Expression and inhibition of il 23 by colon cancer cells a promising approach in prevention of ibd.

Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.4, 2013
Expression and Inhibition
Promising Approach
Dr. Vishal Bhargava
1. Department of Biochemistry, G.R. Medical College
2. Department of Biotechnology DRDO Gwalior
* E-mail of the corresponding author:
The research is financed by G.R. Medical College Autonomous Society
Abstract
Inflammatory bowel disease is chronic uncontrolled inflamma
activated leukocytes. Here we have developed an
epithelial cell line HT-29 expressed elevated IL
strains. In this in-vitro model of IBD, IL
anti-inflammatory drug Sulfasalizine was studied and was found to block the up regulation of IL
Keywords: IBD (Inflammatory Bowel Disease), HT
(Lipopolysaccharide).
1. Introduction
Inflammation can be defined as a series of non
mediators respond to tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterized
in the acute phase by increased blood flow and vascular permeability along with the accumulation of fluid,
leukocytes, and inflammatory mediators such as cy
development of specific humoral and cellular immune responses to the pathogen(s) present at the site of tissue injury
(Carol A. 1997).
Most cytokines involved in the inflammation processes
their effects locally or systemically in an autocrine or paracrine manner (Carol A. 1997). Cytokines are involved in
extensive networks that involve synergistic (proinflammatory cytokines) as
(anti-inflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cells
(Adams G 2003).
Sometimes immune cells fail to distinguish among the foreign and its native cells, as a result
inflammation is targeted towards its own cells, the condition is termed as Auto inflammation.
One such condition is Inflammatory Bowel Disease
interacted with its own underlying immun
there by leading to the destruction of tissue as such
Inflammatory bowel disease (IBD)
Ulcerative colitis (UC) and Crohn’s disease (CD).
uncontrolled inflammation characterized by intense mucosal recruitment of activated leukocytes (Hanauer. S 2006).
Although the diseases have some features in common, there ar
Over the past 15 years wide variety of candidate genes have been studied for IBD. Significant linkages have been
reported on chromosomes 1,3,6,7,12,14,16 and 19. Detailed mapping of chromosome 16 resulted in identification of
the gene responsible, at least in part for this linkage (Podolsky D.K. 2002).This gene encodes a cytoplasmic protein
designated nucleotide-binding oligomerization domain
domain CARD15 (Hanauer B 2006)
immune system. It is the first gene to be clearly associated with IBD, and >60 mutations have been recognized, 3 of
which have been linked to development of CD. The mechanism where
development of IBD remains unclear. The NOD2 gene is expressed mainly in monocyte/macrophage cell lines,
where it has a role in host signaling pathway. Reasonable data suggest th
Crohn’s disease is dominated by CD4+ lymphocytes with a type 1 helper
3208 (Paper) ISSN 2225-093X (Online)
25
nd Inhibition of Il-23 by Colon Cancer Cells: A
Promising Approach in Prevention of Ibd.
Dr. Vishal Bhargava1*
Dr. Neelima Singh1
, Dr.Richa Sijoria
Department of Biochemistry, G.R. Medical College, Gwalior (M.P.) India. (474009)
Department of Biotechnology DRDO Gwalior (M.P) India
mail of the corresponding author: bhargavavishal6@gmail.com
The research is financed by G.R. Medical College Autonomous Society
Inflammatory bowel disease is chronic uncontrolled inflammation characterized by intense mucosal recruitment of
activated leukocytes. Here we have developed an in vitro model for IBD using colon cancer cell line HT
29 expressed elevated IL-23 levels when induced with LPS isolated
model of IBD, IL-23 was playing a key role in inflammation cascade. Action of
inflammatory drug Sulfasalizine was studied and was found to block the up regulation of IL
IBD (Inflammatory Bowel Disease), HT-29, IL-23 (Interleukin-
Inflammation can be defined as a series of non-specific defense mechanism of body, in which cells and different
o tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterized
leukocytes, and inflammatory mediators such as cytokines. In the sub acute/chronic phase it is characterized by the
Most cytokines involved in the inflammation processes are multifunctional. They are pleiotropic molecules that elicit
extensive networks that involve synergistic (proinflammatory cytokines) as well as antagonistic interactions
inflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cells
Sometimes immune cells fail to distinguish among the foreign and its native cells, as a result
Inflammatory Bowel Disease in which the gut micro biota if through any pathway gets
interacted with its own underlying immune cells; the process of inflammation towards these microorganisms begins
there by leading to the destruction of tissue as such
Inflammatory bowel disease (IBD) refers to two chronic diseases that cause inflammation of the intestines:
Crohn’s disease (CD). The hallmark of inflammatory bowel disease is chronic
Although the diseases have some features in common, there are some important differences.
