Toxicon 180 (2020) 31–38

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Toxicon
journal homepage: http://www.elsevier.com/locate/toxicon

A. crassicauda, M. eupeus and H. lepturus scorpion venoms initiate a strong in

vivo anticancer immune response in CT26-tumor mice model
Neda Amirgholami a, Neda Sistani Karampour a, Ata Ghadiri b, Ahmad Tagavi moghadam c,
Mohamad Ghasemi dehcheshmeh b, Mohammad Hassan Pipelzadeh a, *
a
Toxicology Research Centre, Department of Pharmacology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sceinces, Ahvaz, Iran
b
Department of Immunology, Medical School, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
c
Scorpion Research Centre, Razi Vaccine and Serum Research Institute, Ahvaz, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: In the present in vivo study the anticancer efficacy of the venoms from Androctonus crassicauda, Messobuthus
Scorpion venoms eupeus and Hemiscorpius lepturus scorpions was investigated. In addition, we attempted to clarify whether the
CD3þ immune system is involved in this activity. Initially, the LD50 of the venoms from these scorpions were deter­
CD4þ
mined and their 0.1 and 0.2 LD50 were calculated. The toxicity of 0.1 and 0.2 LD50 was tested on healthy mice by
CD8þ
daily SC administration of these venoms for 12 consecutive days. CT26 cells were inoculated by SC route in
IL-12
IFN-γ BALB/c mice to establish a sold tumor, and ten days later, the mice were treated with 0.1 and 0.2 LD50 doses of
the venoms on daily basis for 12 consecutive days. The tumor volume was measured every 4 days. At day 13, the
tumors from untreated-control and venom-treated groups were removed, weighed, and assessed by histopath­
ological and immunohistochemical techniques. In addition, the levels of mRNA expression of IL-12, IFN-γ and IL-
1β were measured by real-time PCR. All the venoms induced anticancer effects as evidenced by significant in­
hibition in tumor growth; significant increases in inflammatory and CDþ-T cells and expression of mRNA IL-12
and IFN-γ in tumor microenvironment of venom-treated as compared to untreated-control. These findings
demonstrated, for the first time, that sub-lethal doses of the venoms from these scorpions induce their in vivo
anticancer effects by stimulating the immune system. Further studies, specifically designed to identify these
active constituents are recommended.

1. Introduction
Cancer is a multifactorial disease and is the second leading cause of
death worldwide. It is caused by uncontrolled proliferation and cellular
escape from the immune system (Fass, 2008). According to the World
Health Organization report, colorectal cancer is the third most common
cancer and the fourth cause of cancer death (Jemal et al., 2011). The
metastatic cases of this disease are commonly fatal. Despite treatment
with different medications, the course of life of these patients is, on
average, 2 years (Barnett et al., 2002).
Recent treatment strategies attempt to employ safer agents with
fewer side effects than currently existing chemotherapeutics, especially
those derived from natural sources. In line with this strategy, in recent
decades, many studies have shown that bio-toxins are potent anti-cancer
agents, and a combination of these substances has turned them into
potentially unique sources for the production of new anticancer drugs
(Liu et al., 2014). Humans have used substances derived from various
toxins such as bee, snake, and scorpion venoms for more than 100 years
(Manetti et al., 1993; Ma et al., 2017).
Scorpions are one of the oldest organisms that have lived on earth for
more than 400 million years. Over 1700 different species of scorpions
have so far been identified (Chippaux and Goyffon, 2008). Despite the
lethal toxicity of some of the venoms from scorpions, they contain a
variety of compounds with important antibacterial, anti-fungal and
anti-tumor effects and regulate immune system (Joseph and George,
2012). Furthermore, many of these compounds have been successful in

* Corresponding author. Toxicology Research Centre, Department of Pharmacology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sceinces,
Ahvaz, Iran.
E-mail addresses: dr.amirgholami@yahoo.com (N. Amirgholami), n.sistani1386@yahoo.com (N.S. Karampour), ata.ghadiri@hotmail.fr (A. Ghadiri), Taghavi84@
gmail.com (A. Tagavi moghadam), Mo.gh1361@gmail.com (M. Ghasemi dehcheshmeh), mhpipelzadeh@yahoo.com, mhp.pipelzadeh@gmail.com, mhp.
pipelzadeh@ajums.ac.ir (M.H. Pipelzadeh).

