CANCER BIOTHERAPY AND RADIOPHARMACEUTICALS

Volume 33, Number 2, 2018

ª Mary Ann Liebert, Inc.
DOI: 10.1089/cbr.2017.2400

Samsum Ant Venom Exerts Anticancer Activity

Through Immunomodulation In Vitro and In Vivo

Jameel Al-Tamimi,1,* Abdelhabib Semlali,2,* Iftekhar Hassan,1 Hossam Ebaid,1 Ibrahim M. Alhazza,1
Syed H. Mehdi,3 Mohammed Al-Khalifa,1 and Mohammad S. Alanazi2

Abstract
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Samsum ant venom (SAV) is a rich repertoire of natural compounds with tremendous pharmacological prop-
erties. The present work explores its antineoplastic activity in different cell lines followed by its confirmation
in vivo. The cell lines, HepG2, MCF-7, and LoVo showed the differential dose-dependent antineoplastic effect
with an increased level of significant cytokines, including Interleukin (IL)-1b, IL-6, and IL-8 and transcription
factor, Nuclear factor-kappa B (NF-jB). However, the venom was more effective on HepG2 and MCF-7 cells
than LoVo cells. Furthermore, the extract was administered to four groups (n = 8) of rats. Group I was taken as a
control without any treatment, whereas group II received CCl4 (1 mL/kg) for induction of mild hepatoma.
Group III was given 100 lg/kg of SAV twice a week for 1 month. Group IV was pretreated with the CCl4 (like
group II) followed by dosing with SAV (100 lg/kg) for 2 months as per the authors’ prestandardized dosing
schedule. Intriguingly, the rats of group IV demonstrated significant decrease in key cytokines, IL-1b and IL-6,
as well as the transcription factors, including Tumor Necrosis Factor-alpha (TNF-a), NF-jB, and Inhibitor-
kappa B (I-jB) as compared with group II. Furthermore, increase in IL-10 and First apoptosis signal (FAS) in
the same group confirmed that SAV induces apoptosis at the given dose through immunomodulation leading to
enhanced tumor killing in vivo. Hence, SAV has an excellent antineoplastic activity that can be directly used to
treat certain types of cancer. Moreover, study of its ingredients can pave ways to design novel anticancer drugs.
However, further in-depth investigation is required before its clinical trials.

Keywords: cancer, cytokines, immunomodulation, samsum ant, toxin

Introduction Nevertheless, lots of literature entails that some of the bio-

active components having various structural repertoire present

A nts are considered as the chemical factories as their

venom may contain over 75 different types of chemical
compounds.1–3 Pachycondyla is a genus having a large group
of ants in which most of them are geographically distributed
in the tropical and subtropical regions. Among them, the
Samsum ant (Pachycondyla sennaarensis) is one of the most
prominent members of the genus. The ant has been held re-
sponsible for the induction of anaphylaxis around the world.4
The venom from these social insects results in mild local re-
actions, including acute pain, inflammation, itching, and irri-
tability in humans. However, some severe allergic reactions
have also been linked to their venom depending on the dose of
the venom and individual’s hypersensitivity toward the same.5
in the venom of many animals/insects have tremendous ther-
apeutic potentials against the different forms of disease and
health crisis.6–11 Moreover, many of their components have
shown high specificity and potency to many established mo-
lecular targets.12 The aqueous solutions of the venoms have
been reported to have various enzymatic and nonenzymatic
proteins, free amino acids, and small biologically active
compounds, such as histamine, 5-hydroxytryptamine, acetyl-
choline, norepinephrine, and dopamine.13 Among many oth-
ers, the samsum ant venom (SAV) has many pharmacological
properties, such as regulation of local inflammation, pain,
triggering an immune response, and enhancing the healing
process of the damaged tissues especially in the liver.14,15

1
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
2
Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia.
3
Department of Geriatrics, Donald W Reynolds Institute of Aging, UAMS Little Rock, Little Rock, Arkansas.
*These authors’ contributed equally to this work.

