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Carcinogenesis vol.30 no.5 pp.799–807, 2009
doi:10.1093/carcin/bgn246
Advance Access publication November 20, 2008
Aldose reductase deficiency in mice prevents azoxymethane-induced colonic
preneoplastic aberrant crypt foci formation
Ravinder Tammali, Aramati B.M.Reddy, Kota V.Ramana,
J.Mark Petrash1,2 and Satish K.Srivastava
To whom correspondence should be addressed. Tel: þ1 409 772 3926;
Fax: þ1 409 772 9679;
Email: [email protected]
Aldose reductase (AR; EC 1.1.1.21), an nicotinamide adenine dinucleotide phosphate-dependent aldo–keto reductase, has been
shown to be involved in oxidative stress signaling initiated by inflammatory cytokines, chemokines and growth factors. Recently,
we have shown that inhibition of this enzyme prevents the growth
of colon cancer cells in vitro as well as in nude mice xenografts.
Herein, we investigated the mediation of AR in the formation of
colonic preneoplastic aberrant crypt foci (ACF) using azoxymethane (AOM)-induced colon cancer mice model. Male BALB/c mice
were administrated with AOM without or with AR inhibitor,
sorbinil and at the end of the protocol, all the mice were euthanized
and colons were evaluated for ACF formation. Administration of
sorbinil significantly lowered the number of AOM-induced ACF.
Similarly, AR-null mice administered with AOM demonstrated significant resistance to ACF formation. Furthermore, inhibition of
AR or knockout of AR gene in the mice significantly prevented
AOM-induced expression of inducible nitric oxide synthase and
cyclooxygenase-2 proteins as well as their messenger RNA. AR inhibition or knockdown also significantly decreased the phosphorylation of protein kinase C (PKC) b2 and nuclear factor kappa
binding protein as well as expression of preneoplastic marker
proteins such as cyclin D1 and b-catenin in mice colons. Our results
suggest that AR mediates the formation of ACF in AOM-treated
mice and thereby inhibition of AR could provide an effective
chemopreventive approach for the treatment of colon cancer.
Introduction
Colon cancer is the third most common form of cancer and the second
leading cause of cancer-related deaths in western countries, including
the USA (1,2). Epidemiological and experimental studies indicate that
colon cancer is usually mediated by dietary and environmental factors
and is more pronounced in genetically predisposed subjects (3–6).
Diet rich in fat, red meat, refined carbohydrates and animal proteins,
along with low physical activity, obesity and hyperinsulinemia are
risk factors, which can predispose colon cancer (3–7). Normal colonic
mucosa is constantly renewed through controlled proliferation and
differentiation of mitotically active cell populations in the lower third
of colonic crypts (8). With normal maturation, the resultant cells lose
their ability to proliferate and migrate to the surface, constantly replacing sloughed cells that have an approximate life span of 6 days
(8). Conditions leading to increased proliferation and loss of differentiation capacity lead to the formation of preneoplastic lesions referred to as aberrant crypt foci (ACF) (9,10). ACF, abnormal crypts,
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; AR, aldose reductase; ARKO, aldose reductase knockout; Cox-2, cyclooxygenase-2; GS-DHN,
glutathionyl-1,4-dihydroxynonene; GS-HNE, glutathionyl-4-hydroxynonenal;
HNE, 4-hydroxy-trans-2-nonenal; IHC, immunohistochemical; IL, interleukin;
iNOS, inducible nitric oxide synthase; mRNA, messenger RNA; NF-jB, nuclear
factor kappa binding protein; PCNA, proliferating cell nuclear antigen; PKC,
protein kinase C; ROS, reactive oxygen species; WT, wild-type.
Ó The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Department of Biochemistry and Molecular Biology, University of Texas
Medical Branch, Galveston, TX 77555-0647, USA, 1Department of
Ophthalmology and Visual Science and 2Department of Genetics, Washington
University School of Medicine, St Louis, MO 63110, USA
are two to three times larger than normal crypts and are characterized
by hyperproliferation, increased and elevated size and expanded pericryptal zones (9). Pretlow et al. (11) have shown the presence of ACF
in the colonic mucosa of colon cancer patients. Over period of time,
they develop into adenomas and carcinomas. Development of ACF
associates with mutations in the colon cancer biomarker genes such as
APC and ras oncogene (12,13).
