Inverse association between phospholipase A2 and COX-2 expression during mouse colon tumorigenesis
Mei Dong1,
Kishore Guda1,
Prashant R. Nambiar1,
Anahita Rezaie2,
Glenn S. Belinsky1,
Gérard Lambeau3,
Charles Giardina4 and
Daniel W. Rosenberg1,5
1 The University of Connecticut Health Center, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3101, USA,
2 The University of Connecticut Health Center, Department of Internal Medicine, Farmington, CT, USA,
3 The Institute de Pharmacologie Moleculaire et Cellulaire, Valbonne, France and
4 The University of Connecticut, Department of Molecular and Cell Biology, Storrs, CT, USA
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Abstract
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Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid (AA) from intracellular phospholipids. We evaluated the status of cPLA2 in azoxymethane (AOM)-induced mouse colon tumors. Despite increased expression of cyclooxygenase 2 (3.7-fold) and PGE2 (3.4-fold) production in tumors, cPLA2 mRNA levels and enzyme activity were significantly reduced (3.6- and 3-fold, respectively). Reduced levels of cPLA2 were also observed in pre-neoplastic aberrant crypt foci (ACF), a distinct morphological alteration that represents an early stage in the pathogenesis of colon tumors. Furthermore, the reciprocal expression patterns of these two genes were found to occur in human colorectal cancers (CRC). Examination of the activity of the secretory phospholipases A2 (sPLA2) and expression of the groups V and X sPLA2s revealed no compensatory increase in tumor tissue. As cPLA2 has been shown to be involved in TNF-
-induced apoptosis in certain cell types, and TNF-
expression is significantly enhanced in AOM-induced tumors (2.8-fold), we examined the role of cPLA2 in TNF-
-induced apoptosis of cultured mouse colonocytes (YAMC). The specific cPLA2 inhibitor, AACOCF3 (arachidonoyl trifluoromethyl ketone), was able to protect colonocytes from TNF-
-induced apoptosis in vitro. In summary, our data demonstrate an inverse relationship between COX-2 and cPLA2 expression in both AOM-induced mouse colon tumors and human CRC and suggest that down regulation of cPLA2 may attenuate TNF-
mediated apoptosis during tumorigenesis and facilitate tumor progression.
Abbreviations: AA, arachidonic acid; ACF, aberrant crypt foci; AOM, azoxymethane; COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; CRC, colorectal cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; iPLA2, Ca2+-independent phospholipase A2; PGs, prostagladins; RPA, RNase protection assay; sPLA2, secretory phospholipase A2; TNF-
, tumor necrosis factor-
.
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Introduction
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An important pathway in the pathogenesis of colorectal cancer is the generation of prostaglandins (PGs) via the cyclooxygenase-2 (COX-2) (1). COX-2, an inducible form of cyclooxygenase, plays a major role in the synthesis of PGs from arachidonic acid (AA) and is rapidly induced by growth factors, inflammatory cytokines and oncogenes (24). AA is provided by the phospholipase A2s through hydrolysis of membrane phospholipids at the sn-2 position. Apart from being a precursor of PGs, intracellular AA can also act as a death mediator via activation of sphingomyelinase, which hydrolyzes sphingomyelin to ceramide, an inducer of apoptosis (5,6). It has been demonstrated in cell lines that this pathway plays a critical role in promoting apoptosis induced by tumor necrosis factor
(TNF-
) (79).
To date, at least 10 mammalian PLA2s have been classified on the basis of their distinct biochemical features. Three important groups include the cytosolic PLA2 (cPLA2), the secretory PLA2s (sPLA2) and a Ca2+-independent PLA2 (iPLA2) (10). cPLA2 (85 kDa) is the major intracellular form of PLA2. It is widely expressed and plays an essential role in stimulus-induced AA release (11,12). Because of its important role in generating PGs, a role for cPLA2 in intestinal tumorigenesis has also been suggested. Two recent studies demonstrated that cPLA2 expression is elevated in small intestinal polyps in both ApcMin and Apc
716 mice (13,14). Furthermore, deletion of the cPLA2 gene significantly suppressed ApcMin or Apc
716-induced tumorigenesis in the small intestine. In both studies, however, elevated cPLA2 was not observed in colon tumors, nor did deletion of the cPLA2 gene reduce tumorigenesis in this organ. On the contrary, cPLA2 deletion was accompanied by increased numbers or size of colon polyps, arguing that the physiological function of cPLA2 and its role in tumorigenesis may differ within specific regions of the gut.