Address correspondence to: Iftekhar Hassan; Department of Zoology, College of Science, King Saud University; P.O. Box 2455, Riyadh
11451, Saudi Arabia
E-mail: iftekharhassan2002@gmail.com; ihassan@ksu.edu.sa

65
 66
Interestingly, this ant venom has been documented to have
strong antineoplastic effect against the breast cancer cells in
a dose- and time-dependent manner without affecting the
neighboring healthy cells.16,17
Despite significant scientific advancements in cancer
management, cancer has been one of the leading causes of
clinical deaths after cardiovascular diseases globally. Today,
the traditional cancer treatment is based on chemotherapy,
radiotherapy, and both as combined modalities. All of these
treatment strategies are either effective against the particu-
lar type of cancer until a particular time, or their serious
side effects force discontinuation of the chemotherapy.18,19
Earlier studies have established the antioxidant and immune-
boosting effects of SAV in vitro and in vivo.16,20
The present study is aimed to confirm if these proper-
ties of SAV attribute in antineoplastic activity through
immunomodulation against the cell lines of three primary
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forms of breast, liver, and colon cancers, such as MCF-7,
HepG2, and LoVo. Also, the authors were interested in
investigating the potential anticancer activity of SAV in
chemical-induced hepatoma in rats.
Materials and Methods
Chemicals
All the chemicals and general reagents, including the cell
culture media were purchased either from Sigma-Aldrich
Chemical Company, USA, or Thermo Scientific (Gibco),
USA. The rest of all other chemicals used were of analytical
grade bought from BDH (United Kingdom) and Merck
(Germany) brands.
Collection and preparation of the SAV
Colonies of samsum ants (including around 2000–2500
workers, with a brood of all stages and multiple queens) were
collected from Al Ehsaa Governorate, East Riyadh (Kingdom
of Saudi Arabia). These collected nests were kept in the in-
sectarium of the Department of Zoology, College of Sciences,
King Saud University (Riyadh). The ants were housed in spe-
cially designed plastic nest bottles with the sufficiently large
surface area (45 · 30 · 18 cm) until venom extraction. The sting
apparatus was removed by grabbing with the help of forceps at
the last segment of the abdomen followed by their detachment
gently. The venom was collected by pinching their venom
gland, and the venom was stored in a small glass tube.20
The samples were homogenized mildly followed by
centrifugation at 1500 rpm for at least 2 min.21 Moreover,
the supernatant thus received was kept at -20C until their
treatment with the cell lines.
Cell culture and treatment
In the present study, there were three most common forms
of human cancer cell lines, breast cancer (MCF-7) cells, liver
cancer (HepG2) cells, and colon cancer (LoVo) cells. All of
them were purchased from ATCC, USA. The cells were
treated with SAV at different concentrations (0.1, 1, 10, 100,
and 1000 ng/mL). All the cells were cultured separately with
standard protocols with medium containing (Dulbecco’s
modified Eagle’s medium [DMEM]), 10% fetal bovine serum,
100 U/mL penicillin, as well as 100 lg/mL streptomycin at
AL-TAMIMI ET AL.
37C with 5% CO2, 95% air, and complete humidity. Once
the cells reached 90% confluence, they were detached using
0.05% trypsin/ethylenediaminetetraacetic acid and counted
using Trypan Blue staining by hemocytometer.
Animal treatment
For confirmation of cell line-based in vitro studies, in vivo
study was also designed in parallel. The experimentation
on animals under the study was approved by the Animal
Ethics Committee of the Zoology Department in College of
Science at King Saud University, Riyadh, KSA. CCl4 was
chosen to induce liver cirrhosis and mild hepatoma in the
rats.22,23 For this, 32 healthy Swiss albino rats (adult,
150 – 20 g) were purchased from Central Animal House
(College of Pharmacy, KSU, Riyadh). They were housed
in the large cages under ethically approved controlled condi-
tions (temperature at 25C – 3C, 12-h day–12-h night cycle
and clean water with pellet diet ad libitum).
They were divided into four groups taking eight rats in
each group (n = 8). The group I was negative control group
without any treatment. Group II received a single dose of
1 mL/kg CCl4 in corn oil (1:1 volume) followed by two
additional half of the same dose once a week through an
intraperitoneal (IP) injection for induction of hepatoma. The
occurrence of hepatoma was confirmed by assessment of
