2(+)], BT-474 [ER(+), HER-2(+)], MDA-MB-231 [ER(−),
HER-2(−)], and normal breast epithelial cells, MCF-10A
[ER(−), HER-2(−)], were chosen. The expression of HER-
2 oncoprotein in MCF-7, MCF-10A, MDA-MB-231, BT-
474, and SK-BR-3-hr cells was verified by western blotting
(Figure 1).
The cell lines (MCF-7, BT-474, MDA-MB-231, and
normal breast MCF-10A) used in this study were purchased
MCF-10A
MCF-7
MDA-MB-231
BT-474
SK-BR-3-hr
HER-2 (185 kDa)
β-actin (42 kDa)
HER2-
unexpressed
HER2-
overexpressed
Figure 1: The expression of HER-2 oncoprotein in various human
breast cancer cell lines. Cells were lysed and analyzed by western blot
as described in methods. As revealed in figure, BT-474 and SKBr-3-
hr were HER-2-overexpressed breast cancer cell lines. Whiles MCF-
7, MCF-10A, and MDA-MB-231 cells were HER-2-unexpressed
breast cancer cells.
from American Type Culture Collection (ATCC). SK-BR-
3-hr, a herceptin-resistant strain, was a kind gift from
Dr. L.-M. Tseng. The resistance of herceptin was induced
by repeated culture of herceptin-treated SK-BR-3 cells,
which were herceptin-sensitive and purchased from ATCC.
The MCF-7 cell line was maintained in RPMI medium
with 10% fetal bovine serum, 50 unit/mL penicillin, and
50 unit/mL streptomycin. The MDA-MB-231 cells were
cultured in L15 medium containing 10% fetal bovine serum,
50 unit/mL penicillin, and 50 unit/mL streptomycin. BT-474
cells were maintained in DMEM/F12 medium with 10% fetal
bovine serum, 50 unit/mL penicillin, and 50 unit/mL strep-
tomycin. SK-BR-3-hr cells were maintained in DMEM/F12
medium with 10% fetal bovine serum, 50 unit/mL peni-
cillin, and 50 unit/mL streptomycin. MCF-10A was main-
tained in MEBM medium with 1% penicillin-streptomycin,
50 μg/mL hydrocortisone, 1 μg/mL EGF, 500 μg/mL insulin,
and 10 μg/mL cholera toxin. Cells were maintained at 37◦C
in a humidified atmosphere in the presence of 5% CO2.
2.2. Compounds. Curcumin (Sigma-Aldrich, Inc., St. Louis,
Mo, USA) was dissolved in DMSO at 50 μg/mL as a stock
solution. Herceptin was purchased from Roche and dissolved
in PBS at 50 μg/mL as stock. Taxol was dissolved in 49.7%
ethanol at 6 mg/mL as stock.
2.3. Growth and Cell Proliferation Analysis. Cell proliferation
was measured using sulforhodamine B (SRB) colorimetric
analysis, which is used for cell density determination, based
on the measurement of cellular protein content [38]. The
method described here has been optimized for the toxicity
screening of compounds to adherent cells in a 96-well
format. After an incubation period, cell monolayers were
fixed with 10% (wt/vol) trichloroacetic acid and stained with
SRB for 30 min after which the excess dye was removed
by washing repeatedly with 1% (vol/vol) acetic acid. The
protein-bound dye was dissolved in 10mM Tris base solution
Evidence-Based Complementary and Alternative Medicine 3
for OD determination at 510 nm using a microplate reader.
The results were linear over a 20-fold range of cell numbers.
The trypan blue exclusion test was also used to confirm
the cell proliferation result in herceptin- and curcumin-
treated BT-474 and SK-BR-3-hr cells. Cells were stained with
4% trypan blue (Sigma), and viable cells counted with a
hemocytometer under a light microscope.
2.4. Determination of Combinatorial Effects. The ability of
herceptin and curcumin to act in a synergistic, additive, or
antagonistic matter with regard to growth inhibition was
determined by a combination index (CI) as proposed by
Chou and Talalay [39,40]. The Calcusyn software (Biosoft,
Great Shelford, Cambridge, UK) was used to determine the
CI for each concentration of drug mixture used. A CI value
<1 represented a case where synergism of herceptin and
curcumin was present. CI values of 1 and >1represented
additive and antagonistic effects, respectively.
2.5. Cell Cycle Analysis. Breast cancer cells were plated at a
density of 5 ×105/dish in 60 ×15-mm culture dishes and
allowed to adhere overnight. Cells were then incubated with
media containing herceptin and/or curcumin at different
concentrations as indicated. Following 72 hours (h) of
incubation, the cells were washed with PBS, trypsinized, and
collected by centrifugation at 1,500 rpm for 10 minutes. After
centrifugation, the supernatant was removed, and the cell
pelletswerefixedwith75%alcoholat−20◦C.After1hof
incubation, the cell pellets were collected by centrifugation
at 1,500 rpm for 10 minutes. The pellets were incubated with
propidium iodide (PI) solution (10 μg/mL) for 30 minutes.
