Leukotrienes induce cell-survival signaling in intestinal epithelial cells

GASTROENTEROLOGY 2000;119:1007-1018

Leukotrienes Induce Cell-Survival Signaling in Intestinal Epithelial Cells JOHN F. C)HD, KATARINA WIKSTROM, and ANITA SJOLANDER Division of Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Maim6, Maim6, Sweden

Background&Aims: Inflammatory bowel conditions, particularly ulcerative colitis, are associated with an increased incidence of neoplastic transformation. High levels of proinflammatory leukotrienes (LTs) and upregulated expression of cyclooxygenase (COX)-2 are characteristic of inflammation. Moreover, COX-2 has been implicated in cell survival and early colon carcinogenesis. Other aspects of interest for intestinal cell viability are the levels of I~-catenin and the antiapoptotic protein Bcl-2. We investigated the possibility that LTs participate in the regulation of these survival factors. Methods: We used the human intestinal epithelial cell line Int 407 and the rat intestinal epithelial cell line IEC-6. Immunoblotting was applied to ascertain protein expression and distribution, and enzyme immunoassay methodology was used to measure prostaglandin E2 (PGE2) production. Apoptotic ability was assessed by trypan blue exclusion, Hoechst staining, DNA fragmentation, and a caspase-3 activity assay. Results: LTD4 and LTB4, but not LTC4, caused a time- and dose-dependent increase in expression and/or membrane accumulation of COX-2, 13-catenin, and Bcl-2, as well as PGE2 production. Apoptosis assays showed that the effects of LTs on these transformation-associated proteins correlated well with the ability of these LTs to reduce programmed cell death. Conclusions:The results suggest that inflammatory conditions are associated with the expression and distribution of proteins that are characteristic of transformed cells; such conditions may involve a signaling mechanism comprising an altered rate of apoptosis. he leukotrienes (LTs) LTB4, LTC4, and LTD4 belong to an important group of proinflammatory mediators derived from arachidonic acid via the 5-1ipoxygenase pathway. ~,2 These eicosanoids affect both inflammatory and noninflammatory ceils via specific cell surface receptors, 2,3 and the LTB4 and the LTD4 receptors have been cloned and identified as 7-transmembrane receptors that are coupled to heterotrimeric G proteins. 4 7 These arachidonic acid metabolites have been recognized as important pathogenic elements in inflammatory bowel diseases. 8,9 In addition, significantly increased levels of cyclooxygenase (COX)-2 (also known as prosta-

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glandin H synthase 2) have been found in tissues from patients with such conditions3 ° The risk of cancer is a major concern in chronic intestinal inflammation; for example, it has been reported that 10 years or more of pancolitis is associated with a 30-fold increased risk of colon carcinoma. 11 Furthermore, several epidemiologic studies have shown that colon cancer is underrepresented in populations treated with nonsteroidal anti-inflammatory drugs (NSAIDs). 12 A possible link between inflammation and the occurrence of cancer was suggested by Sheng et al., 13 who demonstrated that COX-2 is upregulated in colon cancer tissues and in various tumor cell lines, indicating that this protein plays a key role in colon tumorigenesis. Heightened expression of COX-2 has also been shown to increase both cell attachment to extracellular matrix proteins and cell viability--in short, COX-2 impedes apoptosis.~4,a5 After an extensive review of past and current research, the American Cancer Society declared that particular attention should be focused on the study of NSAIDs for the treatment of gastrointestinal cancer.

16

Prescott and White ~v suggested that in colon carcinogenesis, mutations in the adenomatous polyposis coli (APC) gene arise before increased expression of COX-2. The APC gene codes for a protein complexed with 3 other proteins, namely, the serine/threonine kinase GSK3[3, the recently identified linker/promoter protein axin, and [3-catenin, which is also known to associate with E-cadherin/8-22 One of the most important functions of this protein complex seems to be regulation of the intracellular levels and distribution of [3-catenin; if that were not the case, the cellular neoplastic potential would be much higher. 23 Recently, Sheng et al. ~5 observed that COX-2 and Bcl-2 are up-regulated simultaneously in human colon Abbreviations used in this paper: APC, adenomatous polyposis coli; Bcl-2, B-cell lymphoma 2; COX, cyclooxygenase; EIA, enzyme immunoassay; LOX, lipoxygenase; LT, leukotriene; PGE2, prostaglandin E2. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.18141