liver and kidney function tests, and histopathology (data not
shown). Group III was administered with 1 mL of 100 lg/kg
of SAV twice a week for 1 month by IP route. Group IV was
pretreated with the CCl4 (1 mL/kg) like group II. After a
month, the rats of this group were administered IP with
1 mL of 100 lg/kg SAV twice a week for 2 months as per
their prestandardized dosing schedule.9,20,22,23
After completion of the treatment, the animals were sacri-
ficed by cervical dislocation on the next day to the last dose.
Their liver was stored in a suitable quantity of RNA stabili-
zation reagent (RNAlater, Qiagen, Germany) at -80C for
real-time polymerase chain reaction (RT-PCR) and ELISA
analysis. Besides, their serum was collected from freshly
drawn blood of the animals also stored for analysis.
Ethical approval
The current study did not involve any endangered or
protected species. Regarding the experimental animals,
all procedures were conducted by the standard protocols
outlined in the guidelines for the care and use of laboratory
animals by the Committee for the Purpose of Control and
Supervision of Experiments on Animals and the National
Institutes of Health, USA. Also, the study protocol (care
and handling of experimental animals) was approved by
the Animal Ethics Committee of the Department of Zo-
ology in the College of Science at King Saud University,
Riyadh.
MTT assay for cell viability
MTT assay in the present study was performed after 24 h
of treatment of SAV with the cell lines in a 96-well culture
plate with the standard protocol. MTT solution was prepared
at 1 mg/mL in phosphate buffered saline (PBS) that was
filtered through a 0.2 lm filter. Then, 50 lL of MTT plus
200 lL of DMEM (without Phenol Red) were added to each
 ANTICANCER ACTIVITY OF SAMSUM ANT VENOM
well, except the cell-free blank wells. The cells were incu-
bated for 3 h at 37C in a CO2 incubator (Labmed, Korea)
with the maintained condition of 5% CO2, 95% air, and
complete humidity. After 3 h, the MTT solution was removed
and replaced with 200 lL of DMSO and 25 lL Sorenson’s
glycine buffer. The plate was further incubated for 4 min at
room temperature, and a plate reader determined the optical
density of these wells at a wavelength of 570 nm.
RNA extraction and cDNA synthesis
Total RNA from all the three cell lines, MCF-7, HepG2,
and LoVo, were collected in the RNA stabilization reagent.
The liver sample of treated animals was also stored in the
same reagent. From those, RNA was isolated using the
RNeasy Mini Kit (QIAGEN, Germany). The isolation was
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conducted according to the manufacturer’s instructions and
quantified by measuring the absorbance at 260 nm, and RNA
quality was estimated by measuring the 260/280 ratios. The
cDNA synthesis was performed using the High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems,
USA) according to the manufacturer’s instructions.
Around 1.5 lg of the total RNA from each sample was
added to a mixture of 2.0 lL of 10 · reverse transcriptase
buffer, 0.8 lL of 25 · dNTP mix (l00 mM), 2.0 lL of
10 · reverse transcriptase random primers, 1.0 lL of Multi-
Scribe reverse transcriptase, and 3.2 lL of nuclease-free
water.24 Thus, the final reaction mixture was kept at 25C
for 10 min that was heated to 37C for 120 min, and then
85C for 5 sec followed by their cooling to 4C.
Quantification of mRNA expression of Tumor
Necrosis Factor-alpha, interleukin-1, IL-6,
and IL-8 in the cell lines by RT-PCR
The quantitative analysis of mRNA expression of the
target genes was executed by RT-PCR.20,24 For this, cDNA
from the mentioned preparation was subjected to PCR am-
plification using 96-well optical reaction plates in the ABI
Prism 7500 System (Applied Biosystems). The 25-lL re-
action mixture had 0.1 lL of 10 lM forward primer and
0.1 lL of 10 lM reverse primer (40 lM final concentration
of each primer) with 12.5 lL of SYBR Green Universal
Mastermix, 11.05 lL of nuclease-free water, and 1.25 lL of
cDNA sample. All the primers used in this procedure were
chosen from pubmed.com online service. Data were ana-
lyzed using the relative gene expression method as per
Applied Biosystems User Bulletin No. 2. Data were pre-
sented as the fold change in gene expression normalized to
the endogenous reference gene and relative to a calibrator.
Measurement of key immunological factors
in the animal samples by ELISA and RT-PCR
The serum samples from the treated animals were sub-
jected to analysis of many key cytokines interleukin (IL)-1,