https://doi.org/10.1016/j.toxicon.2020.04.001
Received 20 December 2019; Received in revised form 7 March 2020; Accepted 2 April 2020
Available online 8 April 2020
0041-0101/© 2020 Elsevier Ltd. All rights reserved.
N. Amirgholami et al.
inhibiting various cancers, including skin, prostate, breast and brain
tumors under in vivo and in vitro settings (Nabi et al., 2015).
Several studies have found significant success with the use of pep­
tides derived from these toxins for tumor cell cancer. Venom peptides
exhibit high specificity and selectivity towards cancer cells, with effects
on cell proliferation, invasion, migration, and angiogenesis, as well as
modulating immune responses (Manetti et al., 1993Ma et al., 2017).
Despite the wide range of studies on scorpion venoms, only a limited
number of studies specifically assessed the anticancer effects of these
venoms under in vivo settings. A previous in vitro and in vivo study, the
inhibitory potential of BmK scorpion venom on glioma tumor growth
was investigated (Rychahou et al., 2018). The results showed that BmK
induces apoptosis of glioma cell culture, and reduces the growth of
U251-MG tumor in severe combined immunodeficiency mice. In addi­
tion, in a more recent in vitro and in vivo study, the antitumor effects of
the Egyptian scorpion Androctonus amorexi venom in Ehrlich ascites
tumor was evaluated (Salem et al., 2016). The results showed that the
venom from this scorpion produce its cytotoxic effects on tumor cells via
anti-proliferative, apoptotic and anti-angiogenic activities.
Iranian scorpions are divided into two families: Scorpionidae and
Butidae. The medically important Androctonus crassicauda
(A. crassicauda) and Mesoputus eupeus (M. eupeus) belong to Butidae
family (Lourenço, 2001). From A. crassicauda venom, more than 80
peptides have been isolated (Possani et al., 2000). It has been shown that
the venom of this scorpion can induce IL-12 secretion in human
monocytes (Saadi et al., 2015). On the other hand, previous studies have
reported that M. eupeus venom contains many antibacterial bioactive
components (Hmed et al., 2013). While Hemiscorpius lepturus
(H. lepturus) scorpion, the most medically important and dangerous
scorpion in Iran, belongs to Scorpionidae family (Lourenço, 2001).
Studies have shown that the venom of this scorpion also induces IL-12
secretion in isolated human monocytes (Hadaddezfuli et al., 2015).
Moreover, a recent study demonstrated that intratumoural injection of
this venom has significant anti-cancer effects and induces apoptosis
(Moradi et al., 2019).
Although extensive research has been carried out on the venoms
from these three scorpions, no previous study has specifically attempted
to compare and determine the effectiveness of the venoms from these
scorpions under in vivo conditions against the growth of CT26-tumor in
BALB/c mice model. The objectives of this study were, therefore, to
determine whether the venoms from A. crassicauda, M. eupeus and
H. lepturus scorpions can inhibit the growth of CT26 colon cancer cells in
BALB/c male mice model under in vivo conditions. In addition, to assess
whether the immune system has a role in this anticancer activity. For
this purpose, 0.1 and 0.2 LD50 doses of the venoms from these scorpions
were determined and their toxicity was assessed in healthy BALB/c
mice. In the next step, the selected 0.1 and 0.2LD50 doses of the venoms
from these scorpions were assessed for their capacity to inhibit the
growth of CT26-tumors in BALB/c mice model. In order to assess the
possible role of immune system role histopathological, immunohisto­
chemical and mRNA expression of IL-12, IFN-γ and IL-1β in the micro­
environment of the established CT26-tumor between untreated-control
and venom-treated groups were compared.
2. Materials and methods
2.1. Reagents and antibodies
The following antibodies were used as primary antibodies for
immunohistochemical analyses: Anti–CD3 antibody (ab16669),
Anti–CD4 antibody (ab183685), Anti–CD8 antibody (ab217344)
(Abcam, USA) and as secondary antibody: Goat Anti-Rabbit IgG anti­
body (Sigma, USA). For r-PCR: HYBRID-R RNA Mini Kit (GENE ALL,
Korea) the cDNA synthesis 2XRT-PCR PRE-MIX (BIOFACT, Ireland) and
master mix (Ampliqon, Denmark) were used. Specific primers were
designed by Urofine (UK).
32
Toxicon 180 (2020) 31–38
2.2. Cell culture
CT26 cells were purchased from Pasteur Institute of Iran (Tehran).
The cell line was cultured in a RPMI 1640 medium with 10% fetal bovine
serum (FBS) at 5% CO2 and 37 � C to reach the required cell number (4 �
106/mouse) and culture medium was changed every 2–3 days. Cells in
logarithmic phase were used in the tumor development experiments.
2.3. Venom preparation
The lyophilized venom used in this study was obtained from Scor­
pion Research Centre of Razi Institute of Ahvaz. The lyophilized crude
venoms were freshly prepared in normal saline and diluted to the
required concentrations.
2.4. Animals
BALB/c male mice (average 20 g and 6–8 weeks old) were purchased
from Pasture Institute of Iran (Karaj), were fed standard diet, and had
free access to tap water. Animals were maintained under specific
pathogen-free condition. In order to acclimatize, the mice were kept at
25 � C for a week at 12 h light–dark cycle. Ethical code (IR.AJUMS.ABHC.
