Biomedicine & Pharmacotherapy 115 (2019) 108979

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

The effect of earthworm extract on mice S180 tumor growth and apoptosis T
a,b c d e e d a,f,⁎
Zhenhan Deng , Shanshan Gao , Xiang Xiao , Ni Yin , Shiyang Ma , Wenping Li , Yusheng Li
a
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China
b
Department of Sports Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong, China
c
Department of Cardiology, University of Colorado Anschutz Medical Campus, Aurora 800045, CO, USA
d
The Animal Health Inspection Institute of Yuelu District, Changsha 410000, Hunan, China
e
Department of Clinical Medicine (8-Year Program), Xiangya Medicine School, Central South University, Changsha 410013, Hunan, China
f
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410013, Hunan, China

A R T I C LE I N FO A B S T R A C T

Keywords: Great efforts have been made to explore the potential treatment for cancers, and the most common therapies
Earthworm extract include surgery, chemotherapy and radiotherapy. As an alternative medication, earthworms have drawn in-
Sarcoma creased attention considering its abundance in resource, easy access and minor side effects compared to tradi-
Tumor growth tional therapies. However, few studies had focused on the antitumor effect of earthworm-derived components.
Apoptosis
The purpose of this study is to investigate whether earthworm extract has an effect on tumor cell apoptosis and
Bcl-2
growth. Earthworm extract (EE) was purified through multiple steps of centrifugation and chromatography.
Bax
Mice were inoculated with ascitic fluid derived from mice inoculated with S180 sarcoma tumor cells and fed
orally with different amounts of EE for 25 days. Tumor samples were analyzed for size and cell apoptosis. And
we found that the weight and sizes of tumor decreased gradually as the amount of EE administered increased.
More apoptotic cells and lowered level of lactate dehydrogenase (LDH), a biomarker of tumor invasiveness, was
detected in EE-treated group than the untreated group. Our results suggested that EE could dramatically promote
tumor apoptosis and reduce tumor size in vivo, suggesting a novel alternative therapy for cancer patients.

1. Introduction
Cancer is among the leading causes of death world wide. In 2017,
over one million new cases in the United States were diagnosed with
cancer and over 600,000 individuals died of it [1]. Great efforts have
been made for development of new anti-cancer drugs over time.
However, the 5-year survival rates of several types cancers like lung
cancer and pancreatic cancer are still below 20%. Additionally, patients
who underwent chemotherapy and radiation therapy suffer from side
effects such as muscle pain, hair loss, nausea and even the risk of de-
veloping a secondary cancer, due to the fact that besides cancer cells,
healthy active cells were also damaged during the therapy [2].
Earthworms have long been used as a treatment for illness in the
history of traditional medicine. People from Burma applied ashes from
burnt earthworms to alleviate symptoms of fever; in Laos, earthworms
was used to treat small pox; ancient Chinese used earthworm to ease
fever-associated convulsions, hemiplegia and blood clots [3]. In modern
medicine, studies have revealed beneficial effects of earthworm in both
in vitro and in vivo models, with a better understanding of the me-
chanism underlying earthworm’s disease-curing capability. Fu et al.
found the earthworm extract (EE) in culture medium and the ablity of
promoting cell proliferation and activating osteoblasts [4]. Chang et al.
observed stimulated migration in Schwann cells by EE, through in-
creased production of matrix-degrading proteolytic enzymes [5]. Intra-
peritoneal injection of EE could notably reduce pulmonary inflamma-
tion and fibrosis induced by silica inhalation in mice [6]. Isolated active
ingredients from earthworms could protect mice from endoplasmic
reticulum stress-induced liver injury [7]. Our previous study observed
effect of EE on promoting wound healing in mice with excisional
wounds [8].
In terms of cancer-related studies, Augustine et al. found earthworm
coelomic fluid suppressed the proliferation in a squamous cancer cell
line [9]. The earthworm fibrinolytic enzyme from Eiseniafoetida had
shown to significantly inhibit the growth of human hepatoma tumor
xenograft in nude mice [10]. Despite its rising role in development of
alternative anti-cancer therapies and the advantages of low-cost, no

Abbreviation: CAT, catalase; CTX, cyclophosphamide; EE, earthworm extract; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; PBS, phosphate-buffered
saline; RBC, red blood cell; ROS, reactive oxygen species; SOD, superoxide dismutase; WBC, white blood cell
⁎
Corresponding author.
E-mail address: liyusheng@csu.edu.cn (Y. Li).

