Acta Medica Mediterranea, 2020, 36: 3089

ZHIBAI DIHUANG WAN CONTRIBUTES APOPTOSIS OF TUMOR CELLS IN ESTROGEN-RECEPTOR-

POSITIVE BREAST CANCER MICE VIA ERK/AKT PATHWAY

YuLin Li1, Wentao Si1, *, Aihua Hou1, Hongmin Shao1, Rongrong Hu1, Wen Wang1, Yufei Yang2
Oncology Department, Yantai Traditional Chinese Medicine Hospital, Yantai, China - 2Oncology Department, Xiyuan Hospital of
1

China Academy of Chinese Medical Sciences, Beijing, China

ABSTRACT

Background: As an important Chinese medicine, Zhibai Dihuang Wan (ZDW) has been used in clinical treatment. In this study,
we aimed to investigate the potential mechanism of ZDW in the treatment of estrogen receptor-breast cancer.
Methods: The transplanted tumor was established by subcutaneous injection of MCF-7 cells. Forty BALB/c nude mice
were randomly divided into model, tamoxifen citrate (TAM, 2.5 mg/kg), ZDW (10 mg/kg), and TAM+ZDW groups. The levels of
serum estradiol were measured by ELISA kit. Tumor weight and tumor inhibition rate in each group were monitored. Moreover,
histopathological observation of tumors was observed by hematoxylin eosin (HE) and the expression of ERα, Bcl-2 and Bax in tumors
was measured by immunohistochemical staining. The levels of ERα, p-ERK1/2, ERK1/2, p-AKT and AKTprotein in cancer tissues were
analyzed by western blot.
Results: The inhibition rate of tumor in TAM + ZDW group was the highest among groups. HE staining revealed that the tumor
cells in TAM, ZDW and TAM + ZDW group showed a reduction in mitosis together with cell morphology atrophy when compared to
the model group. Meanwhile, after treatment of TAM or ZDW, the positive reactions of ERα and Bcl-2 were weaker. In TAM + ZDW
group, the expressions of ERα and Bcl-2 were the lowest. The levels of ERα, and p-ERK1/2/ ERK1/2 protein in TAM, ZDW and TAM
+ ZDW groups were notably declined than those in model group (P<0.05). Moreover, compared with the model group, the expressions
of p-AKT/AKT in TAM, ZDW and TAM + ZDW group were significantly higher (P<0.05).
Conclusions: ZDW could inhibit the growth of tumor and promote tumor celss apoptosis via adjusting ERK/AKT pathway in
estrogen-receptor-positive breast cancer.

Keywords: Estrogen-receptor-positive breast cancer, Zhibai Dihuang Wan, apoptosis, ERK/AKT pathway.