REC.1398.058)
2.5. Determination of LD50 values of A. crassicauda, H. lepturus and M.
eupeus scorpion venoms
For estimation of LD50 value of the scorpion venoms, serial dilutions
of crude venoms taken from each scorpion were selected on the basis of
100% live to 100% dead mice, with an incremental factor of 1.25. The
venoms were injected via SC route, in 0.1 ml of normal saline per 20 g
body weight, into 4 groups of BALB/c mice (n ¼ 4 each). These doses for
A. crassicauda were: 4.8, 6, 7.5, 9 μg/20 g mouse body weight. While for
H. lepturus venom, the doses were 80,100,125,156 μg/20 g mouse body
weight, and for M. eupeus venom were 19, 23, 29, 37 μg/20 g mouse
body weight. The animals were observed for the first 2 h and then at 6th
and 24th hr after venom administration and the number of dead mice
was counted in each group and time interval and the LD50 was calculated
using the equation suggested by Spearman and Karber and Reed and
Muench statistical method (Hamilton et al.,
P
1977):m ¼ X100 � dn ð r n
2Þ where, m ¼ log LD50, d ¼ log dose
interval, n ¼ number of mice used in each dose; r ¼ number of dead mice
in each dose and X100 ¼ log dosage that caused 100% death.
2.6. In vivo assessment of toxicity of A. crassicauda, M. eupeus and H.
lepturus scorpion venoms by daily SC administration of 0.1 and 0.2 of
LD50 doses for 12 consecutive days in healthy BALB/c mice
In order to investigate whether daily SC administration of 0.1 and 0.2
LD50 doses of the studied scorpion venoms, for 12 consecutive days,
have any adverse toxic effects or cause mortality in healthy BALB/c male
mouse, 28 mice were divided randomly into 7 groups (4 in each): group
C: control normal saline-treated group; groups A1 and A2: recipients of
0.1 and 0.2 LD50 doses of A. crassicauda scorpion venom respectively,
groups M1 and M2: recipients of 0.1 and 0.2 LD50 doses of M. eupeus
scorpion venom respectively; and groups H1 and H2: recipients of 0.1
and 0.2 LD50 dose of H. lepturus scorpion venom respectively. Further­
more, in order to ascertain absence of any delayed toxicity, such as
mortality, change in water and food intake and body weight, or any
abnormal behaviour, the mice were further observed for 50 additional
days after the last dose of venoms administration.
N. Amirgholami et al.
2.7. Establishment of CT26-tumor and in vivo growth inhibition
assessment of the scorpion venoms
In order to establish CT26-tumor, 36 female BALB/c mice were
inoculated subcutaneously with 4 � 106 CT26 cells in a volume of 0.1 ml
of serum free RPMI medium in the right flank. After 10 days, when the
tumors became palpable, the mice were randomly divided into six
groups of 6 mice each: group C: control-normal saline-treated; groups
A1and A2: recipients of 0.1 and 0.2 LD50 doses of A. crassicauda scorpion
venom respectively, groups M1 and M2: recipients of 0.1 and 0.2 LD50
doses of M. eupeus scorpion venom respectively; and groups H1 and H2:
recipients of 0.1 and 0.2 LD50 dose of H. lepturus scorpion venom
respectively. The venoms were administered subcutaneously in volumes
of 0.1 ml for 12 consecutive days. Tumor length and width were
measured every 4 days using vernier caliper and the volume of the tu­
mors were calculated using the equation: 0.5 LW2, where L and W are
the longest and shortest diameter of tumor respectively (Salem et al.,
2016; Hu et al., 2015). Percentages of inhibition in growth of the tumor
at each time point and after excision at day 13, one day after last day of
treatment, were calculated using the following equation:V ¼
ðVt VcÞ x100
, where Vt and Vc are volume of the tumor in venom-treated
Vc
and untreated-control groups respectively.
At day 13 (one day after last dosing), the animals were sacrificed by
neck dislocation, and the tumors tissues were removed and weighed.
The collected samples were subjected to histopathological, immuno­
histochemical and real-time PCR analysis. Percentage of reduction in the
weight of the excised tumor, at day 13, was calculated using the
WcÞ x100
following equation:V ¼ ðWt Wc , where Wt and Wc are weight of
excised tumor in venom-treated and untreated-control groups
respectively.
2.8. Haematoxylin and eosin staining
The collected CT26-tumor samples from untreated-control and
venom-treated groups were fixed in formal saline and then embedded in
paraffin. Sections (4.5 μm) were cut from the paraffin blocks and stained
with haematoxylin and eosin using standard staining protocol (Bancroft
et al., 1996). The stained sections were examined microscopically (Leica
DM4000B, Germany) for the presence of necrosis and inflammatory cells
infiltration. The examiner was blinded to the origin of each sample.
2.9. Immunohistochemical analysis of CD3þ, CD4þ, CD8þ cells
Immunohistochemical detection of CD3þ, CD4þ, CD8þ cells was
performed as described previously (Goudarzi et al., 2017). Briefly,
paraffin tumor sections (4 μm thick) were de-waxed, re-hydrated in a
graded series of ethanol, and equilibrated in PBS for 5 min. Endogenous
peroxidase was removed by incubation in a peroxidase inhibitor for 30
min. Sections were incubated at 4 � C overnight with anti-CD3þ, CD4þ,
CD8þ Ab (1:100 dilution) after blocking with diluted goat serum. Cells
incubated with PBS were used as negative control. After three times
