Helicobacter ISSN 1523-5378

Modulation of Activation-Induced Cytidine Deaminase by

Curcumin in Helicobacter pylori-Infected Gastric Epithelial Cells
Syed Faisal Haider Zaidi,*,† Takeshi Yamamoto,† Alaa Refaat,‡ Kanwal Ahmed,§ Hiroaki Sakurai,‡ Ikuo
Saiki,‡ Takashi Kondo,§ Khan Usmanghani,¶ Makoto Kadowaki† and Toshiro Sugiyama*
*
Department of Gastroenterology and Hematology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama,
Japan, †Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, Toyama, Japan, ‡Division of Pathogenic
Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan, §Department of Radiological Sciences, Graduate School of
Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan, ¶Department of Basic Clinical Sciences, Faculty of Eastern Medicine,
Hamdard University, Karachi, Pakistan

Keywords
H. pylori, activation-induced cytidine
deaminase, curcumin, NF-jB.
Reprint requests to: Toshiro Sugiyama, Depart-
ment of Gastroenterology and Hematology,
Graduate School of Medicine and Pharmaceu-
tical Sciences, University of Toyama, 2630-
Sugitani, Toyama 930-0194, Japan.
E-mail: tsugi@med.u-toyama.ac.jp
Abstract
Background: Anomalous expression of activation-induced cytidine
deaminase (AID) in Helicobacter pylori-infected gastric epithelial cells has
been postulated as one of the key mechanisms in the development of gastric
cancer. AID is overexpressed in the cells through nuclear factor (NF)-jB
activation by H. pylori and hence, inhibition of NF-jB pathway can down-
regulate the expression of AID. Curcumin, a spice-derived polyphenol, is
known for its anti-inflammatory activity via NF-jB inhibition. Therefore, it
was hypothesized that curcumin might suppress AID overexpression via
NF-jB inhibitory activity in H. pylori-infected gastric epithelial cells.
Materials and Methods: MKN-28 or MKN-45 cells and H. pylori strain 193C
isolated from gastric cancer patient were used for co-culture experiments.
Cells were pretreated with or without nonbactericidal concentrations of
curcumin. Apoptosis was determined by DNA fragmentation assay. Enzyme-
linked immunosorbent assay was performed to evaluate the anti-adhesion
activity of curcumin. Real-time polymerase chain reaction was employed to
evaluate the expression of AID mRNA. Immunoblot assay was performed for
the analysis of AID, NF-jB, inhibitors of NF-jB (IjB), and IjB kinase (IKK)
complex regulation with or without curcumin.
Results: The adhesion of H. pylori to gastric epithelial cells was not inhibited
by curcumin pretreatment at nonbactericidal concentrations (£10 lmol ⁄ L).
Pretreatment with nonbactericidal concentration of curcumin downregulated
the expression of AID induced by H. pylori. Similarly, NF-jB activation inhib-
itor (SN-50) and proteasome inhibitor (MG-132) also downregulated the
mRNA expression of AID. Moreover, curcumin (£10 lmol ⁄ L) has suppressed
H. pylori-induced NF-jB activation via inhibition of IKK activation and IjB
degradation.
Conclusion: Nonbactericidal concentrations of curcumin downregulated
H. pylori-induced AID expression in gastric epithelial cells, probably via the
inhibition of NF-jB pathway. Hence, curcumin can be considered as a
potential chemopreventive candidate against H. pylori-related gastric
carcinogenesis.

Helicobacter pylori is a gram-negative, spiral-shaped,
microaerophilic bacterium that colonizes the human
gastric mucosa while neutralizing the hostile gastric
environment [1]. It is estimated to have colonized
about half of the world’s human population and has
been recognized as the causative agent of chronic
gastritis, gastroduodenal ulcers, mucosa-associated
lymphoid tissue lymphoma, and gastric adenocarcinoma
[2]. H. pylori interaction with gastric epithelial cells
resulted in overexpression of pro-inflammatory

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Haider et al.
cytokines and the activation of the transcription factor,
nuclear factor (NF)-jB [3]. NF-jB is a crucial regulator
of many cellular processes and mediators including
regulation of inflammatory cytokines, chemokines,
adhesion molecules, enzymes, and kinases. NF-jB is
localized in the cytoplasm by binding to a family of
cytoplasmic inhibitors, the inhibitors of NF-jB (IjB).