DOI: 10.19193/0393-6384_2020_5_476

Received November 30, 2019; Accepted January 20, 2020

Introduction incidence of side reactions including the relative

Breast Cancer (BC) is an important cause of
cancer-associated mortality in women, which leads
to 23% of cancer cases and 14% of cancer-related
mortalities(1, 2). The expressions of estrogen receptor
(ER) plays a key role in process of BC and 75% of
BCs are ER-positive (ER+). Previous researches
in the treatment of BC have been obtained a lot of
advancements, including surgery, chemotherapy
and radiotherapy. However, endocrine therapies
are the most important treatment for these cancers,
and adjuvant endocrine therapy reduces the
risk of recurrence(4). The estrogen stimulates the
ER signaling pathway and then promotes cell
proliferation. Therefore, endocrine therapies of the
ER signaling pathway are critical and most effective
treatments for estrogen-receptor-positive breast
cancer(5). However, many patients relapse after
endocrine therapy due to resistance. So, new drugs
for estrogen-receptor-positive breast cancer are
needed to find.
Zhibai Dihuang Wan (ZDW) is an important
Chinese medicine, which consists of anemarrhena
(6.9%), phellodendron (6.9%), rehmannia glutinosa
3090 YuLin Li, Wentao Si et Al
(27.6%), cornus, paeoniasuffruticosa (10.3%), po-
ria cocos and alisma (10.3%), and other contains(6).
Among these ingredients, aristolochic acid and lo-
ganin have been reported to be beneficial for ne-
phropathy injury(7, 8). Appropriate dosage of ZDW
improved gentamicin induced apoptotic damage in
renal tubular cells(6). Many components of ZDW also
have a reduced effect on the side effects of steroids.
In recent years, poria cocos has been used in a wider
range of clinical applications, such as treatment of
chronic renal injury(9).
Previous studies have found that rehmannia
glutinosa is regarded as a traditional Chinese med-
icine to treat and prevent cancer(10). Unfortunately,
the molecular mechanism and process of activity of
ZDW in estrogen-receptor-positive breast cancer has
not yet been reported.
In the study, we aimed to investigate the influ-
ence and potential mechanism of ZDW in the treat-
ment of estrogen receptor-breast cancer in mice.
Materials and methods
Animals
Forty adult female BALB/c nude mice, 4-6
weeks, weighting 20-25g, were purchased from
Changzhou Cavens Laboratory Animal Co., Ltd
(SCXK (Su) 20160010). All animals were quaran-
tined as required and observed for their activity and
diet performance during the quarantine period. The
animals were housed (5 mice/cage) in a central facil-
ity, and renewed every 24 h under a 12:12 light-dark
cycle, 20~26°C, humidity 40~70 %.
All animal procedures followed the NIH guide-
lines and have been approved by the Animal Protec-
tion and Use Committee of Yantai Traditional Chi-
nese Medicine Hospital.
Cell culture and animal models
The MCF-7 cells were bought from ATCC and
cultured in DMEM medium (Gibco, Rockville, MD,
USA) with 10 % FBS at 37°C, 5% CO2. The final cell
concentration was adjusted to 1×107/mL.
The right chest wall under the nude mice was
inoculated with 0.2 mL MCF-7 cells. Ten to fifteen
days after inoculation, tumor nodules with hard
texture appeared at the site of the inoculation pad
and the model was established with success.
Animal groups
Forty tumor-bearing mice were randomized
in groups: model group, tamoxifen citrate (TAM,
Shanghai, Fudan Fuhua Pharmaceutical) group (2.5
mg/kg), ZDW (Henan, Wanxi Pharmaceutical) group
(10 mg/kg), TAM (2.5 mg/kg) combined with ZDW
(10 mg/kg) group (TAM+ZDW).
Doses of each group were calculated according
to the dose conversion formulas of human and mice,
and the model group was given intragastric adminis-
tration with the same volume of normal saline. The
drugs were dissolved in saline and administrated
once a day for 21 consecutive days.
ELISA
The mice were killed by cervical dislocation.
Blood was collected and centrifuged at 800 ×g, 15
min to separate the serum. The levels of estradiol
(E2) in serum were measured using ELISA kits
(Huamei Biological Co., Ltd., Wuhan, China).
Tumor weight and inhibition rate
Tumor weights and growth inhibition rate of
tumor were calculated in each group. The inhibition
rate = (average tumor weight in model group - average
tumor weight in treated group)/average tumor weight
in model group × 100%.
Histopathology
4% paraformaldehyde was used to fix cancer
tissues (5 µm) for 24 h, then washed with phosphate-
buffered saline (PBS, Baso, Zhuhai, China), three
times. Alcohol with different concentration was
used to dehydrate. Tissues were transparent in
dimethylbenzene.
The embedded wax blocks were sliced into
thin slices (5 µm) and perfused with hematoxylin
for 15 min, differentiated by 1% hydrochloride-
ethanol and perfused in eosin for another 15 min.