IL-6, and IL-10 by the commercially available kits (Abcam,
United Kingdom, and RayBiotech, USA). Furthermore, the
level of expression of NF-jB, IjB, Tumor Necrosis Factor-
alpha (TNF-a), and first apoptosis signal (FAS) was mea-
sured by RT-PCR gene expression in the liver samples.
67
Statistical analysis
All data of the experiments were analyzed by statistical
analysis software GraphPad Prism. The post-hoc analysis
was conducted by one-way ANOVA with Tukey’s test for
significance selecting p-value <0.05 under the software. In
the figures, the asterisks (*) indicates significantly different
values from the control (group I), whereas # indicates sig-
nificantly different values from group II.
Results
Cell culture
The purpose of these in vitro experiments conducted on
the cancer cells was to assess the effectiveness of SAV in
the inhibition of tumor cell proliferation and its role in in-
flammation involved with them. For this, MTT assay of the
treated cells, as well as the measurement of expression of
critical immune factors, such as TNF-a, IL-1b, IL-6, and IL-
8 gene expression were conducted. The efficacy of SAV was
tested on three cell lines: breast cancer (MCF-7) cells, liver
cancer (HepG2) cells, and colon cancer (LoVo) cells.
Effect of SAV on cellular proliferation. SAV was used in
five different concentrations (0.1, 1, 10, 100, and 1000 ng/
mL) for MTT assay in the established cancer cells for the
assessment of inhibition of cellular proliferation. The extract
demonstrated the dose-dependent inhibitory effect on their
cellular proliferation; however, the statistical significance
was observed in doses of 100 and 1000 ng/mL. In the
present study, they chose 100 ng/mL as the standard dose for
all further studies.
The results showed that SAV at the dose of 100 ng/mL
was capable of inhibiting the growth of MCF-7 and HepG2
by 57.1% and 50.6%, respectively, but it was only 35.8% in
the case of LoVo against their control. Although the ant
extract demonstrated its efficacy in the range of 1–100 ng/
mL in MCF-7 and HepG2, except the LoVo showed 1000 ng/
mL as a statistically significant dose compared with the
control. Hence, the extract was more effective against MCF-
7 and HepG2 cell lines as compared with the LoVo (Fig. 1).
Effect of SAV treatment on the critical cytokines in the cell
lines by RT-PCR. After treatment of the cells with SAV
(dose of 100 ng/mL), their RNA was isolated and subjected
to measurement of essential cytokines, IL-1, IL-6, IL-8, and
TNF-a, taking GAPDH as the reference gene in all the ex-
periments.
IL-1b. After the treatment of all three cell lines with
SAV, the level of IL-1 was found to be increased by 16.1%
and 1166.3% in MCF-7 and HepG2, whereas LoVo showed
a decrease in its level by 25.3% as compared with the
control (Fig. 2).
IL-6. The treatment of SAV with MCF-7, HepG2, and
LoVo cell lines caused an elevation in its level by 112.7%,
1546%, and 30% with respect to their controls (Fig. 3).
IL-8. The ant venom extract led to increasing its level by
755.4%, 1040.1%, and 12.7% in MCF-7, HepG2, and LoVo
cell lines, respectively, in comparison to their control (Fig. 4).
 68
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FIG. 1. MTT assay for the anticancer effects of SAV
against different cancer cell lines. MTT assay for the anti-
cancer effects of SAV against different cancer cell lines (A:
MCF-7, B: HepG2, C: LoVo) with different concentration
(nanogram per milliliter) of SAV. All data have been ex-
pressed in mean – SEM of five independent set of experi-
ments. *Significantly different values from the control.
SAV, samsum ant venom; SEM, standard error of the mean.
TNF-a. The treatment with SAV increased its level by
201.4%, 813.6%, and 16.4% in the cell lines, MCF-7,
HepG2, and LoVo, in the sequence as compared with their
respective controls (Fig. 5).
Animal studies
All the serum samples were subjected to measurement of
key cytokines by the commercially available ELISA Kits to
AL-TAMIMI ET AL.
FIG. 2. Showing level of Interleukin-1b in MCF-7, HepG2,
and LoVo cells. Showing level of IL-1b in MCF-7, HepG2,
and LoVo cells (in nanogram per milliliter of the supernatant
of the cultured cells) by RT-PCR. All data have been expressed
in mean – SEM of five independent set of experiments. *Sig-
nificantly different values from the control. RT-PCR, real-time
polymerase chain reaction; SEM, standard error of the mean.
investigate if any correlation exists between their in vitro
and in vivo findings.
IL-1b. In the present study, group II demonstrated an
