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July 2015⎪Vol. 25⎪No. 7
J. Microbiol. Biotechnol. (2015), 25(7), 1036–1046http://dx.doi.org/10.4014/jmb.1501.01017 Research Article jmbReview
Combined Treatment of Herbal Mixture Extract H9 with Trastuzumab
Enhances Anti-tumor Growth Effect
Sunyi Lee1, Sora Han1, Ae Lee Jeong1, Jeong Su Park1, Seung Hyun Jung2, Kang-Duk Choi3, and Young Yang1*
1Department of Life Systems, Sookmyung Women’s University, Seoul 140-742, Republic of Korea2College of Oriental Medicine, Dongguk University, Seoul 410-773, Republic of Korea3Graduate School of Bio & Information Technology, Hankyong National University, Ansung 456-749, Republic of Korea
Introduction
Breast cancer, which is the most frequent type of cancer
affecting women in the world, is characterized by multiple
molecular phenotypes [6, 7, 10, 14, 16]. Because breast
cancer patients within clinically and morphologically similar
classes show varied response to therapy, the traditional
prognostic factors currently available are insufficient for
patient classification. Thus, the detailed molecular classification
of breast tumors is being elucidated to explain the genetic
heterogeneity of breast tumors, and to develop appropriate
therapeutic strategies. In general, the following four
distinct molecular breast cancer groups were identified
based on gene expression profiles [28]: luminal epithelial/
estrogen receptor (ER) positive, c-erb-B2 (HER2) positive,
basal-like, and normal breast-like. A subsequent study
divided the luminal/ER-positive group into three subtypes:
luminal A, B, and C [33], but existence of the luminal C
group remains uncertain [34].
The combination adjuvant chemotherapy of doxorubicin
and cyclophosphamide (AC) is considered to be the most
effective combination chemotherapy for breast cancer
treatment [22]. These conventional chemotherapeutic drugs
are used without molecular classification of the patients
and often lead to adverse side effects by causing unnecessary
damage to normal and healthy cells. Thus, more selective
therapeutic strategies are under development. Selective
estrogen receptor modulators, including tamoxifen and
raloxifen, are used to treat ER-positive patients, whereas
the HER2-targeted monoclonal antibody drug, trastuzumab,
Received: January 9, 2015
Revised: February 23, 2015
Accepted: March 13, 2015
First published online
March 20, 2015
*Corresponding author
Phone: +82-2-710-9590;
Fax: +82-2-2077-7322;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2015 by
The Korean Society for Microbiology
and Biotechnology
Extracts from Asian medicinal herbs are known to be successful therapeutic agents against
cancer. In this study, the effects of three types of herbal extracts on anti-tumor growth were
examined. Among the three types of herbal extracts, H9 showed stronger anti-tumor growth
effects than H5 and H11 in vivo. To find the molecular mechanism by which H9 inhibited the
proliferation of breast cancer cell lines, the levels of apoptotic markers were examined. Pro-
apoptotic markers, including cleaved PARP and cleaved caspases 3 and 9, were increased,
whereas the anti-apoptotic marker Bcl-2 was decreased by H9 treatment. Next, the combined
effect of H9 with the chemotherapeutic drugs doxorubicin/cyclophosphamide (AC) on tumor
growth was examined using 4T1-tumor-bearing mice. The combined treatment of H9 with AC
did not show additive or synergetic anti-tumor growth effects. However, when tumor-bearing
mice were co-treated with H9 and the targeted anti-tumor drug trastuzumab, a delay in tumor
growth was observed. The combined treatment of H9 and trastuzumab caused an increase of
natural killer (NK) cells and a decrease of myeloid-derived suppressor cells (MDSC). Taken
together, H9 induces the apoptotic death of tumor cells while increasing anti-tumor immune
activity through the enhancement of NK activity and diminishment of MDSC.
Keywords: Apoptosis, breast cancer, doxorubicin/cyclophosphamide, medicinal herbal extracts,
trastuzumab
1037 Lee et al.
J. Microbiol. Biotechnol.
is used on HER2-positive patients [27]. On the other hand,
to overcome the adverse effects of chemotherapy, extracts
from medicinal herbs may also be used to give additive or
synergistic preventive effects for the inhibition of tumor
growth [3]. The combined treatment of chemotherapeutic
drugs with herbal extracts could lower the concentration
of chemotherapeutic drug required to show the same
anticancer effects [26].
