cyclooxygenase-2 generates the endogenous mutagen trans-4 ...€¦ · 15.01.2013 ·...

Cyclooxygenase-2 Generates the Endogenous Mutagen trans-4-Hydroxy-2-

nonenal in Enterococcus faecalis-infected Macrophages

Xingmin Wang1,2, Toby D. Allen1,3, Yonghong Yang1,2, Danny R. Moore1,3,

and Mark M. Huycke1,2,3*

1The Muchmore Laboratories for Infectious Diseases Research, Department of Veterans Affairs

Medical Center, Departments of 2Radiation Oncology and 3Medicine, University of Oklahoma

Health Sciences Center, Oklahoma City, OK 73104, USA

Running title: COX-2 generates 4-HNE in macrophages

Key words: Cyclooxygenase-2; 4-hydroxy-2-nonenal; carcinogenesis; colorectal cancer;

macrophage; commensal.

Grant support: NIH CA127893 (Mark Huycke), OCAST HR10-032 (Xingmin Wang), and

Frances Duffy Endowment

Corresponding author: Mark M. Huycke, M.D.

Veterans Affairs Medical Center

921 N.E. 13th Street

Oklahoma City, OK 73104

E-mail: [email protected]

Conflicts of Interest: The authors disclose no conflicts.

Abbreviations: CIN, chromosomal instability ; CRC, colorectal cancer; 4-HNE, trans-4-

hydroxy-2-nonenal; BSO, buthionine sulfoximine; COX, cyclooxygenase; LOX, lipoxygenases.

Word count (excluding references): 4,984

Number of figures and tables: 6 figures.

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Abstract

Infection of macrophages by the human intestinal commensal Enterococcus faecalis generates

DNA damage and chromosomal instability in mammalian cells through bystander effects. These

effects are characterized by clastogenesis and damage to mitotic spindles in target cells and are

mediated, in part, by trans-4-hydroxy-2-nonenal (4-HNE). In this study we investigated the role

of cyclooxygenase (COX) and lipoxygenase (LOX) in producing this reactive aldehyde using E.

faecalis-infected macrophages and interleukin-10 knockout mice colonized with this commensal.

4-HNE production by E. faecalis-infected macrophages was significantly reduced by COX and

LOX inhibitors. The infection of macrophages led to decreased Cox1 and Alox5 expression while

COX-2 and 4-HNE increased. Silencing Alox5 and Cox1 with gene-specific siRNAs had no

effect on 4-HNE production. In contrast, silencing Cox2 significantly decreased 4-HNE

production by E. faecalis-infected macrophages. Depleting intracellular glutathione increased 4-

HNE production by these cells. Next, to confirm COX-2 as a source for 4-HNE, we assayed the

products generated by recombinant human COX-2 and found 4-HNE in a concentration-

dependent manner using arachidonic acid as a substrate. Finally, tissue macrophages in colon

biopsies from interleukin-10 knockout mice colonized with E. faecalis were positive for COX-2

by immunohistochemical staining. This was associated with increased staining for 4-HNE-

protein adducts in surrounding stroma. These data show that E. faecalis, a human intestinal

commensal, can trigger macrophages to produce 4-HNE through COX-2. Importantly, it

reinforces the concept of COX-2 as a procarcinogenic enzyme capable of damaging DNA in

target cells through bystander effects that contribute to colorectal carcinogenesis.




Introduction Cyclooxygenases (COX) and lipoxygenases (LOX) produce a variety of biologically

important prostanoids, leukotrienes, hydroxyeicosatetraenoic acids, and lipoxins through the

oxidation of polyunsaturated fatty acids such as arachidonic acid (1). These important signaling

molecules help regulate numerous physiological responses including inflammation, cellular

proliferation, vascular and bronchial airway tone, and tissue hemostasis. COX occurs as a

constitutive form (COX-1) in most tissues and an inducible form (COX-2) that is typically

expressed in response to environmental triggers such as infection, physical irritants, growth

factors, and cytokines. There are several LOX isoforms with arachidonate 5-lipoxygenase

(ALOX5 or 5-lipoxygenase) expressed primarily in inflammatory cells and dependent upon the

arachidonate 5-lipoxygenase activating protein. COX and LOX pathways have been implicated

in carcinogenesis. For example, prolonged exposure to aspirin, an irreversible COX inhibitor,

significantly reduces the overall risk for colorectal cancer (CRC) and mortality from CRC in

human trials (2). Observational and randomized clinical trials of NSAIDs and COX-2-specific

inhibitors demonstrate reductions in colorectal adenomas (3). Similar decreases in adenoma

formation are noted in ApcMin/+ and Apc�716 mice when Cox is inactivated (4,5). Finally, ALOX5

is overexpressed in colon polyps and CRC (6), much like COX-2 (7), with inhibition impairing

tumor growth and attenuating polyps in ApcMin/+ mice (8). The mechanisms by which COX-2 or

ALOX5 initiate mutations to drive the adenoma-to-carcinoma sequence, however, remain

unclear.

