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EXPRESSION OF TUMOR-MARKERS AND CYTOKINES IN RESPONSE TO CICHORIUM ENDIVIA L. IN CANCEROUS MICE

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Most of natural products, such as, medicinal plants and herbs confer protective effects against a wide range of cancers, including colon cancer. Cichorium endivia, L. has been shown to have anti-inflammatory and antioxidant properties. Thirty two male mice were divided into four groups. Group I served as control. Groups 2, 3 and 4 were administered freshly prepared DMH for three weeks, twice per week. Group 3 received the anticancer drug 5 FU for two weeks. Half of this group was sacrificed after 2 weeks while the other half continued until the experiment ended. Group 4 received the plant extract which was divided into 3 sub-groups. Each sub group was administered with a different concentration of the extract (200-400-600 mg/kg bodyweight/ day) for 40 days. Animals were sacrificed; blood was collected and the colons were subjected for investigation. P53 expression was nearly the same in both control and all treated tissues. BCl2 expression decreased in mice treated with 5FU and specifically with Cichorium. TNF- expression was lower in tissues treated with 5FU compared with the control. Meanwhile, the expression of dose 200 mg/kg.b.wt, was lower than both the control and the 5FU treated tissues. The real time PCR analysis of blood samples showed that the expression of interleukin IL-12 and IL-4 was higher in blood cells treated with plant extract compared with the 5 FU. The two effective doses were 200 and 600 mg/kg.b.wt. In contrast, therapeutic treatments mute the expression of interferon IFN marker. It is thus concluded that, Cichorium endivia, especially dose 200 mg/ kg.b.wt., represents an effective anticancer and immune response drug especially for colon cancer.
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
EXPRESSION OF TUMOR-MARKERS AND
CYTOKINES IN RESPONSE TO CICHORIUM
ENDIVIA L. IN CANCEROUS MICE
El-Sayd E Hafez1, Effat A Badr2, Yasser M Mabrouk2,
Mohammed A Seehy2 and Sarah A Aggag2*
Research Paper
Most of natural products, such as, medicinal plants and herbs confer protective effects against
a wide range of cancers, including colon cancer. Cichorium endivia, L. has been shown to have
anti-inflammatory and antioxidant properties. Thirty two male mice were divided into four groups.
Group I served as control. Groups 2, 3 and 4 were administered freshly prepared DMH for three
weeks, twice per week. Group 3 received the anticancer drug 5 FU for two weeks. Half of this
group was sacrificed after 2 weeks while the other half continued until the experiment ended.
Group 4 received the plant extract which was divided into 3 sub-groups. Each sub group was
administered with a different concentration of the extract (200-400-600 mg/kg bodyweight/ day)
for 40 days. Animals were sacrificed; blood was collected and the colons were subjected for
investigation. P53 expression was nearly the same in both control and all treated tissues. BCl2
expression decreased in mice treated with 5FU and specifically with Cichorium. TNF- expression
was lower in tissues treated with 5FU compared with the control. Meanwhile, the expression of
dose 200 mg/kg.b.wt, was lower than both the control and the 5FU treated tissues. The real
time PCR analysis of blood samples showed that the expression of interleukin IL-12 and IL-4
was higher in blood cells treated with plant extract compared with the 5 FU. The two effective
doses were 200 and 600 mg/kg.b.wt. In contrast, therapeutic treatments mute the expression of
interferon IFN marker. It is thus concluded that, Cichorium endivia, especially dose 200 mg/
kg.b.wt., represents an effective anticancer and immune response drug especially for colon
cancer.
Keywords: Cichorium endivia, Tumor-markers, P53, Bcl2, TNF-, Cytokines, IL-12, IL-4, IFN
*Corresponding Author: Sarah A Aggag sarah_amgad_8@yahoo.com
ISSN 2250-3137 www.ijlbpr.com
Vol. 3, No. 4, October 2014
© 2014 IJLBPR. All Rights Reserved
Int. J. LifeSc. Bt & Pharm. Res. 2014
1Mubarak C ity for Scientific Research and Technology A pplications, Egypt.
2Department of Genetics, Faculty of Agriculture, Aflat on St., El-Shatby, P.O. Box 21545, Alexandria University, Alexandria, E gypt.
INTRODUCTION
Colon cancer (CRC) is one of the most common
cancers in the developed countries. Due to the
limited prevention and treatment options, colon
cancer is considered to be one of the major
causes of cancer-related death (Alshehri, 2012).