https://doi.org/10.1016/j.biopha.2019.108979
Received 25 March 2019; Received in revised form 20 April 2019; Accepted 8 May 2019
0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Z. Deng, et al.
reported side effects and therefore being possible to be consumed
continuously, the potential outcome of treating cancer with EE still
remains controversial. According to previous studies, activities of an-
tioxidant enzymes such as superoxide dismutase (SOD) and catalase
(CAT) were detected in live earthworms and EE [11,12]. Although
antioxidant enzymes play a positive role in our body and protect our
tissues against damage caused by excess reactive oxygen species (ROS),
other studies advocated that antioxidant enzymes may promote tumor
growth and invasiveness [13–15].
Hence, the effect of EE on cancer should be carefully examined case
by case depending on the purification methods of earthworm compo-
nents, types of cancers and degrees of cancer progression, the way of
drug administration and other factors. S180 is a murine sarcoma cancer
cell line and has been commonly used in cancer research because of its
high proliferation rate in mice. Until today, there have been few studies
on the influence of EE in S180 tumor-bearing mice [16]. Therefore, in
the study we aim to investigate the effect of EE on S180 tumor growth
and apoptosis.
2. Methods and materials
2.1. Extraction and purification of EE
Indian blue earthworms (Perionyx excavates) were collected from
an earthworm breeding farm, and the EE was isolated and purified as
previously described [17]. Briefly, sexually mature earthworms were
rinsed with tap water to remove attached mud, followed by homo-
genization with a tissue homogenizer. The homogenized sample was
ground by a tissue grinder on ice, sonicated and centrifuged with
6000 rpm for 10 min at 4 °C. The supernatant was ultra-filtered with
50KD ultra-filter (Millipore) with 5000 g for 20 min, then the lower
fraction left at the bottom of centrifuge tube was ultra-centrifuged with
10KD ultra-filter (Millipore) at 4 °C. Extract was purified from the ultra-
filtered solution with Sephadex G-200 by chromatography. Desired
protein sample was eluted with phosphate-buffered saline (PBS, 0.1 M,
pH7.8) and stored at −20 °C.
2.2. Cell culture
S180 tumor cells were purchased from Cell Banks of the Chinese
Academy of Sciences (Cat.TCM15) and maintained in RMPI 1640
medium (Gibco) containing 10% FBS at 37 °C in a CO2 incubator. Fresh
medium was changed every 2–3 days.
2.3. Animals
Male Kunming mice (6–8 weeks old) purchased from Changsha
Animal Center were used in the experiment. The mice were housed
under normal laboratory conditions and were allowed to acclimate in
the facility for 1 week prior to experiments. Animal protocol was ap-
proved by the Animal Care and Use Committee of Central South
University and Hunan Agricultural University.
2.4. Ascites tumor-bearing mouse model
To establish a S180-bearing mouse model, we followed a protocol
previously published by other groups [18,19]. Briefly, 20 mice were
injected intraperitoneally with 0.8–1.2 × 106 S180 cells resuspended in
0.4 mL saline solution. One week later, enlarged abdomen was observed
in these mice and ascitic fluid was collected, diluted 3-fold with sterile
saline. 200ul diluted S180 tumor cell solution was subsequently sub-
cutaneously injected into mice used in our experiments for the estab-
lishment of a S180 tumor-bearing model.
2
Biomedicine & Pharmacotherapy 115 (2019) 108979
2.5. Oral administration of EE
The S180 tumor-bearing mice were divided randomly into five
groups. Mice fed with PBS were used as control group.
Cyclophosphamide (CTX) or EE was administered orally to mice for 25
days. CTX was applied at concentration of 30 mg/kg body weight/day,
and EE was used at concentrations of 30, 60, 90 mg/kg body weight/
day, respectively.
2.6. Spleen/thymus index
Immediately after mice were sacrificed, tumor, spleen and thymus
were surgically collected and weighed. The spleen/thymus index was
recorded using the following formula: Spleen/thymus index = the final
weight of spleen/thymus (g)/the final average body weight (g) without
tumor.
2.7. Histological analysis
The collected tumor tissues were fixed with 10% formalin and
embedded in paraffin, sectioned at 7 μm thickness. Hematoxylin and
eosin (H&E) staining was performed to reveal morphology of the tu-
mors. For immunohistochemical staining, Bcl-2 family antibody sam-
pler kit (Boster Technology) was applied to detect Bax and Bcl-2 ex-
pression on tumor sections. The positive cell numbers were counted
manually in 5 random fields from each single slide under a microscope.
2.8. DNA laddering
DNA fragmentation, a key characteristic of apoptosis, was detected
with DNA laddering kit (TianDZ, Inc) following user manual. DNA
samples extracted from S180 tumor tissues were purified and separated
on agarose gel and visualized with imaging system (YLN2000).
2.9. Hematological analysis
Counting of red blood cells (RBCs) and white blood cells (WBCs)
from mice whole blood was performed using a hematology analyzer.
Enzymatic activities of lactate dehydrogenase(LDH) in mice serum were
measured with assay kits (Jiancheng Bioengineering).
2.10. Statistical analysis
The values were expressed as means ± standard deviation (SD). All
statistical analyses were performed using the SPSS 16.0 software