PGE2 and PGD2 are major COX-2-derived products that promote cancer cell growth by

modulating signaling cascades for cellular proliferation, apoptosis, angiogenesis, and immune

surveillance (9). ALOX5 produces 5(S)-hydroperoxyeicosatetraenoic acid, a precursor for




leukotrienes that are signaling molecules in asthmatic, allergic, and inflammatory reactions (1).

Although these lipid mediators promote diverse biological functions, they are not mutagens and

seem unlikely to initiate tumors. Each enzyme, however, produces products that can be reduced

to hydroxyeicosatetraenoic or hydroxyoctadecadienoic acids (1). Arachidonic acid, the primary

ω-6 polyunsaturated fatty acid substrate for COX-2, can also be oxidized by this enzyme to

reactive carbonyl compounds such as 4-oxo-2-nonenal, 4-hydroperoxy-2-nonenal, 4-hydroxy-

2E,6Z-dodecadienal, and trans-4-hydroxy-2-nonenal (4-HNE) (10). These bifunctional

electrophiles can diffuse across cellular membranes to form adducts with proteins,

phospholipids, and DNA. Among these, 4-HNE has been most intensively studied. This α,β-

unsaturated aldehyde is a signaling molecule for cellular stress (11), induces COX-2 (12),

inhibits DNA repair (13), reacts with DNA (1), damages microtubules and mitotic spindles

(14,15), and potentially contributes to chromosomal instability (CIN) (15,16).

The carcinogenic effects of non-prostanoid byproducts derived from COX-2 were

investigated using rat intestinal epithelial cells overexpressing this enzyme (17). A 3-fold

increase in heptanone-etheno-DNA adducts was observed in the presence of ascorbate that

served to promote the decompostion of bifunctional electrophiles that were generated (17).

Stereoisomeric analysis identified these adducts as lipid peroxidation byproducts of arachidonic

acid that were generated by COX-2. Others investigators, however, using colon cancer cell lines

failed to correlate DNA adduct levels with COX-2 activity (18). Finally, as mentioned above,

ALOX5 also generates products that can be converted to lipid hydroperoxides and potentially

decompose into DNA-damaging electrophiles (1).

COX-2 and ALOX5 are not ordinarily expressed in healthy colons. In colonic adenomas,

however, COX-2 expression is found in mucosal macrophages, but not epithelial celss where




cellular transformation leads to cancer (7). In contrast, ALOX5 is abundantly expressed in

epithelial cells in adenomas (6). These temporospatial relationships, when considered in the

context of clinical and animal data (2-6,8), implicate these enzymes in colorectal carcinogenesis.

Using interleukin (IL)-10 knockout mice, we have recently linked the common intestinal

commensal Enterococcus faecalis to COX-2 induction in macrophages and to transforming

events in epithelial cells (19,20). E. faecalis is a minority constituent of the mammalian intestinal

microbiota that generates extracellular superoxide and oxidative stress under conditions of heme

deprivation (21). The unusual redox physiology activates bystander effects (BSE) in

macrophages and causes CRC in IL-10 knockout mice (15,19). BSE is recognized by genomic

damage in target cells that are exposed to diffusible clastogens (or chromosome-breaking factors)

from activated myeloid and/or fibroblast cells (22,23). Historically, these effects occur following

irradiation. However, we discovered that macrophages infected by E. faecalis can also produce

BSE that include double-strand DNA breaks, aneuploidy, tetraploidy, and CIN (15,19,20). This

genomic damage, whether due to BSE from irradiated or infected cells, depends in part on COX-

2 (19,24), although the diffusible mediators remain ill-defined. We and others have suggested 4-

HNE as one such mediator because it can cause DNA damage and act as a spindle poison to

produce tetraploidy in target cells (15,16). As such, this reactive aldehyde represents a potential

link between COX-2 catalysis and CIN.