increase in its level by 116.73% with respect to the control,
group I. However, group III and group IV showed a de-
crease in its level by 42.36% and 48.11%, respectively, as
compared with group II (Fig. 6).
IL-6. In the present in vivo analysis, group II exhibited an
enhancement in its level by 60.19% in comparison to group I,
whereas groups III and IV showed a decline in its level by
38.01% and 43.56%, respectively, with respect to group II
(Fig. 6).
IL-10. To confirm if SAV ceases the inflammation in the
target organ, anti-inflammatory IL-10 cytokine was measured.
FIG. 3. Showing level of IL-6 in the three cell lines.
Showing level of IL-6 in the three cell lines (in nanogram per
milliliter of the supernatant of the cultured cells) by RT-PCR.
All data have been expressed in mean – SEM of five inde-
pendent set of experiments. *Significantly different values
from the control. SEM, standard error of the mean.
 ANTICANCER ACTIVITY OF SAMSUM ANT VENOM
FIG. 4. Showing level of IL-8 in the three cell lines.
Showing level of IL-8 in the three cell lines (in nanogram
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per milliliter of the supernatant of the cultured cells). All
data have been expressed in mean – SEM of five indepen-
dent set of experiments. *Significantly different values from
the control. SEM, standard error of the mean.
Group II showed a decrease in its level by 149.47% with re-
spect to the control, whereas groups III and IV demonstrated
an increase in its level by 61.57% and 103.16% as compared to
group II (Fig. 7).
Measurement of expression of Nuclear Factor-kappa B by
RT-PCR. It is an important marker gene to assess the extent
of necrosis in the target tissue. Group II exhibited an increase
in its expression by 319.49% as compared with the control,
whereas group III and IV showed a decline in its expression
by 40.61% and 52.75% with respect to group II (Fig. 8).
Measurement of expression of Inhibitor kappa B by RT-
PCR. It is a prominent subunit of Nuclear Factor-kappa B
(NF-jB), which is considered as an active form once dis-
sociated from the parent transcription factor, while its
translocation from cytosol to the nucleus. The expression of
this factor was elevated by 208.99% in group II, whereas it
FIG. 5. Showing level of Tumor Necrosis Factor-alpha in
the three cell lines. Showing level of TNF-a in the three cell
lines (in nanogram per milliliter of the supernatant of the
cultured cells) by RT-PCR. All data have been expressed in
mean – SEM of five independent set of experiments. *Sig-
nificantly different values from the control. SEM, standard
error of the mean.
69
FIG. 6. Showing level of IL-1b and 6 in the serum sam-
ples. Showing level of IL-1b and 6 (in nanogram per mil-
liliter of the supernatant of the serum) by ELISA analysis.
All data have been expressed in mean – SEM of five inde-
pendent set of experiments. *Significantly different values
from the control (group I). #Significantly different values
from group II. SEM, standard error of the mean.
was decreased by 54.30% and 52.60% in groups III and IV
in comparison to group II (Fig. 8).
TNF-a and FAS. Both are important markers for apo-
ptosis induction after a cell encounters any toxicant or injury/
insult in which FAS assessment confirms the occurrence of
apoptosis in any cell.
Group II showed an increase in the expression of TNF-a
by 593.44% with respect to the group I, whereas groups III
and IV demonstrated its decrease by 54.84% and 72.57% as
compared with group II (Fig. 9).
FIG. 7. Showing level of IL-10 in the serum samples.
Showing level of IL-10 (in nanogram per milliliter of the
supernatant in the serum) by ELISA analysis. All data have
been expressed in mean – SEM of five independent set of
experiments. *Significantly different values from the control
(group I). #Significantly different values from group II.
SEM, standard error of the mean.
 70
FIG. 8. Showing level of NF-kB and IkB in the liver
samples. Showing level of NF-kB and IkB (in nanogram per
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milliliter of the supernatant of the liver tissue sample) by
RT-PCR. All data have been expressed in mean – SEM of
five independent set of experiments. *Significantly different
values from the control, group I. #Significantly different
values from group II. SEM, standard error of the mean.
Moreover, groups II and III showed an increase in the
expression of FAS by 31.63% and 66.01% with respect to
their control, whereas group IV demonstrated an increase in
its expression by 235.94% as compared with group II (Fig. 9).