binding oligomerization domain 2 NOD2 also known as caspase activation and recruitment
domain CARD15 (Hanauer B 2006). This is a polymorphic gene the product of which is involved in the innate
which have been linked to development of CD. The mechanism whereby defects in the NOD2 gene lead to the
where it has a role in host signaling pathway. Reasonable data suggest that mucosa of the patients with establi
Crohn’s disease is dominated by CD4+ lymphocytes with a type 1 helper-T-cell (Th1) phenotype, characterized by
www.iiste.org
y Colon Cancer Cells: A
f Ibd.
, Dr.Richa Sijoria2
Gwalior (M.P.) India. (474009)
India
bhargavavishal6@gmail.com
tion characterized by intense mucosal recruitment of
model for IBD using colon cancer cell line HT-29. Colon
23 levels when induced with LPS isolated from various bacterial
23 was playing a key role in inflammation cascade. Action of
inflammatory drug Sulfasalizine was studied and was found to block the up regulation of IL-23.
-23), Sulfasalizine, LPS
specific defense mechanism of body, in which cells and different
o tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterized
tokines. In the sub acute/chronic phase it is characterized by the
are multifunctional. They are pleiotropic molecules that elicit
well as antagonistic interactions
inflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cells
Sometimes immune cells fail to distinguish among the foreign and its native cells, as a result the process of
in which the gut micro biota if through any pathway gets
e cells; the process of inflammation towards these microorganisms begins
refers to two chronic diseases that cause inflammation of the intestines:
The hallmark of inflammatory bowel disease is chronic
e some important differences.
also known as caspase activation and recruitment
. This is a polymorphic gene the product of which is involved in the innate
by defects in the NOD2 gene lead to the
at mucosa of the patients with established
cell (Th1) phenotype, characterized by

Vol.3, No.4, 2013
production of IFN-γ, IL-2 IL-8 and IL
by CD4+ lymphocytes with an atypical type 2 helper
transforming growth factor β (TGF-
2. Materials and Methods
2.1 HT-29
These cells are also adherent colonic epithelial cells obtaine
with different cellular products mainly carcinoembryonic antigen (CEA); transforming growth factor beta binding
protein; mucin. The cell line used in the present study was purchased from American Type Cu
(ATCC), Manassas VA 20108, USA.
2.2 Reagents
DMEM Powder, Bovine serum albumin (BSA), DMSO, Sodium bi carbonate, Trizma, EDTA and others were
obtained from Sigma (Sigma Aldrich co. St. Louis MO USA).
2.3 ELISA kit
Kit for both IL-23 was quantitated from the cell supernatants using DuoSet ELISA Development KITS.
ELISAs are sensitive enzyme immunoassays that measure soluble levels of proteins in biological samples. R&D
SYSTEM provides Complete ELISA kits that offer accurate and
DuoSet kits contain the basic components required to develop an immunoassay to measure natural or recombinant
proteins. All these were obtained from
3. Methodology
In our study HT-29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)
and 1% penicillin –streptomycin antibiotic solution .The cells were incubated at 37
of 5% CO2 in air.
3.1 Seeding
Around 80% confluency stage, the cells were sub cultured. Medium contained in the flask was completely discarded
using a sterile disposable pipette. Cells were washed with 1X PBS so as to remove the dead cells and the cell debris.