The transition of normal epithelium to adenoma to carcinoma is
associated with a variety of molecular and biochemical events such as
genetic alterations, intestinal epithelial cell proliferation/differentiation and inflammation (13–15). The major initiators of carcinogenesis
include (i) cells that suffered irreparable DNA damage due to increased free radicals that cause activation of specific nucleases and
damage DNA, RNA, proteins and lipids; (ii) loss of extracellular
stimulation that regulates expression of growth factors and their receptors as well as cell growth and (iii) autosomal dominant inheritance of oncogenes among the multiple family members (9,16–18). In
addition, diet and environmental factors play an important role in
predisposition of carcinogenesis in genetically inherited subjects
(4). Furthermore, many chronic inflammatory diseases such as hepatitis, gastritis and ulcerative colitis are associated with an elevated risk
of colon cancer (14,19,20). However, it is not clear how cancer is
initiated in the setting of chronic inflammation, but accumulating
evidence strongly supports the association between colon cancer
and inflammation. Furthermore, upregulation of cytokines such as
tumor necrosis factor-a, interleukin (IL)-6, growth factors such as
insulin-like growth factor-II, hepatocyte growth factor, hepatocyte
growth factor receptor, epidermal growth factor receptor, insulin growth
factor receptor, vascular endothelial growth factor, fibroblast growth
factor and platelet-derived growth factor and their receptors have
been observed in various human colon cancer cells (17,19–21).
Exposure of cells to inflammatory cytokines and growth factors
triggers upregulation of prostaglandin E2 (PGE2) and nitric oxide
(NO) production via cyclooxygenase-2 (Cox-2) and inducible nitric
oxide synthase (iNOS), respectively (17,22–24). These local messenger
molecules act further in an autocrine and paracrine fashion elevate
reactive oxygen species (ROS). The ROS in turn activate various genes
involved in the expansion of normal epithelial cells to dysplasia
(precancer) and cancer (16). For example, the proinflammatory cytokines tumor necrosis factor-a, IL-1, IL-6 play an important role at
initial stages of cell transformation and tumor development. Among
the proinflammatory cytokines, tumor necrosis factor-a is recognized
as a central mediator in the pathophysiology of chronic inflammatory
bowel diseases such as Crohn’s and ulcerative colitis which cause increased risk of colon cancer (19–21).
The murine azoxymethane (AOM) model of colon cancer shares
many clinical, histopathological and molecular features with human
tumors, but they have a low tendency to metastasize (11,25). AOMinduced form of ACF shows hyperproliferation of colonic mucosal cells
and often mutated on K-ras and b-catenin genes and show microsatellite instability, like human tumors (11,25). This mechanism is associated with the overexpression of iNOS and Cox-2 enzymes with
increased NO and PGE2 production (17,24). Compared with human
tumors, accumulation of b-catenin in the nucleus suggests that Wnt/
b-catenin/Tcf pathway plays a major role in carcinogen-induced rat
tumors (13,26). However, unlike human tumors, AOM-induced tumors
rarely show mutation of APC gene and never mutate p53 gene (25).
Our recent studies in human colon cancer Caco-2 cells, human lens
epithelial cells , vascular endothelial cells and vascular smooth muscle cells suggest that the polyol pathway enzyme,aldose reductase
(AR; AKR1B1 in human, AKR1B3 in mouse and AKR1B4 in rat),
a member of aldo–keto reductase super family, is a regulator of
ROS signals induced by growth factors, cytokines, chemokines and
R.Tammali et al.
Materials and methods
Materials
C57BL/6 ARþ/þ and AR/ mice were maintained in a pathogen-free barrier
facility at Washington University School of Medicine, St Louis, MO. BALB/c
mice (weight 25 g) were obtained from Jackson laboratory (Bar Harbor, ME)
and housed in pathogen-free conditions with free access to food and water at
the institutional animal care facility. The animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by
the National Institutes of Health and in accordance with the Institute’s ‘Guideline of the Animal Care and Use Committee’. Mice were kept in suspended
cages 10 cm above bedding trays with a 12 h light–dark cycle in the animal
facility. Temperature and relative humidity were controlled at 21°C and 55%.
All mice were acclimatized to the above conditions for 1 week with free access
to standard laboratory rodent chow and drinking water until initiation of the
experiment. AOM bought from the Sigma–Aldrich Chemical Company (St
Louis, MO), and sorbinil was gift from Pfizer (Groton, CT). Antibodies against
Cox-2, iNOS, cyclin D1 and phospho-protein kinase C (PKC) b2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). b-Catenin antibodies
obtained from Cell Signaling (Danvers, MA). Mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase antibodies were obtained from Research Diagnostics (Concord, MA). Polyclonal AR and protein adducts of HNE
antibodies raised in rabbit. Other reagents for western blot analysis were obtained from Sigma-Aldrich. All other reagents used were of analytical grade.
AOM-induced colon carcinogenesis and ACF analysis
Approximately, 6-week-old mice were divided into three groups with six mice
in each group. Mice in groups 2 and 3 were treated with AOM in sterile saline,
at a dose of 10 mg/kg body wt intraperitoneally once a week, for 3 weeks. In
group 3, mice were treated with AR inhibitor sorbinil (25 mg/kg body wt/day;
intraperitoneally) for entire period, 10 weeks, after 24 h of first AOM injection.
Mice in group 1 received equal volume of sterile saline. Similarly, ARKO mice
groups also treated with AOM (10 mg/kg body wt; intraperitoneally) once
a week for 3 weeks. After 5 and 10 weeks of first AOM injection, all mice
were euthanized by exposure to CO2. The colons were removed, flushed with
saline and opened from anus to cecum and fixed flat between two pieces of
filter paper in 10% buffered formalin for 24 h. Colons were stained with 0.2%
methylene blue dissolved in saline, and the numbers of ACF were counted
under the microscope.