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
1, 2- Dimethyl hydrazine (DMH) has been
shown to induce colonic carcinomas in rats and
mice (Sitohy and El-S alhy, 2001). Natural
products and related drugs are used to treat 87%
of all categorized human diseases including
cancer and immunological disorders (Newman
and Cragg, 2007). Over 3000 species of plants
have been reported to have anticancer properties.
About 80% of the population in the developing
countries relies on traditio na l plant-based
medicines for their primary health-care needs.
Cichorium sp. belongs to the family Asteraceae
and it is a small aromatic biennial herb. The whole
plant contains a number of medicinally important
compounds such as inulin, esculin, volatile
compounds, coumarins, flavonoids and vitamins
(Alshehri and Hafez, 2012).
Cichorium endivia had been tested in terms
of prevention of ultraviolet B (UVB)-induced
pyrimidine dimer formation and interlukin-6 (IL-6)
mRNA expression in the human keratinocyte cell
line, HaCaT. They found that ethanolic extract of
C. endivia roots absorbed radiation in the UVB
spectrum and partially prevented induction IL-6
expression. They proved that application of the
Cichorium endivia root extract on the skin prior
to UVB irradiation totally prevented erythema (Enk
et al., 2004).
The effect of hydro-alcoholic extract of
Cichorium endivia L. leaves (HCE) has been
elu cid ated against acetaminophen-indu ced
oxidative stress and hepatotoxicity in male rats.
The hepatoprotective activity with C.endivia
leaves-extract in rats was found to be compatible
with the known hepatoprotective drug “Silymarin”
(Marzouk et al., 2011).
The gene expression of breast cancer cell line
(MCF7) have been examined for the DNA cancer
markers; P53, BCl2, TNF-a and interleukin markers
IL-4, IL-6 and IL-2, treated with the root extracts
of Cichorium endivia. They found that due to all
expressions of those markers, C.endivia showed
to be anticancerous (Alshehri and Hafez, 2012).
The aim of the present work was to investigate
Cichorium endivia as an anticancer agent and
an immune response drug especially on CRC.
MATERIALS AND METHODS
Plant Materials
The Cichorium endivia, L. plant material were
collected from the faculty of Agriculture farm,
Alexandria University. The whole plants were
washed with distilled water, and ground using a
blender.
Chemical Materials
DMH (Sigma) was dissolved in double distilled
water and adjusted to pH 6.5 (Sumiyoshi and
Wargovich, 1990). 5-FU, Sigma was diluted in
pyrog en-fre e 0.8 5% sal ine , to be at a
concentration of 100 mg/l0 mL (Kojima et al.,
1999).
Experimental Design
Thirty two male albino mice with initial weight of
(25-30 g) were used in all experiments. Mice were
housed in plastic cages with filter tops (five per
cage) under controlled conditions of light, humidity
and temperature at the Animal House Lab.,
Medical Research Institute, Alexandria University.
They were divided randomly into four groups. The
1st group (n =4) served as control. The 2nd group
(n=8) was separated into two subgroups. Mice
of the second group were injected intraperitoneal
(i.p) of DMH at a dosage of 20 mg/kg b.wt. twice
per week. Subgroup no.1 was treated for only
three weeks (DMH stopped), while subgroup no.2
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
was treated continuously till the end of the
experiment (DMH continued). The 3rd group (n=8)
was treated with DMH for three weeks, then
received 5-fu (80 mg/kg b.wt.) for two weeks. This
third g roup w as then separate d into two
subgroups. The first subgroup was scarified (5-
FU stopped) while the second subgroup were
kept without any treatment till the end of the
experiment (5-FU continued). Mice of forth group
were treated by DMH for three weeks, twice per
week. The individuals of this group were divided
to three subgroups. Each subgroup received a
different concentration of Cichorium extracts 200,
400, 600 mg/kg bodyweight/ day for 40 days. All
animals, except those of 5-FU stopped sub-group,
were sacrificed after 9 weeks from the beginning
of the experiment.
Real time PCR for Cancer DNA Markers
and Immune Response Markers
Cancer DNA markers (p53, Bcl2 and TNF-a) were
carried out on colon tissue which was removed
and washed twice with ice-cold saline solution.
The immune response markers (IL-4, IL-12 and
IFN) was carried on from blood samples which
was collected in EDTA coated tubes, centrifuged
at 2000xg for 10 min to separate plasma and
stored at -80oC until analysis.
Extraction of Total RNA
The RNA extraction was performed using Trizol
re age nt (In vitroge n, Carlsbad, CA, USA)
according to the manufacturer’s p rotocol.