washing in PBS, the cells were incubated for 30 min in secondary affinity
purified biotinylated goat anti-rabbit IgG antibody (1:2000). Immuno­
staining was performed using a DAB kit (3, 30 diaminobenzidine, DAKO)
and manufacturer’s instructions were followed (Abcam,USA). Stained
sections were examined using light microscopy (Leica DM4000B, Ger­
many). Ten areas were randomly selected and the stained cells were
counted at a magnification of � 400. The percentage of CD3þ, CD4þ,
CD8þ was graded on a scale of 1–4: 0–10% of positive cells were scored
as 1; 11–50% as 2; 51–80% as 3; and 81–100% as 4. The total score was
calculated as the product of intensity and percentage scores; 0 was
defined as negative ( ), 1–2 as weak (þ), 3–4 as moderate (þþ), and >4
as strong (þþþ) (Goudarzi et al., 2017).
33
Toxicon 180 (2020) 31–38
2.10. Real-time PCR analysis
Real-time quantitative PCR was performed to determine the
expressionLevel of IL-12, IFN-γ and IL-1β. Total RNA was extracted from
frozen tissue samples using the HYBRID-R RNA Mini Kit according
manufacturer’s instructions (GENE ALL, Korea). The concentration and
purity of RNA were determined using Nano-100. Complementary DNA
(cDNA) was synthesized using the cDNA synthesis 2XRT-PCR PRE-MIX
(BIOFACT, Ireland). Obtained cDNA (2 μg) was amplified using master
mix (Ampliqon, Denmark) and LightCycler® 96 Real-Time PCR System
(F. Hoffmann-La Roche Ltd, Germany). Specific primers for IL-12, IFN-γ,
and IL-1β were from UROFINE (UK). Expression levels of the target
genes were analyzed using the ΔΔCT comparative threshold method.
The glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene was used
as an internal control. Primers were as following:
-IL-12 forward: ATCGATGAGCTGATGCAGTC.
IL-12 reverse: GGTTCATTTTCACTCTGTAAGG.
IFNγ forward: AGTATTGCCAAGTTTGAGGTC.
IFNγ reverse: CTTTTCCGCTTCCTGAGG.
IL-1β forward: CAAGCAACGACAAAATACCTG.
IL-1β reverse: GCTTGCGATCCACACTCTC.
2.11. Statistical analysis
The data were processed using Microsoft Graph Pad Prism 8
(GraphPad Prism Inc., USA) and Excel software. Collected data were
expressed as mean � SEM. ANOVA test, followed by Tukey–Kramer test
for multiple comparisons. P < 0.05 was considered as indicative of
significance for all the comparisons made. Chi-square test was used for
analyzing the difference of mean values between groups in immuno­
histochemical analysis.
3. Results
3.1. LD50 values for the venoms from A. crassicauda, M. eupeus, and H.
lepturus venoms
The results of the calculated 1, 0.1 and 0.2 LD50 values of the studied
venoms are shown in Table 1. The lowest LD50 concentration was
associated with the venom from A. crassicauda with a value of 6.5 μg/20
g of mouse body weight, corresponding to 0.325 mg/kg. While the
highest LD50 concentration value was measured for H. lepturus scorpion
venom, being 16-fold higher than A. crassicauda venom (107 μg/20 g of
mouse body weight, corresponding to 5.35 mg/kg) (Table 1).
3.2. In vivo toxicity assessment results of 0.1 and 0.2 LD50 doses of A.
crassicauda M. eupeus and H. lepturus scorpion venoms in healthy BALB/c
mice
Daily administration of 0.2 LD50 dose of H. lepturus scorpion resulted
in death of all the mice after 7 days. Therefore, this dose group was
omitted from the study. All animals in other groups did not show any
abnormal symptoms as compared to the control normal saline-treated
group. Moreover, after 50 days of the last dose, none of the animals
died and did not show any significant differences in weight gain or
manifested any abnormal behaviour.
Table 1
Summary of the mean concentration values (in μg) of 1, 0.1 and 0.2 LD50/20 g of
mouse body weight of the venoms from A. crassicauda, M. eupeus and H. lepturus
scorpions n BALB/c male mice.
Scorpion species 1 LD50 0.1 LD50 0.2 LD50
A. crassicauda 6.5 0.65 1.3
M. eupeus 12.5 1.25 2.5
H. lepturus 107 10.7 21.4
N. Amirgholami et al.
3.3. Effect of A. crassicauda, M. eupeus and H. lepturus scorpion venoms
on tumor growth in CT26-tumor–bearing mice
The volume of the established tumors on the 10th day after inocu­
lation of CT26 cells was 33 � 2 mm3. All the venoms produced signifi­
cant inhibition in growth of tumors, being evident as from day 4
onwards after their administration (Fig. 1). At day 13 (day 22 from
inoculation), all venoms produced highly significant inhibition (P <
0.001) in the increases of tumor volume when compared with control
tumor-bearing group (Table 2). The average volume of control group, at
day 13, was 2842 mm3. While the average tumor volume for 0.1 LD50
doses of A. crassicauda, M. eupeus, and H. lepturus were 225, 416 and 235
mm3 respectively. At 0.2 LD50 dose, the venoms from A. crassicauda and
M. eupeus scorpions produced higher percentage of growth inhibition.
Maximum inhibition was found with 0.2 LD50 dose of A. crassicauda,
producing 98% inhibition in tumor growth compared with non-treated
control group (Table 2).
Similarly, the tumor weights in all venom-treated groups were highly
significant lower (P < 0.001) than that of the control tumor-bearing
group (Table 2, Fig. 1). The average weight for control group was 1.4
� 0.6 g. In parallel with the level of inhibition of increase in tumor
volume, the maximum inhibition in weight was associated with
A. crassicauda venom producing 85% inhibition when compared with