Secretory phospholipase A2s comprise a family of PLA2 enzymes sharing several biochemical characteristics, including a low molecular mass (14 kDa), the presence of six to eight disulfide bonds, and an absolute catalytic requirement for millimolar concentrations of Ca2+ (10). Among the seven sPLA2 identified in mammals, groups IIA, V and X are expressed in mouse colon (1315). sPLA2 group IIA, the inflammatory sPLA2, has been proposed to play a role in intestinal tumorigenesis; transgenic mice overexpressing group IIA are resistant to ApcMin-induced intestinal tumorigenesis (16). The group V sPLA2 shares the greatest homology with group IIA and functions in a similar manner (10). Studies with immune cells isolated from sPla2g2a-/- mice demonstrate that group V sPLA2 can substitute group IIA for AA production (17,18). Group X sPLA2, a new member of the sPLA2 family, is found to be highly expressed in the large intestine. Like cPLA2, group X sPLA2 mRNA levels are increased in small intestinal polyps in Apc
716 mice, suggesting a concerted role for phospholipases in AA release and subsequent COX-2-mediated PG synthesis, thus contributing to intestinal tumorigenesis (14).
To further characterize the role of the cytosolic and secretory phospholipases in colon tumorigenesis, we examined the expression and functional status of these enzymes in colon tumors induced by the organotropic colon carcinogen, azoxymethane (AOM). Our laboratory has shown that sensitive strains of mice treated with AOM develop up to 40 tumors, restricted primarily to the distal colon (19). The AOM model has thus provided considerable insight into our understanding of signaling pathways that may be dysregulated during colon tumorigenesis (2024). In the present study, a striking reduction in cPLA2 expression was found in carcinogen-induced pre-neoplastic lesions and tumors. In addition, cPLA2 reduction occurred despite enhanced COX-2 expression and PGE2 overproduction, whereas expression of the major secretory phospholipases, group V and group X, were unaltered. The data suggest a dissociation between cPLA2 activity and PG production during colon tumorigenesis. In addition, the reciprocal expression patterns of cPLA2 and COX-2 were also found to occur in human colorectal cancers (CRC). Furthermore, in vitro evidence is obtained that a reduction in cPLA2 levels may compromise the ability of TNF-
to induce apoptosis in colonocytes, raising the possibility that cPLA2 plays an important role in regulating tumor promotion in the colon.
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Materials and methods
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Animals and test materials
A/J mice were purchased from the Jackson Laboratories (Bar Harbor, ME). AOM, cyclohexamide and proteinase inhibitor cocktail were obtained from Sigma (St Louis, MO). The cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Recombinant mouse TNF-
was purchased from Chemicon (Temecula, CA). The RiboQuant multiprobe RNase protection assay system, including the TNF-
template, was obtained from Pharmingen (San Diego, CA). Rabbit anti-mouse cPLA2 was generously provided by Dr A.V.Cybulsky (McGill University, Canada). Monoclonal mouse-anti human cPLA2 antibody was purchased from Upstate (Lake Placid, NY). Mouse anti-ß-actin antibody was purchased from Sigma. Rabbit anti-COX2 antibody, and the assay kits for PGE2 and cPLA2 were purchased from Cayman Chemical (Ann Arbor, MI). The sPLA2 assay kit was purchased from Assay Design (Ann Arbor, MI). Young adult mouse colonocytes were generously provided by Dr R.H.Whitehead (Ludwig Institute for Cancer Research, Switzerland).