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cancer ceils. Bcl-2 is a m e m b e r of a large family of proteins that are involved in regulating apoptosis; that family includes B A D and B A X , which are presumed to form pores in the outer mitochondrial membrane, t h r o u g h which cytochrome c is released to the cytoplasm. 24 Bcl-2 probably effects negative regulation of apoptosis by i m p e d i n g B A D / B A X - i n d u c e d pore formation. 24,25 Little is k n o w n about the roles of lipoxygenases (LOXs) in apoptosis and carcinogenesis, although it has been suggested that 1 2 - L O X and 1 5 - L O X may be important factors both in cell lines and colon cancer tissue.26, 27 In the present study, we addressed the question of whether proinflammatory mediators such as LTs ( 5 - L O X products) can alter the expression and distribution of C O X - 2 , [3-catenin, and Bcl-2 and modify the apoptotic ability in nontransformed intestinal epithelial ceils, as is seen in neoplasia.

M a t e r i a l s and M e t h o d s Materials Rabbit anti-human COX-2 and COX-1 antibodies were a gift from Dr. A. W. Ford-Hutchinson (Merck Frosst Canada, Pointe-Claire-Dorual, Quebec). Rabbit anti-human COX-2 antibody was also obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), as were rabbit anti-human actin and transferrin receptor antibodies. Mouse anti-human [3-catenin antibody was obtained from Transduction Laboratories (Lexington, KY), and mouse anti-human Bcl-2 antibody from Santa Cruz, Biotechnology. ZM198,615 (ICI-198,615) was a gift from Dr. R. Metcalf (Zeneca Pharmaceuticals, Macclesfield, Cheshire, England), and the COX-2-specific inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide (NS398) 28 was purchased from Biomol (Plymouth Meeting, PA). Hoechst 33342 fluorescent chromatin stain was obtained from Molecular Probes (Eugene, OR), caspase-3 fluorometric substratepeptideAc-DEVD-AMC(Ac-Asp-Glu-Val-Asp-7-amino4-methyl coumarin) from Upstate Biotechnology (Lake Placid, NY), and Ac-DEVD-CHO caspase-3-inhibiting peptide from Pharmingen (San Diego, CA). PGE2 assay reagents and LTs were obtained from Cayman Chemical Co. (Ann Arbor, MI). Sep-Pak cartridges were provided by Waters (Milford, MA). All other chemicals were of analytical grade and were obtained from Chemicon International (Temecula, CA) or Sigma Chemical Co. (St. Louis, MO).

Cell Culture We used 2 mammalian intestinal epithelial cell lines: Int 407 and IEC-6. Int 407 cells were derived from an approximately 2-month-old human embryo and exhibit typical nontransformed epithelial morphology and growth. 29 These cells were cultured as a monolayer (to approximately 80% confluence) for 5 days in Eagle's basal medium, as previously

GASTROENTEROLOGYVol. 119, No. 4

described, v,3° IEC-6 cells were obtained from the American Type Culture Collection and have typical intestinal epitheloid morphology, as described by Quaroni et al) 1 These cells were cultured in a medium containing 45% RPMI, 45% Dulbecco's modified Eagle medium, and 10% inactivated fetal bovine serum, supplemented with 0.1 IU/mL insulin, 55 gtg/mL streptomycin, and 55 IU/mL penicillin.