Discussion
Natural compounds or a mixture of such bioactive com-
pounds from any natural source with antineoplastic potential
have been a hot spot for oncological research since long.
The natural compounds are a preferred choice over syn-
thesized chemical-based drugs because of their null or a
very small degree of toxicity with better acceptability in
biological systems under various in vivo studies.13,25–28
Moreover, it is reported that the venom from Brachyponera
sennaarensis has a significant antitumor effect on breast
cancer cells in a dose-dependent and a time-dependent
FIG. 9. Showing level of first apostosis signal and TNF-a
in the liver samples. Showing level of FAS and TNF-a (in
nanogram per milliliter of the supernatant of the liver tissue
sample) by RT-PCR. All data have been expressed in mean –
SEM of five independent set of experiments. *Significantly
different values from the control, group I. #Significantly dif-
ferent values from group II. SEM, standard error of the mean.
AL-TAMIMI ET AL.
manner without affecting the viability of nontumor
cells.16,20 Also, Heinen and da Veiga29 have documented
that many of the ants produce very potent venoms that have
a possibly potent anticancer effect.
The principal components of the venom from Brachy-
ponera sennaarensis, including trimethyl pyrazine and
Phenol-2, 4-bis (1, 1 dimethyl ethyl), have demonstrated
many pharmacologically essential features in various studies
that are very promising for diverse medical and clinical
applications.8,21 Hence, SAV also falls in the same league of
nutraceuticals with prominent pharmacological and medic-
inal properties. It has a rich repertoire of pharmacologically
active compounds that has been found useful in the regu-
lation of pain, inflammation, wound healing, and even
cancer.16,20 Recently, their laboratory has published the
antioxidant and anti-inflammatory properties of SAV in the
rats challenged with CCl4.14,20,24
The present work is aimed to investigate if these features
of SAV can attribute to its potential anticancer activity or
not under in vitro and in vivo studies.
In the present study, the in vitro results indicate that SAV
is differentially effective in all three cell lines, MCF-7,
HepG2, and LoVo. However, the first two cell lines showed
a better response to SAV as their cell proliferation was
decreased up to 50% at a fixed dose, which was quite sig-
nificant as compared with the control cells. The cytokines,
IL-1b, 6, and 8, along with TNF-a, were also found elevated
in HepG2 and MCF-7 significantly post SAV treatment,
although they were not much altered in LoVo cells. These
findings entail that the SAV triggers different mechanisms
of cell death in all three cell lines mediated by differential
immunomodulation.
In MCF-7 and HepG2, SAV might trigger a cellular im-
mune response by elevating the cytokines (IL-1b, 6, and 8)
and the transcription factor TNF-a. However, SAV might
suppress same cellular events in the LoVo cells. This dif-
ference in the pattern of immunomodulation might be at-
tributed to the different origin of the cancer cell lines. This
distinction in SAV-mediated immunomodulation may be
attributed to the differential antineoplastic activity of the
venom in the present study. Additionally, it is well known
that colon has its normal flora of specific microbes on its
mucosal surface that usually assists in its protection from
any antigens or pathogen. It is an important part of its innate
immunity that plays a vital role in controlling infection and
tumorigenesis. It is also reported that toll-like receptors
(TLRs) are major influencing factors in innate immunity,
especially in the mucosal and gastrointestinal portion of the
body, including the colon.30–32 In rodents, 11–13 TLRs have
been reported so far. Their impaired signaling of innate
immunity or any alteration in TLRs has been documented to
trigger an abnormal inflammatory response that might fa-
cilitate even tumorigenesis.30,31
Recently, Semlali et al.30 have reported that TLR4 (one of
the types of TLRs) is overexpressed in the colon cancer
tumor epithelial cells as compared with the other types of
cancers. In the present study, it is highly speculative that the
anticancer potential of SAV might act on colon cancer cell
line, LoVo, through TLRs. The complex interaction between
SAV and TLRs with innate immunity consequently might
compromise the overall anticancer potential of SAV in the
colon cancer cells among all the three cell lines. Earlier,
 ANTICANCER ACTIVITY OF SAMSUM ANT VENOM 71