Animal treatment and tissue sample preparation
Five-week-old male A/J mice were housed in a temperature-controlled environment (23°C ± 1) with a 12 h light/dark cycle. Mice were provided with Purina laboratory rodent chow 5001 and water ad libitum. After an acclimation period of 1 week, mice were divided randomly into two groups. One group was treated with AOM dissolved in saline by i.p. injection at a dose of 10 mg/kg body wt once per week for 6 weeks. The other group received saline and served as vehicle controls. Mice were killed 24 weeks after the last dose. The entire colon was removed and flushed with ice-cold PBS buffer. Colons were slit open longitudinally. Colon tumors from the AOM treatment group and normal distal colons from the control group were dissected and tissues were divided into macroscopically similar portions. The portions for RNA and protein were immediately frozen in liquid nitrogen and stored at 80°C. The remaining portion for paraffin embedding was fixed in 10% neutral-buffered formalin for 6 h and embedded in paraffin for subsequent histopathological examination and immunostaining.
RNase protection assay
Total cellular RNA was isolated from frozen normal colons and tumor tissues using the Qiagen RNeasy kit (Qiagen, Valencia, CA) following the manufacturers instructions. mRNA levels for the genes examined were quantified using the RiboQuant multiprobe RNase protection assay (RPA) system. A customized template set containing TNF-
and GAPDH was used to probe the RNA samples. GAPDH was used as an internal control. To make probes for the other genes, an RTPCR reaction was carried out according to a standard protocol. The primers used in the PCR reaction were as follows: cPLA2, 5'-GGA TGA GCA TGA CCC TGA GT-3' and 5'-GGC AAA CAT CAG CTC TGA CA-3'; COX2, 5'-TCC TCA CAT CCC TGA GAA CC-3' and 5'-CTC ATC ACC CCA CTC AGG AT-3'; sPLA2gX, 5'-GAC CTG GAC CCG GAT TCA GC-3' and 5'-CTT GGG AGA GTC CTT CTC AC-3'; sPLA2 gV, 5'-CGG GGG GCT TGC TAG AAC TCA A-3' and 5'-AAG AGG GTT GTA AGT CCA GAG G-3'; sPLA2 M-type receptor, 5'-TCG CCT ACA CGT CCA GTG GT-3' and 5'-CTG CCA GCC AGC CTT CTC AT-3'; and HPRT (loading control), 5'-GTA ATG ATC AGT CAA CGG GGG AC-3' and 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'. cDNA fragments of different length were cloned into the pGEM-T Easy vector (Promega, Madison, WI). The 32P-labeled anti-sense RNA probes were synthesized using the RiboQuant In Vitro Transcription kit according to the manufacturers instructions. RNA samples (5 µg) were incubated with the anti-sense probes overnight at 56°C and then digested by treatment with RNase. The protected double-stranded RNA fragments were resolved on 7% denaturing gels, which were then exposed to X-ray film at 70°C. Image densitometry was performed using NIH image software.
Western blot analysis
Normal colons and tumor tissue were homogenized in RIPA buffer containing protease inhibitor cocktail and incubated on ice for 30 min. Samples were then centrifuged at 10 000 g for 15 min and the total protein content of the supernatant was quantified with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Protein (30 µg) was separated on a 10% SDSPAGE gel and then electrotransferred onto a nitrocellulose membrane. The membrane was probed with the following primary antibodies: anti-cPLA2 (1:1000), anti-COX2 antibody (1:1000) or anti-ß-actin antibody (1:1000). For cPLA2 and ß-actin, the blot was incubated with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibody, respectively, and visualized using the ECL Western blot analysis system (Santa Cruz Biotechnology, CA). For COX-2, the alkaline phosphatase-conjugated secondary antibody and ECL system provided by the WesternBreeze kit (Invitrogen, Carlsbad, CA) was used according to the manufacturers instructions.
PGE2 levels determination
Normal control colons and AOM-treated tumor tissues were homogenized in ice-cold PBS buffer containing 1 mM EDTA and 10 µM indomethacin (Sigma), mixed with an equal volume of ethanol and then centrifuged at 1500 g for 10 min. The supernatant was acidified to a pH of 4 by addition of acetate buffer, and then applied to a methanol-activated C18 reverse phase column (Waters Corporation, Milford, MA), which was then rinsed with water and hexane. The organic phase was collected by elution with ethyl acetate containing 1% methanol, which was later evaporated under a nitrogen stream. The final purified organic phase was analyzed for PGE2 levels using the Prostaglandin E2 E1A-Monoclonal kit, based on the competition between PGE2 and a PGE2acetylcholinesterase conjugate for a limited amount of PGE2 monoclonal antibody.