The cell cycle phase was determined by Cytomics FC500 flow
cytometry (Beckman Coulter).
2.6. Mobility Test. Cell migration is necessary in many
physiological processes, such as wound healing, and is a
characteristic of cancer cell metastasis [41]. The inhibitory
effect of herceptin and/or curcumin on different breast
cancer cell lines was tested by wound healing assay. Upon
inflicting a scratch wound, these cells were treated with
various concentrations of curcumin and/or herceptin for 4 h
and returned to standard media in an attempt to minimize
any cytotoxic effects that could potentially confound our
observations. Following 20 h of further incubation, the areas
of the wounds were measured using Image J software
(http://rsb.info.nih.gov/ij/).
2.7. Western Blot Analysis. Breast cancer cells were allowed
to incubate with curcumin and/or herceptin at various
concentrations and time points as indicated and harvested
after treatment. Whole cell lysate was prepared by resus-
pending the cells in RIPA buffer supplemented with protease
inhibitors cocktail (PIERCE) and incubating the cells on ice
for 30 minutes. Cell lysates were centrifuged at 13,000 x g
for 10 minutes and the supernatant collected. The protein
concentration was measured using the Bradford assay (Bio-
Rad Laboratories, Hercules, Calif, USA).
An aliquot of protein lysate (10 μg) from each sample
was mixed with 2X Laemmli sample buffer (Bio-Rad,
Hercules, Calif, USA), and the protein lysate was separated
in 10% SDS-polyacrylamide gels for 1 h. After transferring
the sample to a nitrocellulose membrane, the membrane
was blocked with 5% milk in 1X TBST buffer (10 mM
Tris, 150 mM NaCl, 0.5% Tween-20, pH 7.4) for 1 h at
room temperature and immunoblotted using the following
antibodies: HER-2, phosphorylated Akt (p-Akt), total Akt,
phosphorylated MAPK (p-MAPK), total MAPK, and NF-κB
(Cell Signaling Technology). The proteins were probed with
anti-HER-2 (Cell Signaling Technology), anti-p-Akt, anti-
total Akt, anti-p-ERK1/2, anti-ERK 1/2, anti-NF-κB, and
anti-β-actin (Sigma) at 4◦C overnight, followed by incuba-
tion with horseradish peroxidase-conjugated secondary anti-
bodies (Sigma). Protein visualization was performed using
the enhanced chemiluminescence kit (PIERCE) according to
the manufacturer protocol. Equal loading of total protein was
normalized with the β-actin signal.
2.8. In Vivo Model: Xenograft of HER-2-Overexpressed Breast
Cancer in Nude Mice. HER-2-overexpressed BT-474 cells
(1 ×107cells per mice) were injected into 4- to 6-week-
old female, athymic nude mice subcutaneously (s.c.) in the
right flank region to form xenografts. Prior to tumor cell
inoculation, all mice were primed with 17β-estradiol pellets
introduced subcutaneously in a biodegradable carrier binder
7 days before inoculation of the tumor (1.7 mg of estradiol
per pellet, Innovative Research of America, Inc.) to promote
tumor growth. Then, 1 ×107BT-474 cells, suspended in
(200 μL) growth-factor-reduced matrigel (BD Bioscience,
Bedford, MA), were injected s.c. into the right flank region.
Aperiodof14to21dayselapsedtoallowformation
of tumor nodules. Tumor nodules were monitored twice
weekly by a single observer using serial micrometer (mm)
measurements, with tumor volume calculated as the product
of length ×width2/2. Six animals were randomly assigned
to each treatment group. Statistical tests were performed to
assure uniformity in starting volumes between treatment and
control groups at the beginning of each experiment.
Treatment groups included the control, herceptin alone,
curcumin alone, and the combination of herceptin and
curcumin. To evaluate the effect of curcumin and/or her-
ceptin with chemotherapeutic agents, taxol alone, taxol and
herceptin, taxol and curcumin, and taxol + herceptin +
curcumin regimens were tested. Treatment with different
protocols was initiated 21–28 days postxenograft inoculation
status, at which time the xenograft volume was measured
at around 50–100 mm3.Differences in xenograft volume on
the 28th posttreatment day between groups were analyzed by
single-factor analysis of variance (ANOVA). The treatment
protocol is summarized.
(a) Control group: sterile 0.1% DMSO intra-peritoneally
(i.p.) injection once per week for 4 consecutive weeks.
