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: yyang@sookmyung.ac.kr

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).

References

1. Alkreathy HM, Damanhouri ZA, Ahmed N, Slevin M,

Osman AM. 2012. Mechanisms of cardioprotective effect of

aged garlic extract against doxorubicin-induced cardiotoxicity.

Integr. Cancer Ther. 11: 364-370.

2. Bak Y, Ham S, Baatartsogt O, Jung SH, Choi KD, Han TY, et

al. 2013. A1E inhibits proliferation and induces apoptosis in

1045 Lee et al.

J. Microbiol. Biotechnol.

NCI-H460 lung cancer cells via extrinsic and intrinsic

pathways. Mol. Biol. Rep. 40: 4507-4519.

3. Cai Y, Luo Q, Sun M, Corke H. 2004. Antioxidant activity

and phenolic compounds of 112 traditional Chinese medicinal

plants associated with anticancer. Life Sci. 74: 2157-2184.

4. Chen X, Zong C, Gao Y, Cai R, Fang L, Lu J, et al. 2014.

Curcumol exhibits anti-inflammatory properties by interfering

with the JNK-mediated AP-1 pathway in lipopolysaccharide-

activated RAW264.7 cells. Eur. J. Pharmacol. 723: 339-345.

5. Chu Q, Satoh K, Kanamoto T, Terakubo S, Nakashima H,

Wang Q, Sakagami H. 2009. Antitumor potential of three

herbal extracts against human oral squamous cell lines.

Anticancer Res. 29: 3211-3219.

6. Douma KF, Meiser B, Kirk J, Mitchell G, Saunders C,

Rahman B, et al. 2014. Health professionals' evaluation of

delivering treatment-focused genetic testing to women newly

diagnosed with breast cancer. Fam Cancer. 14: 265-272.

7. Euhus DM, Diaz J. 2015. Breast cancer prevention. Breast J.

21: 76-81.

8. Fei XF, Wang BX, Li TJ, Tashiro S, Minami M, Xing DJ,

Ikejima T. 2003. Evodiamine, a constituent of Evodiae Fructus,

induces anti-proliferating effects in tumor cells. Cancer Sci.

94: 92-98.

9. Han S, Jeong AL, Lee S, Park JS, Kim KD, Choi I, et al. 2013.

Adiponectin deficiency suppresses lymphoma growth in

mice by modulating NK cells, CD8 T cells, and myeloid-

derived suppressor cells. J. Immunol. 190: 4877-4886.

10. Iakovou IP, Giannoula E. 2014. Nuclear medicine in

diagnosis of breast cancer. Hell. J. Nucl. Med. 17: 221-227.

11. Jamal S, Casley-Smith JR. 1989. The effects of 5,6 benzo-[a]-

pyrone (coumarin) and DEC on filaritic lymphoedema and

elephantiasis in India. Preliminary results. Ann. Trop. Med.

Parasitol. 83: 287-290.

12. Ji Y, Rao Z, Cui J, Bao H, Chen C, Shu C, Gong JR. 2012.

Ginsenosides extracted from nanoscale Chinese white

ginseng enhances anticancer effect. J. Nanosci. Nanotechnol.

12: 6163-6167.

13. Jin H-J, Kim S-H, Lee S-W. 2012. Patent analysis of Sasang

constitution medicine. J. Sasang Constitutional Med. 24.

14. Kanavos P. 2006. The rising burden of cancer in the

developing world. Ann. Oncol. 17(Suppl 8): viii15-viii23.

15. Kenarova B, Neychev H, Hadjiivanova C, Petkov VD. 1990.

Immunomodulating activity of ginsenoside Rg1 from Panax

ginseng. Jpn. J. Pharmacol. 54: 447-454.

16. Kummerow KL, Du L, Penson DF, Shyr Y, Hooks MA. 2014.

Nationwide trends in mastectomy for early-stage breast

cancer. JAMA Surg. 150: 9-16.

17. Kwon HK, Hwang JS, So JS, Lee CG, Sahoo A, Ryu JH, et al.

2010. Cinnamon extract induces tumor cell death through

inhibition of NFkappaB and AP1. BMC Cancer 10: 392.

18. Lee J, Kim KA, Jeong S, Lee S, Park HJ, Kim NJ, Lim S.

2009. Anti-inflammatory, anti-nociceptive, and anti-psychiatric

effects by the rhizomes of Alpinia officinarum on complete

Freund's adjuvant-induced arthritis in rats. J. Ethnopharmacol.

126: 258-264.

19. Lee S, Han S, Jeong AL, Park JS, Yang Y. 2014. Depletion of

IK causes mitotic arrest through aberrant regulation of

mitotic kinases and phosphatases. FEBS Lett. 588: 2844-2850.

20. Lee S, Han S, Park JS, Jeong AL, Jung SH, Choi K-D, et al.

2013. Herb mixture C5E aggravates doxorubicin-induced

apoptosis of human breast cancer cell lines. J. Kor. Soc. Appl.