control non-treated group.
3.4. Histopathological findings
As shown in Fig. 2, venom-treated groups showed sings of necrosis/
apoptosis as evidenced by shrinkage (pyknosis) of the nucleus, in
addition to aggregated denatured cytoplasm. The regions of infiltration
of inflammatory cells were greater in A. crassicauda and M. eupeus
groups. For further assessment of the possible mechanism of this inhi­
bition and the role of immune system, we carried out immunohisto­
chemical analysis on all the collected tumor samples.
4000
3500 Control
H studied venoms from these scorpions, when administered by SC route, at
3000 A1
***
Tumor volume (mm3)
A2
2500 M1
M2
2000
***
1500
1000 ***
500
0 assess whether similar immune stimulating action does occur under in
10 14 18
Days after tumor inoculation
Fig. 1. Effect of A. crassicauda, M. opeus, and H lepturus scorpion venoms on the
trend of increase in volume of the established CT26-tumor in BALB/c mice
compared to control untreated group. 4 � 106 cells/100 μl were implanted
subcutaneously at the right flank of the mice 10 days before administration of
the venoms. A1 and A2 (0.1 and 0.2LD50 A. crassicauda), M1 and M2 (0.1 and
0.2 LD50 M. eupeus) and H (0.1LD50 H lepturus). ***P < 0.001 between
untreated-control and venom-treated groups. All data are expressed as mean �
SEM (n ¼ 6 in each group).
Toxicon 180 (2020) 31–38
3.5. Immunohistochemical findings for detection of CD3, CD4 and CD8 T
cells in CT26-tumor microenvironment
The immunohistchemical analysis revealed an extensive infiltration
with CD3þ, CD4þ and CD8þ T cells in venom-treated groups compare to
untreated BALB/c mice group (Table 3 and Fig. 3). The highest scores of
CD8þ- and CD4þ-T cells were associated with A. crassicauda scorpion
venom. Fig. 3 shows a photo-microscopic illustration of the related
specific biomarker-stained samples for detection of the CD3þ-, CD4þ-
and CD8þ-T cells. The overall pattern of prevalence of all CDþ-T cells
was similar, being most prevalent for the group received 0.2LD50 dose of
A. crassicauda venom (Table 3 and Fig. 3).
3.6. IL-12, IFN-γ and IL-1β mRNA expression profile in BALB/c CT26-
tumor microenvironment after12 daily treatment with A. crassicauda, M.
eupeus and H. lepturus scorpion venoms
Considering the critical role of cytokines in inducing and executing a
nti-tumor immunity, we explored the presence of IL-12, IFN-γ, IL-1β in
BALB/c mice CT26-tumors microenvironment of untreated-control and
venom-treated samples by real-time quantitative PCR. The level of IL-
12-mRNA expression was significantly upregulated compared with
that of untreated-control group (P < 0.001) (Fig. 4 A). Maximum
upregulation in gene expression of IL-12 belonged 0.2LD50 doses of
M. eupeus scorpions venoms (64 fold) followed by A. crassicauda (43.5
fold), while those for H lepturs was 3.3 fold (Fig. 4A). While maximum
increase in gene expression in IFN-γ was associated with tumor micro­
environment of H lepturus-treated group (Fig. 4B). On the other hand,
gene expression of IL-1β was highest in tumor microenvironment treated
with 0.1 LD50 dose of A. crassicauda (3.29 fold) (P < 0.01), but was not
significantly different, as compared to untreated-control group, after
treatment with 0.1 LD50 dose of H lepturus and 0.2 LD50 dose
A. crassicauda (Fig. 4C).
4. Discussion
The present in vivo study was designed to achieve two main objec­
tives: Firstly to determine the capacity of the venoms from A crassicauda,
M eupeus and H. lepturus scorpions to inhibit the growth of the estab­
lished tumor in CT26-tumor mice model. Secondly to clarify the role of
the immune system in this growth inhibitory activity. Regarding the first
objective, the morphometric results showed that treatment with the
0.1 and 0.2LD50 doses highly significantly inhibited the growth of CT26-
tumor. As for the second objective, histopathological, immunohisto­
chemical and real time PCR investigations provided evidence in support
of the hypothesis that the observed growth inhibitory action is mediated
by increasing the recruitment of inflammatory and inducing necrosis
increasing CDþ-T cells and upregulating the expression of IL-12, IFN-γ
and IL-1β in CT26-tumor tissue.
A recent in vitro study demonstrated that the venoms from H. lepturus
and A. crassicauda scorpions promoted the secretion of IL-12 in human
polymorphic monocytes (Saadi et al., 2015; Hadaddezfuli et al., 2015).
These observations prompted the present investigations, and aimed to
vivo settings? The results showed that all the venoms from these selected
22
scorpions have strong local immune stimulating within the microenvi­
ronment of CT26-tumor. These observations come in agreement with
recent reports which advocate scorpion venoms as potential sources in
suppression of different types of cancers (Ding et al., 2014). Moreover,
there is ample evidence that show a close association between
dysfunction in the immune system with progression and negative
prognosis in patients with cancer (Lippitz, 2013).
Our initial experiments aimed to allocate the LD50 concentrations of
the selected venom scorpions. This was an essential step for the next
stage of our investigations and has been used for achieving various
34
N. Amirgholami et al. Toxicon 180 (2020) 31–38