Cell stimulation triggers specific intracellular signaling
pathways consequently leading to the activation of the
IjB kinase complex (IKK complex). The activated IKK
complex phosphorylates the NF-jB-bound IjB proteins
and targets them for poly-ubiquitination and rapid
degradation. The dissociated NF-jB is translocated into
the nucleus and regulates NF-jB-dependent gene
expression [4,5].
Recently, Matsumoto et al. has reported the involve-
ment of NF-jB in the abnormal expression of activation-
induced cytidine deaminase (AID) in H. pylori-infected
gastric epithelial cells [6]. AID is an enzyme that
produces immune diversity by inducing somatic hyper-
mutations (SHM) and class-switch recombinations (CSR)
in human immunoglobulin (Ig) genes. The impaction of
AID as a genome mutator could aim at the generation of
somatic mutations in various host genes of nonlymphoid
tissues like TP53 tumor suppressor gene and contribute
to tumorigenesis. Thus, NF-jB and NF-jB-regulated
gene products, particularly AID, have been closely
linked with H. pylori-induced gastric carcinogenesis.
Therefore, agents that suppress NF-jB pathway might
have potential efficacy in preventing the occurrence of
H. pylori-related cancerous lesions [7,8].
Since the role of H. pylori infection is to promote car-
cinogenesis rather than to act as a direct carcinogen,
the eradication in long-standing infected gastric mucosa
might be insufficient to halt or reverse the pathogenic
changes of gastric inflammation and tumorigenesis
[9,10]. A recent surge in nonantimicrobial approach
such as anti-oxidants, probiotics, vitamins, plant
extracts, and phytochemicals has opened unique
dimensions to control H. pylori-related inflammation
and precancerous lesions [11,12]. The anti-H. pylori
effects of phytomedicines and their phytochemicals
have been documented in the literature [13–15].
Curcumin, a major constituent of the spice named
Curcuma longa, is cited earlier for its anti-inflammatory,
cancer chemopreventive, and anti-H. pylori activity
[16,17]. Various biological and signaling effects of
curcumin have been reported such as cell shrinkage,
chromatin condensation, and inhibition of nitric oxide
synthase, protein kinase C, NF-jB, and cyclin D1
[18,19]. In this study, we have examined the effect of
non-bactericidal concentrations of curcumin on the
expression of AID in H. pylori-infected gastric epithelial
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 588–595
Curcumin Downregulates AID in H. pylori-Infected Cells
cells and whether or not this effect is the result of the
inhibition of NF-jB pathway.
Materials and Methods
Reagents
RPMI-1640 culture medium was procured from Wako
(Osaka, Japan). Curcumin was purchased from Nacalai
Tesque, Inc. (Kyoto, Japan). NF-jB activation inhibitor
SN-50 and proteasome inhibitor MG-132 was obtained
from Calbiochem (EMD Biosciences, Inc., CA, USA).
Human tumor necrosis factor (TNF)-a was purchased
from R&D systems (Minneapolis, MN, USA). Brucella
broth (BBLTM) was procured from BD (Sparks, MD,
USA).
Bacterial Strains Culture Conditions
H. pylori strain 193C derived from gastric cancer patient
[20], was cultured in Brucella broth medium supple-
mented with 10% fetal bovine serum (FBS; i.e.,
BB-FBS). The strains were subcultured before co-culture
experiments in 10 mL Brucella broth liquid culture for
24–48 hours under microaerophilic conditions (5% O2,
10% CO2, and 85% N2 at 37C; Sanyo-Multigas Incuba-
tor; SANYO Electric Co., Ltd. Tokyo, Japan) on a gyra-
tory shaker at 160 rpm with 100% humidity. The
concentration of bacteria was estimated by using the
formula as an absorbance of 0.1 = 108 bacteria per mL
[21].
