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Cytoplasmic Phospholipase A₂ Levels Correlate with Apoptosis in Human Colon Tumorigenesis

¹ Center for Molecular Medicine, Program in Colorectal Cancer; Departments of ² Internal Medicine, ³ Pathology, and ⁴ Medicine, Division of Gastroenterology, Program in Colorectal Cancer, University of Connecticut Health Center, Farmington, Connecticut; and ⁵ Department of Molecular and Cellular Biology, University of Connecticut-Storrs, Storrs, Connecticut

Requests for reprints: Daniel W. Rosenberg, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3101. Phone: 860-679-8704; Fax: 860-679-7639; E-mail: rosenberg{at}nso2.uchc.edu.

	ABSTRACT

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Colon cancers often display perturbations in arachidonic acid metabolism, with elevated levels of cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) production frequently observed. Whereas COX-2 and PGE₂ are associated with cancer cell survival and tumor angiogenesis, arachidonic acid itself is a strong apoptotic signal that may facilitate cancer cell death. To further explore how cancer cells exploit the progrowth actions of prostaglandins while suppressing the proapoptotic actions of intracellular arachidonic acid, we determined the cytoplasmic phospholipase A₂ (cPLA₂) and COX-2 expression levels in a panel of human colon tumors by immunohistochemistry. Although high levels of cPLA₂ and COX-2 expression are predicted to facilitate maximal prostaglandin production, tumors frequently displayed a high-COX-2/low-cPLA₂ phenotype. The least represented phenotype was the high expression of cPLA₂, a characteristic predicted to generate the highest levels of intracellular arachidonic acid. The potential proapoptotic role of cPLA₂ was supported by a higher frequency of terminal deoxynucleotidyl transferase–mediated nick end labeling staining in cPLA₂-positive tumors. Moreover, analysis of preneoplastic aberrant crypt foci from high-risk patients suggests that acquisition of the high-COX-2/low-cPLA₂ phenotype may arise at an early stage of colon carcinogenesis. We additionally inhibited cPLA₂ in HT-29 cells using antisense oligonucleotides. Our results indicate that cPLA₂ plays an important role in tumor necrosis factor –induced apoptosis in human colon cancer cells. Our data further support the model in which colon cancer growth is favored when intracellular arachidonic acid levels are suppressed by inhibition of cPLA₂ or by a high-COX-2/low-cPLA₂ phenotype.

Key Words: cytosolic phospholipase A2 • cyclooxygenase 2 • TNF • apoptosis • arachidonic acid

	INTRODUCTION

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Prostaglandin endoperoxide synthase, commonly referred to as cyclooxygenase (COX), is the key regulatory enzymatic step in the conversion of arachidonic acid to prostaglandins. There are two known isoforms, COX-1 and COX-2. COX-1 is constitutively expressed within the human intestine and plays a fundamental role in maintaining gastrointestinal integrity. COX-2 is normally present at relatively low levels, but can be rapidly induced during infection and inflammation (1). COX-2 is also overexpressed in 70% to 80% of human colorectal cancers (2, 3). The connection between COX-2 expression and carcinogenesis was first established in studies that showed the efficacy of aspirin and other nonsteroidal anti-inflammatory drugs to reduce the relative risk of colon cancer and to promote tumor regression in a subset of patients with cancer as well as in animal tumor models (4–8).

There is growing evidence that COX-2 and its key metabolite, prostaglandin E₂ (PGE₂), a major eicosanoid produced within the intestine, are associated with enhanced cell proliferation and survival (1). An important source of arachidonic acid for COX-2 is a ubiquitously expressed family of phospholipases (PLA). This complex gene family consists of eight secretory and three cytoplasmic phospholipases (9). The group IV cytoplasmic phospholipases are composed of three forms, , ß, and (10). Our focus is on the Ca²⁺-sensitive form, referred to as cytoplasmic phospholipase A₂ (cPLA₂), which is ubiquitously expressed in most tissues and preferentially hydrolyzes phospholipids in the sn-2 position (10). cPLA₂ plays a central role in cytokine-induced release of arachidonic acid (11). In cells stimulated by tumor necrosis factor (TNF-), cPLA₂ undergoes immediate activation via mitogen-activated protein kinase–mediated phosphorylation and Ca²⁺-directed translocation from the cytoplasm to the endoplasmic reticulum and nuclear membrane (12, 13). In the endoplasmic reticulum, it hydrolyzes phospholipids to arachidonic acid, which in turn are metabolized by the actions of colocalized COXs to produce prostaglandin (PG) metabolites (9).