Cells were treated with trypsin-EDTA solution (0.25%W/V) and kept at37
cells were detached from the substratum, as monitored under the phase contrast microscope. Once the cells were
completely detached, immediately excess amount of
prolonged exposure of cells with the trypsin
the complete medium acts as an inhibitor of trypsin. Flushing gently with a pipette t
From this cell suspension, a sample was subjected to counting using a hemocytometer. The desired no. of cells was
selected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the resp
plate. (12, 24, or 96 well plate) according to the requirement of the experiment. Incubate the respective plates over
night at 37°C in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.
3.2Treatment with LPS and the drug addition
Specific cell line was selected based on the experiment and the cells were allowed to get confluent. Post confluent
cells were seeded in 96 well plate as described above, with a concentration of 30,000
then left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to be
pre-incubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in different
concentrations. Based on the solubility of the drug if soluble in water was directly diluted with medium and applied
to the cells or if insoluble was the diluted in DMSO and the applied. 3 hours later of drug incubation cells were
treated with LPS to get inflamed. LPS treatment was optimiz
efficient strain to cause inflammation was chosen for the experiment. Based on the volume of the cell suspension in
the well LPS was introduced respectively. Followed this plate containing cells were incub
overnight. Next day supernatant from each well was collected by centrifuging plate at 1500 rpm for 15 min.
Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA at
plate with the adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess the
maximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISA
per the user manual provided with the R
Observations of ELISA are depicted graphically as follows with interpretations.
26
8 and IL-23. In contrast the mucosa in patients with ulcerative colitis may be dominated
th an atypical type 2 helper-T-cell (Th2) phenotype, characterized by the production of
-β) and IL-5 (Podolsky. D.K 2002).
are also adherent colonic epithelial cells obtained from the patients of colorectal adenocarcinoma but
protein; mucin. The cell line used in the present study was purchased from American Type Cu
(ATCC), Manassas VA 20108, USA.
(Sigma Aldrich co. St. Louis MO USA).
quantitated from the cell supernatants using DuoSet ELISA Development KITS.
SYSTEM provides Complete ELISA kits that offer accurate and reproducible results with no development time
proteins. All these were obtained from R&D Systems, Inc 614 McKinley Place NE, Minneapolis, USA.
29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)
streptomycin antibiotic solution .The cells were incubated at 37°C under a humidified atmosphere
EDTA solution (0.25%W/V) and kept at37°C in the CO2 incubator for 2
completely detached, immediately excess amount of complete medium was added to the cells. This is because a
prolonged exposure of cells with the trypsin -EDTA solution is known to cause cell death. The serum contained in
the complete medium acts as an inhibitor of trypsin. Flushing gently with a pipette tip mixed the contents of the flask.
selected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the resp
C in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.
and the drug addition
cells were seeded in 96 well plate as described above, with a concentration of 30,000-40,000 cells/well. Plate was
hen left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to be
incubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in different
ility of the drug if soluble in water was directly diluted with medium and applied
treated with LPS to get inflamed. LPS treatment was optimized using different strains of bacteria and maximum
the well LPS was introduced respectively. Followed this plate containing cells were incub
Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA at
the adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess the
maximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISA
per the user manual provided with the R&D Duo kit system.
Observations of ELISA are depicted graphically as follows with interpretations.
www.iiste.org
23. In contrast the mucosa in patients with ulcerative colitis may be dominated
cell (Th2) phenotype, characterized by the production of
d from the patients of colorectal adenocarcinoma but
protein; mucin. The cell line used in the present study was purchased from American Type Culture collection
quantitated from the cell supernatants using DuoSet ELISA Development KITS. Sandwich
reproducible results with no development time
, Inc 614 McKinley Place NE, Minneapolis, USA.
29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)
C under a humidified atmosphere
C in the CO2 incubator for 2-5 mins. The
complete medium was added to the cells. This is because a
EDTA solution is known to cause cell death. The serum contained in
ip mixed the contents of the flask.
selected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the respective
C in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.