Determination of Nonbactericidal Concentration
of Curcumin
The anti-H. pylori concentration of curcumin was evalu-
ated by the method described earlier [22] with minor
modifications. Briefly, the BB-FBS medium routinely
used for growing H. pylori was prepared by adding
various concentrations of curcumin as 1, 5, 10, 20, 40,
and 80 lmol ⁄ L. Equal amounts of H. pylori liquid
culture were added in each curcumin-supplemented
media after adjusting the absorbance at A600. Control
was prepared for each concentration by adding similar
concentrations of curcumin in liquid media but without
H. pylori inoculation. Cultures were grown in microaer-
ophilic conditions as described before for different time
periods and the absorbance was measured at 600 nm.
Determination of DNA Fragmentation
Quantitative DNA fragmentation assay was carried out
according to the method of Sellins and Cohen [23].
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Curcumin Downregulates AID in H. pylori-Infected Cells
Briefly, cells pretreated with or without curcumin
were lysed in a lysis buffer (10 mmol ⁄ L Tris,
1 mmol ⁄ L EDTA, 0.2% Triton X-100, pH 7.5) and
centrifuged at 13,000 ·g for 10 minutes. Then, each
DNA sample in the supernatant and the resulting
pellet were precipitated in 12.5% trichloroacetic acid
(TCA) at 4C, and quantified using a diphenylamine
reagent after hydrolysis in 5% TCA at 90C for
20 minutes. The percentage of fragmented DNA for
each sample was calculated as the amount of DNA in
the supernatant divided by the total DNA for that
sample (supernatant plus pellet).
Co-Culture of Helicobacter pylori with Gastric
Epithelial Cells
Human gastric carcinoma-derived MKN-45 epithelial
cells (Riken Cell Bank, Tsukuba, Japan) were grown in
RPMI 1640 containing 2 mmol ⁄ L of l-glutamine and
10% FBS at 37C in 5% CO2. MKN-45 cells were
routinely passaged every 3 days. Cells were seeded into
6-cm culture dish and grown for 24 hours followed by
washing with phosphate-buffered saline (PBS) thrice.
The medium RPMI 1640, without antibiotics and FBS,
was added finally 3 hours before addition of H. pylori.
H. pylori were cultured overnight in BB-FBS (10%)
under the conditions mentioned before and then
washed twice with PBS. Bacteria were then directly
added to MKN-45 cells for the indicated times.
Anti-Adhesion Activity Assay by ELISA
MKN-28 and MKN-45 cells, derived from human gas-
tric carcinomas, were used for the analysis of H. pylori
adhesion by the method described earlier [24,25].
Briefly, cells were preincubated with or without Curcu-
min (2.5–10 lM) in 96-well plates at 37C under 5%
CO2 for 90 minutes and H. pylori was added in the trea-
ted and untreated wells. The cells were then fixed at
4C for 60 minutes and after washing, 100 lL of rabbit
anti-H. pylori polyclonal antibody (DakoCytomation,
Glostrup, Denmark) was added to each well, and the
plates were incubated for 2 hours at 37C. After wash-
ing, 100 lL of peroxidase-conjugated goat anti-rabbit
Igs (Wako) diluted 1 : 1000 in PBS was added to each
well and incubated for 2 hours at 37C. After final
washing, 100 lL of substrate reagent pack (DY999;
R&D System Inc., Minneapolis, MN, USA) was added
for 15 minutes followed by addition of 100 lL of
1 mol ⁄ L H2SO4 to terminate the reaction. The optical
density (OD) of the reaction was measured at 490 nm
with a microplate reader. The OD represents the
amount of H. pylori adhering to target cells.
590
Haider et al.
Quantitative Analysis of mRNA Expression
For the evaluation of AID gene expression, cells were
co-cultured with H. pylori in the presence or absence of
curcumin, SN-50, and MG-132 for 24 hours, then
washed thrice with PBS, scrapped by rubber policeman
and the total RNA was extracted by RNeasy Plus Mini
kit (Qiagen, Germany) according to the manufacturer’s
instructions. Reverse transcription was performed using
the Exscript RT reagent kit (Takara Bio Inc., Shiga,
Japan) and random primers, followed by real-time
polymerase chain reaction (RT-PCR). Amplification of
AID and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was performed using SYBR Premix Ex Taq
(Takara Bio Inc.). The following primer pairs were used:
AID, forward 5¢-AATTAGCCAGGCGTGGTAGCAG-3¢
and reverse 5¢-TGCAACGGCACGATCTCAG-3¢; GAPDH,
forward 5¢-GCACCGTCAAGGCTGAGAAC-3¢ and
reverse 5¢-TGGTGAAGACGCCAGTGGA-3¢. Real-time
(RT)-PCR was carried out using the Takara Thermal
Cycler Dice Real time system TP800 (Takara Bio Inc.).