cPLA2 activity determination
Equal amounts of normal untreated control colons and tumor samples were homogenized in 500 µl ice-cold PBS buffer containing 1 mM EDTA and centrifuged at 10 000 g for 15 min at 4°C. The supernatant was treated with 5 µM bromoenol lactone for 15 min at 25°C to inhibit the activity of iPLA2 and then filtered through a cellulose membrane filter with a molecular weight cut-off of 30 000 (Millipore) to remove any residual sPLA2. The purified sample was then incubated with the substrate, arachidonoyl Thio-PC. Hydrolysis of the substrate at the sn-2 position by PLA2 releases free thiol, which was then detected colorimetrically using Ellmans reagent on a microplate reader (405 nm).
sPLA2 activity determination
Equal amounts of normal control colon tissues and tumor samples from AOM-treated mice were homogenized in 500 µl ice-cold PBS buffer and centrifuged at 10 000 g for 15 min at 4°C. Samples of 50 µl of the supernatant were incubated with HEPC (2-hexadecanoylthio-1-ethylphosphorylcholine), a specific substrate for the sPLA2s, but not metabolized by cPLA2 (25). HEPC is converted into a thiol molecule, which is detected colorimetrically using Ellmans reagent (405 nm).
Immunohistochemistry
5-µM formalin-fixed, paraffin-embedded mouse and human colon tissue sections were incubated with primary antibody (anti-COX-2 1:200, anti-mouse cPLA2 1:300, anti-human cPLA2 1:100) overnight at 4°C. Sections were washed with PBS and incubated with corresponding biotinylated secondary antibody (1:100) (Vector Laboratories, Burlingame, CA) at room temperature for 30 min. After washing, the sections were incubated with avidinbiotin peroxidase complex provided by Vectastain Elite ABC kit (Vector Laboratories) at room temperature for 30 min. Color was developed with 3,3-diaminobenzidine as the substrate. Sections were then counterstained with hematoxylin. As a negative control, the duplicate sections were immunostained in parallel with 10% goat serum in place of the primary antibody.
Analysis of cPLA2 and COX-2 in human colon tumors
Human colorectal cancer tumors and paired normal adjacent mucosal tissues were collected from surgically resected specimens of primary CRC patients following approval by the Institutional Ethics Committee. Tissue samples were immediately frozen in liquid nitrogen and stored at 80°C for subsequent RNA isolation and semi-quantitative RTPCR analysis. The primers used in the PCR reaction were as follows: cPLA2, 5'-CTC ATG CCC AGA CCT ACG ATT-3' and 5'-TAA TAC GAC TCA CTA TAG GGC GTC AGG TTT GAC-3'; COX-2, 5'-GAT TCA AAT GAG ATT GTG GAA AAA TTG CTT-3' and 5'-GAT AGC CAC TCA AGT GTT GCA CA-3'; GAPDH, 5'-CCA TGG AGA AGG CTG GG-3' and 5'-GGT CAT CCA TGA CAA CTT TG-3'.
Cell culture and treatments
Experiments were carried out using the conditionally immortalized murine colonic epithelial cells (YAMC) (26). YAMC cells express the IFN-
-induced heat-labile SV40 large T antigen, which is active only at 33°C. All cells were grown to 95% confluence at 33°C with 5% CO2 on 24-well plate in RPMI 1640 media supplemented with 5% FBS, ITS (insulin 6.25 ng/ml, transferrin 6.25 µg/ml, selenious acid 6.25 ng/ml, linoleic acid 5.35 mg/ml and bovine serum albumin 1.25 mg/ml), 5 IU/ml of murine IFN-
, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were pre-incubated with different concentrations of AACOCF3 or BPB for 2 h, and then sensitized to TNF-
-induced cell death by incubating with 25 µg/ml cycloheximide for 1 h. TNF-
(25 ng/ml) was added to each well and cells were then further incubated for 6 h. For quantification of apoptotic cells, both attached and collected floating cells were briefly trypsinized into a single-cell suspension. Cells were then collected by centrifugation at 1000 r.p.m. for 10 min, fixed in Carnoys solution (1:3 v/v glacial acetic acid:methanol, prepared fresh), dropped on a glass slide and stained with Hoechst 33258 (Sigma) for 10 min at room temperature. Cells with condensed nuclei were counted as apoptotic cells. For determination of cPLA2 activity, YAMC cells cultured in a 35 mm dish were incubated with TNF-
following pre-treatment with AACOCF3 and CHX. One hour after the incubation, cells were washed twice with ice-cold PBS and homogenized in PBS containing 1 mM EDTA. cPLA2 activity was immediately assayed as described above.