Immunohistochemistry
After microscopic evaluation of ACF, the colons were Swiss-rolled and embedded in paraffin. Immunohistochemical (IHC) analyses serial sections of
800
colon (5 lM) were performed as described elsewhere. Briefly, slides were
warmed at 60°C for 1 h and deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by boiling
slides in 10 mM sodium citrate (pH 6.0) for 10 min followed by peroxidase
reaction was blocked with 3% H2O2. After that, sections were rinsed in phosphate-buffered saline twice and incubated with blocking buffer (2% bovine
serum albumin, 0.1% Triton X-100 and 2% normal goat serum) for overnight at
4°C. The sections were incubated with primary antibodies against proliferating
cell nuclear antigen (PCNA), Cox-2, iNOS, cyclin D1, AR, protein–HNE and
b-catenin for 1 h at room temperature. Antigen–antibody binding was detected
by using DakoCytomation LSABþSystem-HRP kit. Sections were examined
by bright-field light microscopy (EPI-800 microscope; Nikon, Tokyo, Japan)
and photographed with a camera (Nikon) fitted to the microscope. Photomicrographs of the stained sections were acquired using an EPI-800 microscope
(bright-field) connected to a Nikon camera. Percent staining was determined
by measuring positive immunoreactivity per unit area. Arrows represent the
area for positive staining for an antigen. The intensity of antigen staining was
quantified by digital image analysis and the values are represented as fold
change versus control in arbitrary units.
Determination of lipid peroxidation
The levels of lipid peroxides (total a, b-unsaturated aldehydes) were estimated
in colon tissues by using lipid peroxidation assay kit. Briefly, the colon tissue
homogenized in phosphate-buffered saline in the presence of butylated hydroxytoluene (5 mM) and aldehydes were quantified colorimetrically using a lipid
peroxidation kit (Bioxytech LPO-586TM) obtained from Oxford Biomedical
Research, Oxford, MI, as per the supplier’s instructions. The determination is
based on the reaction of the chromogenic reagent, methanesulfonic acid with a,
b-unsaturated aldehydes such as HNE at 45°C. One molecule of aldehyde
reacts with two molecules of reagent to yield a stable chromophore with
maximal absorbance at 586 nm.
Reverse transcription–polymerase chain reaction
Total RNA from colon samples was isolated using the RNeasy kit as per
supplier’s instructions (Qiagen, Valencia, CA). Equal aliquots of RNA (1.0 lg)
isolated from each sample were reverse transcribed with Omniscript and
Sensiscript reverse transcriptase One-Step Reverse Transcriptase–PCR system with HotStar Taq DNA polymerase at 55°C for 30 min followed by
polymerase chain reaction amplification. The oligonucleotide primer sequences were as follows: 5#-GCATTGCCTCTGAATTCAACACAC-3# (sense) and
5#-GGACACCCCTTCACATTATTGCAG-3#
(antisense)
for
Cox-2,
5#-CTGCAGGTCTTTGACGCTCG-3# (sense) and 5#-GTGGAACACAGGGGTGATGC-3# (antisense) for iNOS and 5#-GTGGGCCGCTCTAGGCACCAA3# (sense) and 5#-CTTTAGCACGCACTGTAGTTTCTC-3# (antisense) for b-actin. Polymerase chain reaction was carried out in a GeneAmp 2700 thermocycler
(Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95°C for 15 min; 35 cycles of 94°C for 45 s, 61°C for 45 s, 72°C for
1 min and then 72°C for 5 min for final extension. Equal amounts of polymerase
chain reaction products were electrophoresed with 2% agarose, 1 tris acetate
ethylene diamine tetra acetic acid gels containing 0.5 lg/ml ethidium bromide.
Bands were quantified using Kodak Image Station 2000R loaded with Kodak
one-dimensional image analysis software and the average fold change intensities
were calculated.
Western blot analysis
Colon extracts were prepared in RIPA cell lysis buffer and an equal amount of
protein was separated on 12% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis, electroblotted on nitrocellulose membranes and probed with
specific antibodies against AR, Cox-2, phospho-PKCb2, cyclin D1, b-catenin
and glyceraldehyde-3-phosphate dehydrogenase. Antibody binding was detected by enhanced pico chemiluminescence (Pierce, Rockford, IL). Immunopositive bands were quantified using Kodak Image Station 2000R loaded with
Kodak one-dimensional image analysis software and the average fold change
intensities were calculated.
Statistical analysis
Data are presented as mean ± SE and the P values were determined using
the Wilcoxon rank-sum test. P values ,0.05 were considered as statistically
significant.