Table 2
CT26-tumor volume and weight and percentage of growth inhibition in control-untreated as compared to venom-treated BALB/c mice groups, following the estab­
lishment of the tumor, after 12 daily SC administrations of 0.1 and 0.2 LD50 doses of the venoms from A. crassicauda, H. lepturus and M. eupeus scorpions.
Parameter Control A. crassicauda M. eupeus H. lepturus

0.1LD50 0.2LD50 0.1LD50 0.2LD50 0.1 LD50

Volume (mm3) 2842 � 950*** 225 � 90 65 � 15 416 � 266 89 � 48 235 � 80

(% of inhibition) * (92) (98) (85) (96) (91)
Weight (g) 1.4 � 0.6*** 0.3 � 0.1 0.2 � 0.1 0.5 � 0.1 0.2 � 0.1 0.3 � 0.1
(% of inhibition) (78) (85) (64) (85) (78)

***P < 0.001 between untreated-control and venom-treated groups.

Fig. 2. Photomicroscopic illustrations of the histopathological findings of tumor samples from (C) control untreated CT26-bearing mice after 12 consecutive days of
SC administration as compared to venom-treated groups: M2: 0.2 LD50 M. eupeus scorpion venom; A2: 0.2 LD50 A. crassicauda scorpion venom, and H: 0.1 LD50
H. lepturus scorpion venom (H&E x400). Filled arrow point to the nucleus in each group; the venom-treated undergone pyknosis (shrinked), arrow heads point to
aggregated denatured cytoplasm and yellow arrows point to inflammatory cells. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)

The results from our volume and weight of tumor growth assess­
Table 3
ments showed that all the venoms from these scorpions were potent
Immunohistochemical scores for CD3þ-, CD4þ- and CD8þ-T cells in CT26-tumor
inhibitors of growth of CT26-tumor in BALB/c mice model. The per­
microenvironment in BALB/c mice after 12 daily SC administrations of 0.1 and
0.2LD50 doses of the venoms from M. eupeus and A. carssicauda and H lepturus centage of inhibition in both the volume and weight of tumor ranged
scorpions compared to untreated-control group. from 98 to 85% for 0.2LD50 dose of the venom from A. crassicauda
scorpion, to 85, and 64% for the 0.1LD50 dose of the venom from M.
CDþ-T cell Marker Score
eupeus scorpion respectively. These data show that the venom from these
CD8þ CD4þ CD3þ scorpions are more effective than those reported for A. amoreuxi scor­
Group – þ þþ – þ þþ – þ þþ pion against Ehrlich ascites carcinoma and for Buthus martensii Karsch
Control 10 0 0 10 0 0 10 0 0 scorpion against H22 cells orthotopic transplantation tumor mouse
A1 2 6* 2 2 6** 2 0 2 8 model in which the volume of these tumors was reduced by 25.4 and
A2 1 2 7* 0 4 6*** 0 10*** 0 30.7% respectively (Hu et al., 2015; Chen et al., 2016). On the other
M1 2 8** 0 2 8** 0 4 6* 0 hand, treatment with BmK venom, the tumor volume was nearly 1/8 of
M2 2 8** 0 2 4* 4 0 4 6
H 0 10*** 0 4 6* 0 6 4* 0
that of control and the tumor weight decreased by 50% after 21 days of
treatment (Wang and Ji, 2005). Furthermore, in a recent study (Moradi
- ¼ absence, þ ¼ presence of the related to a specific CD marker. A1 and A2: et al., 2019) the tumor growth rate inhibition after intratumoral injec­
0.1and 0.2LD50 doses of A. crassicauda venom, M1 and M2: 0.1 and 0.2 LD50
tion of 0.1 LD50 of H lepturus scorpion venom in CT26-bearing mice was
doses of M. eupeus venom, and H: 0.1 LD50 dose of H. lepturus venom. *P < 0.05,
41%, which is much less than our 91% inhibition achieved by SC route
**P < 0.01 and ***P < 0.001 between untreated-control and venom-treated
groups.
and after the same dose. These findings suggest that these venoms are
effective in inducing their anticancer effects when administered
subcutaneously.
objectives in different in vivo and in vitro toxicological studies (Hu et al.,
The result from the morphometric observation was unexpected and
2015; Salem et al., 2016; Chen et al., 2016; Wang and Ji, 2005). One
showed a high degree of inhibition of growth, especially when achieved
probable reason may be related to their anticipated differences in the
under in vivo settings. These observations are, therefore, encouraging
extraction process. For this reason, in order to ensure consistent potency
and open the door for more detailed investigations to characterize the
of the selected venoms, we used the same batches throughout this study.
active constituents and develop them into novel anticancer agents.
Despite these precautions and employment of standard experimental
Although we did not carry out detailed molecular investigations on the
conditions, the allocated 0.2LD50 dose of the venom from H lepturus
possible mechanisms underlying this anticancer activity, previous
scorpion, during the toxicity assessment experiments, caused the death
studies have shown that the venom from A. crassicauda inhibited human
of all the mice by day 7. These findings suggest that a 24-hr observation
neuroblastoma cell line by arresting cell growth in the S-phase and
allocated for determination of the LD50 concentrations for the venom
induced apoptosis (Zargan et al., 2011). In addition, a previous in vitro
from H. lepturus scorpion is not robust enough, simply because this
report showed that the venom from H lepturus scorpion induced its
venom seems to have delayed toxic manifestations (Pipelzadeh et al.,
cytotoxic action by apoptosis and necrosis in HT29 cells, mitochondria
2006).