(Chicago, IL, U.S.A.). Data were analyzed by the independent samples t-
test compared between two groups and a one-way analysis of variance
(ANOVA) was conducted to assess differences between multiple groups.
P-value < 0.05 was considered statistically significant.
3. Results
3.1. In vivo antitumor activity of EE
To investigate whether EE has antitumor effect, mice injected with
S180 tumor cells were sacrificed on day 25 post-implantation. No tumor
formation was observed in control group. The collected tumors from
S180 tumor-bearing mice were photographed and weighed. It was
found that tumors in CTX treated group and all the EE treated group
were significantly smaller in size (Fig. 1A) and lower in weight (Fig. 1B)
compared to control group. There was an inverse correlation between
the amount of EE orally administrated and the weight of tumors, in-
dicating the antitumor activity of EE is dose-dependent. However,
tumor weight in 30 mg/kg EE treated group was significantly higher
than that in CTX (30 mg/kg) treated group, suggesting that CTX acts
more efficiently in inhibiting tumor growth than EE at the same dosage.
Z. Deng, et al.
Fig. 1. Tumor size and weight in S180 tumor-bearing mice with different treatment. (A) Gross view of tumors from inoculated mice in different treated groups. (B)
Quantification of tumor weight in different treated groups (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs CTX.
Fig. 2. Spleen and thymus index of S180 tumor-bearing mice with different
treatment (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs Uninoculated.
Ψ < 0.05 vs CTX.
Spleen and thymus are the two primary immune organs involved in
antitumor activity [20,21], and the spleen and thymus indexes were
measured to evaluate the impact of tumor formation and subsequent
drug treatment on these immune organs (Fig. 2). Compared to control
group, a significant enhancement of both spleen and thymus index was
observed in CTX and EE treated groups, but more prominent in CTX
treated group. However, it has been reported that CTX treatment leads
to splenomegaly in mice [22]. Interestingly, while S180 cell inoculation
reduced the thymus index, no difference was found in spleen index
between uninoculated mice and control group, indicating S180 cell-
induced tumor formation didn’t exert much influence on spleen size.
3.2. The effect of EE on activities of serum LDH
Previous studies had detected enhanced expression of LDH in sev-
eral types of human cancers compared to normal tissues [23]. LDH has
been used as a prognostic biomarker for evaluation of tumor progres-
sion, invasiveness and patient survival rate [24]. Serum LDH level is
served as a useful test to determine outcome of antitumor treatments
[25,26]. The S180 tumor-bearing mice without drug treatments showed
significantly increased LDH level compared to uninoculated group
(Fig. 3). LDH level in the CTX treated group was significantly lower
than the control group, indicating that CTX treatment could efficiently
inhibit tumor growth. This result was consistent with the results in
Fig. 1, showing reduced size and weight of tumors in CTX treated
group. All groups of mice that fed with EE exhibited a suppression of
S180 tumor-induced LDH level in a dose-dependent manner.
3.3. Oral administration of EE induces S180 tumor cell death in vivo
In H&E staining, the tumor tissues in EE and CTX treated groups
showed cells with pyknotic nuclei and hypereosinophilic cytoplasm,
which served as an indicator of cell apoptosis. More apoptotic cells
were observed in 90 mg/kg EE treated mice than those treated with 30
3
Biomedicine & Pharmacotherapy 115 (2019) 108979
Fig. 3. Serum levels of LDH in S180 tumor-bearing mice with different treat-
ment (N = 5/group).*p < 0.05, **p < 0.01. vs Uninoculated; #p < 0.05,
##p < 0.01 vs Ctrl; Ψp < 0.05, ΨΨp < 0.01 vs CTX.
or 60 mg/kg EE (Fig. 4). Bax is an essential promoter for apoptosis and
Bcl is considered as an apoptosis inhibitor. Immunohistochemical
analysis of Bax and Bcl-2 revealed increased Bax-positive cells in mice
treated with CTX or EE, while cells stained with Bcl-2 antibody de-
creased in tumor tissues from these drug-treated groups (Fig. 5A).
Statistical analysis of Bax- and Bcl-2- positive cells from tumor sections
showed a significantly higher ratio of Bax-positve cells to Bcl-2 positive
cells in CTX and EE treated mice than control group (Fig. 5B). Analysis
of DNA fragmentation, a feature of early stage apoptosis, was per-
formed by DNA laddering kit with agarose gel electrophoresis. Se-
quential degraded DNA fragments were detected in tumor tissues from
CTX and EE treated mice, but not in control group (Fig. 5C). These
results suggested that EE be able to induce apoptosis in S180 tumors.
3.4. Blood cell counts
Blood cell number counts were performed in normal and S180 cells-
implanted mice (Fig. 6). No significant difference in RBC count or WBC
count was found between uninoculated mice and control mice, sug-
gesting tumor formation has little effect on the numbers of these two
types of blood cells. RBCs were dramatically increased in CTX-fed mice
compared to control mice or S180 tumor-bearing mice treated with
30 mg/kg EE. Oral administration of EE raised the RBC counts in a
dosage-dependent manner, but none of the EE treated group showed
statistic difference compared to uninoculated group, indicating EE has