AID mRNA was normalized to GAPDH mRNA as an
internal control in each sample. Results were expressed
as the ratio relative to the average of uninfected cells.
Immunoblot Analysis
MKN-45 cells were maintained in RMPI-1640 medium
as mentioned before. Cultures were kept at 37C in a
humidified atmosphere of 5% CO2 ⁄ 95% air. MKN-45
cells were co-cultured with H. pylori for 45 minutes.
Curcumin was added for 60 minutes before the addi-
tion of H. pylori. Whole cell lysates from MKN-45 cells
were prepared with lysis buffer (25 mmol ⁄ L of HEPES
pH 7.7, 0.3 mol ⁄ L of NaCl, 1.5 mmol ⁄ L of MgCl2,
0.2 mmol ⁄ L of EDTA, 0.1% Triton X-100, 20 mmol ⁄ L
of b-glycerophosphate, 0.1 mmol ⁄ L of sodium ortho-
vanadate, 0.5 mmol ⁄ L of phenylmethylsulfonyl
fluoride, 1 mmol ⁄ L of dithiothreitol, 10 lg ⁄ mL of apro-
tinin, and 10 lg ⁄ mL of leupeptin). Cell lysates were
resolved by SDS-PAGE and transferred to an
Immobilon-P nylon membrane (Millipore, MA, USA).
The membrane was treated with BlockAce (Dainippon
Pharmaceutical Co. Ltd., Suita, Japan) overnight at 4C
and probed with antibodies against human AID from
ProSci Inc. (Poway, CA, USA) and NF-jB p65 (C-20-G),
IjBa (C-21), actin (C-11) from Santa Cruz Biotechnol-
ogy (CA, USA), and NF-jB phospho-p65 (Ser536;
93H1), IKKa, and phospho-IKKa ⁄ b (ser176 ⁄ 180) from
Cell Signaling Technology (Boston, MA, USA). The pri-
mary antibodies were detected using horseradish perox-
idase-conjugated anti-rabbit, or anti-goat IgG (Dako)
and visualized with the enhanced chemiluminescent
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 588–595
Haider et al.
system (Amersham Biosciences, Buckinghamshire, UK).
Ramos cell lysate (ProSci Inc.) was employed as a posi-
tive control for AID expression.
Statistical Analysis
The results are expressed as the mean ± SD. Statistic
significance (p < .01, p < .05) was evaluated by one-
way ANOVA followed by Bonferroni post-hoc test for
real-time PCR analysis of AID expression.
Results
Nonbactericidal Concentration of Curcumin
As a first step, we analyzed the growth of H. pylori in
10% BB-FBS medium supplemented with different curc-
umin concentrations (1–80 lmol ⁄ L), to determine the
nonbactericidal concentration of curcumin. A previous
study by Foryst-Ludwig et al. demonstrated no effect on
H. pylori viability upto 80 lmol ⁄ L of curcumin when
preincubated for 1 and 3 hours [19]. The results of our
study coincides with these results as curcumin
(£ 80 lmol ⁄ L) showed no significant effect on H. pylori
growth up to 5 hours culture. However, H. pylori
growth was prominently inhibited after 5 hours culture
at a concentration ‡ 20 lmol ⁄ L (data not shown).