Treatments, Cell Fractionation, Gel Electrophoresis, and Immunoblotting The ceils were washed and allowed to rest for 30 minutes in serum-free medium and then exposed to 40 nmol/L LTD4, LTB4, or LTC4. In some experiments using LTD4, the cells were preincubated with the LTD4 antagonist ZM198,615 (40 ~mol/L) or NS-398 (100 btmol/L) for 30 minutes. Cells were scraped into ice-cold lysis buffer or buffer A and homogenized, and membrane/cytosolic fractions were prepared by ultracentrifugation, v,3° All samples were assayed and compensated for protein content by using the Coomassie blue protein assay (Pierce, Rockford, IL) to ensure equal protein loading. The cellular fractions or whole-cell lysates were solubilized by boiling at 100°C for 5 minutes in a sample buffer, v Samples were then electrophoresed on 8 % - 1 2 % homogeneous polyacrylamide gels in the presence of sodium dodecyl sulfate. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane, which was then blocked overnight at 4°C with dry milk (5%, wt/vol) for the COX-1 and COX-2 antibodies or with 3% bovine serum albumin for the other antibodies. The membrane was subsequently incubated for 2 hours at 20°C with specific antibodies against COX-l, COX-2 from Merck Frosst (1:5,000), COX-2 from Santa Cruz (1:1000), and Bcl-2 and [3-catenin (both 1:500). Thereafter, the membrane was washed extensively, incubated for 1 hour at room temperature with peroxidaselinked antibodies (dilution 1:10,000; Dako A/S, Glostrup, Denmark), and washed and incubated with SuperSignal (Pierce). Visualization and densitometric analysis were performed with a Fluor-S quantitative imaging system (Bio-Rad Laboratories, Hercules, CA).

Prostaglandin E2 Assay Prostaglandin production was measured with an enzyme immunoassay (EIA), performed according to the manufacturer (Cayman Chemical Co). In short, the cells were cultured in 25-cm 2 flasks, in the presence or absence of 100 btmol/L NS-398 (30 minutes' preincubation), and stimulated with the indicated concentrations of LTD4, LTB4, or LTC4 for 30 minutes, 1 hour, or 5 hours using nonstimulated ceils as control. The cell media were collected, and prostaglandins were separated by solid-phase extraction using Sep-Pak Vac RC (C18-500 mg) cartridges. The samples were transferred to a 96-well plate for the EIA and absorbance measurements at 405 nm with a BMG plate reader (Offenburg, Germany). Triplicate samples were prepared for all time points in each experiment.

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Assessment of Apoptosis in Adherent Cells: Trypan Blue Exclusion, Hoechst Staining, and Phase-Contrast Microscopy The cells were cultured for 3 days on glass coverslips in 35 X 1 0 - m m Petri dishes and subsequently exposed for 5 days to 100 btmol/L NS-398, 40 nmol/L LTD4, or both, using unchallenged cells as controls. During this time, the culture media were changed once and supplemented with fresh portions of the abovementioned additives. To determine viability, the cells were stained with 0.2% trypan blue in phosphate-buffered saline (PBS), and the percentage of viable cells was calculated. To assess apoptosis, the cells were washed once in PBS and thereafter incubated for 15 minutes in 10 Dg/mL Hoechst 33342 stain (PBS). The coverslips were then washed and mounted on glass slides, and the cells were examined in a Nikon fluorescence microscope at 350 and 460 nm (excitation and emission wavelengths) and 60 × magnification. Images were obtained and processed using a Hamamatsu (Photonics kk Hamamatsu City, Japan) digital camera and Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). A Leica phase-contrast microscope (Wetzlar, Germany) was used to examine confluent ceils that had been exposed to 100 btmol/L NS-398, 40 nmol/L LTD4, or both, for 24 and 48 hours; unstimulated cells served as controls. Photomicrographs were taken with an Olympus SC35 type 12 camera (Japan) at 4 0 x magnification.

Assessment of Apoptosis: DNA Fragmentation Confluent cells were treated with NS-398 for 24 hours and then lysed in a buffer containing 5 mmol/L Tris-HCl, 20 mmol/L EDTA, and 0.1% (vol/vol) Triton X-100 (pH 8.0). The cell lysates were exposed to RNase (5.0 mg/mL) for 60 minutes at 37°C to eliminate RNA and then to proteinase K (4 mg/mL) for 90 minutes at 50°C to cleave proteins. The samples were mixed with DNA loading buffer (89 mmol/L Tris, 89 mmol/L boric acid, 2 mmol/L EDTA, 10% sucrose, and 0.06% bromphenol blue), electrophoresed on 1.8% agarose gel in Tris-borate-EDTA at 60 V for 3 hours, and then stained with ethidium bromide (0.5 Dg/mL).