weaker effect on RBC number than CTX at the same dosage. WBCs were
significantly increased in CTX treated mice compared to uninoculated
group and 30 mg/kg EE group. In mice treated with 90 mg/kg EE, WBC
Z. Deng, et al.
Fig. 4. H&E staining of the tumor tissues in S180 tumor-bearing mice with different treatment (N = 5/group, 200×). Ctrl stands for mice without any drug
treatment.
count was much higher than the uninoculated group, suggesting EE
treatment stimulates the production of WBCs in vivo.
4. Discussion
In this study, we investigated the antitumor potential of EE and
compare its effect on tumor growth and progression with CTX, a
medication to treat several types of cancers including leukemia, lym-
phoma, breast cancer and sarcoma. We used a sarcoma mouse model
inoculated with S180 tumor cells and administrated CTX or EE orally
for a defined period of time to observe the outcome of drug treatment.
When the tumor tissues were collected at the endpoint of the experi-
ment, it was found that at the same dosage, CTX treated mice had
smaller tumors size compared to EE treated group (Fig. 1), but lower
thymus and spleen index (Fig. 2), which could be due to the thymus
atrophyor splenomegaly caused by CTX 27,28]. It was shown that CTX
has an inhibitory effect on the growth of all the lymphocyte subsets
[29]. It also needs to be taken into consideration that expansion and
activation of effector T cells induced by CTX could overcome the de-
crease in exact T cell counts [29,30] and therefore, exhibit a better
antitumor effect in CTX treated animals. It is interesting to notice that
all the EE treated mice had higher thymus and spleen indexes than the
uninoculated and control groups, although how EE led to the increased
indexes of both immune organs remains unknown and needs to be
further explored.
Upregulated in many types of cancers, LDH has been used as a
biomarker [31] and potential therapeutic target for cancer treatment
[32,33]. LDH catalyzes the conversion of pyruvate to lactate during
glycolysis, a process critical for providing energy supply in cancer cells
due to their increased cellular proliferation metabolism [34]. In sar-
coma, high serum LDH level has been associated with poor outcome of
radiotherapy and chemotherapy [35]. Previous studies have found that
incubation with LDH inhibitor in culture medium inhibits the growth of
cancer cells [36]. Additionally, inactivation of LDH gene in mice
models of lung cancer suppresses tumor initiation and progression [37].
In our studies, we discovered that oral administration of EE deceased
the serum LDH level in S180-inoculated mice in a dosage-dependent
manner, but as dramatically as the CTX treated group (Fig. 3). More
4
Biomedicine & Pharmacotherapy 115 (2019) 108979
experiments should be performed to investigate whether EE can sensi-
tize cancer cells or animal models of cancer for responses to radio- or
chemotherapy in the future.
Apoptosis in cancer has been extensively studied for many years and
cytotoxic drugs that target certain apoptotic pathways have been de-
veloped for clinical cancer therapies [38,39]. So far there are mainly
two principle types of apoptotic pathways identified: the intrinsic
pathway and the extrinsic pathway [40], both involved in tumorigen-
esis [39]. The intrinsic pathway is mitochondria-dependent, in which
the Bcl-2 protein family members, including pro-apoptotic Bax and
anti-apoptotic Bcl-2, interact with each other to trigger or inhibit
apoptosis [41]. Sabah et al [42] detected Bcl-2 expression in soft tissue
sarcoma samples, and the protein expression of Bcl-2 is more prevalent
in high-grade sarcoma tissues. Liu et al [43] have shown that direct
activation of Bax protein induces apoptosis of cancer cells, which may
provide clues for development of new anti-cancer drugs. Nevertheless,
whether and how treatment with EE affects the apoptosis of tumor cells
in vitro and in vivo remains elusive. One group has found that earth-
worm coelomic fluid leads to apoptosis of Hela cells [44], a cervical
cancer cell line; another group has also detected apoptosis-like cell
death in tumor cells incubated with coelomocyte lysates from earth-
worm [45]. On the other hand, EE exhibit an inhibitory effect on car-
diomyoblast apoptosis induced by lipopolysaccharides [45]. It will be
interesting to investigate why components derived from earthworms
show different impacts on distinct types of cells. In this study we ex-
amined the histology of tumor samples from S180-inoculated mice
treated with or without EE, and found that EE leads to apoptosis in
tumors, partially via regulation of Bax and Bcl-2 proteins. We noticed
that although in the tumor samples the ratio of Bax-positive cells to Bcl-
2-positive cells was the highest in CTX treated animals compared to all
the EE treated groups, mice fed with 90 mg/kg EE had the most obvious
DNA fragmentation among all the groups, a hallmark of apoptosis
(Fig. 5). This indicates there may be other apoptotic pathways EE is
involved with and more studies need to be done in order to further
explore the mechanism underlying the apoptosis-inducible effect of EE
in cancer.
Strong chemotherapies come with unpleasant side effects including
inhibition of blood cell production [46,47]. Lowered white blood cell
Z. Deng, et al. Biomedicine & Pharmacotherapy 115 (2019) 108979