Herbal extracts exhibit a variety of activities, including
anti-allergic, antimicrobial, anti-oxidant, and anticancer
effects, due to the presence of various components [2], and
usually exert cytotoxic effects on tumor cells directly, or
through enhancement of the immune response. For this
reason, the anti-tumor activity of various herbal extracts is
currently being studied [5, 32]. In the present study, the
most effective herbal extracts were screened, and their
combined effects with chemotherapeutic drugs and a
targeted drug were examined. To determine the molecular
mechanism underlying the anti-tumor growth effects, the
immune cell population was also analyzed.
Materials and Methods
Cell Cultures
The human breast cancer cell lines MDA-MB-231 and MCF-7, as
well as the murine breast cancer cell line 4T1, were obtained from
the American Type Culture Collection (USA). The human fibroblast
cell line HFCH8 was a kind gift from Dr. Myeong-Sok Lee
(Sookmyung Women’s University, Korea). MDA-MB-231, MCF-7,
and HFCH8 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (HyClone Laboratories, USA) at 37°C in a
humidified 5% CO2 incubator as described previously [19]. The
4T1 cells were maintained in RPMI 1640 supplemented with 10%
heat-inactivated FBS.
Oriental Medicinal Herbs and Formulation of H5, H9, and H11
The medicinal herb ingredients included in H5, H9, and H11
are listed in Table 1. The herbal ingredients were obtained from
the Oriental Medical Hospital, Dongguk University (Korea), and
were kindly authenticated by Dr. Seong Hyun Jung (College of
Oriental Medicine, Dongguk University). Ethanol extracts from
the listed plants were prepared as follows. The dried and
pulverized medicinal herbs were mixed together, and the
indicated amounts of each herb were mixed, soaked in 10× (w/v)
of 40% ethanol, and then extracted for 3 h at 95°C. The ethanol
extracts were concentrated using a rotary evaporator, lyophilized,
and then reconstituted in distilled water for the in vitro studies.
Cell Viability Assay
Cell proliferation was measured by direct cell counting. Briefly,
MDA-MB-231, MCF7, HFCH8, and 4T1 cells were collected and
seeded at a density of 5 × 104 cells/well in 24-well plates, and then
treated with various concentrations of H5, H9, and H11. After
incubation for 48 h, cells were trypsinized, and the viable cells
were counted using a hemacytometer after trypan blue staining to
exclude dead cells, as described previously [20].
Animals
All animals were obtained from Harlan Laboratories (USA).
Six-week-old Balb/c female mice were subdivided, acclimatized
for one week, and divided randomly into experimental groups.
Bedding was changed once a week, and the temperature and
humidity were controlled. Mice were housed under 12 h light/
12 h dark conditions and allowed free access to food and water.
The plans and protocols for animal experiments were approved
by the Institutional Animal Care and Use Committee of Sookmyung
Women’s University, Seoul, Korea.
Syngeneic 4T1 Tumor-Bearing Mice Model
4T1 cells at a concentration of 2 × 105 per 0.1 ml of PBS were
injected subcutaneously into mice, and tumor growth was monitored
by measuring the tumor volumes with calipers [Volume = (length
Table 1. The compositions of H5, H9, and H11.
H5 (%) H9 (%) H11 (%)
Cyperus rotundus L. (22.2) Ginseng Radix (20) Ginseng Radix (9)
Evodiae Fructus (22.2) Cinnamomi Cortex (12) Cinnamomi Cortex (9)
Psoraleae Semen (22.2) Evodiae Fructus (8) Evodiae Fructus (9)
Sparganii Rhizoma (22.2) Foeniculi Fructus (12) Foeniculi Fructus (9)
Curcumae Radix (11.1) Psoraleae Semen (12) Psoraleae Semen (9)
Myristicae Semen (12) Citri Unshius Pericarpium (9)
Alpiniae Officinari Rhizoma (8) Zingiberis Rhizoma (9)
Sparganii Rhizoma (4) Atractylodis Rhizoma Alba (9)
Curcumae Radix (12) Paeoniae Radix (9)
Glycyrrhizae Radix et Rhizoma (9)
Aconiti Lateralis Radix Preparata (9-18)
Synergistic Effect of H9 Combined with Trastuzumab 1038
July 2015⎪Vol. 25⎪No. 7
× width2)]. H9 and H11 were administered daily at a concentration
of 400 mg/kg by oral injection. For the vehicle treatment group,
the same volume of distilled water was administered. Trastuzumab
was administered every third day at a concentration of 1 mg/kg
by intraperitoneal injection, and IgG (Sigma, USA) was given as
an experimental control. For AC treatment, adriamycin (Sigma,
USA) and cyclophosphamide (Sigma, USA) were mixed and
administered intraperitoneally once, 7 days after tumor injection.