Assessment of Apoptosis: Caspase-3 Activity Assay The cells were cultured in 12-well plates and then exposed to NS-398 for 2, 4, or 6 hours in the presence or absence of 40 nmol/L LTD4. Thereafter, the cells were lysed on ice for 20 minutes in a sterile-filtered lysis buffer containing 1% (vol/vol) Triton X-100, 20 mmol/L Tris-HC1 (pH 7.5), and 150 mmol/L NaC1. Samples were suspended in a reaction buffer containing 20 mmol/L HEPES, 2 mmol/L dithiothreitol, and 10% glycerol on Nunc Polysorp 96-well, white fluorescence measurement plates (Nalge Nunc Int., Naperville, IL); Ac-DEVD-AMC was subsequently added with or without Ac-DEVD-CHO (caspase-3-inhibiting peptide) for control purposes. The plate was incubated at 37°C for 1 hour, and the

LTD4SIGNALINGAND CELLSURVIVAL 1009

fluorescence was measured at 390 and 460 nm (excitation and emission wavelengths) using a BMG plate reader (Offenburg, Germany). Triplicate samples were prepared for all time points.

Statistical Analysis Results are expressed as means + SEM. Differences between experimental groups were assessed by Student t test. P values of <0.05 were considered significant.

Results Expression and Distribution of COX-1 and COX-2 in LTD4-Treated Cells I n t 407 cells were s t i m u l a t e d w i t h LTD4 and then analyzed for C O X - 2 and C O X - 1 by W e s t e r n b l o t t i n g (Figure 1A and B, respectively). T h e results show t h a t LTD4 i n d u c e d a t i m e - d e p e n d e n t increase in C O X - 2 in the m e m b r a n e fraction (Figure 1A), and d e n s i t o m e t r i c analysis of these blots s h o w e d an initial peak in C O X - 2 between 0 and 30 m i n u t e s and a second, m o r e substantial increase between 120 and 300 m i n u t e s (Figure 1C). T h e c o n s t i t u t i v e l y expressed e n z y m e C O X - 1 was also present in the m e m b r a n e fraction, b u t neither expression nor d i s t r i b u t i o n of this p r o t e i n was altered by exposure to LTD4 (Figure 1B). T h e increase in C O X - 2 in the m e m b r a n e fraction b e g a n at a relatively low c o n c e n t r a t i o n of LTD4 (Figure 1D), in contrast to the effects of this LT on ~ - c a t e n i n and Bcl-2 (Figures 2C and 3C). T h e observed effects of LTD4 were abolished by the specific LTD4 receptor a n t a g o n i s t Z M 1 9 8 , 6 1 5 (Figure 1C and D). Finally, the L T D 4 - i n d u c e d increase in C O X - 2 was also observed in whole lysates o f l n t 407 ceils (data not shown).

Effects of LTD4 on Expression and Distribution of I~-Catenin In light of a report indicating close connections between C O X - 2 and free ~-catenin, 17 we performed experiments to determine whether the inflammatory mediator LTD4 could alter the subcellular distribution of ~-catenin in intestinal epithelial cells. W e found that the LT caused a transient increase in [3-catenin in the m e m brane fraction of Int 407 cells (Figure 2A), which, according to densitometric analysis, reached a m a x i m u m (2-fold accumulation) after approximately 60 minutes' stimulation (Figure 2B). A similar effect was observed in the cytosolic fraction (Figure 2A). Exposure to different concentrations of LTD4 for 60 minutes resulted in a concentration-dependent effect in Int 407 cells (Figure 2C). The described influence of LTD4 was eliminated by

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the specific LTD4 receptor antagonist ZM198,615 (Figure 2B and C). The LTD4-induced increase in [3-catenin was also observed in whole lysates of Int 407 ceils (data not shown).

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Int 407 intestinal epithelial cells were stimulated with LTD4 and then analyzed for Bcl-2 by Western blotting (Figure 3). Densitometric analysis of the blots obtained from human Int 407 cells (Figure 3A) revealed an approximately 6-fold increase in Bcl-2 in the membrane fraction after exposure to LTD4 for 300 minutes (Figure 3B). The effect of LTD4 was concentration dependent (Figure 3C) in much the same way as seen with ~-catenin and was completely blocked by ZM198,615 (Figure 3B and C). Whole-cell lysates were similar to the membrane fraction in regard to LTD4-induced accumulation of Bcl-2 (data not shown).