(b) Herceptin-only group: loading dose of 4mg/kg her-
ceptin in sterile PBS, administered by i.p. injection,
then a dose of 2 mg/kg maintained at once per week,
for 4 consecutive weeks.
4 Evidence-Based Complementary and Alternative Medicine
(c) Curcumin only: curcumin dissolved in 0.1% DMSO
injected i.p. at a dose of 45 mg/kg twice per week for
4 consecutive weeks.
(d) Combined curcumin and herceptin: curcumin dis-
solved in 0.1% DMSO injected i.p. at a dose of
45 mg/kg twice per week, combined with a loading
dose of 4 mg/kg herceptin in sterile PBS, admin-
istered by i.p. injection, then a dose of herceptin
2 mg/kg maintained at once per week, for 4 consec-
utive weeks.
(e) Taxol-alone group: taxol 10 mg/kg i.p. once per week
for 4 consecutive weeks.
(f) Taxol + herceptin: taxol 10mg/kg i.p. once per week,
combined with a loading dose of 4 mg/kg herceptin in
sterile PBS, administered by i.p. injection, then a dose
of herceptin 2 mg/kg maintained at once per week,
for 4 consecutive weeks.
(g) Taxol and curcumin: taxol 10 mg/kg i.p. once per
week, curcumin dissolved in DMSO injected i.p. at
a dose of 45 mg/kg twice per week for 4 consecutive
weeks.
(h) Taxol, curcumin, and herceptin: taxol 10 mg/kg i.p.
once per week, curcumin dissolved in 0.1% DMSO
injected i.p. at a dose of 45 mg/kg twice per week,
combined with a loading dose of 4 mg/kg herceptin in
sterile PBS, administered by i.p. injection, then a dose
of herceptin maintained at 2 mg/kg once per week,
for 4 consecutive weeks.
After completing 4 weeks of treatment, the mice were
sacrificed before recording body weight and tumor volume.
The tumor was harvested and processed for various ana-
lyticalpurposes.Theanimaluseprotocolwerereviewed
and approved by the Institutional Animal Care and Use
Committee of Changhua Christian Hospital, Changhua,
Taiw a n .
2.9. Statistical Analysis. Analyses were performed using the
Statistical Analysis System (SAS 9.1). Data are presented
as mean ±standard deviation, except where indicated.
Comparisons between groups were analyzed using the chi-
square test, Student’s two-tailed t-test, or one-way ANOVA
with Bonferroni’s correction, as appropriate. A value of P<
0.05 is considered statistically significant.
3. Results
3.1. Effects of Herceptin or Curcumin on Cell Proliferation.
To examine the biological effect of herceptin and curcumin,
breast cancer cell lines were treated with different con-
centrations of herceptin (0.1–10 μg/mL) or curcumin (1–
25 μg/mL) for 72 h. Cell proliferation change was assayed
with an SRB assay. As shown in Figure 2(a), in HER-2-
overexpressed BT-474 breast cancer cells, cell proliferation
was inhibited by herceptin in a dose-dependent manner.
Cell proliferation decreased to 60% after treatment with
1μg/mL of herceptin and reached a plateau when with
0
20
40
60
80
100
120
012345678910
Cell growth (%)
Herceptin (μg/mL)
(a)
0
20
40
60
80
100
120
Cell growth (%)
0 5 10 15 20 25
Curcumin (μg/mL)
MCF-7 MDA-MB-231
BT-474 SK-BR-3-hr
MCF-10A
(b)
Figure 2: The effect of herceptin and curcumin on growth of
different breast cancer cell lines. Cells were incubated with different
levels of herceptin (a) or curcumin (b) for 72 h, respectively, and cell
growth was measured using SRB assay.
a concentration >1μg/mL. In SK-BR-3-hr breast cancer
cells, an HER-2-overexpressed and herceptin-resistant breast
cancer cell line, cell growth was not inhibited by herceptin,
even at a 10 μg/mL high concentration. The growth of MCF-
7 and MCF-10A, which were non-HER-2-overexpressed
cells, was not affected by herceptin treatment. The growth
of MDA-MB-231 cells decreased to 64% of the control after
10 μg/mL of herceptin treatment (Figure 2(a)).