Biol. Chem. 56: 567-573.

21. Lu B, Yu L, Xu L, Chen H, Zhang L, Zeng Y. 2010. The

effects of radix curcumae extract on expressions of VEGF,

COX-2 and PCNA in gastric mucosa of rats fed with

MNNG. Curr. Pharm. Biotechnol. 11: 313-317.

22. Mamounas EP, Bryant J, Lembersky B, Fehrenbacher L,

Sedlacek SM, Fisher B, et al. 2005. Paclitaxel after

doxorubicin plus cyclophosphamide as adjuvant chemotherapy

for node-positive breast cancer: results from NSABP B-28. J.

Clin. Oncol. 23: 3686-3696.

23. Marshall ME, Conley D, Hollingsworth P, Brown S,

Thompson JS. 1989. Effects of coumarin (1,2-benzopyrone)

on lymphocyte, natural killer cell, and monocyte functions

in vitro. J. Biol. Response Mod. 8: 70-85.

24. Marshall ME, Riley LK, Rhoades J, Eichhorn T, Jennings

CD, Cibull M, Thompson J. 1989. Effects of coumarin (1,2-

benzopyrone) and cimetidine on peripheral blood lymphocytes,

natural killer cells, and monocytes in patients with

advanced malignancies. J. Biol. Response Mod. 8: 62-69.

25. Mo SJ, Son EW, Lee SR, Lee SM, Shin DH, Pyo S. 2007.

CML-1 inhibits TNF-alpha-induced NF-kappaB activation

and adhesion molecule expression in endothelial cells through

inhibition of IkBalpha kinase. J. Ethnopharmacol. 109: 78-86.

26. Ng PL, Rajab NF, Then SM, Mohd Yusof YA, Wan Ngah

WZ, Pin KY, Looi ML. 2014. Piper betle leaf extract

enhances the cytotoxicity effect of 5-fluorouracil in

inhibiting the growth of HT29 and HCT116 colon cancer

cells. J. Zhejiang Univ. Sci. B 15: 692-700.

27. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R,

Langer R. 2007. Nanocarriers as an emerging platform for

cancer therapy. Nat. Nanotechnol. 2: 751-760.

28. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS,

Rees CA, et al. 2000. Molecular portraits of human breast

tumours. Nature 406: 747-752.

29. Rahman MA, Kim NH, Yang H, Huh SO. 2012. Angelicin

induces apoptosis through intrinsic caspase-dependent

pathway in human SH-SY5Y neuroblastoma cells. Mol. Cell

Biochem. 369: 95-104.

30. Shan BE, Zeki K, Sugiura T, Yoshida Y, Yamashita U. 2000.

Chinese medicinal herb, Acanthopanax gracilistylus, extract

induces cell cycle arrest of human tumor cells in vitro. Jpn. J.

Cancer Res. 91: 383-389.

31. Shiao HY, Kuo CC, Horng JT, Shih SR, Chang SY, Liao CC,

et al. 2012. An efficient, mild and scalable synthesis of

bioactive compounds containing the angelicin scaffold. J.

Synergistic Effect of H9 Combined with Trastuzumab 1046

July 2015⎪Vol. 25⎪No. 7

Chin. Chem. Soc. 59: 1548-1554.

32. Shoemaker M, Hamilton B, Dairkee SH, Cohen I, Campbell

MJ. 2005. In vitro anticancer activity of twelve Chinese

medicinal herbs. Phytother. Res. 19: 649-651.

33. Sorlie T, Sexton HC, Busund R, Sorlie D. 2000. Predictors of

satisfaction with surgical treatment. Int. J. Qual. Health Care

12: 31-40.

34. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel

A, et al. 2003. Repeated observation of breast tumor

subtypes in independent gene expression data sets. Proc.

Natl. Acad. Sci. USA 100: 8418-8423.

35. Yang HL, Chen SC, Chen CS, Wang SY, Hseu YC. 2008.

Alpinia pricei rhizome extracts induce apoptosis of human

carcinoma KB cells via a mitochondria-dependent apoptotic

pathway. Food Chem. Toxicol. 46: 3318-3324.

36. Yoo JH, Lee EJ, Ko BH, Song IB, Kim TY. 2003. The study

on the upgrade of QSCC II (I) - a study on comparison of

responses to the questionnaire based on Sasang Constitution's

differences-questionnair of Sasang Constitution Classification

II. J. Sasang Constitutional Med. 15: 99-108.

37. Zhao S, Wu J, Wang C, Liu H, Dong X, Shi C, et al. 2013.

Intraoperative fluorescence-guided resection of high-grade

malignant gliomas using 5-aminolevulinic acid-induced

porphyrins: a systematic review and meta-analysis of

prospective studies. PLoS One 8: e63682.