Extracted RNA was dissolved in DEPC-treated
water and analyzed on 2% agarose gel. Stored
at -80°C until used.
The Quantitative Real Time-PCR
The extracted RNA from colon tissues was used
as template to examine the expression level of
three different specific genes (P53, Bcl2, TNF-)
in the presence of housekeeping gene primers
(GPDH). While the RNA extracts from the blood
samples was used to examine the expression
level of three different specific genes (IL-4, IL-12,
and IFN) also in the presence of housekeeping
gene primers (GPDH). All RNA samples were
treated with DNaseI to remove residual DNA. The
re ver se transcription fr om mRNA to
complementary DNA (cDNA) was made using 4
L of RNA per sample, 2.5 L Buffer enzyme, 5.5
L oligo d (t), 2.5 L dNTPs, 0.2 L taq enzyme
and 5.3 L RNase-free water. The mixture was
incubated in a thermocycler at 25°C for 5 min.
followed by 42°C for 60 min. and 70°C for 15 min.
The Real time reaction consists of 12.5 L for 2X
Green Dye RT Mix (ROVALAB), 2 L of the
extracted cDNA, 1 L of 25 pM/l forward primer,
1 l of 25 pM/l reverse primer (Table 1), 9.5 l of
Table 1: The Primers Used in this Study
Primer Name Primer Sequence from 5 to 3
TNF-F TTC TGT CTA CTG AAC TTC GGG GTG
ATG GGT CC
R GTA TGA GAT AGC AAA TCG GCT GAC
GGT GGT GG
BCL2F ATG TGT GTG GAG AGC GTC AAC C
R TGA GCA G AG TC T TCA GAG ACA
GC C
P53 F AGG GAT ACT ATT CAG CCC GAG GTG
R ACT GCC ACT CCT TGC CCC ATT C
IL-4 F TCT GTG GTG TTC TTC GTT GC
R TCA ACC CCC AGC TAC TTG TC
IL-12 F AAC TGG AGG GAG GAG TAC GGA
GAA TGG
R GGA AGC ACG GCA GCA GAA TA
IFN F GTC ACA GTT TTC AGG TGT ATA GGG
R AGC GGC TGA CTG AAC TCA GAT TGA
AG
GAPDH F ATT GAC CAC TAC CTG GGC AA
R GAG ATA CAC TTC AAC ACT TTG ACC
OLIGO d(t) (t)12-18
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
RNase-free water to make a total of 25 l.
Samples were spun before loading in the rotors
wells. The real time PCR program was performed
as follows: initial denaturation at 95°C for 15 min;
45 cycles of 96°C for 15 s, annealing at 60°C for
30 s and extension at 72°C for 30 s (Alshehri and
Hafez, 2012). Data acquisition was performed
during the extension step. This reaction was
performed using Rotor-Gene 6000 system
(Qiagen, USA).
DATA ANALYSIS
The data set of both samples and control for real-
time PCR was analyzed with Rotor-Gene-6000
version 1.7.94. for estimation of the relative
expression of genes using Real Time PCR and
the results normalized to GPDH gene (Reference
gene). Comparative quantitation were statistically
evaluated, interpreted and analyzed by Livak and
Schmittgen (2001).
RESULTS
The real time PCR analysis of colon tissue
revealed that P53 expression was nearly the same
in the control and the treated tissues, except in
the stopped 5-fluorouracil (5FU) where it was low
(Figure 1). The Bcl2 gene showed low expression
in the treated tissues compared with the control,
especially in tissues treated with plant extract
(Fig ure 2). TNF- expressio n was lower in
tissues treated with 5 FU than the control.
Meanwhile the TNF- expression was so high in
the tissues treated with plant extract at the two
dos es 400 and 600 mg/kg .b. wt, bu t th e
expression at the dose 200 mg/kg.b.wt was very
low (Figure 3).
The real time PCR analysis of blood samples
revealed that interleukin IL-12 and IL-4 showed
Figure 1: Real Time PCR for the DNA
cancer marker, P53, expression in colon
tissue treated with 5FU and different
doses of Cichorium extract
Figure 2: Real Time PCR for the DNA
cancer marker, Bcl2, expression in
colon tissue treated with 5FU and
different doses of Cichorium extract
Figure 3: Real Time PCR for the DNA
cancer marker, TNF-á, expression in
colon tissue treated with 5FU and
different doses of Cichorium extract
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
high expression in samples treated with 200 and
600 mg/kg.b.wt of plant extract compared with 5
FU (Figures 4 and 5). Whereas, 400 mg/kg.b.wt
of plant extract didn’t show expression of
interleukins. IFN marker exhibited extremely low
expression for 200 and 400 mg/kg.b.wt of plant
extract and 5FU treatments compared with the
control. But the expression of the dose 600 mg/
kg.b.wt was high (Figure 6).