Statistical analysis
The Generalized linear model procedure using SAS software was applied. Significant differences were determined by the probability of difference (PDIFF) between the means. A P-value <0.05 was considered statistically significant.
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Results
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COX-2 and PGE2 levels in AOM-induced colon tumors
COX-2 levels are typically elevated in rodent colon tumor models (4,27). As an initial step to determine COX-2 status in AOM-induced A/J colon adenocarcinomas, we quantified COX-2 mRNA expression by quantitative RPA. As shown in Figure 1A and B
, COX-2 mRNA levels were below the level of detection in vehicle-treated control colon tissue, but significantly increased (3.7-fold, P < 0.01) in the tumors. Consistent with elevated mRNA levels, western blot analysis (Figure 1C
) also indicated a marked increase in COX-2 protein in adenocarcinomas compared with vehicle-treated controls. This was also confirmed by enhanced COX-2 immunostaining in tumors compared with adjacent normal-appearing crypts and vehicle-treated mouse colons (Figure 2A and C
). Furthermore, markedly enhanced COX-2 immunostaining in the large dyplastic ACF (Figure 2B
) demonstrates that COX-2 overexpression is an early event during colon tumorigenesis. To determine if overexpression of COX-2 was associated with overproduction of PGE2, a major eicosanoid product believed to be involved in COX-2-mediated colon tumorigenesis (28,29), PGE2 levels were measured by ELISA and found to be significantly elevated (3.4-fold, P < 0.01) within excised adenocarcinomatous tissue (Figure 3
). The overproduction of PGE2 mediated by COX-2 may therefore participate in AOM-induced mouse colon tumorigenesis.

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Fig. 1. COX-2 expression in normal mouse colon and tumor tissue. (A) Comparison of COX-2 mRNA levels in normal colon (N) and tumor tissues (T) by RPA analysis. HPRT was used as a loading control for normalization. (B) Quantitative analysis of RPA data was performed using NIH image analysis software. Statistical analysis was performed using the Generalized linear model procedure followed by PDIFF post-hoc analysis for comparing the means. Each column represents the mean ± SE. *P < 0.05. (C) Comparison of COX-2 protein levels in normal colon and tumor tissues. Equivalent amounts of total protein extract from normal colon and tumor tissues were separated by SDSPAGE and immunoblotted with antibody against COX-2 or ß-actin.
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Fig. 2. Immunohistochemical analysis of COX-2 expression and localization in normal mouse colon, ACF and tumor tissue. (A) Normal colon section from a vehicle-treated control mouse. Note the low level of staining for COX-2 in columnar epithelial cells and within the lamina propria. (B) Large dysplastic ACF induced by AOM. Note the marked enhancement in staining for COX-2 within the ACF relative to adjacent normal-appearing colonic crypts. (C) Polypoid adenocarcinoma induced by AOM. Note the increased staining for COX-2 within the tumor crypts. (D) Negative control tumor section immunostained with 10% goat serum in place of the primary COX-2 antibody showing no positive staining.
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Fig. 3. Comparison of PGE2 levels in normal mouse colon and tumor tissue. Tissue samples (normal, n = 3; tumor, n = 5) were homogenized in PBS buffer containing 1 mM EDTA and 10 µM indomethacin. Lipids were extracted using a C18 reverse phase column and PGE2 levels were determined by ELISA using PGE2 monoclonal antibody (*P < 0.01).