40,000 cells/well. Plate was
hen left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to be
incubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in different
ility of the drug if soluble in water was directly diluted with medium and applied
ed using different strains of bacteria and maximum
the well LPS was introduced respectively. Followed this plate containing cells were incubated in CO2 incubator for
Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA at -80°C. The
the adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess the
maximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISA was performed as

Vol.3, No.4, 2013
4. Observations and Results.
The Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disease where intestinal epithelial cells (IEC)
gets differentiated as cancer cells when induced by some inflammatory agents. Normally in
remain separated from underlying immune cells by a membrane lining. In most of the cases of IBD the underlying
immune cells comes in direct contact
(Hendrickson BA 2002), thereby leading to uncontrolled tissue destruction (Weber CR 2007).
The process of inflammation could be studied with the help of certain tumour markers. In prepa
model for IBD several cytokines were used as prominent markers whose upregulation determined the stages of
inflammation (Carol A 1997).
In-vitro model for IBD could be developed by using colon cancer cell line like HT
which show some inflammatory responses. In our experiment we have used HT
proteins indicating inflammatory responses (Haller D 2004).
Development of inflammatory conditions in HT
studies the bacterial DNA or bacteria as a whole were confirmed to express inflammatory responses (Haller D 2000
& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell wal
as an inflammatory agent is a new approach of our research laboratory for this study since no study in this regard has
been made so far.
In normal intestinal flora (in-vivo) numbers of bacterial strains exist but few of them have been found to be
pathogenic in nature. LPS was isolated from different bacterial strains and were introduced in HT
selection of LPS we used strains of E.coli B
E.coli B-4 did not induce much inflammatory conditions in HT
of bacteria like Salmonella Minnesota (S.Minn.), Salmonella Enteritidis (S.Ent.) and Pseudomonas to study the
inflammatory reactions. It was observed that S.Minn and
After developing inflammatory conditions in colon cancer cell line HT
S.Minn and S.Ent we then checked inflammatory responses with cytokines as a marker (Neurath MF 2003).
Cytokines are categorized under pro
Interleukin-23 (IL-23) cytokine for observing the effect of LPS. LPS induction expressed different levels of IL
with respect to each bacterial strain (Puleston J 2005). Upregu
the inflammatory conditions in given cell line.
Further in our study to check the inflammation process developed, the effect of certain anti
studied in the same (Botoman VA 1991).
concentration was introduced to inhibit the upregulated cytokine IL
inhibition of upregulated IL-23 with minimum concentration of Sulfasalizine was
mimic the in-vivo model of IBD (Mulder J 1988) (Fig.1).
Dose response curve (DRC) was set up for each given concentration of drug with respect to bacterial strain and cell
line to study the cytotoxic effect and most effect
4.1Figure.1
4.2 Figure.2
5. Conclusion
HT-29 cells expressed different levels of inflammatory processes for in
Lipopolysaccharide (LPS) isolated from strains of
gram-negative bacteria induced inflammation in the monolayer and co
were highly expressed in the process of inflammation. Sulfasalizine at different concentration was found effective in
treatment of inflammatory bowel disease.
6. References
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plausible mechanisms for dextran sulfate sodium
Bisping, G. Lügering, N. Lütke-Brintrup, S. Pauels, H.G. Schürmann, G, Domschke, W. & Kucharzik, T. (2001)
Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular
interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co
27
rentiated as cancer cells when induced by some inflammatory agents. Normally in
immune cells comes in direct contact with micro-biota of intestine resulting in the process of inflammation
The process of inflammation could be studied with the help of certain tumour markers. In prepa
vitro model for IBD could be developed by using colon cancer cell line like HT-29, CaCo
which show some inflammatory responses. In our experiment we have used HT-29 cell which expressed various
proteins indicating inflammatory responses (Haller D 2004).
Development of inflammatory conditions in HT-29 cell line could be achieved through various ways. In several
& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell wal
vivo) numbers of bacterial strains exist but few of them have been found to be
nic in nature. LPS was isolated from different bacterial strains and were introduced in HT
selection of LPS we used strains of E.coli B-4 (present in abundance in intestine) to elicit infection. It was found that
much inflammatory conditions in HT-29 cells. Then LPS was isolated from different strains
inflammatory reactions. It was observed that S.Minn and S.Ent were most potent inflammatory agents.