In examining the effect of curcumin treatment on these
cell lines, we found that the cell proliferations of these five cell
lines (MCF-7, BT-474, SK-BR-3-hr, MCF-10A, and MDA-
MB-231) were all decreased after treatment with curcumin,
with different sensitivities (Figure 2(b)). The SK-BR-3-hr,
MCF-10A, and MDA-MB-231 cells were more sensitive to
curcumin than BT-474 and MCF-7 cells. After treatment
Evidence-Based Complementary and Alternative Medicine 5
0
20
40
60
80
100
012345678910
Cell growth (%)
Herceptin (μg/mL)
BT-474
No curcumin 5μg/mL curcumin
15 μg/mL curcumin
0
20
40
60
80
100
012345678910
Cell growth (%)
Herceptin (μg/mL)
10 μg/mL curcumin
No curcumin 5μg/mL curcumin
15 μg/mL curcumin
10 μg/mL curcumin
SK-BR-3-hr
(a)
BT-474
No curcumin 5μg/mL curcumin
10 μg/mL curcumin
0
5
10
15
20
25
30
35
40
45
50
55
0 0.1 1 2 5 10
Cell number (1 ×104cells)
Herceptin (μg/mL)
SK-BR-3-hr
0
5
10
15
20
25
30
35
40
45
50
55
00.11 2 5 10
Cell number (1 ×104cells)
Herceptin (μg/mL)
15 μg/mL curcumin
No curcumin 5μg/mL curcumin
10 μg/mL curcumin 15 μg/mL curcumin
(b)
Figure 3: Cell growth of HER-2-overexpressed breast cancer cells with combined treatment of herceptin and curcumin. (a) The combined
effects of herceptin and curcumin on the growth of BT-474 and SK-BR-3-hr cells. Cell growth was analyzed by SRB assay after drug treatment
for 72 h. (b) The combinational effects of herceptin and curcumin on BT-474 and SK-BR-3-hr cells were analyzed by trypan blue exclusion
assay after drug treatment for 72 h.
with 10 μg/mL of curcumin, the cell proliferation of HER-
2-overexpressed BT-474 cells and herceptin-resistant SK-BR-
3-hr cells decreased to 65% and 20%, respectively.
3.2. Combination Treatment of Cells with Herceptin and
Curcumin. Figure 3(a) shows the proliferation of different
cell lines after treatment with a combination of herceptin
and curcumin. In the HER-2-overexpressed BT-474 cells,
we found that the antiproliferative effect of herceptin was
not affected by the addition of curcumin. Curcumin could
further decrease the proliferation of BT-474 cells when
combined with herceptin. No apparent synergistic effect was
presented when herceptin was combined with curcumin in
the SK-BR-3-hr cells. The antiproliferative effect was mainly
from curcumin, and the combination of herceptin with
curcumin did not reveal a more antiproliferative effect than
curcumin alone.
Trypan blue exclusion assay confirmed that both her-
ceptin and curcumin inhibited the BT-474 cells dose-
dependently. The combination of herceptin and curcumin
further effectively decreased the cell viability of BT-474
cells (Figure 3(b)). The SK-BR-3-hr cells that we used here
were resistant to herceptin but sensitive to curcumin. The
combination of herceptin and curcumin effectively decreased
6 Evidence-Based Complementary and Alternative Medicine
Tab le 1: The combination index of herceptin and curcumin treatment of the growth of BT-474 cells.
Herceptin (μg/mL) Curcumin (μg/mL) Fa CI Effect
0.1 5 0.34 0.669 Synergistic
0.1 10 0.45 0.836 Synergistic
0.1 15 0.54 0.900 Synergistic
1 5 0.52 0.393 Synergistic
1 10 0.52 0.713 Synergistic
1 15 0.54 0.953 Synergistic
2 5 0.52 0.467 Synergistic
2 10 0.53 0.750 Synergistic
2 15 0.54 1.013 Additive
5 5 0.5 0.794 Synergistic
5 10 0.52 1.007 Additive
5 15 0.56 1.075 Additive
10 5 0.49 1.354 Antagonistic
10 10 0.54 1.195 Antagonistic
10 15 0.57 1.241 Antagonistic
AvalueofCI<1 represents a case where synergism of herceptin and curcumin was present. CI values of 1 and >1 represent additive and antagonistic effects,
respectively. Fa, fraction affected; CI, combination index.
the cell viability of SK-BR-3-hr cells. However, no apparent
synergistic or antagonistic effect was present in SK-BR-3-hr
cells when a combination of herceptin and curcumin was
used.
The combination effectofherceptinandcurcuminonthe
growth of BT-474 breast cancer cells was further analyzed
with Calcusyn software for calculation of the CI. The
combination of herceptin with curcumin exerted a biphasic
interaction in BT-474 cells. The CI was less than 1 and
showed a synergistic effect in the following conditions: a
lowdoseofherceptin(0.1–1μg/mL) with curcumin (5–
15 μg/mL), 2 μg/mL herceptin with curcumin (5–10 μg/mL),
or 5 μg/mL herceptin with curcumin 5 μg/mL. The CI was
larger than 1 and showed an antagonistic effect when using
a high dose of herceptin (>10 μg/mL) with curcumin (5–
15 μg/mL). The CI and interaction between herceptin and
curcumin in BT-474 cells are summarized in Ta b l e 1 .