Results
Inhibition of AR prevents AOM-induced ACF formation in BALB/c
mice
The impetus for studying the effect of AR inhibition on AOM-induced
ACF formation comes from our recent observation that AR inhibitors
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lipopolysaccharide (27–30). AR efficiently reduces one of the most
abundant and toxic lipid aldehyde, 4-hydroxy-trans-2-nonenal (HNE),
to 1,4-dihydroxynonene and its glutathione conjugate, glutathionyl-4hydroxynonenal (GS-HNE), to glutathionyl-1,4-dihydroxynonene
(GS-DHN) (31). Recently, we have demonstrated that the reduced
glutathione-lipid aldehydes such as GS-DHN may be one of the regulators involved in nuclear factor kappa binding protein (NF-jB)
activation (19,31). Inhibition of AR prevented protein kinase C, NFjB and activator protein (AP)-1 activation and the increase in cell
growth caused by HNE and GS-HNE, but not GS-DHN suggesting
that the already reduced form of GS-DHN is insensitive to AR inhibition (31). Further, in in vivo rat model, we have shown that inhibition of AR significantly decreases neointima formation in
balloon-injured carotid arteries and in situ activation of NF-jB during
restenosis (29,32).
In human colon cancer Caco-2 cells, we have shown that inhibition
of AR prevents the cytokines- and growth factors-induced Cox-2
expression, activation of NF-jB and PGE2 production (17,19). In
addition, in in vivo nude mice xenograft model, we have shown that
inhibition of AR by AR–small interfering RNA (siRNA) prevents the
human colon adenocarcinoma SW480 cells-induced tumor growth
(17). Therefore, to further understand the role of AR in the pathogenesis of colon cancer, we investigated the role of AR in AOM-induced
ACF formation and inflammatory markers such as Cox-2 and iNOS
expression by using BALB/c mice. The results obtained from BALB/c
mice were further confirmed by using aldose reductase knockout
(ARKO) C57BL/6 mice and its wild-type (WT) littermates. Our studies indicate that inhibition or knockout of AR prevents AOM-induced
ACF formation and expression of inflammatory markers in mice.
Aldose reductase in ACF formation
Inhibition of AR prevents AOM-induced inflammatory markers
expression in BALB/c mice
Since both Cox-2 and iNOS are known to be involved in chronic inflammation, which creates a microenvironment contributing to the development of preneoplastic lesions in the colon carcinogenesis and
inhibitors of Cox-2 and iNOS have been shown to reduce ACF formation in rodents (11,14,24), we next investigated if inhibition of AR
could prevent AOM-induced Cox-2 and iNOS expression in mice colons. As shown in Figure 2A, AOM-induced expression of Cox-2 and
iNOS was significantly (68% and 86%) prevented by AR inhibition.
These results were further confirmed by immunohistochemistry using
antibodies against Cox-2 and iNOS (Figure 3A and B). The colon from
AOM-treated mice showed a significant intensity of antibody staining,
whereas treatment of mice with sorbinil showed a marked decrease in
antibody staining, suggesting that inhibition of AR prevented Cox-2
and iNOS expression. We next determined the effect of AR inhibition
on Cox-2 and iNOS expression at messenger RNA (mRNA) level
(Figure 2B). Treatment of mice with AOM caused a significant increase in the expression of both Cox-2 and iNOS mRNA and inhibition
of AR prevented it by 60 and 42%, respectively, suggesting that
AR could regulate transcriptional activation of Cox-2 and iNOS.
Inhibition of AR by sorbinil prevents AOM-induced phosphorylation
of PKCb2 and expression of AR in BALB/c mice
Carcinogen-induced preneoplastic lesions in the colonic epithelium are
known to be associated with the activation of PKCb2 that could cause
hyperproliferation (33,34). We therefore examined the effect of AR
inhibition on the activation of AOM-induced PKCb2 in mice colons.
A dramatic increase in PKCb2 phosphorylation was observed in the
AOM-treated mice, whereas inhibition of AR in AOM-treated mice
showed a significant decrease in the phosphorylation of PKCb2 (Figure
2C). Since AR is an oxidative stress response protein that is elevated in
various pathological conditions including cancer (29,35), we next examined the expression of AR in AOM-treated mice colons. As shown in
Figure 2C, treatment of mice with AOM increased the expression of AR
that was prevented by AR inhibition. IHC staining of colon sections
with anti-AR antibodies further confirmed the increased expression of
AR in AOM-treated mice as evidenced by a strong staining of AR in colon epithelium compared with colon from untreated controls (Figure 3C).