PBS was given as a vehicle treatment.
Flow Cytometry Analysis
Splenocytes were separated from cell debris using a 70 µm
nylon cell strainer (BD Bioscience, USA) after removal of red
blood cells (RBC) using RBC lysis buffer (Sigma, USA). The
prepared splenocytes were stained in phosphate-buffered saline
with 1% FBS for 30 min at 4°C with the following fluorescein
isothiocyanate-, phycoerythrin-, PECy7-, or allophycocyanin-
conjugated anti-mouse antibodies: anti-CD3e, -DX5, -CD8, -CD4,
-CD11b, and -Gr1. All antibodies were purchased from eBioscience
(USA). The labeled cells were analyzed on a FACSCanto II
cytometer equipped with FACSDiva software (BD Bioscience,
USA), and data were analyzed using FlowJo software (USA).
NK Cell Purification and Cytotoxicity Assay
NK cells were isolated from freshly obtained mouse splenocytes
using an NK cell isolation kit (Miltenyi Biotec, Germany),
according to the manufacturer’s protocol. For NK cell cytotoxicity
assay, viable NK cells were counted with the trypan blue dye
exclusion method and used as effectors in the cell-mediated
cytotoxicity assay. YAC-1 target cells were incubated with
carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen,
USA) at 37°C for 10 min. After the washing step, CFSE-labeled
YAC-1 cells were used as target cells, and the NK cells were
distributed in triplicate at the effector-target cell ratios from 5:1 to
1:1. The cells were incubated for 3 h at 37°C in 5% CO2. At the end
of the incubation, dead target cells were labeled with fluorescence
labeled caspase-3 (BD Bioscience, USA), and the percentage of
YAC-1 cell death was analyzed on a FACSCanto II cytometer
equipped with FACSDiva software. Data were analyzed with
FlowJo software, as described previously [9].
Immunoblot Analysis
The 4T1, HFCH8, and MCF7 cells were lysed with protein
lysis buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 5 mM
ethylenediaminetetraacetic acid, 1% NP-40, and 1 mM
phenylmethylsulfonyl fluoride). Proteins were resolved by sodium
dodecyl sulfate polyacrylamide gel electrophoresis and transferred
to nitrocellulose (Amersham Pharmacia Biotech, USA). Blocking
and antibody incubations were then performed in TBST (150 mM
NaCl, 20 mM Tris (pH 8.0), and 0.05% Tween 20) containing 5%
low-fat milk. Blots were developed by ECL (Amersham Pharmacia
Biotech, USA), as described previously [20].
Statistical analysis
The paired Student’s t-test and one-way factorial ANOVA were
employed for statistical analysis, along with the Tukey and Scheffe
tests. Values of p < 0.05 were considered as significant differences.
Data were presented as the mean ± standard deviation (SD).