In this study we show that E. faecalis-infected macrophages produce increased amounts of 4-

HNE in a COX-2, but not ALOX5, dependent fashion. In addition, using IL-10 knockout mice

colonized by E. faecalis, we demonstrate increased COX-2 expression in colonic macrophages in

association with 4-HNE-protein adducts. These data suggest that colonic macrophages, when

triggered by an intestinal commensal such as E. faecalis, can induce COX-2 and thereby increase




the production of 4-HNE, a diffusible mutagen. These findings reinforce the role of COX-2 as a

pro-carcinogenic enzyme that is able to endogenously initiate transforming events leading to

CRC.




Materials and Methods

Cells, bacteria, and chemicals

Murine macrophages (RAW264.7 cells; American Type Culture Collection) were maintained

in high glucose Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with

10% FBS, penicillin G, and streptomycin at 37ºC in 5% CO2. E. faecalis strain OG1RF, a human

oral isolate, and the spectinomycin- and streptomycin-derivative strain OG1RFSS were grown in

brain heart infusion (BD Diagnostics) and used for macrophage infection and to colonize mice,

respectively, as previously described (15). In brief, RAW264.7 cells were treated with OG1RF at

a multiplicity of infection of 1000 in antibiotic- and serum-free Dulbecco’s for 2 hrs at 37ºC.

After washing with PBS, cells were incubated in complete medium supplemented with 100 μg

ml-1 gentamicin for 24 hrs at 37ºC. Supernatants were collected for 4-HNE analysis and cell

pellets lysed for protein and RNA analyses. The COX-2-specific inhibitor celecoxib was kindly

provided by C.V. Rao. The ALOX5 specific inhibitor AA861 (2-(12-hydroxydodecane-5,10-

diynyl)-3,5,6-trimethyl-p-benzo-quinone), and COX and LOX inhibitor ETYA (eicosatetraynoic

acid) were purchased from Enzo Life Sciences (25). Buthionine sulphoximine (BSO) was

obtained from Sigma.

4-HNE and PGD2 analysis

Lipids were extracted from supernatants by the Folch procedure and reconstituted in 100%

ethanol as previously described (15). Extracts were analyzed by high performance liquid

chromatography using reversed-phase chromatography coupled to a 4-cell electrochemical

detector as previously described (15).




COX-2 catalysis of arachidonic acid

Reactions were performed in a thermostatic cuvette (Gilson Medical Electronics) with 1 ml

of 100 mM KH2PO4 (pH 7.4) containing 100 �M arachidonic acid (Cayman), 1 �M hematin

(Sigma), and 2 units of human recombinant COX-2 (Cayman) at 37°C. Reactions were

terminated at 5 min by adding butylated hydroxytoluene and depletion of oxygen. Mixtures were

extracted and separated by reversed-phase high performance liquid chromatography with 4-HNE

and arachidonate metabolites determined by electrochemical detection as previously described

(15). Enzyme activity was measured by O2 uptake using an Oxygen Probe Amplifier equipped

with a Clarke-type electrode (Harvard Apparatus).

siRNA and RT-PCR

Cox1, Cox2, and Alox5 were silenced by RNA interference using siGENOME SMARTpool

siRNAs or with siCONTROL Non-Targeting siRNA Pool (Dharmacon). Transient transfections

were performed using DharmaFECT 4 Transfection Reagent according to the manufacturer’s

protocol and gene silencing confirmed by RT-PCR and Western blot.

Total RNA was isolated from RAW264.7 cells using the NucleoSpin RNA II Kit (BD

Biosciences). Complementary DNA (cDNA) was synthesized at 37°C using TaqMan® Reverse

Transcription Reagents according to manufacturer’s instructions (Life Technologies). Primers

and cycling parameters are shown in the Supplementary Table.

Western blots

Protein extraction, SDS-PAGE, and Western blotting were performed as previously

described (20). Goat polyclonal antibody for COX-2 (Santa Cruz Biotechnology) and anti-




ALOX5 rabbit monoclonal antibody (Thermo Scientific) were used as primary antibodies.

Rabbit anti-goat IgG HRP conjugate (Millipore) and goat anti-rabbit IgG HRP conjugate (Cell

Signaling Technology) were used for secondary antibodies. Signals were generated by

Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare) and images

captured using the ChemiDoc™ XRS+ System (Bio-Rad).

Colonization of Il10-/- mice

Conventionally housed Il10-/- knockout mice (C57Bl/6J, Jackson Laboratory) were

orogastrically inoculated with 1 × 109 colony forming units of E. faecalis OG1RFSS, or PBS as

sham, as previously described (26). Colonization was confirmed by plating stools on

enterococcal agar (Becton Dickinson) supplemented with streptomycin and spectinomycin. After

9 months, mice were sacrificed and colons fixed for immunostaining. Animal protocols were

approved by the University of Oklahoma Health Sciences Center IACUC and Oklahoma City

Department of Veterans Affairs Animal Studies Committee.