Effects of LTD4 on Expression and Distribution of COX-2, ~-Catenin, and Bcl-2 in IEC-6 Cells To further establish the observed effects of LTD4 on COX-2, [3-catenin, and Bcl-2 protein levels in Int 407 cells, we investigated its effects on the expression and distribution of these proteins in an additional intestinal epithelial cell line, IEC-6. LTD4 induced increased accumulation of the 3 proteins in the membrane fraction from IEC-6 cells (Figure 4). The effects were similar to those observed in Int 407 ceils (Figures 1-3).

Effects of LTB4 and LTC4 on Expression and Distribution of COX-2, ~-Catenin, and Bcl-2 For comparison, we also examined the effects of LTB 4 and LTC4 on expression and distribution of COX-2, [3-catenin, and Bcl-2 in Int 407 ceils. Similar to LTD4, LTB4 increased the levels of the 3 proteins (Figure 5A-C), causing an even more pronounced increase in [3-catenin (approximately 4-fold after 120 minutes of stimulation; Figure 5B). On the other hand, LTC4 had no effect on the amounts of these proteins in the membrane fraction (Figure 5A-C).

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LTD 4 (nM) Figure 1. LTD4-induced accumulation of COX-2 in membrane fractions of Int 407 cells. (A and B) Representative Western blots of (A) COX-2 and (B) COX-1 in membrane fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. (C) Densitometric analysis of COX-2 levels in cells exposed to LTD4 for different periods in the absence (0) or presence (©) of the LTD4-receptor antagonist ZM198,615. (D) Concentrationdependent effects of LTD4 on COX-2 levels in cells exposed to different concentrations of LTD4 for 60 minutes in the absence (0) or presence (©) of ZM198,615. The densitometric values represent means + SEM of 4 separate experiments. *P < 0.05; * * P < 0.01.

Effects of LTD4 and NS-398 on Membrane Accumulation of COX-2, ~-Catenin, and Bcl-2 and Production of PGE2 in Intestinal Cells To get an indication whether prostaglandin production could influence membrane levels of COX-2, ~-catenin, and Bcl-2, we added the COX-2-specific inhibitor NS-398 to Int 407 ceils 30 minutes before stimulation with LTD4. NS-398 did not alter the membrane accumulation of COX-2, whereas [3-catenin was negatively affected by this inhibitor, indicating an essential role of prostaglandin formation in the regulation of [3-catenin expression/distribution in these cells (Figure

LTD4 SIGNALING AND CELL SURVIVAL 10:/.1

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LTD 4 (nM) Rgure 2. LTD4-induced accumulation of ~-catenin in membrane and cytosolic fractions of Int 407 cells. (A) Representative Western blot of !~-catenin in membrane (top panels) and cytosolic (lower panels) fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. As loading controls, we used the presence of transferrin receptors in the membrane fractions and the presence of actin in the cytosolic fractions. (B) Densitometric analysis of 13-catenin levels in membrane fractions from cells exposed to LTD4 for different periods in the absence (0) or presence (©) of the LTD4-receptor antagonist ZM198,615. (C) Concentration-dependent effects of LTD4 on 13-catenin levels in membrane fractions from cells exposed to different concentrations of LTD4 for 60 minutes in the absence (0) or presence (0) of ZM198,615. The densitometric values represent means + SEM of 4 separate experiments. *P < 0.05; * * P < 0.01.

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LTD 4 (nM) Figure 3. LTD4-induced accumulation of Bcl-2 in membrane fractions of Int 407 cells. (A) Representative Western blot of Bcl-2 in membrane fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. As loading control we used the presence of transferrin receptors in the membrane fractions. (B and C) Densitometric analysis of Bcl-2 protein levels in cells treated in the same way as described in Figures I and 2. The densitometric values represent means _+ SEM of 4 separate experiments. *P < 0.05; * * P < 0.01.