The results thus show that, Cichorium endivia,
especially dose 200 mg/kg.b.wt, represents an
effective means for anticancer activity as well as
an immune response drug especially for Colonic
carcinoma. DISCUSSION
Colorectal cancer is the third most commonly
diagnosed cancer in the world, but it is more
common in the developed countries (Ferlay et
al., 2010). Organic compounds from plants have
extensive past and present use in the treatment
of many diseases. Natural compounds from
plants having antioxidant and immuno-modulatory
activities have potential as therapeutic agents
(Devasagayam and Sainis, 2002).
Family Asteraceae contained medical ly
important plants such as Cichorium endivia, due
to their polyphonic components. The expression
of tumor suppressor gene (P 53) showed no
variability in treated and control tissues. P53 can
act to arr est cell cycle progression, help to
preserve the integrity of the cellular genome and
activate directly the process of programmed cell
death. Some studies obtained similar results
(Alshehri and Hafez, 2012).
The effect of Cichorium extract on colon
cancerous tissues decreased the expression of
the apoptosis regulator protein (BCl2), even more
than 5FU, compared with the control tissues. We
assume that plant extract especially its phenolic
Figure 4: Real Time PCR for cytokines,
IL-12, expression in blood samples
from mice treated with 5FU and different
doses Cichorium extract
Figure 5: Real Time PCR for cytokines,
IL-4, expression in blood samples from
mice treated with 5FU and different
doses Cichorium extract
Figure 6: Real Time PCR for cytokines,
IFN, expression in blood samples
from mice treated with 5FU and
different doses Cichorium extract
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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sarah A Aggag et al., 2014
compounds play a role in decreasing BCl2 in
treated tissues. These results agree with those
obtained by others [Sitohy and El-Salhy (2001);
Kokawa et al. (2001) and Tudor et al. (2000)]. They
reported that a large portion of these plants show
great potential for targeting cancer through the
down regulation of anti-apoptotic proteins (e.g.,
bcl-2, bcl-xL).
On the other hand, the expression of tumor
necrosis factor alpha TNF-in tissues treated
with 5 FU and 200 mg/kg.b.wt was lower than
the control tissues, with the plant extract treatment
being lower than 5 FU treatment. Recent studies
proved that the TNF-a levels increase in cancer
cells with end-stage disease (Nakashima et al.,
1998).
Both Interleukins IL-12 and IL-4 expression
increased in the treated blood cells with plant
extract than with 5 FU. In case of interferon IFN
marker, the plant extract and the 5 FU treated
were extremely low compared with the control.
These results agree with several previous studies
[(Milowsky and Nanus (2001); Glaspy (2002) and
Yang et al. (2011)]. They reported that interleukins
12 and 4 induces interferon secretion by T cells
and natural killer cells. The above process
enhances the proliferation of activated T cells and
natural killer cells. IFN upgraded the apoptosis
rate. Interleukin 12, 4 stimulates In vivo antitumor
activity in many murine tumor models.
CONCLUSION
The present findings imply that the promising data
obtained with Cichorium endivia in the pre-clinical
models of anti-tumor immunotherapy have raised
much hope that this c ou ld be a powerf ul
therapeutic agent against cancer and immune
reactions. However, the effective dose of 200 mg/
kg.b.wt needs further studies to reach the exact
optimum dose.
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... Phytochemicals are promising modulators of interleukins in the management of cancer (Hamsa and Kuttan, 2010, 2011Chen et al., 2011;Alonso-Castro et al., 2012;Hafez et al., 2014;Du et al., 2016;Sun et al., 2018). Bioactive polysaccharides isolated from plants were efficacious in anticancer treatments and are promising anticancer nutraceuticals (Huang et al., 2015;Yu et al., 2018) Hafez et al. (2014. ...
... Phytochemicals are promising modulators of interleukins in the management of cancer (Hamsa and Kuttan, 2010, 2011Chen et al., 2011;Alonso-Castro et al., 2012;Hafez et al., 2014;Du et al., 2016;Sun et al., 2018). Bioactive polysaccharides isolated from plants were efficacious in anticancer treatments and are promising anticancer nutraceuticals (Huang et al., 2015;Yu et al., 2018) Hafez et al. (2014. reported that Cichorium endivial. ...
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