Immunohistochemical and immunofluorescent staining

Immunohistochemical staining was performed as previously described. For COX-2 staining,

slides were blocked with 5% normal rabbit serum, incubated with goat anti-COX-2 polyclonal

antibody (Santa Cruz), and stained with rabbit anti-goat IgG HRP conjugate (Millipore). Slides

were developed with DAB Enhanced liquid substrate and counterstained with Mayer’s

hematoxylin solution (Sigma).

Immunofluorescent staining for macrophages (F4/80) and COX-2 or 4-HNE was performed

as previously described (27). Goat anti-COX-2 antibody (Santa Cruz), rabbit anti-4-HNE




antibody (Alpha Diagnostic International), and rat anti-F4/80 antibody (eBioscience) were used

as primary antibodies. Anti-goat IgG FITC, anti-rabbit IgG FITC, and anti-rat IgG Texas Red

(Santa Cruz) were used as secondary antibodies. Cells were counterstained with 4'-6-diamidino-

2-phenylindole and images collected using a fluorescent microscope (Nikon Instruments).

Statistical analysis

Data are shown as means with SDs. Groups were compared using Student’s t test with P

values < 0.05 considered significant.




Results

LOX and COX inhibitors decrease 4-HNE production by macrophages

E. faecalis-infected macrophages produce increased 4-HNE, a lipid peroxidation byproduct

of arachidonic acid (15). Since cyclooxygenases and lipoxygenases catalyze reactions using this

�-6 polyunsaturated fatty acid, and 4-HNE is a known breakdown product, we explored the role

of these enzymes in increased 4-HNE production from infected murine macrophages. Initially,

we treated uninfected macrophages with an ALOX5-specific inhibitor and a dual COX/LOX

inhibitor. Both inhibitors decreased background levels of 4-HNE in supernatants from 252 ± 35

to 85 ± 18 and 87 ± 16 nM for AA861 and ETYA, respectively (Fig. 1A, P < 0.01). These

inhibitors also decreased 4-HNE production when macrophages were infected with E. faecalis

(439 ± 104 to 105 ± 28 and 128 ± 34 nM for AA861 and ETYA, respectively, P = 0.01). Of note,

COX-2, which is not measurably expressed by these macrophages under ordinary culture

conditions, is strongly induced by E. faecalis (19), and was less strongly induced in the presence

of these inhibitors following infection (Fig. 1B). In contrast, COX-1 and ALOX5 were

constitutively expressed by uninfected RAW264.7 cells and each decreased following E. faecalis

infection (Fig. 1C-F). These changes made it difficult to determine whether the reduction in 4-

HNE production caused by these inhibitors was due to enzyme inhibition vs. changes in enzyme

expression in infected macrophages.

COX-1 and ALOX5 are not sources of 4-HNE for infected macrophages

To assess potential contributions of COX-1 or ALOX5 to 4-HNE production in E. faecalis-

infected macrophages, Cox1 and Alox5 were silenced using siRNA and 4-HNE production




measured. RT-PCR showed a 38% decrease in Cox1 expression for cells transfected with Cox1-

specific siRNA compared to cells transfected with non-targeting siRNA (Fig. 2A and 2B).

Similarly, a 60% reduction was noted in Alox5 expression using Alox5-specific siRNA compared

to control (Fig. 2C and 2D). Although the expression of Cox1 and Alox5 decreased slightly in E.

faecalis-infected macrophages transfected with gene-specific siRNAs compared to non-targeting

siRNA, these effects were largely overwhelmed by the strong inhibition of gene expression for

these enzymes in infected macrophages (Fig. 2A-2D). No decrease was observed in 4-HNE

production for Cox1-silenced macrophages compared to controls (P = 0.24 and 0.22, for either

uninfected or E. faecalis-infected macrophages, respectively). Similarly, no change was noted in

4-HNE production for Alox5-silenced macrophages following E. faecalis infection compared to

control. Of note, 4-HNE production modestly increased in Alox5-silenced uninfected

macrophages compared to non-transfected controls (P = 0.04, Fig. 2E). Although ALOX5

expression is typically associated with increased lipid peroxidation (28), this observation may

represent a perturbation and increase in non-enzymatic lipid peroxidation from the loss of

ALOX5 endproducts.