3.3. Effect of Herceptin and/or Curcumin on HER-2-Related
Akt and MAPK Pathways in BT-474 and SK-BR-3-hr Cells.
In the HER-2-overexpressed BT-474 and SK-BR-3-hr cells,
phosphorylation of Akt and MAPK was observed (Figure 4).
Herceptin dose-dependently inhibited the phosphorylation
of Akt and MAPK in BT-474 breast cancer cells (left
panel, Figure 4(a)). HER-2 oncoprotein was not depleted by
herceptin treatment, even at high concentrations (10 μg/mL).
The addition of herceptin on SK-BR-3-hr cells did not
decrease the expression of HER-2 oncoprotein nor decrease
the phosphorylation of Akt and MAPK, even at a 10 μg/mL
concentration (right panel, Figure 4(a)). This was compati-
ble with the fact that this SK-BR-3-hr cell line was a strain
resistant to herceptin. When treated with curcumin, the
phosphorylation of Akt and MAPK was decreased, combined
with the downregulation of HER-2 oncoprotein, in a dose-
dependent manner in both the BT-474 and SK-BR-3-hr cells
(Figure 4(b)).
The level of NF-κB in BT-474 cells was decreased along
with the decreased phosphorylation of Akt and MAPK after
treatment with herceptin. The level of NF-κB was increased,
even in high concentrations of herceptin-treated SK-BR-3-
hr cells. When treated with curcumin, the level of NF-κB
was decreased in a dose-dependent manner in both BT-474
and SK-BR-3-hr cells. The combination of herceptin and
curcumin treatment resulted in a decreased level of HER-2
oncoprotein, p-Akt, p-MAPK, and NF-κB in both BT-474
and SK-BR-3-hr cells (Figure 4(c)).
3.4. Effect of Curcumin and/or Herceptin on BT-474 Breast
Cancer Cell Cycle. To further characterize the effects of
curcumin and/or herceptin on breast cancer cell growth,
analyses of cell cycle phase distribution were conducted. The
cell cycle of BT-474 cells without drug treatment showed a
G0/G1 phase of 74%, S phase of 19%, and G2/M phase of
7%. When treated with herceptin, the G0/G1 phase increased
from 74% to 79%, the S phase decreased from 19% to 11%,
and the G2/M phase increased from 7% to 10%. When
BT-474 cells were treated with curcumin, the G0/G1 phase
remained at 74%, the S phase decreased from 19% to 9%,
and G2/M increased from 7% to 20%. The combination
of 1 μg/mL of herceptin with 10 μg/mL curcumin showed
a decrease in the S phase (from 19% to 12%) without an
apparent change in the G0/G1 (from 74% to 78%) and G2/M
phases (7% to 10%). The results indicated that no significant
change in cell cycle progression was observed in BT-474 cells
after treatment with herceptin and/or curcumin.
In SK-BR-3-hr cells, the cell cycle was not changed
after treatment with herceptin, which is compatible with
Evidence-Based Complementary and Alternative Medicine 7
0 0.1 1 2 5 10
1 0.9 0.9 0.9 0.9 0.9
1 0.5 0.5 0.9 0.9 0.3
1 1 1 0.9 0.5 0.1
1 1 0.5 0.5 0.25 0.25
Herceptin (μg/mL)
HER-2
p-Akt
Total Akt
p-MAPK
Total MAPK
NF-κB
β-actin
BT-474 SK-BR-3-hr
0 0.1 1 2 5 10
1 0.9 0.9 0.9 0.9 0.9
1 0.5 0.5 0.9 0.9 1.3
1 1 1 1.5 1.5 1.5
1 1 1 1.5 2 2.5
(a)
0 1 2.5 5 10 25 0 1 2.5 5 10 25
11110.10
1 1 1 0.5 0.3 0.3
111110.5
1 1.5 1 1 0.5 0.1
11110.50.1
1 1 1 0.5 0.3 0.1
1 1 1 0.5 0.5 0.5
1 1.5 1 1 1 0.1
Curcumin(μg/mL)
HER-2
p-Akt
Total Akt
p-MAPK
Total MAPK
NF-κB
β-actin
BT-474 SK-BR-3-hr
(b)
10 μg/mL herceptin
10 μg/mL curcumin
1 0.8 0.8 0.5
1 0.1 0.2 0.1
1 0.5 0.9 0.5
1 0.01 0.1 0.01
1 0.9 0.9 0.5
1 0.5 0.9 0.5
1 1.5 2 1
1 0.9 0.5 0.5
−+−+−+−+
−−++−−++
HER-2
p-Akt
Total Akt
p-MAPK
Total MAPK
NF-κB
β-actin
BT-474 SK-BR-3-hr
(c)
Figure 4: The level of HER-2, phosphorylated Akt, MAPK, and NF-κB after treatment with herceptin and/or curcumin in HER-2-
overexpressed breast cancer cells. The dosage effects of herceptin (a), curcumin (b), and the combination of herceptin and curcumin (c)
on the BT-474 and SK-BR-3-hr cells, respectively. The number at the bottom of each lane indicates the relative fold change of control.