Fig 1. Inhibition of AR prevents AOM-induced ACF formation in BALB/c
mice. (A) After 10 weeks of AOM treatment, mice were killed and colons
were fixed in formalin and stained with 0.2% methylene blue for 5 min. ACF
were identified under light microscope (400 magnification) with colon
mucosal side up; 1, singlet; 2, doublet; 3, triplet and 4, multiaberrant crypts
with well-defined eye were identified and counted. (B) Balb/c mice were
divided into three groups: (i) control; (ii) AOM (10 mg/kg body wt, weekly
for 3 weeks) and (iii) received sorbinil (25 mg/kg body wt, intraperitoneally
per day) throughout the study after 24 h of first AOM injection. Mice were
euthanized 10 weeks after first AOM injection and colons were stained and
examined for ACF formation. For each group of animals, n 5 6;
P , 0.016 as compared with WT þ AOM alone. Wilcoxon rank-sum test
was performed for the statistical analysis of ACF data obtained from AOM
versus AOM þ sorbinil-treated animals. (C) Colon sections were stained
with PCNA antibodies and total intensity of sections was measured using
metamorph software. Bars represent mean ± SE (n 5 3); P , 0.001 as
compared with control and P , 0.01 as compared with AOM alone. Open
circle (s) indicates number of ACF and closed circle indicates (d) average
number of ACF.
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prevent colon cancer cell growth in culture as well as nude mice xenografts (17). Since ACF formation in animals is recognized as early
preneoplastic lesions in the colon and mimics putative precursor lesions
from which adenomas and carcinomas are formed in humans (11),
we first examined the effect of an AR inhibitor on AOM-induced
ACF formation in mice. The body weights of AOM and vehicle or
AR inhibitor-treated groups were comparable and no significant
changes were observed throughout the study (data not shown). At the
early preneoplastic stage (10 weeks after first AOM injection), mice
were killed and their colons were removed and analyzed microscopically for the presence of ACF. ACF were distinguished from the
surrounding normal crypts by increased thickening of the crypt walls
and aberrant change in the shape of the crypt lumen (Figure 1A). The
parameters used to assess the ACF were their occurrence/colon and
the number of aberrant crypts/foci. All the colons were scored by
three observers who were masked to the identity of the samples. In
mice, AOM treatment induced ACF, 9–17/colon (Figure 1B). No evidence of ACF was observed in vehicle-treated control animals. Administration of sorbinil to AOM mice significantly suppressed the
formation of ACF, 2–4/colon, indicating that inhibition of AR prevents
preneoplastic lesion formation in mice. Since inhibition of AR
prevents the AOM-induced ACF formation, we next measured the
proliferation index by staining colon sections with anti-PCNA antibodies. Significant expression of PCNA was observed in mice treated
with AOM alone (Figure 1C). Treatment of mice with AR inhibitor
significantly (70%) reduced the expression of PCNA. A good correlation was observed between the ability of AR inhibitor to prevent
ACF formation and decreased PCNA-labeling index.
R.Tammali et al.
Collectively, these results suggest that AR inhibition prevents its
signaling events responsible for its own gene expression.
We next measured the effect of AR inhibition on unsaturated lipid
aldehydes to determine the status of lipid peroxidation in AOM-treated
colons. As shown in Figure 2D, increased levels of fluorescence in
AOM-treated mice indicated increased levels of a, b-unsaturated aldehydes in colons and inhibition of AR significantly prevented the
increase in the formation of unsaturated aldehydes. Since lipid aldehydes are highly electrophilic and can form conjugates with cellular
proteins (16,32,36), we next measured the effect of AR inhibition on the
levels of protein–HNE adducts in colon sections. Treatment of mice
with AOM increased the formation of protein–HNE adducts as evidenced by strong staining with HNE antibodies (Figure 3D). Inhibition
of AR significantly prevented it. These results suggest that inhibition of
AR prevents AOM-induced lipid peroxidation, protein–HNE adducts,
activation of PKCb2 and expression of Cox-2 and iNOS, which could
be considered as major factors for the development of ACF.
Inhibition of AR prevents AOM-induced cyclin D1 and b-catenin
expression in BALB/c mice
We next measured the expression of key colon cancer-related markers
such as cyclin D1 and b-catenin in mice colon. Cyclin D1 is an
important cell cycle-regulated protein involved in G1–S phase transition. This cyclin is overexpressed in the presence of growth factors
and carcinogens and causes epithelial cell proliferation in various
malignancies, including colon cancer (37). b-Catenin is involved in
Wnt-signaling pathway. During normal conditions, it binds to axin–
GSK-3–APC complex and undergoes proteolytic degradation,
whereas during carcinogenesis b-catenin stabilizes in the cytoplasm
by deregulated Wnt-signaling pathway and translocates into the nucleus and interacts with T-cell factor/lymphoid enhancer factor family
of transcription factors to promote expression of various oncogenes
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(26,38). IHC examination of cyclin D1 and b-catenin showed that the
abundance of these two enzymes was markedly elevated when mice
were treated with AOM alone as evident by dark brown staining using
antibodies (Figure 3E and F). Inhibition of AR by sorbinil resulted in
lower levels of AOM-induced expression of cyclin D1 and b-catenin,
indicating that AR regulates the expression of these proteins.