We tested the hypothesis that 4-HNE produced by macrophages derives in part from non-

enzymatic lipid peroxidation using �-tocopherol, a potent terminator of lipid peroxidation chain

reactions with no effect on COX-2 expression (19). We found that �-tocopherol significantly

decreased 4-HNE production from both uninfected and E. faecalis-infected macrophages,

suggesting non-enzymatic lipid peroxidation was in part a source for the basal production of 4-

HNE by these cells (Fig. 2F). In summary, the expression of Cox1 and Alox5 decreased in E.

faecalis-infected macrophages as these cells otherwise were producing increased amounts of 4-

HNE. Partial silencing of these genes resulted in either no change, or a trend toward increased 4-




HNE production. These observations indicate that Cox1 and Alox5 were not significant sources

for 4-HNE in E. faecalis-infected macrophages.

COX-2 produces 4-HNE

The lack of association of Cox1 or Alox5 with 4-HNE production led us to more closely

investigate the role of Cox2. Initially, we pretreated macrophages with celecoxib—COX-2-

specific inhibitor—prior to infection with E. faecalis. RT-PCR showed a 94% decrease in Cox2

expression compared to untreated E. faecalis-infected controls (P = 0.01, Fig. 3A). These

findings suggest that celecoxib not only blocks the active site of COX-2 but also suppresses

Cox2 transcription. Western blotting showed a 46% decrease in COX-2 expression in E. faecalis-

infected macrophages that were treated with celecoxib (Fig. 3B). This observation is consistent

with previous reports (29). Of note, the inhibition of COX-2 by celecoxib was associated with a

45% decrease in 4-HNE production following E. faecalis infection (P < 0.001, Fig. 3C). Since

celecoxib may have off-target effects, we transfected macrophages with Cox2-specific siRNA

and observed a 35% decrease in Cox2 expression and 67% decrease in COX-2 protein after E.

faecalis infection (P = 0.001, Fig. 3D and 3E). This was associated with a 68% decrease in 4-

HNE production compared to infected macrophages that were transfected with non-targeting

siRNA (P < 0.001, Fig. 3F). These findings confirm COX-2 as the predominant source for

increased 4-HNE in E. faecalis-infected macrophages.

To directly assess the ability of COX-2 to generate 4-HNE, we tested recombinant human

COX-2 in vitro using arachidonic acid as substrate. We discovered that 4-HNE was produced in

a concentration-dependent manner as a significant byproduct of catalysis. When the reaction was

allowed to go to completion using 50, 100, or 2000 μmol arachidonate as substrate, 18, 36, and




1,560 μmols 4-HNE were detected, respectively, in final reaction mixtures. Controls using buffer

and substrate, but no enzyme, did not produce detectable 4-HNE. Celecoxib decreased 4-HNE

production by 71% when 50 μmol arachidonic acid was used as the substrate (Fig. 3G). This was

associated with 27% decrease in PGD2 production—a decomposition product of PGH2 (Fig. 3H).

These observations offer additional support for COX-2 as the primary source of 4-HNE from

infected macrophages.

Glutathione depletion increases 4-HNE production by E. faecalis-infected macrophages

We previously showed that 4-HNE mediates genotoxicity through macrophage-induced BSE

(15). Since glutathione is required for enzymatic scavenging of 4-HNE by glutathione S-

transferase, we investigated the effect of BSO, a �-glutamylcysteine synthetase inhibitor that

depletes intracellular glutathione and enhances genotoxicity (20,30), on 4-HNE production. In

comparison to E. faecalis-infected controls, a 2.5-fold increase in 4-HNE production was seen

for BSO-treated macrophages that were infected with E. faecalis (P = 0.003, Fig. 4A). A 1.3-

fold increase in Cox2 expression was evident in these same macrophages (P = 0.03, Fig 4B).

Since 4-HNE can specifically induce COX-2, this response might represent positive feedback by

this electrophile (12). Although Cox2 expression appeared slightly suppressed by BSO in

uninfected macrophages (Fig. 4B), this was not confirmed at the protein level (Fig. 4C). Finally,

COX-2 activity, as measured by production of PGD2, increased 5.7-fold following BSO

treatment of E. faecalis-infected macrophages compared to 3.3-fold for untreated macrophages

infected with E. faecalis (P = 0.03, Fig. 4D).

Co-localization of COX-2 and 4-HNE in colonized Il10-/- mice




Il10-/- mice colonized with E. faecalis develop colon inflammation and CRC (31). These

pathological changes are associated with increased 4-HNE-protein adducts in colonic mucosal

cells and stroma (15). To further examine the mechanism for these events, we stained colon

biopsies from E. faecalis-colonized Il10-/- mice for COX-2 and 4-HNE-protein adducts.