scientists have shown33,34 that insulin-like growth factor
(IGF) plays a major role in cellular proliferation, survival,
and chemoresistance in various forms of cancer, including
MCF-7 and HepG2 cells.
Expression of its receptor, IGF-1R is one of the consistent
hallmarks of malignant transformation in preclinical breast
cancer models in vitro as well in vivo.35,36 Furthermore,
Badr et al.16 have reported that SAV significantly abrogated
the level of IGF-1-mediated breast cancer cell proliferation
and actin polymerization. However, in the case of hepato-
cellular carcinoma and HepG2 cells, it has been published
that IGF-II is more involved than IGF-I in its spread and
malignancy.37,38 Hence, the antineoplastic activity of SAV
might involve in the downregulation of IGF-I and IGF-II in
all the cell lines. As the antineoplastic activity of SAV was
more pronounced in MCF-7 and HepG2 as compared with
LoVo, it seems the downregulation of IGF by the venom
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a member of the TNF-receptor superfamily. The activation
of FAS induces apoptosis by its ligand through activation of
the caspase cascade, which is a hallmark of apoptosis.43 It is
noteworthy that a moderately higher level of NF-kB and IL-
10 is assumed to increase the sensitivity of many chemo-
therapeutic agents and even directs cellular machinery to
undergo apoptosis.42,44
Also, Badr et al.16 have documented that SAV abrogates
IGF-1-triggered phosphorylation of ERK and AKT signaling
modulation that consequently flags strong apoptotic signals
through p38MAPK phosphorylation. Hence, the present study
along with the previously published works confirm that SAV
has biologically active molecules of immunomodulatory ef-
fects that orchestrate the immune system favoring apoptosis in
the target tissues over the necrotic mode of cell death. Al-
ternatively, SAV has demonstrated that its therapeutic dose
organizes the antioxidant machinery to nullify the free radi-