Arachidonic acid has been shown to be an important mediator of TNF- induced apoptosis, possibly via activation of the ceramide pathway, or by a generalized disruption of membrane integrity (1, 14–18). In addition, Scorrano et al. (19) recently showed that Ca²⁺ release from the endoplasmic reticulum via arachidonic acid is a key mechanism for controlling apoptotic death. In contrast, PGs, especially PGE₂, have been shown to possess potent proliferative and tumorigenic capacity. Given the central role of cPLA₂ and COX-2 in maintaining the balance of arachidonic acid and PG levels, the coordinated regulation of these two proteins may be a critical mechanism for maintaining the balance between proliferation and apoptosis. In our previous study, we reported that cPLA₂ levels were barely detectable in murine colon tumors induced by the organotropic colon carcinogen, azoxymethane (15). Despite the minimal expression of cPLA₂, COX-2 levels were markedly increased with concomitant overproduction of PGE₂ (15). We hypothesized that the absence of coordinated regulation between cPLA₂ and COX-2 expression may attenuate the apoptotic signals that are mediated by arachidonic acid while further potentiating the proliferative signals elicited by PGE₂. To further explore the potential for coordinated regulation of cPLA₂ and COX-2 expression in human colon tumors, a total of 27 human colon cancer specimens were analyzed for cPLA₂ and COX-2 expression and correlated with apoptotic index. In addition, the role of cPLA₂ in TNF-–induced apoptosis was clearly shown in a human colon tumor cell line.

	MATERIALS AND METHODS

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Colorectal Tumor and Aberrant Crypt Foci Samples. A total of 27 matched human colorectal cancer specimens collected from surgically resected primary colorectal cancers were provided by the Department of Pathology at UCHC after approval by the Institutional Ethics Committee. A summary of the human tumor data is provided under Table 1. For the analysis of human preneoplastic aberrant crypt foci, close-focus, magnifying colonoscopy (Olympus Corporation, Lake Success, NY) was done to identify aberrant crypt foci in the colons of high-risk patients. Methylene blue staining was done in vivo to reveal colonic crypts. Biopsy specimens were immediately frozen in ornithine carbamyl transferase and sectioned for subsequent histopathologic and immunohistochemical analyses.

Terminal Deoxynucleotidyl Transferase–Mediated Nick-End Labeling Assay. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay was carried out according to the instructions provided by the ApopTag peroxidase in situ Apoptosis Detection Kit (Intergen Company, Purchase, NY). Briefly, deparaffinized tissue sections were treated with proteinase K followed by incubation with terminal deoxynucleotidyltransferase enzyme in the presence of digoxigenin-labeled deoxynucleotides (dUTPs) at 37°C for 60 minutes. The integrated dUTPs were then conjugated to antidigoxigenin peroxidase and color was developed with 3,3'-diaminobenzidine. Sections were counterstained with hematoxylin. For each section, five tumor crypts were randomly selected and the number of apoptotic cells were counted at 200x magnification and recorded as a percentage of total cells within one crypt. In addition to brown staining, cells classified as apoptotic also displayed condensed nuclei with integral cell membrane.

Cell Culture and Treatments. Equal numbers of HT-29 cells were seeded on 24-well plates and grown to 85% confluence in DMEM supplemented with 10% fetal bovine serum and 100 IU/mL penicillin/streptomycin. High-performance liquid chromatography–purified cPLA₂ antisense oligonucleotides or control oligonucleotides (Biomol, Plymouth Meeting, PA) were transfected according to the LipofectAMINE 2000 protocol (Life Technologies, Inc., Gaithersburg, MD) into HT-29 cells. Twenty-four hours after transfection, cells were sensitized to TNF-–induced cell death by incubating with 25 µg/mL cyclohexamide for 1 hour. TNF- (25 ng/mL) was added to each well and the cells were incubated for an additional 6 hours. Data represent the result of experiments done in triplicate.