Light microscope (Sigma-Aldrich, USA) was used to
observe pathological changes of tumor tissue.
Immunohistochemical staining
Serial graded ethanol was used to dewax and
hydrate for cancer tissues (5µm). Following that,
PBS washed the sections 15 min, 5 min each time.
Antigen was renovated at 95 °C, 60 min and cooled
at 37°C, 5 min, three times.
Primary antibodies ERα, Bcl-2, Bax (1:1000,
R&D systems, Shanghai, China) were cultured at
4°C, 12 h, and incubated at 25°C, 30 min.
After washing with PBST, horseradish
peroxidase labeled secondary antibody (1:500, R&D
systems, Shanghai, China) was added at 37°C for 30
min. The slices were cultured with DAB Horseradish
Zhibai Dihuang Wan contributes breast tumor cells apoptosis 3091
Peroxidase Color Development Kit (R&D systems,
Shanghai, China) for 15 min. Microscope (DMi8,
Feica Microsystems, Germany) was used to observe
positive expression.
Western blot
The proteins of tumor tissues were performed
by BCA kit (Thermo Fisher Scientific, Shanghai,
China). Transmembrane was performed to transfer
proteins to PVDF and blocked by 5% skimmed
milk. After washing with PBS, membranes were
incubated with anti-ERα antibody (1: 800, Abcam,
Shanghai, China), anti- ERK1/2 antibody (1: 1200,
Abcam), anti- p-ERK1/2 antibody (1: 1200, Abcam),
anti-AKT antibody (1:1500, Abcam), anti-p-AKT
antibody (1:1500, Abcam) and anti-β-actin (1:1200,
Abcam) at 4°C for overnight.
Then washed with PBS, and incubated with
IgG-HRP secondary antibody (1:1000, Abcam) at
37°C for 60 min. Signals detection was performed
using. ECL method (Sigma-Aldrich, USA) was used
to detect signals.
Statistical analysis
All experimental data were expressed as
̅
“X±SD”, and statistical differences were examined
by software SPSS 20.0.
One-way analysis of variance (ANOVA) was
used for the comparison of multiple groups followed
by Dunnett test. Significance was P<0.05.
Results
Influence of ZDW on weight of mice and es-
tradiol levels in serum
The general status of mice was observed.
In model group, the mice manifested as obvious
weight loss, slow action, loss of appetite, and de-
creased activity. Due to tumor depletion, the body
weight also decreased in mice (Fig. 1 A).
However, the state of mice kept it better in
therapy groups. The average weight in each group
was increased from 1 to 21 days. After the admin-
istration, the average weight in TAM+ZDW group
was significantly higher when compared with in
model group (P<0.05) on 10 and 21 days (Fig. 1 A).
The effects of ZDW on estradiol level in serum
were analyzed, and the results showed the level of
estradiol was significantly decreased in drug thera-
py groups compared to model group (p<0.05, Fig.1
B). The level of estradiol in TAM+ZDW group was
the lowest.
Fig. 1: The effect of drugs therapy on mice weight (A)
and estradiol in serum (B). TAM: tamoxifen citrate;
ZDW: Zhibai Dihuang Wan. The data was expressed as
mean ± standard (SD), n=10. vs. model group, *p<0.05.
ZDW inhibited tumor growth
To reveal the effects of ZDW on tumor growth,
we found the tumor weights in the TAM, ZDW and
TAM+ZDW group were 1.38±0.11g, 1.69±0.41g and
1.21±0.33g, which were significantly lower than that
in model group (P<0.05, Fig. 2 A).
The inhibition rate of tumor in TAM, ZDW and
TAM+ZDW group reached to 47.13%, 35.25%, and
53.64%, respectively (Fig. 2 B).
It was suggested that the growth of tumor had
been inhibited by ZDW treatment.
Fig. 2: The effect of drugs therapy on tumor weight (A)
and inhibition rate of tumor (B). The data was expressed
as mean ± standard (SD), n=10. vs. model group, *p<0.05.
ZDW suppressed tumor cell proliferation in
tumor tissue
As shown in Fig. 3 A, the tumor cells in the
model group were larger, round or polygonal, and
some cells had lipid vacuoles in cytoplasm.
Furthermore, tumor cells were arranged in a
nest, flake, or cord. Compared to model group, the
tumor cells in TAM, ZDW and TAM + ZDW group
showed a reduction in mitosis, together with cell
morphology atrophy (Fig. 3 B-D).
Simultaneously, many tumor cells grew with
hyperplastic connective tissue and a small number of
catheters. In TAM + ZDW group, the cell morphol-
ogy of cancer tells were obviously atrophied, when
compared with TAM and ZDW groups.
3092 YuLin Li, Wentao Si et Al
Fig. 3: The histopathology of tumor tissues in groups.
Microscopic images of 100-magnification were acquired
(n=5). A: Model group; B: TAM group; C: ZDW group;
D: TAM + ZDW group.
ZDW contributed apoptosis in cancer tis-
sues
The expression of ERα, Bcl-2 and Bax in can-
cer tissues was measured by immunohistochemistry
(Fig. 4). The positive reactions of ERα and Bcl-2
protein were the highest in model group, when com-
pared to other groups (Fig. 4 A,B).
After treatment of TAM or ZDW, the positive
reactions of ERα and Bcl-2 were weaker. In TAM +