35
N. Amirgholami et al. Toxicon 180 (2020) 31–38

Fig. 3. Photomicroscopic illustrations of the immunohistochemical analysis results of CT26-tumor samples taken from BALB/c mice, for detection of CD3þ, CD4þ
and CD8þ T cells in venom-treated groups as compare to untreated-control. A1 and A2: 0.1 and 0.2LD50 doses of A. crassicaud scorpion venom, M1 and M2: 0.1 and
0.2 LD50 doses of M. eupeus scorpion venom, and H: 0.1 LD50 dose of H. lepturus scorpion venom ( � 400).

A B

70 6
IFN- (fold change)
Gene expression of IL-

Gene expression of

60 5
12 (Fold change)

50 4
40
3
30
20 2
**
10 ** 1
0 0
C A1 A2 M1 M2 H C A1 A2 M1 M2 H
Scoprion venom Scorpion venom

***
Gene expression of IL-

3.5
1 (fold change)

3
2.5 ***
2 ***
1.5
1
0.5
0
C A1 A2 M1 M2 H
Scorpion venom

Fig. 4. Fold increases in gene expression of (A) IL-12, (B) IFN-γ and (C) IL-1β cytokines in the tumor microenvironment after SC administration of scorpion venoms
for 12 consecutive days in CT26 bearing BALB/c mice. A1 and A2: 0.1 and 0.2LD50 doses of A. crassicauda scorpion; M1 and M2: 0.1 and 0.2 LD50 doses of M. eupeus
scorpion, and H: is 0.1 LD50 dose of H. lepturus scorpion. **P < 0.01, ***P < 0.001 in comparison with untreated-control.

dysfunction and ROS over production, which play important roles in the
process of cell injury and death (Pipelzadeh et al., 2015). Moreover, a
recent in vivo study showed that intratumoral administration of the
venom from H lepturus scorpion induced its anticancer effect through
increment of mRNA expression of Bax, Trp53 and caspase-3 apoptosis
markers in the tumor microenvironment (Moradi et al., 2019). Put
together, these observations propose the notion that these scorpion
venoms are potential sources for novel anticancer agents that act by a
variety of mechanisms leading to caner cell death.
Furthermore, histopathological examinations demonstrated an
extensive sign of necrosis within the tumor tissue in venom-treated mice.
A recent study has proposed that tumor shrinkage occurs as a