Western Blot Analysis. Twenty-four hours after transfection, cells were harvested, washed in ice-cold PBS, and lysed by sonication in radioimmunoprecipitation assay buffer. The mixture was centrifuged at 10,000 x g for 10 minutes at 4°C. Protein concentrations in the supernatant were measured using the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of protein were separated on a 10% SDS-PAGE gel and electrotransferred onto a nitrocellulose membrane. The membrane was probed with 2 µg/mL anti-cPLA₂ antibody overnight at 4°C. The membrane was washed, incubated with anti-mouse horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and visualized using the ECL Western blot analysis system (Santa Cruz Biotechnology).

Cytoplasmic Phospholipase A₂ Activity. cPLA₂ activity was measured upon incubation of cell lysates with the substrate, arachidonyl thiophosphatidylcholine, according to instructions provided with the cPLA₂ assay kit (Cayman, Ann Arbor, MI). Briefly, equal numbers of cells transfected with either control or antisense oligonucleotides were homogenized in 500 µL ice-cold PBS buffer containing 1 mmol/L EDTA and centrifuged at 10,000 x g for 15 minutes at 4°C. Supernatants were treated with 5 µmol/L bromoenol lactone for 15 minutes at 25°C to inhibit the activity of calcium-independent PLA₂ and filtered through a cellulose membrane filter with a molecular weight cutoff of 30,000 (Millipore, Bedford, MA) to remove any residual secretory PLA₂. The purified sample was then incubated with the substrate, arachidonoyl thiophosphatidylcholine. Hydrolysis of the substrate at the sn-2 position by the PLA₂ enzyme releases free thiol, which is then detected colorimetrically using Ellman's reagent on a microplate reader (405 nm).

Caspase-3 Activity. After a 6-hour incubation with TNF-, cells were harvested, washed in ice-cold PBS, and lysed by sonication in lysis buffer. The mixture was centrifuged at 10,000 x g for 10 minutes at 4°C. After determining the protein concentrations in the supernatant, cell lysates containing 80 µg of protein were incubated with the colorimetric caspase-3 substrate I (Calbiochem, San Diego, CA) for 30 minutes at 37°C. Absorbance at 405 nm was recorded using a microplate reader.

Statistical Analysis. A two-tailed unpaired Student's t test was done to determine statistical significance between levels of apoptosis.

	RESULTS

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Expression Analysis of cPLA₂ and COX-2 Human Colorectal Cancers. To examine the relative expression levels of cPLA₂ and COX-2 in human colorectal cancers, 27 archival human tumor specimens with matching normal control tissue were examined by immunohistochemical analysis. As summarized under Table 1, the tumors were grouped into four distinct phenotypes as follows: COX-2+/cPLA₂+, COX-2+/cPLA₂–, COX-2–/cPLA₂+, and COX-2–/cPLA₂–. Shown in Fig. 1 are representative serial tumor sections and normal colonic mucosa stained for COX-2 and cPLA₂. When both proteins are present in normal tissues, our data show perinuclear colocalization (Fig. 1A and B), an observation that is consistent with their presumed functional coupling (19, 20). Figure 1C highlights a crypt-specific staining pattern that suggests monoclonality, a feature of cPLA₂ staining that we consistently observe in adjacent normal colonic epithelium. This crypt-specific cPLA₂ staining, however, was accompanied by uniform COX-2 staining, as shown in a serial section (Fig. 1D). Those crypts that stained positive for cPLA₂ in adjacent normal tissue showed intense COX-2 staining as well (Fig. 1E and F). Finally, Fig. 1G and H shows representative serial sections of colon tumor that are cPLA₂ negative and COX-2 positive. Further analysis of the frequency of cPLA₂ staining show its absence in 84.6% (11/13) of the COX-2–overexpressing tumors. These observations suggest that COX-2 and cPLA₂ expression are not coordinately regulated within a significant percentage of human colorectal cancers.