extract might also be one of the attributing factors for the cals that further decrease the aggression of the inflammatory
differential anticancer activity. response promoting tissue healing in the damaged tissues and
In addition, each cell line has unique characteristics, in- apoptosis in the unrepairable sections.20,45–47
cluding mechanism action of any drug, metabolism, immune Additionally, it is also established that cellular redox
response, sensitivity toward any chemical/drug, activation, status determines the fate of a cell if it will undergo cell
and expression of cell death receptors, etc. When a cell line is cycle or die through apoptosis or necrosis.18,48 It is evitable
treated with an antineoplastic drug, all the mentioned factors that SAV in the treated cell lines and the rodents might
impart together making an intricate network of upregulation dictate the redox status that favors cell death by apoptosis at
and downregulation of various genes and proteins involved in the given dose as found in several studies (Fig. 10).
cell division and cell death. The outcome of all these interac-
tions finally decides the fate of the cell, if it should enter into
cell cycle or undergo programmed cell death.39 From results, it
seems that SAV might induce an immune response to a dif-
ferent extent in the three cell lines leading to the differential
degree of cell death in the cells in the present investigation.
Under the in vivo study, the ELISA of the serum sample
from CCl4-treated animals demonstrated a high level of
inflammatory cytokines, IL-1b and 6 that were found sig-
nificantly lowered in the animals with hepatoma after
treatment with SAV. Furthermore, the level of anti-
inflammatory cytokine, IL-10 was also elevated to a sig-
nificant extent in the same group. All these results confirm
the anti-inflammatory properties of SAV that is a bit con-
tradictory to the in vitro findings. Moreover, the RT-PCR of
TNF-a, NF-kB, and its active form, Inhibitor kappa B (Ij-B)
in the liver samples of the fourth group, demonstrated a
vivid decline in their expression as compared with the group
II. Furthermore, Fujii et al.40 have shown that IL-10 can help
CD8(+) T cells in the sustenance of anticancer activity.
Earlier, IL-10 has been reported to decrease the tumor
burden in the rodents.41 The elevated level of NF-kB and its
subunit Ij-B are essential requisites in cellular proliferation
in malignant condition as they are supposed to abrogate the
caspase-mediated apoptosis.42 However, decreased level of
these transcription factors indicates ceasing of inflammation
and tissue damage. Furthermore, to confirm the mode of cell
death in the damaged tissues, the expression of FAS and
TNF-a were assessed. The pattern of both the parameters
clearly entails that SAV triggers apoptosis in the target or-
gan significantly by controlling the inflammation and tissue
injury. These findings are, by their previous studies, show- FIG. 10. Concept arrow-diagram on efficacy of anticancer
ing SAV at the given dose as a strong antioxidant agent that activity of SAV. Showing concept arrow-diagram on effi-
has helped the CCl4-challenged animals in the restoration cacy of SAV after cell line and animal-based studies high-
of tissue damage and retaining of normal function.20,24 It is lighting the major events. >Indicates more incidences of the
well known that FAS, type I membrane receptor, belongs to mode of cellular death. SAV, samsum ant venom.
 72
Moreover, the differential antiproliferative effect on dif-
ferent cell lines also exhibits its preferential specificity for
specific target tissues, such as breast and liver cancer cells
over the colon cancer cell line. Their current findings also
correlate well with their earlier work20,24 in which SAV has
shown potent antioxidant nature of its biocomponents in
the rodent model study. The malignancy and propagation
of any cancer in vivo is attributed to free radical-mediated
oxidative stress and related extent of inflammation.48,49 In
the present study, SAV seems to moderate the extent of
inflammation up to the level in which only the healthy cells
can thrive, whereas the cancerous cells are rerouted toward
apoptosis at the same time.
Hence, the antioxidant potential of SAV might dictate
the CCl4-induced tissue injury and mild tumorigenesis in
the treated animals in such a way that apoptosis became the
preferred mode of cell death over the necrosis.20,47 How-
Downloaded by Laval University e-journal package from www.liebertpub.com at 03/25/18. For personal use only.
ever, the study needs to be done with other cell lines and
cancer models of different organs in vivo to confirm the
efficacy of SAV as an anticancer agent.
It is noteworthy that SAV exerts its anticancer activity by
enhancing inflammatory response in vitro leading increased
cell death, whereas the venom extract demonstrates its anti-
neoplastic activity by ceasing inflammation in vivo orches-
trating the condition that favors apoptosis. Nevertheless, SAV
is a complex mixture of diverse types of biologically active
compounds that have not been fully elucidated so far. Hence,
further study is warranted to identify these essential bioactive
components for their exact mechanism and target-specific
application under clinical trials for effective cancer treatment.
Conclusion
The present study establishes anticancer activity of SAV
in vitro as well as in vivo. The venom extract at the standard
dose is antioxidant in nature that triggers immunomodula-
tion (IL-1b, IL-6, IL-10) and regulates the key transcription
factors (NF-jB, Ij-B, TNF-a) leading to the ceasing of the
inflammation concomitant with an increased rate of tissue
healing. All these events promote apoptosis in the tumorous
and damaged cells that consolidates SAV as an important
anticancer agent with promising clinical implication. How-
ever, the venom is a mixture of complex chemical ingredi-
ents of the diverse type that can trigger various apoptosis
and cell death pathways that needs further in-depth research
to pave the exact mode of pathways in vitro as well in vivo.
Authors’ Contribution
J.A. had conducted animal handling and treatment and
preparation of samples for in vivo experiments. A.S. orga-
nized the RT-PCR experiments that were executed with
J.A., I.H., and H.E. had analyzed the results and discussion.
I.H. and J.A. had prepared the graphical representation of
the results. The article was written by I.H., J.A., and S.H.M.
that was crosschecked by H.E., I.A., and M.A., and M.A.
supervised and provided the laboratory facilities for con-
ducting experiments, and animal and cell culture treatment.
Acknowledgment
The authors would like to extend their sincere apprecia-
tion to Deanship of Scientific Research at King Saud Uni-
versity for funding the research group no. RGP-1438-004.
AL-TAMIMI ET AL.
Disclosure Statement
No competing financial interests exist.
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