Concentrations of curcumin at £ 10 lmol ⁄ L showed no
bactericidal effect on the growth of H. pylori till 36 hours;
hence, these concentrations were employed for further
assays. To test the effects of curcumin on the viability of
the bacteria, cultures containing different concentrations
of curcumin (2.5–10 lmol ⁄ L) were plated after serial
dilutions onto H. pylori-selective agar plates under micro-
aerophilic conditions, and the numbers of CFU
(CFU ⁄ mL) were determined, as previously described
[22]. The results revealed that the numbers of CFUs
recovered from the liquid cultures containing curcumin
£10 lmol ⁄ L (curcumin 2.5 lmol ⁄ L; 55 · 106 CFU ⁄ mL,
curcumin 5 lmol ⁄ L; 61 · 106 CFU ⁄ mL, and curcumin
10 lmol ⁄ L; 57 · 106 CFU ⁄ mL) were similar to that of the
liquid culture without curcumin (62 · 106 CFU ⁄ mL).
Nontoxic Concentration of Curcumin Evaluated by
DNA Fragmentation
To evaluate whether nonbactericidal concentration of
curcumin possess some toxicity against MKN-45 cells,
DNA fragmentation, a feature characteristic of apopto-
sis, was measured concentration dependently at 6 and
24 hours of incubation. The results revealed no signi-
ficant induction of DNA fragmentation on any concen-
tration (5–50 lmol ⁄ L) of curcumin at 6 hours of
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 588–595
Curcumin Downregulates AID in H. pylori-Infected Cells
incubation when compared with untreated cells. With
24 hours of incubation, curcumin showed high level of
DNA fragmentation at 50 lmol ⁄ L compared with the
control (data not shown). Hence, concentrations of
curcumin less than 20 lmol ⁄ L were deemed suitable
for co-culture experiments.
Determination of Anti-Adhesion Activity of
Curcumin against Helicobacter pylori Binding to
Gastric Epithelial Cells
Previous studies have demonstrated that colonization of
H. pylori is an initial step for H. pylori-induced gastric
inflammation. After adhesion, H. pylori inject various
factors by type IV secretion system that are responsible
for gastric carcinogenesis. Hence, an agent that could
inhibit the adhesion of H. pylori to gastric epithelial cells
might hamper H. pylori-related pathogenesis in stomach
[25,26]. To examine the anti-adhesion activity of
different nonbactericidal concentrations of curcumin
(2.5–10 lmol ⁄ L), enzyme-linked immunosorbent assay
(ELISA) was employed in two gastric cancer cell lines,
MKN-28 and MKN-45. The results revealed that
adhesion of H. pylori to gastric epithelial cells was not
inhibited by curcumin pretreatment at nonbactericidal
concentrations (Fig. 1). These results indicate that
possible effects of curcumin (£ 10 lmol ⁄ L) in H. pylori-
infected gastric epithelial cells will not be either a result
of alteration of bacterial viability or by inhibition of
H. pylori adhesion to epithelial cells.
Curcumin Downregulated AID Expression in
Helicobacter pylori-Infected Cells
The mRNA expression of AID was analyzed by real-
time PCR. As described earlier [6], AID expression is
Figure 1 Evaluation of anti-adhesion activity of curcumin against
Helicobacter pylori binding to MKN-28 and MKN-45 cells. Cells were
treated with various concentrations of curcumin (2.5–10 lmol ⁄ L) for
90 minutes. The amount of adherent H. pylori is expressed as the
percentage of the amount of H. pylori adhering to untreated cells.
Each value represents the mean ± SD (n = 3).
591
Curcumin Downregulates AID in H. pylori-Infected Cells Haider et al.

upregulated via NF-jB activation in H. pylori-infected A

cells; hence, we evaluated the effect of curcumin on
AID expression. Initially, H. pylori was co-cultured with
cells for 6, 12, and 24 hours followed by AID mRNA
expression analysis. AID was not upregulated in 6 and
12 hours co-culture with H. pylori; however, after
24 hours co-culture the AID mRNA was significantly
(p < .01) overexpressed (Fig. 2A). Further, gastric epi-
thelial cells were pretreated with curcumin (5 and
10 lmol ⁄ L) for 1 hour followed by H. pylori co-culture
for 24 hours, and the extracted RNA was employed for
B
real-time PCR analysis. After pretreatment of curcumin
at 5 and 10 lmol ⁄ L, the expression of AID was down-
regulated significantly (p < .05 and p < .01, respec-
tively) in a concentration-dependent manner (Fig. 2B).