Immunohistochemistry showed intense staining for COX-2 in macrophages in areas of

inflammation (Fig. 5A). In comparison, we saw little to no staining for sham-colonized mice.

Immunofluorescence showed similarly increased COX-2 expression in colonic macrophages

(Fig. 5B) with markedly increased 4-HNE-protein adduct staining in macrophages and,

compared to shams, associated intercellular spaces (Fig. 5C).




Discussion These data demonstrate COX-2 as a predominant source for 4-HNE following macrophage

infection by E. faecalis. In addition, staining for 4-HNE-protein adducts was noted in the colonic

stroma and tissue macrophages of Il10-/- mice colonized by E. faecalis. This intestinal

commensal is a trigger for colitis and CRC in this model (15,31). By comparison, COX-1 and

ALOX5 did not contribute to 4-HNE production. These findings help link COX-2 to

clastogenesis caused by BSE (19,24). In the context of associations between this enzyme and

carcinogenesis (1,3-5,32), COX-2 should be considered an autochthonous source for

mutagenesis leading to cellular transformation.

The role of COX-2 in CRC has been recognized for many years (2,9,33). Several lines of

evidence derive from animal models where tumorigenesis is curtailed after Cox is deleted or

inhibited (4,5). Although major products downstream from COX-2—PGE2 and PGD2—are

diffusible and promote cellular growth (9), these lipid mediators are not mutagenic and seem

unlikely to initiate cellular transformation. COX-2, however, can produce less well recognized

byproducts that include 4-oxo-2-nonenal, 4-hydroperoxy-2-nonenal, 4-hydroxy-2E,6Z-

dodecadienal, and 4-HNE (1). As amphiphilic aldehydes, these compounds can readily form

adducts with proteins, phospholipids, and DNA (1,10,11,14).

4-HNE and related carbonyl compounds are most often thought of as being derived from �-6

polyunsaturated fatty acids due to oxidative stress (10,11). These reactive aldehydes can form in

cellular membranes from fatty acids via non-enzymatic pathways and potentially represent a

major source of 4-HNE in tissue culture for cells not expressing COX-2 (28). Our findings,

however, describe COX-2 as another important source for 4-HNE. We propose that the induction

of this enzyme in macrophages, and subsequent increase in 4-HNE production, overwhelms




scavenging mechanisms for this aldehyde in epithelial cells, viz., glutathione S-transferases

(15,34), and results in ongoing genomic damage that leads to cellular transformation.

Any plausible theory for sporadic CRC should address the role of COX-2 in carcinogenesis,

describe the origin of CIN, and elucidate the relationship between reactive stromal cells and

colonic epithelial cells. Our proposed model for autochthonous mutagenesis mechanistically

links these characteristics for inflammation-associated and sporadic CRC (Fig. 6). The lack of

COX-2 expression in normal colonic mucosa, with strong expression in tissue macrophages in

human adenomas, is consistent with a theory for human CRC that is caused by commensal-

triggered and macrophage-induced BSE (7). We propose that specific commensals such as E.

faecalis serve as triggers for innate immune responses that drive mutagenesis through BSE.

This hypothesis joins the oxidative physiology of E. faecalis to the expression of COX-2 in

colonic macrophages. We postulate that extracellular superoxide from E. faecalis generates

clastogens by chronically activating macrophages through ongoing infection. E. faecalis must

translocate the intact intestinal epithelium for this to occur. This is a trait that has been

previously described for this genus (35). In addition, enterococci must persist in macrophages

despite the otherwise lethal effects of phagocytosis—again, this is a recognized phenotype for E.

faecalis (36). Macrophages respond to E. faecalis infection by activating nuclear factor-�B and

inducing COX-2 (37). Following this sequence of signaling events, as shown here, COX-2 is

induced and generates mutagenic 4-HNE (and possibly related congeners although they were not

specifically investigated in this study). As an amphiphilic aldehyde, 4-HNE can readily diffuse

into nearby epithelial cells to stochastically damage DNA that, over long periods, leads to CIN.

The phenomenon of BSE is readily demonstrated using supernatants from irradiated cells to

induce CIN in non-irradiated cells (22). Congenic sex-mismatch bone marrow transplant studies




kin mice have confirmed that BSE occurs in vivo (38). BSE is not exclusively induced by

irradiation. Superoxide also triggers this phenomenon with DNA damage prevented by

superoxide dismutase (19,23). Mediators for radiation- or superoxide-induced clastogenesis,

however, have remained elusive. Potential candidates include long-lived lipid radicals,

byproducts of lipid peroxidation, inositol, and TNF-� (16,23,39). A clue into these mediators

was reported by Zhou et al. who described their dependence on COX-2 (24). Our laboratory

similarly implicated this enzyme using infected macrophages (19,20). In this paper, we expand

on these earlier observations by showing COX-2 is a source for 4-HNE. Further evaluation of

this using COX-2 inhibitors in IL-10 knockout mice would be difficult since these drugs

paradoxically cause severe colitis (40). Genetically inactivating COX-2 in tissue macrophages

using Cre mice is a potential alternative approach. In sum, these results link an intestinal

commensal to BSE and provide additional evidence for 4-HNE as a bona fide mediator of

clastogenesis (15,16).