Fig. 5. Evaluation of apoptosis in tumor tissues from S180 tumor-bearing mice with different treatment (N = 5/group). (A) Immunohistochemical analysis of Bax and
Bcl-2 expression in tumor tissues (400×). (B) Quantification of Bax and Bcl-2 positive cells in tumor tissues. (C) DNA fragmentation from tumor tissues demonstrated
by agarose gel electrophoresis. *p < 0.05., **p < 0.01., ***p < 0.001.vs Ctrl, #p < 0.05., ##p < 0.01.vs CTX.

count can weaken the immune system of cancer patients, rendering
them susceptible to infections [48]. In this study lowering of white
blood cells or red blood cells was not detected in EE treated animals
(Fig. 6), suggesting EE does not exert immune suppression effect when
it’s orally administered.
In conclusion, our results demonstrated that EE is able to inhibit
tumor growth, as well as induce apoptosis of tumor cells via Bax/Bcl-2
regulation, without significantly reducing blood cells in S180-in-
oculated mice. This study sheds new insights into the potential of EE as
an alternative antitumor drug; however, thorough investigations are
required in the future to identify and purify the active antitumor
component(s) from earthworm due to the complex composition of EE.

Fig. 6. RBC counts (A) and WBC counts (B) in whole blood obtained fromS180 tumor-bearing mice with different treatment (N = 5/group). *p < 0.05 vs CTX,
#p < 0.05 vs Uninoculated.

5
Z. Deng, et al.
Authors’ contribution
LY, WL, and SG designed and directed the study. ZD and XX per-
formed the experiments. ZD, NY, and SM analyzed the data. ZD and SG
prepared the manuscript. YL was responsible for funding acquisition.
Conflicts of interest
None.
Acknowledgments
This research was funded by National Natural Science Foundation of
China [grant numbers 81874030, 81402224]; the Provincial Science
Foundation of Hunan [grant number 2015JJ3139]; the Health and
Family Planning Commission of Hunan Province [grant number
B2016105]; the Administration of Traditional Chinese Medicine of
Hunan Province [grant number 2015116]; and the China Scholarship
Council [grant number 201606375101].
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