Immunoblot analysis showed that AID protein expres-
sion was increased substantially in H. pylori-infected
MKN-45 cells which was considerably inhibited by
curcumin (10 lmol ⁄ L) pretreatment (Fig. 2C). To clarify
whether NF-jB transcription pathway might have
involved in the regulation of AID, NF-jB activation
inhibitor (SN-50) and proteasome inhibitor (MG-132)
C
were examined by real-time PCR at the concentrations
(250 ng ⁄ mL and 0.15 lmol ⁄ L, respectively) mentioned
earlier [6]. The cells were preincubated with SN-50 and
MG-132 for 2 hours followed by addition of H. pylori.
The results revealed almost complete inhibition of AID
expression with both the inhibitors as shown in Fig. 3,
which is suggestive of NF-jB involvement in AID
Figure 2 MKN-45 cells co-cultured with Helicobacter pylori strain
expression in MKN-45 cells.
193C induces AID mRNA and protein expression. (A) Expression of AID
mRNA in H. pylori-infected cells at different time intervals (6, 12, and
Curcumin Suppressed NF-jB Activation by 24 hours). Cells were incubated with or without H. pylori and RNA
was extracted. Data represent the mean values of three independent
Inhibition of IKK Activity and IjB Degradation
experiments. **p < .01 (compared with that of the control). (B) Effect
As previously reported [19] that curcumin inhibits of curcumin on the expression of AID in H. pylori-infected cells.
NF-jB activation by hampering IKK activity and subse- Cells were plated in culture dishes till 80% confluence and incubated
with or without curcumin (5 and 10 lmol ⁄ L) for 60 minutes followed
quent IjB destruction, we further examined this effect
by the addition of H. pylori. Total RNA was isolated after 24 hours of
by Western blot at nonbactericidal concentrations of co-culture and AID mRNA expression was normalized to glyceralde-
curcumin (5 and 10 lmol ⁄ L). As shown in Fig. 4, it can hyde-3-phosphate dehydrogenase mRNA as an internal control in each
be seen that IjBa is phosphorylated and degraded in sample. Results were expressed as the ratio relative to the uninfected
H. pylori-co-cultured and TNF-a-treated cells; in con- cells. Data represents the mean values of three independent experi-
trast, IjBa degradation was inhibited by pretreatment ments; **p < .01 and *p < .05 (compared with H. pylori-infected cells
of curcumin in a concentration-dependent manner. without curcumin). (C) Immunoblot of lysates from MKN-45 cells either
left untreated or treated with curcumin followed by infection with
Similarly, H. pylori- and TNF-a-induced NF-jB p65
H. pylori for 24 hours using human AID antibody. Ramos cell lysate
phosphorylation was also contained by curcumin (RCL) was used as positive control for AID expression.
pretreatment at 5 and 10 lmol ⁄ L. To evaluate whether
this phosphorylation of NF-jB p65 and IjBa is
regulated by IKK activity, we further carried out immu-
Discussion
noblot analysis using phospho-IKKa ⁄ b antibody. The
results showed inhibition of IKK activity by curcumin Previous global cancer studies reported 876,000 new
pretreatment particularly at a concentration of cases and more than 649,000 deaths from gastric cancer
10 lmol ⁄ L while there was no effect on the expression which remains still the second leading cause of cancer
of IKKa. deaths worldwide [27,28]. H. pylori plays a key role in

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Haider et al.
Figure 3 Co-culture of Helicobacter pylori with MKN-45 cells in the
presence or absence of nuclear factor (NF)-jB activation inhibitors
SN-50 (250 ng ⁄ mL) and MG-132 (0.15 lmol ⁄ L). Cells were incubated
with or without inhibitors for 2 hours before H. pylori inoculation.
RNA was extracted after 24 hours of co-culture and AID mRNA expres-
sion was examined. Data represent the mean values of three indepen-
dent experiments; **p < .01 (compared with H. pylori-infected cells
without inhibitors).
gastric diseases and its presence increases the risk of
gastric cancer 20-fold, compared with controls [9].