AR-deficient mice are resistant to AOM-induced ACF formation
Our results with AR inhibitor strongly suggest that AR mediates AOMinduced ACF formation. If this is true, the AR-deficient mice should be
resistant to AOM-induced ACF formation. To investigate this, we examined the effect of AOM on ACF formation in ARKO C57BL/6 mice
and their WT controls. The results as shown in Figure 4A demonstrate
that ARKO mice had a significant decrease in the total number of ACF
per colon at both 5 and 10 weeks as compared with WT controls. The
low numbers of ACF were found in ARKO at both 5 weeks (3–5 ACF/
colon) and at 10 weeks (5–6 ACF/colon) as compared with WT (8–10
and 12–16 ACF/colon, respectively). These results suggest that AR-null
mice are less susceptible to AOM-induced ACF formation. Expression
of proliferation index marker, PCNA was significantly lowered in
AOM-treated ARKO mice compared with WT mice (Figure 4B). These
results suggest that ARKO mice are less susceptible to AOM-induced
growth of colonic cells.
AR-deficient mice are resistant to AOM-induced inflammatory
markers expression and formation of protein–HNE adducts
We have also examined the effect of AOM on inflammatory
markers expression in AR-null mice. Our results show a significant
decrease in Cox-2 and iNOS expression in ARKO mice as compared with WT mice at 5 and 10 weeks after AOM injection (Figure
4C). Similar results were observed in IHC analysis of colon sections with Cox-2 and iNOS antibodies (Figure 5A and B).
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Fig. 2. Inhibition of AR prevents expression of inflammatory markers in AOM-treated BALB/c mice. (A and C) Ten weeks after first AOM injection, colons were
removed and homogenized. Equal amounts of cell lysates were subjected to western blot analysis using antibodies against Cox-2, iNOS, phospho-PKCb2, AR and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Reverse transcription–polymerase chain reaction analysis of colon samples obtained 10 weeks after
AOM treatment. Densitometry analysis was performed using Kodak 1D image analysis software. A representative blot is shown (n 5 3). (D) The levels of a,
b-unsaturated aldehydes were measured as described in Material and Methods. Bars represent mean ± SE (n 5 3); P , 0.001 as compared with control and
P , 0.01 as compared with AOM alone.
Aldose reductase in ACF formation
Consistent with the protein levels, the mRNA levels of Cox-2 and
iNOS were significantly increased in WT mice treated with AOM
alone (Figure 4D). However, AOM-treated ARKO mice show a significantly (50%) reduced expression of Cox-2 and iNOS mRNA
suggesting that AR regulates the expression of these inflammatory
proteins at transcriptional level. Further, as shown in Figure 5C, the
levels of protein–HNE adducts are significantly increased in AOM
WT mice but not in AOM AR-null mice, suggesting that AR deficiency reduces the formation of protein–HNE adducts. Further,
we also examined the effect of AOM on the expression of cyclin D1
and b-catenin in WT and ARKO mice. As shown in Figure 5D and
E, treatment of WT mice with AOM significantly increased the
expression of cyclin D1 and b-catenin as compared with ARKO
mice, suggesting that AR deficiency prevents AOM-induced expression of cyclin D1 and b-catenin in mice.
We next measured the extent of AOM-induced inflammation in
colon sections. WT and ARKO control mice displayed normal crypts
and colonic architecture with no signs of apparent abnormality (Figure 5F). WT mice treated with AOM alone showed enlarged nuclei
and thickened mucosal layer with densely packed inflammatory cell
infiltrations, whereas no infiltration of inflammatory cells was observed in ARKO mice treated with AOM. These results suggest that
ARKO mice are resistant to AOM-induced inflammation.
AR-deficient mice are resistant to AOM-induced phosphorylation of
PKCb2 and p65 and general inflammation
Since the expression of Cox-2 and iNOS depends on the activation of
NF-jB and its upstream signals, we next measured the phosphorylation of PKCb2 and p65 in ARKO and WT mice treated with AOM. As
shown in Figure 6A, WT mice at 5 and 10 weeks after first AOM
injection showed increased phosphorylation of PKCb2 and p65,
whereas ARKO mice showed a significant resistance to AOM-induced
activation of PKCb2 and p65. Similar results were observed with IHC
analysis (data not shown).
Discussion
Colon cancer is a multistep process that involves sequential transformation of normal epithelial cells into malignant cells through various
progressive stages such as preneoplastic, neoplastic and malignant
(17,37). This multistep carcinogenesis is referred as a progressive
disorder in signal transduction mechanism. Altered regulations of redox stress signal transduction pathway intermediates such as PKCb2
and NF-jB, whichpromote the expression of various inflammatory
markers, are early events in the colon carcinogenesis (17,33,34). In
this mechanism, identification of the earliest detectable preneoplastic
lesions may provide appropriate way of screening and prevention
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Fig. 3. Inhibition of AR prevents the expression of inflammatory and preneoplastic markers in AOM-treated BALB/c mice. (A–F) Colon sections were stained
with antibodies against Cox-2, iNOS, AR, protein–HNE, cyclin D1 and b-catenin. Immunoreactivity of the antibody was assessed by quantifying the evident as
a dark brown stain in the cells, whereas the non-reactive areas displayed only the background color. Photomicrographs of the stained sections were acquired using
an EPI-800 microscope (bright-field) connected to a Nikon camera (400 magnification). Percent staining was determined by measuring positive
immunoreactivity per unit area. Arrows represent the area for positive staining for an antigen. The intensity of antigen staining was quantified by digital image
analysis and the values are represented as fold change versus control in arbitrary units.