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matin (Figure 8A, middle), compared with untreated cells (Figure 8A, left). This pattern, which is indicative of apoptosis, was absent in cells exposed to both 40 nmol/L LTD4 and NS-398 (Figure 8A, right); that observation was confirmed statistically, using data based on nuclear changes that occurred in adherent cells after 5 days of NS-398 treatment (Figure 8B). These changes were significant, despite the massive detachment of cells 24 hours after the addition of NS-398 (shown in Figure 7). Therefore, such changes were better characterized by total activity studies, as shown in Figures 7 and 9.

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Phase-Contrast Microscopy of Cells Treated With NS-398 and LTD4 Int 407 cells exposed to 100 b~mol/L NS-398 displayed considerable shrinkage, rounding up, and detachment leading to decreased cell density (Figure 7A, middle) compared with control cells (Figure 7A, left). The indicated changes were not observed after cotreatment with NS-398 and 40 nmol/L LTD4 (Figure 7A, right), or LTB4 (data not shown). Similar results were observed in IEC-6 ceils, although the features were less pronounced, possibly because these ceils had a much denser growth pattern (Figure 7B). LTs alone caused no visible morphologic alterations in these cells (data not shown).

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both Int 407 and IEC-6 ceils (Figure 6B and C). LTB4 had a similar but more pronounced effect, whereas LTC4 did not affect the level of PGE2 (data not shown). The level of PGE2 was still elevated 5 hours after stimulation with LTD4 (data not shown). The COX-2 inhibitor NS-398 decreased the basal PGE2 level in both cell lines, which is in good agreement with the findings that both cell lines exhibit COX-2 immunoreactivity at baseline (Figures 1A and 4). Subsequent stimulation with LTD4 only partially reversed the NS-398-induced reduction of the basal PGE2 level in Int 407 cells; such an effect was not detected in IEC-6 ceils (Figures 1A and 4).

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LTD4 SIGNALING AND CELL SURVIVAL 1013

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In the trypan blue exclusion assay, lower viability was seen in ceils treated with NS-398 than in control ceils (Figure 8C). This effect was reversed by the addition of LTD4, as well as LTB4, whereas LTs alone produced a minor increase in cell viability.

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Caspases are a large group of proteolytic enzymes that play a role early in the apoptotic cascade. 32 One of the most thoroughly characterized members of this protein family is caspase-3, and it has been reported that Bcl-2 is an upstream inhibitor of this caspase. 33 Consequently, we investigated the effects of NS-398 on caspase-3 activity in the presence and absence of LTD4 (Figure 9A.) NS-398 (100 ~mol/L) caused an increase in caspase-3 activity (compared with spontaneous activity) that was evident after 2 hours and reached a maximum after 4 - 6 hours. This increase was significantly inhibited by 40 nmol/L LTD4; LTD4 alone induced a minor decrease in spontaneous caspase-3 activity after 6 hours, which is analogous to the effect seen in the cell viability assay. Caspase-3 activity was reduced to a similar extent by 40 nmol/L LTB4 (data not shown). To determine the effect of NS-398 and LTD4 on endonucleosomal DNA fragmentation, which is an indicator of programmed cell death, we incubated intestinal epithelial cells with 100 ~mol/L NS-398 in the absence or presence of 40 nmol/L LTD4 for 24 hours and then separated the extracted DNA on an agarose gel. NS-398 caused increased DNA lad&ring in the absence of LTD4 (Figure 9B, lane 3), whereas DNA fragmentation was clearly reduced in the presence of the LT (Figure 9B, lane 4). These results indicate that LTD4 decreases N S - 3 9 8 induced apoptosis.