View larger version (137K): [in this window] [in a new window] [Download PPT slide]   Fig. 1 Immunohistochemical analysis of cPLA2 and COX-2 expression and localization in human colon adenocarcinoma. A and B, punctate perinuclear staining of cPLA2 and COX-2, respectively, in normal colon crypts (magnification x400). C, shows a crypt specific staining of cPLA2; D, COX-2 staining often found in normal crypts adjacent to the tumor (magnification x200). E and F, colocalization of cPLA2 and COX-2 in normal colon crypts surrounded by a tumor, which does not express either protein (magnification x200). G, absence of cPLA2; H, perinuclear localization of COX-2 in a tumor (magnification x200).

Involvement of cPLA₂ in TNF--Induced Apoptosis in Human Colon Tumor Cells.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 2 TNF-–induced cell death was attenuated by blocking cPLA2 expression in HT-29 cells. A, HT-29 cells were transfected with increasing concentrations of cPLA2 antisense or control oligonucleotides. cPLA2 protein levels were determined 24 hours after transfection by Western blot analysis. Note the dose response reduction of cPLA2 protein levels. B, cPLA2 activity in each cell lysate was assessed using a colorimetric PLA2 substrate (arachidonoyl thiophosphatidylcholine) and expressed as absorbance at 405 nm. C, cells were sensitized to TNF-–induced apoptosis by incubating with 25 µg/mL cyclohexamide for 1 hour and then treated with TNF- (25 ng/mL) for 6 hours. Cells were collected and caspase-3 activity was determined using a colorimetric substrate. Mean of three independent experiments. Oligo Conc, oligonucleotide concentration.

View larger version (121K): [in this window] [in a new window] [Download PPT slide]   Fig. 3 Comparison of apoptotic index in COX-2–overexpressing tumors in the presence/absence of cPLA2. TUNEL staining was done on serial sections of tumor tissue. A and B, cPLA2-negative tumors (magnification x200). Note the minimum TUNEL staining within most of the tumor crypts. C, representative of a cPLA2-positive tumor (magnification x100). Note the abundance of apoptotic nuclei within the tumor crypts (arrows). D, higher magnification (x400) of C. E, graphical representation of five randomly selected tumor crypts counted and presented as a percentage of total cells. Note that cPLA2-positive tumors scored significantly more (P < 0.05) apoptotic cells than the tumors without cPLA2.

View larger version (119K): [in this window] [in a new window] [Download PPT slide]   Fig. 4 Identification of aberrant crypt foci and COX-2 immunohistochemistry. A total of 24 aberrant crypt foci were identified in 17 high-risk patients by close-focus, magnifying colonoscopy. A and B, photomicrographs of colonic mucosa examined using close-focus, magnifying colonoscopy (magnification x40). Methylene blue staining was done to distinguish aberrant crypt foci from normal colonic epithelium. C and D, H&E-stained sections of a hyperplastic (original magnification x100) and dysplastic (original magnification x200) aberrant crypt foci, respectively, removed during colonoscopy. E, COX-2 immunostaining in a serial section of the hyperplastic aberrant crypt foci showing minimal staining (original magnification x400). F, COX-2 immunostaining in a serial section of the dysplastic aberrant crypt foci (original magnification x200). Inset, dysplastic aberrant crypt foci at higher magnification (x400), with intense perinuclear COX-2 staining, consistent with the staining patterns observed in tumors and adjacent normal tissue. cPLA2 staining was not present in the 24 aberrant crypt foci examined (data not shown).

	DISCUSSION

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Apc⁷¹⁶

Previously, the levels of cPLA₂ were examined in human colon tumors, with somewhat ambiguous results. Using reverse transcription–PCR, Dimberg et al. (25) reported that cPLA₂ expression was sometimes increased, although there was no correlation with COX-2 expression. Soydan et al. (26) also reported elevated cPLA₂ protein levels in 6 of 17 human colorectal tumors, although the data were highly variable. Using a dot-blot method to estimate cPLA₂ expression, Osterstrom et al. (27) found a significant increase in cPLA₂ levels in human tumors. Wendum et al. (28) recently compared COX-2, secretory PLA2, and cPLA₂ protein expression in small bowel adenocarcinomas and colorectal adenocarcinomas. Consistent with our findings, they reported that only 35% of tumors with moderate to strong staining of COX-2 had strong cPLA₂ positivity. Using an immunohistochemical approach, our findings clearly show that cPLA₂ protein is markedly reduced in 11 (84.6%) of 13 human colorectal tumors expressing high levels of COX-2 (Table 1). The relative infrequency of the low-COX-2/high-cPLA₂ phenotype suggests that cells harboring these alterations are less likely to progress to tumors.