R.Tammali et al.
method for colon cancer. In mice model, AOM-induced formation of
ACF was first described by Bird (10). ACF are described as enlarged,
thicker epithelial lining, slightly elevated from the surrounding mucosa and darkly stained with methylene blue. In humans and experimental animal models, ACF are considered putative preneoplastic
lesions (11). Formation of a large number of ACF with increased cell
proliferation, genetic alteration in K-ras, APC and p53 and microsatellite instability is observed in patients with familial adenomatous
polyposis as well as in patients with sporadic colon cancer (25,39).
Numerous reports show that treatment of colon cancer patients with
non-steroidal anti-inflammatory drugs effectively reduces the formation of ACF (24,40). Most drugs with antioxidant property prevent the
generation of ROS thereby inhibit the formation of ACF (4,16,40,41).
However, antioxidant drugs, which have good solubility in both hydrophilic and hydrophobic milieu and whichdo not become pro-oxidants when given in therapeutic doses, are currently unavailable. We
have shown previously that inhibition of AR can prevent growth
factors-, cytokines-, chemokines- and hyperglycemia-induced ROS
signals (27–30). Indeed, our present report on AOM-induced ACF
formation in mouse model shows that inhibition of AR by pharmacological agents could prevent AOM-induced ROS generation as well as
its dependent activation of NF-jB. In human colon cancer cells, in-
804
hibition of AR prevented growth factors such as fibroblast growth
factor- and platelet-derived growth factor-induced ROS formation
(17). The ROS generated during growth factors and cytokines signaling could induce formation of a wide range of cytotoxic, lipid
peroxidation-derived aldehydes such as HNE (31). These aldehydes
could readily conjugate with reduced glutathione and form glutathionyl
aldehydes such as GS-HNE (31). We have reported that AR efficiently
reduces GS-HNE to GS-DHN, which in turn activates transcription
factors such as NF-jB and AP-1 to induce expression of various inflammatory markers such as Cox-2 and iNOS via signaling cascade
axis of PKC/phosphotidyl inositol 3-kinase/mitogen-activated protein
kinase/inhibitory kappa kinase (17,31). Uncontrolled production of
these inflammatory markers would lead to cellular cytotoxicity leading
to ACF formation (Figure 6B).
To further understand the role of AR in colonic epithelial cell proliferation and colon carcinogenesis, we have used AOM-induced ACF
formation in BALB/c mouse model. Treatment of mice with AR inhibitors significantly prevented the AOM-induced ACF formation.
Various reports show that AR is a growth-responsive enzyme and is
overexpressed in various forms of cancer such as hepatocarcinogenesis, lung, breast and colon cancers (29,35,42). We are first to report
the role of AR in AOM-induced ACF formation. Consistent with
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Fig. 4. AR-deficient mice are resistant to AOM-induced ACF formation and exhibit decreased expression of AOM-induced inflammatory markers. (A) Six-weekold ARKO and WT mice were injected with AOM (10 mg/kg body wt) weekly for 3 weeks. After 5 and 10 weeks of first AOM injection, mice were killed, colons
were fixed in formalin and stained with 0.2% methylene blue for 5 min. Colons were analyzed for the presence of ACF counted under light microscope (400
magnification) with colon mucosal side up. For each group of animals, n 5 6; P , 0.015 as compared with WT þ AOM alone. Wilcoxon rank-sum test was
performed to statistically analyze ACF data obtained from WT þ AOM versus ARKO þ AOM. (B) Colon sections, obtained after 10 weeks after first AOM, were
stained with PCNA antibodies and total intensity of sections was measured using metamorph software. Bars represent mean ± SE (n 5 3); P , 0.001 as
compared with control and P , 0.01 as compared with WT þ AOM alone. (C) Total cell lysate of colons subjected to western blot analysis by using antibodies
against Cox-2, iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Reverse transcription–polymerase chain reaction analysis of colon samples.
Bands were quantified by densitometry analysis using Kodak 1D image analysis software. A representative blot is shown (n 5 3). Open circle (s) indicates number
of ACF and closed circle (d) indicates average number of ACF.
Aldose reductase in ACF formation
prevention of AOM-induced ACF formation by AR inhibitor, we have
shown protection of AR-null mice from AOM-induced ACF formation. Kawamura et al. (43) using a mice model showed that inhibition
of AR by ponalrestat prevents the cachexia syndrome induced by
colon 26 adenocarcinoma cells. Further, they showed that inhibition
of AR prevents the lipopolysaccharide-induced IL-1 secretion in
monocytes. In nude mice xenograft model, we have shown that inhibition of AR by AR–siRNA prevented the progression of human
adenocarcinoma SW480 cells-induced tumor growth (17). In addition, our results further confirm that inhibition or deletion of AR
prevents AOM-induced expression of Cox-2, iNOS, phospho-PKCb2
and phospho-p65. The inducible form of Cox-2 enzyme plays an
important role in the pathogenesis of colon cancer by generating
hyperalgesic, proinflammatory prostaglandins and leukotrienes.