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Expression and functional status of cPLA2 in AOM-induced colon tumors
cPLA2 plays a key role in the generation of AA from internal cell membranes. To investigate the possibility that COX-2-induced PGE2 overproduction is coordinately regulated with cPLA2 activity, we examined the expression of cPLA2 using both RPA and western blot analysis. Despite high levels of PGE2, a significant reduction (3.6-fold, P < 0.01) in cPLA2 mRNA levels was observed in AOM-induced colon tumors compared to vehicle-treated controls (Figure 4A and B
). This reduction in transcript was associated with a decrease in protein levels as shown by western blot analysis (Figure 4C
). To determine whether the observed reduction in cPLA2 mRNA and protein was accompanied by altered enzymatic activity, we examined the functional status of cPLA2 in both tumors and vehicle-treated control colons. As shown in Figure 4D
, a significant decrease in cPLA2 activity was observed in adenocarcinomas (3.5-fold, P < 0.01). Taken together, these results indicate that cPLA2 expression is not correlated with high levels of COX-2 and PGE2 overproduction in AOM-induced colon tumors. cPLA2 immunostaining further demonstrates a relative reduction in protein levels in the pre-neoplastic lesions, including hyperplastic (Figure 5B
) and dysplastic ACF (Figure 5C
), relative to normal colonic crypts (Figure 5A
). This finding indicates that reduction of cPLA2, like COX-2 overexpression, occurs early during colon tumorigenesis.

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Fig. 4. Expression and functional analysis of cPLA2 in normal colon and tumor tissue. (A) cPLA2 mRNA levels in normal colon and tumor tissue were determined by RPA. HPRT was used as a loading control for normalization. (B) Quantitative analysis of RPA data using NIH image software (*P < 0.05). (C) Comparison of cPLA2 protein levels in normal colon and tumor tissue. Total protein extracts were separated by SDSPAGE. Membrane was probed with antibody against cPLA2 or ß-actin. (D) cPLA2 activity in normal colon (n = 3) and tumor tissue (n = 5). Tissue homogenates were treated with 5 µM bromoenol lactone to inhibit the activity of iPLA2 and filtered through a cellulose membrane with a molecular weight cut-off of 30 000 to remove residual sPLA2. cPLA2 activity was then measured using arachidonoyl Thio-PC as a substrate (*P < 0.05).
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Fig. 5. Immunohistochemical analysis of cPLA2 expression and localization in normal mouse colon, ACF and tumor tissue. (A) Normal colon tissue section from a vehicle-treated control mouse. Note the intense perinuclear staining of cPLA2. (B) Hyperplastic ACF from AOM-treated mouse colon. Note the decreased staining intensity for cPLA2 in the ACF relative to adjacent normal colonic crypts. (C) Large dysplastic ACF from AOM-treated mouse colon. Note the decreased staining intensity for cPLA2 in the ACF. (D and E) Represent a flat adenoma and a polypoid adenoma from AOM-treated mouse colons. Note the markedly reduced staining for cPLA2 in tumor crypts relative to adjacent normal-appearing colonic crypts. (F) Represents a negative control colon section immunostained with 10% goat serum in place of the primary cPLA2 antibody showing no positive staining.
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Status of sPLA2s in AOM-induced colon tumors
A reduction of cPLA2 in the tumors suggests alternative mechanisms for the supply of AA to COX-2 for PG production, possibly mediated by the sPLA2s. As A/J mice carry a nonsense mutation in the major colon sPLA2 gene, group IIA sPLA2 (30), it was of interest to determine whether additional secretory phospholipase(s) may be compensatory induced in colon tumors. As an initial step to evaluate this possibility, we examined total sPLA2 activity in AOM-induced tumors and vehicle-treated control colons using a general sPLA2 substrate, HEPC, which is not reactive to cPLA2 (25). As shown in Figure 6A
, no significant differences were found between the two groups. Furthermore, RPA analysis showed equivalent expression of group V and group X sPLA2 mRNA in tumor and control tissue. Constitutive expression of the group X gene, however, was markedly higher relative to the group V, although the significance of these observations to the pathophysiology of the colon is not clear. Finally, we examined the expression of the M-type sPLA2 receptor, which binds to group IB, IIA and group X sPLA2s and has been proposed to play an important role in regulating the activity of these enzymes (31,32). As shown in Figure 6B and C
, results by RPA indicated no significant difference between the normal colon and tumor tissues. These results indicate that sPLA2 levels are not correspondingly elevated in A/J colon tumors that have sustained a significant reduction in cPLA2 expression. The data suggest that AA production may be compromised as a result of the down regulation of cPLA2. Reduced AA production, combined with increased AA utilization resulting from up regulation of COX-2, might actually lead to reduced levels of AA within the tumor cells.