There is accumulating evidence that cellular arachidonic acid balance plays a key role in regulating apoptosis (1). For example, diminished production of arachidonic acid as a result of reduced cPLA₂-dependent generation or enhanced utilization through elevated COX-2 may deplete intracellular arachidonic acid pools, thereby attenuating apoptotic signals and facilitating tumorigenesis (1, 14). In an earlier study, we reported that in young adult mouse colon cells, pharmacologic inhibition of cPLA₂ with arachidonyltrifluorometylketone attenuated apoptosis in response to TNF- (15). In the present study, we show for the first time that cPLA₂ is an important mediator of TNF- induced apoptosis in human colon tumor cells as well.

We have also evaluated the possibility that the balance of cPLA₂ and COX-2 may be disrupted during early stages of colon tumorigenesis, perhaps providing a stimulus for tumor cell proliferation. The development of human colorectal cancer has been proposed to follow a stepwise progression, with the formation of dysplastic aberrant crypt foci constituting an important transitional event (29, 30). Genetic alterations occurring during this stage may allow the transformed tumor cells to escape apoptosis and undergo uncontrolled proliferation. Several key genetic alterations that are associated with colorectal cancer, including loss of Apc or K-ras activating mutations, have been identified in aberrant crypt foci and may represent important initiating events in the pathogenesis of colon cancer (31). Until recently, however, there have been only limited efforts to more fully characterize aberrant crypt foci at the molecular level, largely due to limitations in imaging technology that has the requisite sensitivity to distinguish aberrant crypt foci within the colonic epithelium. In the present study, we have used close-focus magnifying colonoscopy to identify aberrant crypt foci. We found that COX-2 expression was markedly up-regulated in a subset of aberrant crypt foci procured from the colons of high-risk individuals (12/24). This is the first time that COX-2 overexpression has been shown within these early precursor lesions and raises important questions about the actions of COX-2 as an early driving force in colorectal cancer. Considering the importance of COX-2 in colon cancer, these initial observations may provide some insight as to why aspirin is able to prevent colon cancer within the general population, including those individuals that are not necessarily at high risk. More importantly, we also found that within aberrant crypt foci expressing COX-2, there was no concomitant elevation in cPLA₂, a coordinated response that we established often occurs within the normal colonic epithelium.

PLA₂s and COX-2 are commonly activated in response to immune stimulation and their coordinate activation plays a key role in regulating cell survival or death. Evasion from immune surveillance may play an important role in the early stages of tumorigenesis. Obviously, transformed cells that have acquired genetic alterations that enable survival against immune-mediated attack will be able to undergo further clonal expansion. Our findings of COX-2 elevation without concomitant cPLA₂ induction potentially provides a pathway that may confer a survival advantage to early precancerous lesions in the colon, thereby facilitating aberrant crypt foci expansion and the ultimate development into adenocarcinomas. Thus, this model predicts enhanced cell survival in the presence of immune activation by attenuated apoptotic response (15). In summary, our results show that within the normal colonic epithelium, COX-2 and cPLA₂ expression are coordinately regulated; that is, COX-2 up-regulation is generally accompanied by cPLA₂ overexpression. However, in most colon tumors, this coordinate induction response is obscured. It is argued that up-regulation of COX-2 without a corresponding increase in cPLA₂ activity may lead to diminished arachidonic acid production and enhanced arachidonic acid utilization. This imbalance may lead to an environment in which arachidonic acid levels are depleted within tumor cells, potentially removing several key apoptotic pathways that ultimately favor tumor promotion.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 2/04; revised 11/30/04; accepted 12/15/04.

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