the resistance of this cell line. When treated with curcumin
for 48 h, the cell cycle of SK-BR-3-hr showed a decrease in
the G1 phase (65% to 37%) and an increase in the S phase
(28% to 37%) and G2/M phase (6% to 25%). Compared
with the control, the combination of 1 μg/mL herceptin and
10 μg/mL curcumin showed a decrease in the G1 (65% to
53%), no apparent change in the S phase (28% to 27%), and
an increase in the G2/M phase (6% to 19%).
3.5. Effect of Curcumin and/or Herceptin on the Mobility of
Different Breast Cancer Cells. The inhibitory effect of her-
ceptin and/or curcumin on different breast cancer cell lines
was tested by wound-healing assay. Curcumin showed an
apparent antimobility effect as illustrated in the MCF-7,
MDA-MB-231, and SK-BR-3-hr cells (Figure 5). No effect
of herceptin and/or curcumin on the mobility of BT-474
cells was observed due to the lack of migration of these
cells. In the SK-BR-3-hr cells, cell migration persisted despite
treatment with herceptin, which is consistent with herceptin
resistance. When treated with curcumin, cell migration was
greatly inhibited as compared with the control, revealing that
curcumin had an apparent antimobility effect on SK-BR-3-
hr cells. The combination of herceptin and curcumin did
not have a better antimobility effect than curcumin alone.
In the wound healing assay, curcumin showed an apparent
antimobility effect in MCF-7, SK-BR-3-hr, and MBA-MB-
231 breast cancer cells.
3.6. Curcumin and/or Herceptin Inhibited Tumor Growth
in the Xenograft Animal Model. Compared with the con-
trol group, the mice treated with curcumin, herceptin,
or combined herceptin and curcumin all had a smaller
mean xenograft tumor volume after 4 weeks of treatment
(control group 273.6 ±190.1 mm3, curcumin group 63.6 ±
25.7 mm3, herceptin group 36.3 ±7.8 mm3, and combined
herceptin and curcumin group 34.1 ±25.0 mm3,P=0.001)
8 Evidence-Based Complementary and Alternative Medicine
0h
24 h
0h
24 h
0h
24 h
MCF-7
MDA-MB-231SK-BR-3-hr
−100 102030405060708090100
Mobility (μm)
Control
10 μg/mL herceptin
10 μg/mL curcumin
10 μg/mL herceptin +10 μg/mL curcumin
∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗
∗∗∗
∗∗∗
∗
10 μg/mL herceptin
10 μg/mL curcumin
20
9
9
19
8
8
33
72
39
38
97
38.5
−+−+
−− ++
Figure 5: Changes in mobility of MCF-7, MDA-MB-231, and SK-BR-3-hr cells after treatment with herceptin and/or curcumin. The
antimobility effect of herceptin and/or curcumin on breast cancer cells were tested by a wound-healing assay. The anti-mobility effect was
illustrated in the MCF-7, MDA-MB-231 and SK-BR-3-hr cells, respectively. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001.
(Figure 6(a)). The curcumin alone group had a larger mean
xenograft tumor size than the herceptin-alone group (63.6 ±
25.7 mm3versus 36.3 ±7.8 mm3,P=0.003). The combined
herceptin and curcumin group showed a smaller mean tu-
mor volume than the curcumin-alone group (34.1 ±25.0
versus 63.6 ±25.7 mm3,P=0.079) or herceptin-alone
group (34.1 ±25.0 versus 36.3 ±7.8 mm3,P=0.324),
although without statistical significance.