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Fig. 6. Examination of total sPLA2 activity and expression of group X and group V sPLA2 and the M-type sPLA2 receptor. (A) Total sPLA2 activity in normal colon and tumor tissues was determined using HEPC, a general sPLA2 substrate that is not reactive to cPLA2. (B) Group X and group V sPLA2 and M-type sPLA2 receptor (M-R) mRNA levels in normal colon and tumor tissue were determined by RPA. HPRT was used as a loading control for normalization. (C) Quantitative analysis of RPA data was performed using NIH image software.
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cPLA2 and COX-2 expression in human colorectal tumors
To examine the relationship between of cPLA2 and COX-2 in human CRC, mRNA levels were determined by semi-quantitative RTPCR analysis. Reduced mRNA levels of cPLA2 was found in all five of the tumor samples examined, whereas overexpression of COX-2 was found in four out of five of the samples (P < 0.01, Figure 7
). Immunohistochemistry also demonstrated diminished cPLA2 staining in human colon tumors compared with adjacent normal-appearing epithelium (Figure 8
). The intense perinuclear staining of cPLA2 observed in our study is in agreement with an earlier report by Schievella et al. (33) in which the subcellular localization of cPLA2 was localized to the nuclear envelope and endoplasmic reticulum (33). The data from human tissues are consistent with the expression patterns observed in carcinogen-exposed mouse colon, indicating that alterations in AA levels may also occur in human colon tumors.

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Fig. 7. Examination of cPLA2 and COX-2 in human colorectal tumors. mRNA levels of cPLA2 and COX-2 in human colon tumor and matched normal colon specimens was determined by semi-quantitative RTPCR analysis as described under Materials and methods. GAPDH was used as a loading control for normalization.
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Fig. 8. Immunohistochemical analysis of cPLA2 expression and localization in human colon. (A) Human colon tissue section showing both longitudinal and transverse staining of normal colon crypts. Note the intense perinuclear staining of cPLA2. (B) There is decreased cPLA2 staining within the adenocarinoma relative to adjacent normal appearing colon tissue. (C) Negative control colon section immunostained with 10% goat serum in place of the primary antibody.
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cPLA2, AA and TNF-
-induced cell death
TNF-
treatment results in the activation of cPLA2 (11). In a number of instances, cPLA2 activation by TNF-
has been shown to be critical for TNF-
-induced apoptosis, potentially through the ability of AA to activate sphingomyelinase and increase ceramide production (5), although this phenomenon has not yet been demonstrated in colon cells. In the present study, we found a 2.8-fold increase in TNF-
expression within the mouse colon tumors (Figure 9A
). To test the potential significance of these findings in tumors with compromised cPLA2 activity, cultured immortalized mouse colonocytes (YAMC) were pre-treated with the cPLA2 specific inhibitor, AACOCF3, prior to a 6 h incubation with TNF-
. As shown in Figure 9C and D
, a clear doseresponse was observed. Cells treated with increasing concentrations of AACOCF3 demonstrated more resistance to TNF-
-induced cell death. We also examined the activity cPLA2 1 h after TNF-
treatment and found that, in the presence of 10 mM AACOCF3, there was an
87% decrease in cPLA2 activity (Figure 9B
).
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Discussion
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COX-2 is overexpressed in
85% of human colon adenocarcinomas (34). Several lines of evidence have demonstrated that COX-2 and its product, PGE2, play an important role in the promotion of intestinal tumorigenesis. For example, inactivation of COX-2 by the introduction of a null mutation in the COX-2 gene or the use of COX-2 selective inhibitors markedly reduces intestinal tumor formation in Apc
716 mice (26). Sulindac, a general COX inhibitor, has also been shown to prevent colon tumorigenesis and reduce polyp size in familial adenomatous polyposis patients (35). The pro-tumorigenic effect of COX-2 has been attributed to its ability to enhance cell proliferation and angiogenesis, while inhibiting cell differentiation and apoptosis. The role of COX-2 and PGE2 in AOM-induced colon tumorigenesis has also been reported (3639). In the present study, we show that COX-2 overexpression can actually occur at an early stage during colon tumorigenesis and its overexpression is accompanied by overproduction of PGE2 in the tumors.