Results
Selection of Effective Herbal Extracts with Anti-Breast
Cancer Activity
Osuyubujaijungy-tang described in Donguisusebowon was
the typical prescription used to cure Shao Yin (Lesser Yin)
disease. The hepatoprotective effects of Osuyubujaijung-tang
were demonstrated in a chronic liver cirrhosis model [36],
and it was also shown to improve the symptoms of
peripheral T-cell lymphoma patients, including fever,
myalgia, poor performance status, neck pain, and headache
[13]. The extracts of each herb in Osuyubujaijung-tang also
have anti-tumor effects. Crude extract of Cinnamomi
cortex showed anti-tumor activity against Sarcoma 180 in
mice, and suppressed adjuvant-induced arthritis in rats
[30]. Crude extract of Evodiae Fructus is known to inhibit
the proliferation of several tumor cell lines and to induce
tumor cell death through the caspase pathway [8]. Crude
extract of Alpinia officinarum rhizomes is known to be a
viable therapeutic or preventive candidate for the treatment
of acute and chronic arthritis patients [18]. In addition, crude
extract of Curcumae Radix is a potential chemopreventive
agent for gastric cancer, because it suppresses the expression
of VEGF, COX-2, and PCNA in the gastric mucosa of
rats [21]. These facts stimulated us to examine whether
Osuyubujaijung-tang has anti-tumor growth effects.
Ethanol extraction was used instead of water to extract
the many active ingredients from Osuyubujaijung-tang. The
ethanol extract of Osuyubujaijung-tang was named H11. To
make a new extract with the highest anticancer efficacy
against breast cancer, the additional herbal extracts H5 and
H9 were designed. Each component of H5 and H9 was
selected based on its ability to induce apoptosis and immune
activation from H11 (Table 1).
H5, H9, and H11 Inhibit the Proliferation of Breast Cancer
Cells
To evaluate the effects of the herbal extracts on the
proliferation of breast cancer cells, breast cancer cell lines,
including human MDA-MB-231 and MCF-7 cells, as well as
mouse 4T1 cells, were treated with H5, H9, and H11 at the
indicated concentrations for 48 h. The viable cells were
then counted using a hemocytometer after trypan blue
1039 Lee et al.
J. Microbiol. Biotechnol.
staining to exclude dead cells. All three extracts were
observed to inhibit the proliferation of breast cancer cell
lines. Interestingly, H5 demonstrated the strongest inhibition
of proliferation of all three cell lines at the concentration of
0.5 mg/ml, and H9 also showed stronger inhibition than
H11 (Figs. 1A-1C). It is well known that herbal extracts
that demonstrate anti-proliferative ability in vitro sometimes
fail to suppress tumor growth in vivo. Thus, the ability of
the three extracts to suppress tumor growth was next
examined in vivo. The mouse breast cancer cell line 4T1 was
subcutaneously injected into the mammary pad of female
Balb/c mice. Daily oral administration of the herbal extracts
was then carried out from 3 days after 4T1 injection.
Although H5 showed the highest effectiveness for inhibition
in vitro, the anti-tumor growth effects of both H9 and H11
were superior to H5 in vivo (Fig. 1D). Thus, the remaining
investigation focused on H9.
H9 Treatment Induces Apoptosis in Breast Cancer Cells
To determine the mechanism underlying the anti-tumor
growth effect of H9, the morphological changes of 4T1 cells
were photographed after the H9 treatment for 24 and 48 h
(Fig. 2A). The untreated cells appeared to be intact, whereas
cell shrinkage resembling an apoptotic phenotype was
observed after H9 treatment. Thus, to determine whether
the cell death was related to apoptosis, the levels of
expression of apoptotic markers were examined. The
apoptotic markers cleaved poly-ADP ribose polymerase
Fig. 1. Effects of H5, H9, and H11 on the proliferation of breast cancer cells in vitro and in vivo.
(A-C) MDA-MB-231, 4T1, and MCF7 cells were treated with the indicated concentrations of H5, H9, and H11 for 48 h, and then viable cells were
measured by direct counting. Data are expressed as the mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, compared with control at 48 h. #p <
0.05, ##p < 0.02, ###p < 0.002, compared with control at 0 h. (D) Mice were subcutaneously injected with 2 × 105 4T1 cells, and 1,000 mg/kg of each
extract was orally administered daily. Tumor volume was measured on the indicated day. One-way factorial ANOVA was used for the
comparison of multiple groups (n = 7). *, p < 0.05 for vehicle (Veh) versus H9 treated mice. #, p < 0.05 for Veh versus H11 treated mice.