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We found that the inflammatory mediators LTD4 and LTB4, but not LTC4, up-regulated COX-2 in intestinal epithelial cells. Moreover, LTB4 and LTD4 had almost identical effects on protein expression and distribution, suggesting that other inflammatory mediators probably up-regulate proteins associated with carcinogenesis in a similar way. On the other hand, we found that only 1 of 2 closely related cysteinyl LTs (LTD4 but not LTC4) caused this effect, which shows that inflammatory mediators also display a certain degree of specificity. Using ZM198,615 (a specific LTD4 receptor antagonist), we could clearly demonstrate that the effect of LTD4 on COX-2 levels is mediated by a specific ligandreceptor interaction. Further support for this assumption is provided by the observation that LTD4 had no effect on COX-1 levels. These results are noteworthy, not only as controls, but also because these 2 enzymes have been found to be partly counterregulated, both constitutively and inducibly, in mouse knockout models) 4 The role of COX-2 in cell transformation is not yet clear, although

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Control

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Figure 7. (A) Int 407 and (B) IEC-6 cell morphology visualized by phase-contrast microscopy. The micrographs show control cells and cells treated with 100 Fmol/L NS-398 or 100 i~mol/L NS-398 and 40 nmol/L LTD4. Each micrograph is representative of 4 separate experiments (original magnification 40×).

it has been shown that inhibition of this enzyme by both selective and nonselective inhibitors dramatically impairs the cellular neoplastic potential. 12'35 In addition, Oshima et al. 35 studied mice bearing an APC mutation causing altered ~-catenin homeostasis and observed that inhibition of COX-2 significantly decreased colon tumorigenesis. We found that LTs had approximately the same effect on ~-catenin levels in intestinal epithelial ceils that they had on the amounts of COX-2. LTD4 and LTB4, but not LTC4, induced a dose- and time-dependent increase in ~-catenin in the membrane as well as in the cytosolic fractions. How is [3-catenin involved in cell transformation, and what is known about the regulation of this protein? In most cases of colon cancer, familial or spontaneous, the APC gene is somehow altered, rendering the protein product nonfunctional or dysfunctional38 Studies concerned with the effect of the APC gene on colon cancer and melanoma cell lines have shown increased levels of free, stable [3-catenin and abnormal distribution of that protein within the ceils. 36,19 Rubinfeld et al. 2° concluded that the APC protein may be involved in maintaining intracellular homeostasis of [3-catenin through continuous phosphorylation by GSK-3[3. Lack of such phosphorylation leads to an increase in free [3-catenin and activation of the transcription factor family tcf/lef. 23 These momentum events also occur during embryogenesis and are commonly referred to as the wnt signaling pathway because they are induced by the polypeptide wnt, which acts through G protein-coupled receptors. 22 In this context, our finding that LTD4 and LTB4, but not LTC4, altered [3-catenin homeostasis implies that these 2 LTs trigger a signal cascade that is at least partly analogous to the wnt signaling pathway.

PGE2, a product of COX-2, has been reported to change the distribution of Bcl-2 and the apoptotic activity in human colon cancer ceils, 15 which strongly indicates a link between COX-2, Bcl-2, and increased cell survival. Therefore, we studied the effects of LTs on the expression and distribution of the cell-survival protein Bcl-2 and on programmed cell death induced by inhibition of COX-2. We found that LTD4 and LTB4 caused an up to 4-fold increase in Bcl-2 in the membrane fraction and in whole-cell lysates; as seen with COX-2 and ~-catenin, LTC4 had no significant effect on Bcl-2 levels. A connection between activation of caspase-3 and Bcl-2 has previously been established by findings showing that Bcl-2 inhibits both the release of cytochrome c, which in turn activates caspase-3, and the conversion of membrane-bound procaspase-3 to the active f o r m ) 7,24 We noted that exposing epithelial cells to the C O X - 2 specific inhibitor NS-398 caused a rapid and sustained activation of caspase-3 that was partially reversed by LTD4. Interestingly, we also observed that LTD4 alone caused a small decrease in spontaneous caspase activity, suggesting that this LT exerts an effect on elements that participate in the regulation of spontaneous apoptosis. Although this finding itself is intriguing and may indicate more specific roles for LTs in the control of caspase activity, in the present study the caspase-3 activity assay was used solely as an early biochemical indicator of the apoptosis cascade. Exposure to the COX-2 inhibitor NS-398 for 24 hours induced further cellular changes indicative of progressing apoptosis, for example, distinct rounding-up and massive detachment of cells, D N A laddering, and typical nuclear condensation and frag-