The effect of combining curcumin and/or herceptin
with taxol was tested by comparing the effects of taxol,
taxol + herceptin, taxol + curcumin, and taxol + herceptin
+ curcumin regimens in the BT-474 xenograft model. In
another set of experiments, 24 mice inoculated with 1 ×
107BT-474 cells were randomized into 4 groups and treated
with taxol, taxol + herceptin, taxol + curcumin, or combined
taxol + herceptin + curcumin. After 4 weeks of treatment, the
mean xenograft tumor volumes were taxol 58.3 ±11.2 mm3,
taxol+herceptin35.0±13.4 mm3, taxol + curcumin 44.5
±6.2 mm3, and combined taxol + herceptin + curcumin
group 31.3 ±27.7 mm3.Theantitumoreffect of taxol +
herceptin was apparent, and combined taxol and curcumin
had a comparable antitumor effect (44.5 ±6.2 versus
Evidence-Based Complementary and Alternative Medicine 9
0
50
100
150
200
250
300
01234
Time after treatment (week)
Tumor volume (mm)
Control Herceptin + curcumin
Herceptin Curcumin
(a)
01234
Time after treatment (week)
0
20
40
60
80
100
Tumor volume (mm)
Taxo l Taxol+herceptin
Taxol + curcumin Taxol + herceptin + curcumin
(b)
0
3
6
9
12
15
18
21
24
Weight (g)
01234
Time after treatment (week)
Control Herceptin + curcumin
Herceptin Curcumin
(c)
Figure 6: In vivo effects of curcumin on the herceptin and/or taxol-treated HER-2-overexpressed breast cancer xenografts. The HER-2-
overexpressed BT-474 cells were injected in 4–6-week-old, female, athymic, nude mice subcutaneously at 1 ×107cells/tumor in the right
flank region to form xenografts. Six mice per group were treated with different protocols with tumor volume monitored biweekly for
consecutive 4 weeks. (a) The mean xenograft tumor volume change of control (0.1% DMSO), herceptin, curcumin, and combined curcumin
and herceptin. (b) The mean xenograft tumor volume of taxol, taxol + herceptin, taxol + curcumin, and combined taxol + herceptin +
curcumin. (c) The body weight change of these mice treated with herceptin and/or curcumin. DMSO, dimethyl sulfoxide.
35.0 ±13.4 mm3,P=0.884). The combination of taxol,
herceptin, and curcumin resulted in the smallest tumor
volume, but this was not statistically different from that of
the taxol and herceptin regimen (31.3 ±27.7 versus 35.0
±13.4 mm3,P=0.079) (Figure 6(b)). The body weight of
these mice treated with herceptin and/or curcumin was quite
stable during the 4-week period (Figure 6(c)).
4. Discussion
This study was designed to test the efficacy of curcumin
in HER-2-overexpressed breast cancer, with a direct com-
parison with herceptin in the in vitro cell line and in vivo
xenograft animal model. Our preclinical result revealed that
curcumin reduced the cell viability of different breast cancer
cell lines, including MCF-7 (ER-positive, HER-2-negative),
MDA-MB-231 (ER-negative, HER-2-negative), HER-2-over-
expressed BT-474 (ER-positive, HER-2-positive), and her-
ceptin-resistant SK-BR-3-hr (ER-negative, HER-2-positive)
cells. The level of HER-2 oncoprotein, p-Akt, p-MAPK,
and NF-κB were decreased in a dose- and time-dependant
manner in BT-474 and SK-BR-3-hr cells when treated with
curcumin. The cell cycle perturbation by curcumin was
mainly found in the increase in the G2/M phase. The
apparent antimobility effect of curcumin was also revealed
in the MCF-7, MDA-MB-231, and SK-BR-3-hr cells. The
combinational effect of herceptin with curcumin was a
biphasic interaction on the growth of BT-474 cells. When
a low dose of herceptin was used with curcumin, there was
a synergistic effect, but an antagonistic effect was observed
when a high dose of herceptin was used. In the BT-474
xenograft model, curcumin treatment effectively decreased
10 Evidence-Based Complementary and Alternative Medicine
the tumor size, and the combination of taxol with curcumin
had an antitumor effect comparable with that of taxol and
herceptin treatment.
The action of herceptin in inhibiting ErbB2 (HER-
2) signaling involves the reduced phosphorylation of Akt
but not endocytic downregulation of ErbB2 [42]. HER-2
oncoprotein was not depleted by herceptin, even at a high
concentration (Figure 4). The exact mechanism of herceptin
resistance is not clear, and possible mechanisms include
obstacles to herceptin-binding to HER-2, upregulation of
HER-2 downstream signaling pathways, signaling through
alternative pathways, or failure to trigger immune-mediated
mechanisms to destroy tumor cells [14]. In the herceptin-
resistant SK-BR-3-hr cells, the persistent activation of
PI3K/Akt and MAPK pathways, despite treatment with
herceptin, may have been acquired through repeated cultures
of herceptin-treated SK-BR-3 cells.
NF-κB activation played an important role in chemother-
apy resistance [29,30], and targeting NF-κB showed im-
proved benefits in some preclinical HER-2-overexpressed
cancer cells [28,43]. In HER-2-overexpressed breast cancer,
ErbB2 activates NF-κB via signaling that includes PI3K,
PDK1, Akt, protein kinase 2 (CK2), and CKBBP1 [44]. In
herceptin-sensitive HER-2-overexpressed BT-474 cell lines,
herceptin treatment effectively decreased the phosphoryla-
tion of Akt, MAPK and the expression of NF-κB(Figure 4).