Synergistic Effect of H9 Combined with Trastuzumab 1040
July 2015⎪Vol. 25⎪No. 7
(PARP) and caspases, as well as the anti-apoptotic marker
Bcl-2, were examined after treatment with H9 at the
indicated concentration for 48 h. Cleavage of PARP, caspase
3, and caspase 9 was observed after 48 h of treatment with
150 µg/ml H9, whereas decrease of the anti-apoptotic
protein Bcl-2 was observed (Fig. 2B). This finding implies
that H9 directly induces apoptosis in 4T1 cells. To know
whether the H9-induced apoptotic effect is specific to
tumor cells, HFCH8 cells (a normal human fibroblast cell
line) were treated with H9 for 48 h and no apoptotic cells
were observed, unlike breast cancer cells. Moreover,
apoptotic and anti-apoptotic proteins were not altered
(Figs. 2C-2D). These results indicate that the apoptotic
effect of H9 is more selective to breast cancer cells than
normal fibroblast cells.
H9 and H11 Do Not Show Combined Effects with
Chemotherapeutic Drugs
Herbal extracts are generally used as an adjuvant
therapy with anticancer drugs to relieve the pain or to
enhance the cytotoxicity of the anticancer drugs and fight
tumor immunity. To examine the combined effects of H9
and H11 with chemotherapeutic drugs, adriamycin and
cyclophosphamide (AC) were chosen for analysis, because
these two drugs are commonly used as an anticancer drug
cocktail for the treatment of breast cancer patients. The 4T1
cells were injected into the mammary pad of female Balb/c
mice and AC was administered intraperitoneally once,
7 days after 4T1 injection. Oral administration of H9 and
H11 was carried out daily after an additional 7 days.
Tumor growth was measured every other day, and the data
Fig. 2. H9-induced apoptosis of breast cancer cells.
(A) 4T1 cells were treated with the indicated concentrations of H9 for 24 and 48 h. Representative photographic images are shown. (B) 4T1 cells
were treated with the indicated concentrations of H9 for 48 h, after which cell lysates were prepared. The levels of PARP, caspase 3, caspase 9, and
Bcl-2 were examined using immunoblot assay. (C) HFCH8 cells were treated with the indicated concentrations of H9 for 24 and 48 h.
Representative photographic images are shown. (D) HFCH8 cells were treated with the indicated concentration of H9 for 48 h and then the levels
of PARP and Bcl-2 were examined using immunoblot assay.
1041 Lee et al.
J. Microbiol. Biotechnol.
were used for generation of a graph (Fig. 3A). On the 18th
day after treatment with herbal extracts, the mice were
sacrificed and the tumor masses were weighed and
photographed (Figs. 3B-3C). The combined treatment of
AC with herbal extract did not show enhanced anti-tumor
effects, although separate treatment with H9 and H11 also
showed anti-tumor growth effects, respectively. However,
it cannot be ruled out that the effect of AC was too strong
to show the combined effect with H9 and H11.
Combined Treatment of H9 and Trastuzumab Shows the
Most Remarkable Anti-Tumor Growth Effect
Because the combined treatment of AC with H9 and H11
did not show enhancement of the anti-tumor growth effects,
examination was carried out to determine whether H9 and
H11 could show additive anti-tumor growth effect with
trastuzumab, a HER2-targeting monoclonal antibody. The 4T1
cells were injected into the mammary pad of female Balb/c
mice and trastuzumab was administered intraperitoneally
every third day from 7 days after 4T1 injection. After an
additional 7 days, H9 and H11 were orally administered
daily. Tumor growth was measured every other day and
data were used for the generation of a graph. Trastuzumab
treatment significantly suppressed tumor growth, while
the combined treatment of trastuzumab and H9 showed
the most effective suppression. The tumor weight was also
greatly reduced in the trastuzumab and H9 combination
treatment, without body weight change (Figs. 4A-4C). In
general, tumor-bearing mice showed increased spleen
weight because of immune activation. The H9 treatment
reduced the spleen weight in 4T1 tumor-bearing mice
(Fig. 4D), indicating the possibility that H9 could regulate
immune cell function.
Combined Treatment of H9 and Trastuzumab Shows
Increased Natural Killer (NK) Cell Population
To determine the mechanism underlying the anti-tumor
growth effects of the combined therapy of H9 and
Fig. 3. Combined effects of H9 and H11 with AC on 4T1 tumor growth.