There are numerous non-steroidal anti-inflammatory drugs, designed
based on the structure and function of the Cox-2 enzyme for the
treatment of colon carcinogenesis (41). The de novo synthesis of
Cox-2 is triggered by the exposure of epithelial cells to endotoxin,
cytokines and growth factors, whereas constitutive Cox-1 enzyme is
involved in eicosanoid metabolism and homeostasis of tissues (17).
Moeckel et al. (44) in Cox-2 knockout mice demonstrated that expression of AR mRNA is dramatically reduced during hypertonic
stress indicating that AR is involved in the signal transduction path-
way of Cox-2 enzyme. In addition, other inflammatory markers such
as iNOS, PKCb2 and p65 play important roles in the progression of
colon carcinogenesis (17,24,33). Overexpressing PKCb2 transgenic
mice have been shown to be more sensitive to AOM-induced ACF
formation than its WT littermates and overexpression of PKCb2 is
early step to promote colon carcinogenesis (33,34). Various reports
have demonstrated that AR-deficient mice are protected from increased c-Jun-N-terminal kinase activation, depletion of reduced glutathione, increased superoxide accumulation and DNA damage during
oxidative stress conditions such as hyperglycemia (45). Similar to
diabetic ARKO mice, inhibition of AR in diabetic and WT mice
showed decreased activation of mitogen-activated protein kinase suggesting that AR is involved in oxidative stress (46). Our results
in vascular smooth muscle cells, human lens epithelial cells, vascular
endothelial cells and Caco-2 cells also show that inhibition of
AR prevents the growth factors-, cytokines-, chemokines- and
hyperglycemia-induced oxidative signals such as generation of
ROS, activation of poly (ADP) – ribose polymerase and mitogenactivated protein kinase via NF-jB (17,27–30). Since AR gene promoter has binding site for NF-jB, AR is regulated by NF-jB (28).
Our results show that inhibition of AR by sorbinil prevents AOMinduced colonic AR expression suggesting that AR inhibition responsible for signaling events regulates its own gene expression. Our
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Fig. 5. AR-deficient mice exhibit decreased expression of AOM-induced preneoplastic markers. (A–E) Colon sections were stained with antibodies against Cox-2,
iNOS and protein–HNE, cyclin D1 and b-catenin. Immunoreactivity of the antibody was assessed by quantifying the evident as a dark brown stain in the cells,
whereas the non-reactive areas displayed only the background color. Photomicrographs of the stained sections were acquired using an EPI-800 microscope (brightfield) connected to a Nikon camera (400 magnification). Percent staining was determined by measuring positive immunoreactivity per unit area. Arrows
represent the area for positive staining for an antigen. The intensity of antigen staining was quantified by digital image analysis and the values are presented as fold
change versus control in arbitrary units. (F) Histopathological examination of colon sections stained with hematoxylin and eosin (H&E). Photomicrographs of the
stained sections were acquired using an EPI-800 microscope (bright-field) connected to a Nikon camera (400 magnification). Arrows in black and pink color
indicate densely packed inflammatory cell infiltration and enlarged nuclei in AOM-treated mice.
R.Tammali et al.
results further suggest that inhibition or deletion of AR prevents
AOM-induced expression and nuclear translocation of b-catenin and
decreases the expression of cyclin D1 and PCNA. Altered expression
of b-catenin seems to be one of the earliest genetic events critical in
colonic hyperproliferation through regulation of cyclin D1 (13,37).
Further, number of reports shows that targeted downregulation of bcatenin or its downstream regulator, cyclin D1, plays an important
role in chemoprevention of colon carcinogenesis (38). In addition, it
has been shown that numerous non-steroidal anti-inflammatory drugs
have chemopreventive effect by suppressing the expression of b-catenin and cyclin D1 (38).
In summary, our results show for the first time that inhibition or
deletion of AR significantly prevents AOM-induced ACF formation
by inhibiting the expression of important inflammatory markers and
that the use of AR inhibitors may provide an effective chemopreventive approach for the treatment of colon cancer.
Funding
Pearle Vision Research Foundation; Washington University Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc.(CA 129383, DK 36118 to S.K.S., GM 71036 to
K.V.R.); EY05856, EY02687 to J.M.P.
Acknowledgements
The authors thank Dr Stephen Chung (University of Hong Kong, Hong Kong,
China) for providing the ARKO mice and Theresa Harter (Washington University School of Medicine) for assistance with animal studies. The authors
also thank Dr Heidi Weiss (Biostatistics Core, Department of Comprehensive
Cancer Center, University of Texas Medical Branch, Galveston, TX) for helping in performing statistical analysis.
Conflict of Interest Statement: None declared.
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