The source of AA for COX-2 metabolism can be potentially derived from a number of phospholipase A2 enzymes. A critical pathway is via the intracellular phospholipase (cPLA2) that provides AA for COX-2 synthesis of PGs. Immune cells isolated from cPLA2 knockout mice fail to produce eicosanoids during either the immediate- or delayed-phase response to pro-inflammatory stimuli, indicating that cPLA2 can be a major source of AA for PG production (12). Apart from its role in the inflammatory response, two recent studies have demonstrated that cPLA2 also plays an important role in tumorigenesis. Targeted deletion of cPLA2 can protect against the formation of small intestinal tumors in both ApcMin and ApcD716 mice. Interestingly, colon tumor formation was unexpectedly increased in both of these mouse strains. The potential role of cPLA2 in colon tumorigenesis uncovered by these studies prompted us to explore the expression and activity of cPLA2 in a colon-specific cancer model. We found that cPLA2 expression and activity were markedly reduced in AOM-induced colon tumors. Moreover, this reduction occurred at a relatively early stage of AOM-induced tumorigenesis. Thus, three independent studies using different mouse tumor models, each illustrate the lack of correlation between cPLA2 and COX-2 during colon tumorigenesis. The data further suggest that the role of cPLA2 in colon tumorigenesis may be fundamentally different from its protective role in the small intestine. Indeed, the tumorigenic process within different regions of the intestine may be driven by distinct pathophysiological mechanisms.
The question of whether secreted phospholipases may compensate for the observed decrease in cPLA2 activity was also examined in the present study. No enhanced protein activity or expression was observed between tumor and normal colon tissues, indicating that sPLA2s are not correspondingly up regulated as a compensatory response to the loss of cPLA2. Thus, the loss of cPLA2 without a compensatory increase in sPLA2, combined with increased COX-2 production of PGs, suggests the possibility that there may be a reduction in the steady-state levels of AA within the tumor cells.
To establish whether a similar pattern of alteration in these key AA-metabolizing enzymes may also be present in the human colon, an analysis of cPLA2 and COX-2 expression was undertaken in colon tumors and adjacent normal tissue. A similar pattern of alteration was found, suggesting that an imbalance between AA production and utilization may also occur during human colon tumorigenesis. Soydan et al. (40) have also reported a decrease in cPLA2 in 11 out of 17 human colon tumors compared with matching normal controls. In contrast to these findings, however, Dinberg et al. (41) demonstrated a 3-fold increase in cPLA2 expression using RTPCR analysis. However, it is not clear from this study how many of the 44 samples examined actually showed the increase.
A substantial amount of evidence indicates that cPLA2-generated AA can serve a second messenger function, signaling apoptosis by stimulating formation of ceramide production and activation of caspase (5). Alternatively, generation of AA from phospholipids may disrupt the integrity of various cellular membranes, resulting in cell death (5). Lending support to this mechanism, it has been shown that cPLA2 inhibitors, or overexpression of an AA-modifying enzyme, fatty acid-CoA ligase 4 (FACL4), render cells resistant to TNF-
-induced apoptosis (6,7). On the other hand, overexpression of cPLA2 can enhance cell death (6,7). In the present study, we demonstrate that by specifically blocking cPLA2 activity in cultured mouse colonocytes (YAMC), cells become resistant to TNF-
-induced apoptosis. This activity of cPLA2 may be particularly important in tumors, which maintain high levels of TNF-
expression. Therefore, it is possible that decreased AA release as a result of diminished cPLA2 protein levels in colon may contribute to attenuated apoptotic response in vivo, thereby facilitating tumorigenesis.
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Notes
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5 To whom correspondence should be addressed Email: rosenberg{at}nso2.uchc.edu 
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Acknowledgments
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We thank Dr Jim Hewitt for his excellent technical advice on the analysis of COX-2. This work was supported in part by NIH grant CA 81428-01, and NIEHS grant ES 10547-03.
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References
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Received June 12, 2002;
revised September 30, 2002;
accepted October 14, 2002.