Whether the resistance to herceptin was also related to NF-
κB activation is not clear. In the herceptin-resistant SK-BR-
3-hr cells, the expression level of NF-κB increased in parallel
with the incremental dose of herceptin. When treated with
curcumin, the expression of NF-κBwasdecreased,accom-
panied with increased cell death. The ability of curcumin to
inhibit herceptin-resistant SK-BR-3 cells may be related to
the downregulation of HER-2 oncoprotein, and suppression
of related Akt, MAPK, and NF-κB signaling pathways. The
ability of curcumin to downregulate EGFR and HER-2
oncoproteins and inhibit the phosphorylation of Akt and
MAPK and NF-κB activation suggests that curcumin has
potential in the treatment of HER-2-overexpressed and/or
herceptin-resistant breast cancer.
The interaction of curcumin and herceptin in HER-2-
overexpressed cancer has rarely been reported. Whether the
combination of herceptin with curcumin had any therapeu-
tic advantage over herceptin alone is unknown. In the in
vitro BT-474 cell line study, the combination of herceptin and
curcumin showed an advantage over either treatments alone
(Figure 3). The combination of herceptin and curcumin
exerted a biphasic interaction on the growth of BT-474 cells.
A synergistic effect was present when a low dose of herceptin
(0.1–1 μg/mL) was combined with curcumin, while a high
dose (>10 μg/mL) of herceptin would exert an antagonistic
effect when combined with curcumin (Tabl e 1 ). In the SK-
BR-3-hr cells, the combination of herceptin with curcumin
exerted neither a synergistic nor antagonistic effect. This
biphasic interaction observed in vitro warrants caution when
herceptin is to be used with curcumin or other medications.
In the xenograft animal study, curcumin treatment effec-
tively reduced the tumor volume by 76.7%, compared with
the control; however, it was not as effective as of herceptin,
which achieved an 86.7% tumor reduction (Figure 6(a)). The
combination of herceptin and curcumin showed a greater
antitumor effect than curcumin alone (87.5% versus 76.7%
in tumor regression) but a similar effecttothatofherceptin
(87.5% versus 86.7%). The anticipated synergistic effect of
combining herceptin with curcumin was not observed in
our in vivo xenograft animal model. Although curcumin
might not interfere with herceptin in the normal physiologic
concentration, from our in vivo study, combined herceptin
and curcumin was not better than herceptin alone. More
solid evidence might be needed to support the rationale of
combing herceptin with curcumin in the treatment of HER-
2-overexpressed breast cancer.
It has been reported that curcumin could suppress taxol-
induced NF-κB, and curcumin combined with taxol showed
greater antitumor effects than taxol alone [31,33]. In our
in vivo xenograft study, the combination of curcumin and
taxol had therapeutic effects comparable with taxol and
herceptin, one of the current preferred regimens for HER-2-
overexpressed breast cancer (Figure 6(b)). The combination
of taxol + herceptin + curcumin was associated with the
smallest mean tumor volume although this was not statis-
tically different from that of the taxol and herceptin regimen.
Curcumin was rated safe and well-tolerated [45]; how-
ever, the application of curcumin might be limited by its
low bioavailability [16,17,45,46]. To reduce the impact
of the low bioavailability of oral intake, we used an intra-
peritoneal injection to treat xenograft nude mice. In our
study, the body weight of herceptin and curcumin-treated
mice did not vary greatly during the entire treatment
period (Figure 6(c)), demonstrating the relative safety and
tolerability of curcumin as previously reported in animal
and human studies [16,47,48]. The main limitation of
our in vivo study was that we had only one fixed dose
protocol for herceptin and/or curcumin treatment, and the
serum concentration of the drugs was unknown. The dose
of herceptin used here was in accordance with the current
practice guideline [26], while the dose of curcumin was
derived from a previous report [49]. The optimal dose of
curcumin for HER-2-overexpressed breast cancer is unclear
and needs to be determined for maximum therapeutic effect.
In this study, we showed that curcumin could reduce
the cell viability of both HER-2-overexpressed herceptin-
sensitive BT-474 cells and herceptin-resistant SK-BR-3-hr
breast cancer cells. In the BT-474 xenograft model, though
not as much as herceptin, curcumin did effectively decrease
the tumor size. The combination of curcumin with her-
ceptin was not better than herceptin alone; however, the
combination of taxol and curcumin had an antitumor effect
comparable with taxol and herceptin. The results, both in
vitro and in vivo, suggested that curcumin has the treatment
potential for HER-2-overexpressed breast cancer.
Conflict of Interests
The authors declare that they have no conflict of interests.
Evidence-Based Complementary and Alternative Medicine 11
Acknowledgment
C.-W. Chi and D.-R. Chen equally contributed to the paper.
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