A focus on E. faecalis in the Il10-/- model should not be construed to mean that other

intestinal commensals do not have the potential to serve as triggers for BSE and clastogenesis.

Conversely, it is unlikely that any member of the colonic microbiota can cause CRC. Indeed, the

vast majority of commensals co-exist as symbiotes and promote health while excluding

potentially pathogenic exogenous bacteria (41). Commensals may even lower the risk for CRC.

Strains of Escherichia coli express enterotoxins with anti-proliferative properties (42). Despite

these caveats, accumulating evidence provides a strong rationale for considering colonic

commensals as sources for endogenous DNA damage in colorectal carcinogenesis. Similar to E.

faecalis, E. coli can generate double-strand DNA breaks in mammalian cells and CRC in

monoassociated Il10-/- mice when expressing an unusual hybrid peptide-polyketide toxin (43).




Strains of Bacteroides fragilis can produce an enterotoxin that induces colonic tumors in ApcMin/+

mice (44). Finally, a role of commensals in colorectal carcinogenesis is found in diverse murine

models for CRC where, in nearly every instance, including the IL-10 knockout model, germ-free

or pathogen-free derivatives have a reduced tumor burden or fail to develop CRC (31,45-49).

Collectively, these studies suggest that certain commensals or, more accurately, pathobioants

can damage epithelial cell DNA and help drive colorectal carcinogenesis. Notwithstanding, these

models have weaknesses. Commensals do not cause inflammation or CRC in healthy wildtype

mice. Deficiencies in host immunity (e.g., Il10-/-) are typically necessary for cellular

transformation by E. faecalis. Despite this caveat, the Il10-/- model, is useful for studying

mechanisms that may contribute to human carcinogenesis.

In sum, our findings support a novel mechanism of inflammation-associated and sporadic

CRC. This theory involves specific pathobioants (e.g., E. faecalis) that trigger abnormal innate

immune responses that induce BSE, cause DNA damage in epithelial cells, and initiate CIN. The

key effector in this scheme is COX-2 with its ability to generate 4-HNE. This theory, if

confirmed in human tissue, will open new avenues for preventing these cancers.

Acknowledgements

Grant Support: NIH CA127893 (to M.M.H.), the Oklahoma Center for the Advancement of

Science and Technology HR10-032 (to X.W.), and the Frances Duffy Endowment.




Figure Legends

Figure 1. Inhibitors for ALOX5 and COX decrease 4-HNE production from macrophages.

A, AA861 (ALOX5 inhibitor) and ETYA (inhibitor for ALOX5 and COX) significantly decrease

4-HNE production in supernatants from uninfected (open bar) and E. faecalis-infected

macrophages (solid bar, **P < 0.01; #P = 0.01 compared to control). B, Western blots show

decreased COX-2 in macrophages by AA861 and ETYA following treatment with E. faecalis. C,

RT-PCR shows decreased Cox1 expression in E. faecalis-infected macrophages (lower panel)

compared to uninfected macrophages (upper panel). D, Normalized Cox1 expression increases in

untreated macrophages (open bar) while decreases following E. faecalis-infection (solid bar). E,

Western blots for ALOX5 in uninfected macrophages (upper panel) and E. faecalis-infected

macrophages (lower panel). F, Normalized ALOX5 production decreases at 24-72 hrs following

E. faecalis-infection (solid bar) compared to uninfected macrophages (open bar; NS, not

significant; *P < 0.05; **P < 0.01 compared to untreated control at zero time point for D and F).

Data represent mean ± SD for 3 independent experiments.