Recent advances in cancer research has opened new
insights in the molecular mechanism of gastric carcino-
genesis especially cancer initiation and promotion. The
involvement of AID enzyme in tumorigenesis is one of
the milestones in understanding the process of carcino-
genesis providing a novel link between inflammation,
mutagenesis, and cancer development [29]. The causa-
tive factors for upregulation of AID can be a viral infec-
tion, lipopolysaccharides from bacteria, inflammation
and, more recently, H. pylori itself [6,7]. AID has been
identified as a key enzyme for CSR and SHM leading to
chromosomal translocations and genetic mutations.
Therefore, the blockade of AID can be a promising
chemopreventive approach to control H. pylori-associ-
ated gastric cancers.
Curcumin, a spice-derived polyphenol, is known for
its anti-inflammatory and chemopreventive activity.
Several lines of evidence, including epidemiological
data, preclinical and clinical trials, suggest that curcu-
min may be used as a potential candidate for cancer
chemoprevention in diseases like pancreatic cancer,
colorectal cancer, familial adenomatous polyposis, and
inflammatory bowel disease [30–36]. These studies also
demonstrated low bioavailability of curcumin after oral
administration suggesting that the anticancer activity of
oral curcumin may be limited to the gastrointestinal
tract. To evaluate the toxicity of curcumin, three
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 588–595
Curcumin Downregulates AID in H. pylori-Infected Cells
Figure 4 Effects of nonbactericidal concentration of curcumin on
Helicobacter pylori-induced nuclear factor (NF)-jB activation via
modulating degradation of NF-jB inhibitors (IjB), and IjB kinase (IKK)
activation. MKN-45 cells were pretreated with or without curcumin at
the indicated concentrations for 60 minutes followed by infection with
H. pylori for 45 minutes or tumor necrosis factor (TNF)-a stimulation
(10 ng ⁄ mL, 10 minutes). NF-jB phosphorylation, IjBa degradation,
and IKK activation in the cells were evaluated by immunoblotting using
antibodies against NF-jB phospho-p65 (p-p65), NF-jB p65 (p65),
phospho-IjBa (p-IjBa), IjBa, IKKa, and phospho-IKKa ⁄ b (p-IKKa ⁄ b).
different Phase I clinical trials performed to determine
safety have reported no toxicity of curcumin at doses as
high as 15 g ⁄ day [37]. A recent study by Mario et al.
comprising of 30 mg ⁄ day curcumin-based 1-week triple
therapy for H. pylori eradication mentioned significant
improvement of dyspeptic symptoms and reduction of
gastric inflammatory response even after 2 months of
the therapy [11]. These propitious sequels of curcumin
prompted us to investigate its effect on AID regulation
in H. pylori-infected gastric epithelial cells. The results
obtained from real-time PCR and immunoblot analysis
vividly revealed that pretreatment with curcumin even
at low concentrations, downregulated the expression of
AID in H. pylori-treated MKN-45 cells. To elaborate that
this downregulation of AID might be because of NF-jB
inhibition, we have added NF-jB activation inhibitor
(SN-50) and proteasome inhibitor (MG-132) before
infecting cells with H. pylori. The results showed almost
complete downregulation of AID. Although, these
inhibitors are not specific for only NF-jB pathway but
the results are suggestive that NF-jB might be responsi-
ble for AID regulation in H. pylori-infected MKN-45
cells as demonstrated earlier in AGS gastric cancer
cell line [6]. Next, we examine the regulation of
molecules which are involved in NF-jB pathway by
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Curcumin Downregulates AID in H. pylori-Infected Cells
low concentrations of curcumin. NF-jB is mainly com-
posed of a heterodimer of p65 and p50 subunits in most
cell types and IKK can directly phosphorylate NF-jB
p65 at Ser-536 residue which is suggested to be a useful
marker for detecting the constitutive activation of
NF-jB in cancer cells [38,39]. As demonstrated in this
study, curcumin suppressed H. pylori-induced NF-jB
p65 and IjB phosphorylation by inhibiting IKK activity
in a concentration-dependent manner.
Taken together, low concentrations of curcumin
downregulated H. pylori-induced AID expression which
might be because of the inhibition of NF-jB activation
in H. pylori-infected MKN-45 gastric cancer cells. Further
studies are required to explicit this potential role of
curcumin in preventing the development of cancer that
can be employed in countries with high gastric cancer
prevalence like East Asian countries including Japan.
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