(A) Mice were subcutaneously injected with 2 × 105 4T1 cells. AC was intraperitoneally administered 7 days after tumor injection, and each extract
was orally administered daily 7 days after the AC treatment. Tumor size (B) and volume (C) were measured at 35 days post-4T1 injection. One-
way factorial ANOVA was used for the comparison of multiple groups (n = 5). *, p < 0.05 for the indicated groups.
Synergistic Effect of H9 Combined with Trastuzumab 1042
July 2015⎪Vol. 25⎪No. 7
trastuzumab, the immune cells with anti-tumor function
were analyzed. The 4T1 tumor-bearing mice were subjected
to H9 and trastuzumab treatment, as in Fig. 4A, and then
the immune cells were analyzed using harvested spleens
after sacrifice of the mice. The population of NK cells was
increased from 3% at basal level to about 6% by the H9 and
trastuzumab combination treatment (Figs. 5A-5B). To
determine whether the increased NK cells had enhanced
NK cytotoxicity, the NK cytotoxicity was examined. The
NK cells isolated from spleen and Yac-1 target cells were
mixed together at the indicated ratio and incubated for 3 h
to allow the NK cells to kill the Yac-1 target cells. The
combined treatment of H9 and trastuzumab indeed
increased the cytotoxicity of the NK cells (Fig. 5C).
In addition to NK cells, the CD8-positive T-cell population
was also examined. This population was not affected by the
combined treatment of H9 and trastuzumab (Figs. 5D-5E).
However, analysis of the MDSC population demonstrated
great reduction by the combined treatment of H9 and
trastuzumab (Figs. 5F-5G). The results from the immune
cell profile analysis indicate that the combined treatment of
H9 and trastuzumab decreased the immune-suppressing
MDSC while increasing tumor-killing NK cells.
Discussion
Traditional medicinal herb extracts usually exert their
cytotoxic effects on tumor cells directly, or by enhancement
of the immune response. Herbal extracts can also lower the
cytotoxic effects of conventional chemotherapy drugs. In
Fig. 4. Combined effects of H9 and trastuzumab on 4T1 tumor growth.
(A) Mice were subcutaneously injected with 2 × 105 4T1 cells. Trastuzumab (1 mg/kg) was intraperitoneally administered every third day starting
from 7 days post-4T1 injection. H9 (400 mg/kg) was orally administered daily starting 7 days after trastuzumab treatment began. (B) Tumor
weight (left) and size (right) were measured 30 days post-4T1 injection. Body weight (C) and spleen weight (D) were also measured 30 days post-
4T1 injection. One-way factorial ANOVA was used for the comparison of multiple groups (n = 6). *, p < 0.05; **, p < 0.01 for IgG+Vehicle (Veh)
versus IgG+H9 treated mice. #, p < 0.05 for Her+Veh versus Her+H9 treated mice. Veh was treated as the control for H9, and IgG was treated as
the control for Her. Her indicates trastuzumab.
1043 Lee et al.
J. Microbiol. Biotechnol.
Fig. 5. Combined effects of H9 and trastuzumab on anti-tumor immune cells, including NK, CTL, and MDS cells.
(A, B) NK cell population (CD3-/DX5+) among splenocytes was analyzed by fluorescence-activated cell sorting analysis and shown as a dot blot
(A) and bar graph (B). (C) Ex vivo NK cell cytotoxicity assay was performed. (D, E) The CD8+ T-cell population (CD3+/CD8+) among splenic
lymphocytes was analyzed by fluorescence-activated cell sorting analysis and shown as a dot blot (D) and bar graph (E). (F, G) MDSC population
(Gr-1+/CD11b+) among splenocytes was analyzed by fluorescence-activated cell sorting analysis and shown as a dot blot (F) and bar graph (G).
Splenocytes from mice were stained with the indicated antibodies toward cell surface markers and analyzed by flow cytometry. *, p < 0.05. **, p <
0.01 by ANOVA.