Figure 2. Silencing Cox1 and Alox5 fail to reduce 4-HNE production. A and B, RT-PCR

shows that Cox1-specific siRNA (siCox1) significantly decreases Cox1 expression in uninfected

(open bar) and E. faecalis-infected macrophages (solid bar) compared to non-targeting siRNA

(siCtrl). C and D, Western blots show Alox5-specific siRNA (siAlox5) decreases ALOX5 in both

uninfected (open bar) and E. faecalis-infected macrophages (solid bar) compared to siCtrl. E,

Significantly increased 4-HNE production is seen in E. faecalis-infected macrophages (solid bar)

compared with uninfected macrophages (open bar). Of note, silencing Cox1 and Alox5 does not

decrease 4-HNE production by E. faecalis-infected macrophages (P = 0.2 and P = 0.8,




respectively). F, �-tocopherol significantly decreases 4-HNE production in uninfected (open bar)

and E. faecalis-infected macrophages (solid bar). Data represent mean ± SD for 6 independent

experiments for 4-HNE assays and 3 independent experiments for other measures.

Figure 3. COX-2 is the predominant source for 4-HNE. A, Celecoxib significantly decreases

E. faecalis-induced Cox2 expression in macrophages. B. Western blots confirm significantly

decreased COX-2 in E. faecalis-infected macrophages treated with celecoxib. C, Celecoxib

significantly decreases 4-HNE production in E. faecalis-infected macrophages. D, Cox2-specific

siRNA (siCox2) decreases Cox2 expression in E. faecalis-infected macrophages compared to

non-targeting siRNA (siCtrl). E, Western blots shows remarkably decreased COX-2 production

in E. faecalis-infected macrophages transfected with Cox2 siRNA (siCox2) compared to non-

targeting siRNA (siCtrl). F, siCox2 decreases 4-HNE production in E. faecalis-infected

macrophages compared to siCtrl. G, Celecoxib significantly inhibits 4-HNE production by

recombinant human COX-2 with 50 �M arachidonic acid as the substrate. H, Similarly,

celecoxib significantly decreases PGD2 production by recombinant human COX-2.

Figure 4. 4-HNE increases COX-2 via positive feedback. A, Normalized to uninfected

macrophages, buthionine sulfoximine (BSO) increases 4-HNE production in E. faecalis-infected

and uninfected macrophages. B, Normalized to uninfected macrophages, Cox2 expression

decreases 2-fold following BSO treatment. Normalized to control, BSO treatment of E. faecalis-

infected macrophages increases Cox2 expression by 50-fold. Cox2 expression is further

increased when BSO is added to E. faecalis-infected macrophages compared to control. C,

Western blots show no effect on COX-2 production when BSO is added. D, BSO significantly

increases PGD2 in E. faecalis-infected macrophages when normalized to control, suggesting that




4-HNE increases COX-2 activity. Fold changes represent values normalized to untreated

macrophages. Data are means ± SD for experiments performed in triplicate.

Figure 5. 4-HNE co-localizes to macrophages expressing COX-2. A, Immunohistochemical

staining for COX-2 (brown) in colon sections from Il10-/- mice colonized with E. faecalis (left)

or sham (right) for 9 months. COX-2 is seen in macrophages (green arrows) in areas of

inflammation. No COX-2 is noted in colon sections from sham-colonized Il10-/- mice. B,

Immunofluorescence staining for COX-2 (ii, green) and F4/80 (iii, red) confirm co-localization

of COX-2 to macrophages (iv, yellow). DNA is counter-stained using DAPI (i, blue).

Significantly increased COX-2-positive cells are seen in sections from E. faecalis-colonized

Il10-/- mice (left) compared to sham (right). C, Immunofluorescence staining for 4-HNE-protein

adducts (ii, green) and F4/80 (iii, red) shows focal staining of 4-HNE in macrophages (iv,

yellow) and diffuse staining on crypts (green) for E. faecalis-colonized mice (left) compared to

minimal staining for sham-colonized mice (right).

Figure 6. Il10-/- model for bystander effects due to E. faecalis-infected macrophages.

Translocating E. faecalis are phagocytosed by tissue macrophages with superoxide contributing

to peroxidation oxidative stress, NF-�B signaling, and COX-2 expression that generates

clastogenic lipid peroxidation byproducts (e.g., trans-4-hydroxy-2-nonenal [4-HNE]). 4-HNE

diffuses to nearby colonic epithelial cells to cause bystander effects (BSE). 4-HNE reacts with

DNA to form mutagenic adducts and to cause CIN (1,11,15); prostanoids are derived from COX-

2 and limit apoptosis, enhance angiogenesis, and promote proliferation in epithelial cells but are

not mediators of BSE.




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Published OnlineFirst January 15, 2013.Cancer Prev Res Xingmin Wang, Toby Allen, Yonghong Yang, et al. Macrophages

-infectedEnterococcus faecalis-4-Hydroxy-2-nonenal in transCyclooxygenase-2 Generates the Endogenous Mutagen

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