36
N. Amirgholami et al.
consequence of cell cycle arrest, apoptois and necrosis as well as by
activating the immune system (Salem et al., 2016). In order to clarify
and correlate these histopathological findings, immunohistochemical
staining was carried out. These analyses were specifically designed for
detection of CD3þ-, CD4þ- and CD8þ-T cell subtypes. The results
showed that, in contrast to tumor tissues in control untreated group,
which showed low count of positive CD-T cells, their the prevalence in
all venom-treated counterparts were significantly higher.
One of the major mechanisms that enables cancer escape results from
defect in the IL-12- IFNγ-HLA-DR (or HLA-class II) axis. This axis is
central in immunodetection and immunological eradication of cancer
cells by CD4þ-T lymphocytes (Trinchieri, 2003). IL-12 is recognized to
play a central role in TH-1 response and regulates IFN-y production by
CD-T cells (Manetti et al., 1993). In addition, IL-12 enhances the gen­
eration and activation of cytotoxic T-lymphocytes. More importantly, it
promotes the production of IFN-γ by CD4þ-, CD8þ-T cells and NK cells.
In turn, IFN-γ, not only inhibits the production of immunosuppressive
factors, it also plays an essential role in MHC expression as a central step
in immunodetection (Beatty and Paterson, 2001; Harty et al., 2000). The
combined results from our immunophenotyping and real-time PCR ex­
periments showed that, in concordance with the increase in CD4þ- and
CD8þ-T cells, the expression of IL-12 and IFN-γ were increased signifi­
cantly in a similar manner. The results of the present study revealed that,
in correlation with the degree of inhibition in tumor growth activity, the
maximum increase was associated with IL-12 after the administration of
all the venoms, but being higher after administration of 0.2 LD50 doses of
M. eupeus and A. carssicauda scorpion venoms. These findings suggest
that activation of the immune system by these venoms seems to play an
important role in prevention of progression of the CT26-tumor in mice.
Furthermore, while exogenously administered IL-12 was found to
induce excessive toxicity (Colombo and Trinchieri, 2002), it was also
found to be less effective than endogenous IL-12 in cancer immuno­
therapy (Lippitz, 2013). Put together, these observations support the
notion that the venoms from M. eupeus and A. carssicauda and H lepturus
scorpions, besides their intracellular disruptive mechanisms, seem to
produce their anticancer effects by restoring the functional activity of
IL-12- IFNγ-HLA-DR axis (Lippitz, 2013).
The results for IL-1β mRNA expression after treatment with these
studied scorpion venoms were variable and did not correlate with the
changes in the expression of mRNA IL-12 and IFN-γ. The level of
expression of IL-1β was significantly higher after treatment with 0.1
LD50 of A. crassicauda scorpion venom, but was not significant after H
lepturus scorpion venom. A similar observation has been reported for the
peptides derived from Tityus discrepans scorpion, where a high level of
expression of IL-1β mRNA was measured after exposure of human im­
mune cells to low concentration of Centruroides noxious toxin 5, but was
not significant after exposure to discrepin, another peptide from this
scorpion (Corzo and Espino-Solis, 2017). These discrepancies may be
due to the regulatory interactions between immune cells and/or their
soluble mediators (Manetti et al., 1993). Furthermore, different cellular
signaling pathways have been reported to operate in response to varying
levels of IL-1 β leading to genotoxic damage, cell apoptosis or cell
growth of estrogen-dependent tumors (Roy et al., 2006). In present
study, the level of IL-1β was non-significantly lower than in the control
with the venom from H lepturus scorpion, but significantly increased by
2.5-fold with M eupeus scorpion. Furthermore, these finding come in
contrast to a previous report, in which the level of IL-1β was found to
increase by 10 and 23-fold in the serum of human subjects accidental
envenomed with M eupeus or H lepturus scorpions respectively (Jalali
et al., 2011). One plausible explanation for these discrepancies is that
these venoms may induce different immune responses in CT26
mice-model as compared to those in scorpion-envenomed human
subjects.
What are the clinical implications of the present study? Although the
results from our present study are encouraging, however, extrapolating
these experimental findings into clinical settings needs to be carefully
37
Toxicon 180 (2020) 31–38
guarded. Since uncontrolled cellular proliferation is considered as a
hallmark for progression of cancer, therefore, has been considered as a
target for potential anticancer agents. The results from the present study
revealed that the selected scorpion venoms, at sub-lethal doses, from
M. eupeus and A. carssicauda and H lepturus drastically inhibited the
growth of CT26-tumor in BALB/c mice. This action seems to be partly as
a consequence of activation of the immune system, specifically by
increasing production of pro-inflammatory cytokines namely IL-12 and
IFN-γ which result in recruitment of CD8þ-, CD4þ- and CD3þ-T cells. On
the other hand, although exposure to scorpion venoms can produce toxic
effects, in this study we demonstrated that the studied scorpion venoms,
at sub-toxic doses, by activating the immune system, and without pro­
ducing any serious complication, contain active constituents with anti­
cancer potential which deserve to be studied in future investigations.
Ethics statement
This project was approved by the Ethics and Research committee of
Ahvaz Jundishapur University as part of Ph.D. project by Mrs Neda
Amirgholami (Research project number: TRC-9823).
Declaration of competing interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Neda Amirgholami: Investigation, Writing - original draft, Formal
analysis. Neda Sistani Karampour: Funding acquisition, Writing - re­
view & editing. Ata Ghadiri: Methodology, Validation, Formal analysis.
Ahmad Tagavi moghadam: Methodology, Writing - review & editing.
Mohamad Ghasemi dehcheshmeh: Resources. Mohammad Hassan
Pipelzadeh: Supervision, Project administration, Writing - review &
editing.
Acknowledgements
The data presented in this communication are taken from the results
collected from N.A. Ph.D. research project. The authors gratefully
acknowldege the useful scientific commnets of Professor Abdul-Rahman
Dezfulian and Dr Mohammad-Amin Behmanesh on the histological
findings. Financial support by the Deputy of Research Affairs of Ahvaz
Jundishapur University of Medical Sciences is gratefully acknowledged.
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