Synergistic Effect of H9 Combined with Trastuzumab 1044
July 2015⎪Vol. 25⎪No. 7
this study, H9 showed the most effective anti-tumor
activity against breast cancer cells in vivo, although H5 was
the most effective in vitro. Because several components of
H9 have anticancer effects [12, 17, 35], it is conceivable that
the total H9 extract would show anti-tumor activity. The
marker compounds of H9 are evodiamine, ginsenosides
Rg1 and Rb1, and cinnamic acid. Gas chromatography/
mass spectrophotometry showed that the H9 extract
contained coumarin, isoeugenol, isoelemicin, angelicin, and
psolarene as the major compounds. Angelicin, which is
structurally related to psoralens, has apoptosis-inducing
effect and is a well-known chemical class of photosensitizers,
used for its anti-proliferative activity in the treatment of
different skin diseases. Angelicin was also reported to be
an effective apoptosis-inducing natural compound of human
neuroblastoma cancer, and its analogs have been shown to
have potent anti-influenza and anticancer activities [29, 31].
Thus, angelicin would be one of the major compounds
conferring the anti-tumor activity of H9. Coumarin and its
derivatives have multiple biological activities, such as
antifungal, antiviral, anticancer, anti-inflammatory, and
antidiabetic activities. Coumarin treatment also enhances
the macrophage migration activity in the presence and
absence of LPS and increased nitric oxide release. This
suggests that the immunomodulatory activity of coumarin,
a component of H9, would contribute to the anti-tumor
effect by enhancing the anti-tumor immune function.
Although the combined treatment of coumarin with
cimetidine was reported to increase the percentage of
monocytes without NK cell population change [11, 23, 24],
it is possible that coumarin, along with other compounds
present in H9 or trastuzumab, could be involved in the
population change of NK cells and MDSC. Taken together,
angelicin and coumarin may be the biologically active
compounds in H9.
Although the molecular mechanisms of herbal extracts
have not been well-characterized, the safety and efficacy of
many medicinal herb extracts have been validated [1, 37].
Medicinal herb extracts can be used as a strategy to reduce
the side effects caused by chemotherapy or to enhance the
effects of chemotherapeutic drugs. Therefore, the combined
effect of H9 and AC was examined in vivo, in addition to
the anti-proliferative effect of H9 observed in vitro. Co-
treatment of H9 and AC failed to show additive or
synergistic anti-tumor growth effects. However, the lack of
a combined effect may be due to the strong anti-tumor
effect of AC treatment as a primary chemotherapy. Thus,
the combined effect of H9 and trastuzumab was also
examined. Trastuzumab, which is a HER2-targeted monoclonal
antibody, is also used as an adjuvant therapy for breast
cancer. The combination of H9 and trastuzumab was found
to demonstrate enhanced anti-tumor effects through
increase in the NK cell population and decrease in the
MDSC population. On the other hand, it is possible that the
MDSC population was decreased in H9/trastuzumab-
treated mice because of the loss of tumor weight. Therefore,
it is valuable to find out whether H9/trastuzmab directly
suppresses MDSC differentiation from the precursor cells
or MDSC expansion.
There are many immune modulating compounds in the
H9 extract. Ginsenoside Rg1, one of the compounds in H9,
increases the number of T helper cells and the splenocyte
NK cytotoxicity. CML-1 oriental herbal extract containing
Cinnamoni Cortex inhibits TNF-α-induced inflammation
[15, 25]. Curcumol, one of the major components of the
essential oil of Rhizoma Curcumae, inhibits lipopolysaccharide-
activated RAW264.7 cells through the suppression of TNF-
α, IL-1, IL-6, and iNOS [4]. In addition to these known
immune modulating compounds, further study is needed
to identify the compounds related to the change of NK cell
and MDSC number and activity.
In summary, H9 inhibited the proliferation of breast cancer
cells, eventually leading to apoptotic death. Furthermore,
H9 suppressed 4T1 tumor growth in a syngeneic tumor
model, and the combined treatment of H9 and trastuzumab
further enhanced the anti-tumor growth effect through
increase of the NK cell number and activity and decrease in
the MDSC number. Although some of the compounds from
the H9 extract have anti-tumor activity and immune
modulating activity, exactly which compound of H9 was
responsible for the synergistic effect with trastuzumab for
inhibition of tumor growth remains to be elucidated.
Acknowledgments
This work was supported by a grant from the Korea
Health Industry Development Institute (B110053-1101-
0000200), funded by the Ministry of Health and Welfare,
and a grant from Sookmyung Women’s University (2015).
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