ZDW group, the expressions of ERα and Bcl-2 were
the lowest (Fig. 4 A,B). By contrast, the levels of Bax
were the highest in TAM + ZDW group, the lowest
in model group (Fig. 4 C).
Fig. 4: The expression of ERα, Bcl-2 and Bax in cancer
tissues were measured by immunohistochemistry. Micro-
scopic images of 100-magnification were acquired (n=5).
The effect of ZDW on ERK/AKT pathway in
cancer tissues Berberine and palmatine are known anti-inflamman-
Western blot was used to detect the expression
of ERα, p-ERK1/2/ ERK1/2, and p-AKT/ AKT in
cancer tissues (Fig. 5). When compare to model
group, ERα, and p-ERK1/2/ ERK1/2 protein expres-
sion in TAM, ZDW and TAM + ZDW group were
obviously decreased (P<0.05). Moreover, compared
with model group, p-AKT/AKT protein expression
was distinctly higher in TAM, ZDW and TAM +
ZDW groups (P<0.05).
The results suggested ZDW promoted apopto-
sis of cancer cells, which mechanism might be asso-
ciated with the ERK/AKT pathway.
Fig. 5: The effect of drugs therapy on ERK/AKT pa-
thway in tumor tissues. (A) the expressions of ERα, ERK,
p-ERK, AKT, p-AKT proteins were analyzed by Western
blot; B-D, An anti-β-actin antibody was used to confirm
equivalent protein loading. The data was expressed as
mean ± standard (SD), n=5. vs. model group, *p<0.05.
Discussion
In this study, we found ZDW treatment de-
creased the levels of estradiol and inhibited tumor
growth. Treatment of ZDW also promoted apoptotic
signals in estrogen-receptor-positive breast cancer
mice, including Bcl-2, Bax increase.
In cancer tissues of mice, the ZDW treatment
obviously reduced the expression of ERα and p-ERK
proteins, but at the same time ZDW administration
significantly increased the expression of p-AKT.
These results suggested that ZDW could be beneficial
for treatment patients with estrogen-receptor-posi-
tive breast cancer. As known, multiple components
of ZDW had been used to treat various diseases. The
components include aristolochia acid, loganin, ber-
berine, palmatine, and jatrorrhizine and so on(8, 11, 12).
tory agents and reduce oxidative stress in different
diseases. In Chinese traditional medicines, Anemar-
rhena asphodeloides and Rehmannia glutinosa are
widely used to treatment diseases, such as kinney
Zhibai Dihuang Wan contributes breast tumor cells apoptosis 3093
injury, diabetes, and ischemia-induced brain injury.
Furthermore, ZDW treatment reduced gentamicin-in-
duced apoptotic nuclei in kidney injury mouse, and
the mechanism was associated with the apoptotic
signals(6). These studies supported our research that
ZDW could inhibit estrogen-receptor-positive breast
cancer growth and induce apoptosis of cancer cells.
Breast cancer is a heterogeneous disease which
is composed of different histologic subtypes. Estro-
gen receptor-positive breast cancer is the most com-
mon estrogen-dependent cancer(3). A large number
of clinical experiments have shown that E2, as an
important mitogen, could bind to estrogen receptors
in estrogen receptor-positive MCF-7 cells(13-15). E2
could activate the receptor to exert its biological ac-
tivity and promote the metastasis and proliferation of
breast cancer cells by activating the ERK/AKT signal
pathway. The higher level of estrogen, the higher risk
of breast cancer recurrenced in cancer patients(16). In
this study, ZDW therapy decreased the expression
of ERα in mice with receptor-positive breast cancer.
This result suggested the protective effect of ZDW
could result from blocking E2 signal. AKT/ERK
signals are crucial for progression of breast cancer,
which take part in the regulation of cell survival and
proliferation(17, 18). In a study, tamoxifen was found to
induce apoptosis in estrogen receptor-negative breast
cancer cells by inactivating Akt phosphorylation(19). A
phospho-protein analysis revealed that the phospho-
rylation of ERK considered as key regulator of estro-
gen receptor-positive MCF-7 breast cancer cells(20).
In the current study, our results were consistently
with previous reports. ZDW management down-reg-
ulated the expression of p-ERK and up-regulated the
expression of p-AKT in breast cancer tissues, sug-
gesting that ZDW induced brance cells apoptosis
through regulating ERK/AKT pathway. Thereby, the
results suggested that ZDW could be a novel drug for
estrogen-receptor-positive breast cancer.
Conclusions
In conclusion, our research revealed that ZDW
contributed the apoptosis of breast cancer cells in
estrogen-receptor-positive breast cancer mice, and
increased the expression of ERα, and p-ERK1/2/
ERK1/2, decreased the expression of p-AKT/AKT
in cancer tissue.
These data suggested that ZDW suppression of
cancer growth was associated with ERK/AKT path-
way. ZDW may have a therapeutic potential for pa-
tients with estrogen-receptor-positive breast cancer.
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