After developing inflammatory conditions in colon cancer cell line HT-29 with LPS isolated from bacterial strains of
Cytokines are categorized under pro-inflammatory and anti-inflammatory in nature. In our present study we selected
23) cytokine for observing the effect of LPS. LPS induction expressed different levels of IL
with respect to each bacterial strain (Puleston J 2005). Upregulation of IL-23 cytokine (proinflammatory) confirmed
the inflammatory conditions in given cell line.
Further in our study to check the inflammation process developed, the effect of certain anti
studied in the same (Botoman VA 1991). The popularly used drug for IBD like Sulfasalizine at different
concentration was introduced to inhibit the upregulated cytokine IL-23 (Bonner GB 1996). Complete or above 70%
23 with minimum concentration of Sulfasalizine was regarded as efficiency of drug to
vivo model of IBD (Mulder J 1988) (Fig.1).
line to study the cytotoxic effect and most effective concentration with least cytotoxic effect was analyzed (Fig 2).
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www.iiste.org
rentiated as cancer cells when induced by some inflammatory agents. Normally in-vivo these epithelial cells
biota of intestine resulting in the process of inflammation
The process of inflammation could be studied with the help of certain tumour markers. In preparing the in-vitro
29, CaCo-2, SW-620 and others
29 cell which expressed various
ough various ways. In several
& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell wall
vivo) numbers of bacterial strains exist but few of them have been found to be
nic in nature. LPS was isolated from different bacterial strains and were introduced in HT-29 cells. In the
4 (present in abundance in intestine) to elicit infection. It was found that
29 cells. Then LPS was isolated from different strains
S.Ent were most potent inflammatory agents.
29 with LPS isolated from bacterial strains of
inflammatory in nature. In our present study we selected
23) cytokine for observing the effect of LPS. LPS induction expressed different levels of IL-23
23 cytokine (proinflammatory) confirmed
Further in our study to check the inflammation process developed, the effect of certain anti-inflammatory drugs was
The popularly used drug for IBD like Sulfasalizine at different
23 (Bonner GB 1996). Complete or above 70%
regarded as efficiency of drug to
ive concentration with least cytotoxic effect was analyzed (Fig 2).
vitro model of inflammatory bowel disease.
Salmonella Minnesota of
culture system of inflammation. IL-23 levels
Araki, Y. Sugihara, H. & Hattori T. (2006) In vitro effects of dextran sulfate sodium on a Caco-2 cell line and
.
Brintrup, S. Pauels, H.G. Schürmann, G, Domschke, W. & Kucharzik, T. (2001)
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Vol.3, No.4, 2013
Figure 1.
Legend 1.
The above graph shows induction of IL
and Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of IL
by LPS strain. From third plot onwards different concentration of drug is provided to down regulate the IL
production and almost 100% inhibition
Figure 2.
Legend 2.
Cell viability for the above drug concentration was as
drug can be safely used till a concentration of around 1mM and there after number of cell death was found to be
more.
0
100
200
300
400
500
600
700
800
900
HT con
IL-23
(pg/ml)
Inhibition of IL
0
20
40
60
80
100
120
Percentageviability
Cell viability upon treatment of HT29 cells with Sulfasalizine
29
The above graph shows induction of IL-23 in HT-29 cells by LPS isolated from the strains of Salmonella Minnesota
and Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of IL
From third plot onwards different concentration of drug is provided to down regulate the IL
inhibition is seen around 1mM – 2.5 mM drug concentration
Cell viability for the above drug concentration was assayed simultaneously using MTT assay and it was found that
No drug 25uM 50uM 100uM 500uM 1mM
Inhibition of IL-23 in HT 29 cells by Sulfasalizine
S.minn
Sal.ent
concentration of drug
Cell viability upon treatment of HT29 cells with Sulfasalizine
sal.minn LPS
Sal.ent LPS
www.iiste.org
29 cells by LPS isolated from the strains of Salmonella Minnesota
and Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of IL-23
From third plot onwards different concentration of drug is provided to down regulate the IL-8
2.5 mM drug concentration
sayed simultaneously using MTT assay and it was found that
1mM 2.5mM
S.minn
Sal.ent
sal.minn LPS
Sal.ent LPS

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