October 2000

LTD4 SIGNALING AND CELL SURVIVAL 1015

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Figure 8. (A) Fluorescence micrographs of Int 407 cells stained with Hoechst 33342 (original magnification 6 0 x ) . Control cells, cells treated with 100 l~mol/L NS-398, and cells exposed to 100 t*mol/L NS-398 and 40 nmol/L LTD4 are shown. (B) Percentage apoptotic nuclei (calculated as Hoechst-positive cells per total cells) in untreated cells (control) and cells exposed to NS-398 alone or with 40 nmol/L LTD4. (C) Results of the trypan blue viability assay in control cells and cells treated with LTD4, NS-398, or NS-398 and 40 nmol/L LTD4. The data are representative of 4 separate experiments and are means _+ SEM. *P < 0.05; * * P < 0.01.

mentation. All of these changes were significantly suppressed by cotreatment with LTD4, suggesting that this LT has an antiapoptotic effect. The described effects of NS-398 are most likely related to the observed basal levels of COX-2 protein and prostaglandin formation in both Int 407 and IEC-6 ceils. Other investigators have observed low basal COX-2 levels in epithelial cells from tissue sections of normal human colon and small bowel, as well as in cultured nontransformed rat intestinal epithelial cells. 38-4° In contrast, other studies of normal human colon and small bowel showed no basal level of COX-2 in these cells41; there is no obvious explanation for these conflicting results. Nevertheless, recent work by Inaba et al. 42 show that unaltered basal PGE2 levels are sufficient to promote carcinogen-induced colon carcinogenesis in the rat. To block this induction of cancer, PGE2 levels had to be brought down well below the endogenous. The well-known beneficiary effects of NSAID treat-

ment on colon cancer also suggest that baseline variations in prostaglandin formation may be of importance for cell viability in the bowel. 43 However, it should be pointed out that no studies have yet shown that COX-2 inhibition induces apoptosis in normal human colon and small bowel. Inasmuch as LTB4 and LTD4 are c o m m o n and important mediators of inflammation, our data shed new light on the cross-talk between inflammatory and survival/death signaling. In addition, considering the temporal pattern of COX-2 and Bcl-2 induction, our results imply that there is an initial mechanism that up-regulates COX-2 and that a subsequent increase in arachidonic acid metabolites can further potentiate changes in cancer-related proteins associated with intestinal cell survival. This is further substantiated by our findings that LTD4 induces increased prostaglandin production and that inhibition of this formation by NS-398 is paralleled by an increased apoptosis rate.

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GASTROENTEROLOGYVol. 119, No. 4

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Figure 9. (A) Caspase-3 activity illustrated as percentage of baseline activity in lysates of Int 407 cells exposed to LTD4 and NS-398 for the indicated periods. The fluorescence intensity data represent means _+ SEM of 4 separate experiments; *P < 0.05; * * P < 0.01. (B) DNA fragmentation over 24 hours in Int 407 cells. Results are shown for untreated cells (lane 1) and for cells treated with 40 nmol/L LTD4 (lane 2), 100 ~mol/L NS-398 (lane 3), and both NS-398 and LTD4 (lane 4). The pattern is representative of 4 separate experiments.

However, the LTD4-induced Bcl-2 response was only partly blocked by NS-398, suggesting that an LTD4initiated PGE2-independent signaling pathway mediates the induction of Bcl-2. Thereby, our data shed new light on the earlier findings that PGE2 enhance Bcl-2 expression in intestinal epithelial cells, 15 whereas McEntee et al. 44 found that Bcl-2 levels remained unchanged in sulindac-treated Min mice. There is considerable uncertainty in the results of previous studies as to whether apoptosis induced by inhibition of COX-2 is caused by suppressed prostagiandin formation or by prevention of some other effect of this enzyme. However, it was recently suggested that reduced tumorigenesis caused by inhibition of COX-2 may be the result of accumulation of arachidonic acid, which yields neutral sphingomyelinase, an enzyme that catalyzes the conversion of sphingomyelin to ceramide, a known inducer of apoptosis. 45 NS-398, the specific COX-2 inhibitor used in our study, has been found to reduce rat colon carcinogenesis and stimulate apoptosis in prostate cancer cells, while Bcl-2 is simultaneously down-regulated. 46,4v Moreover, blocking of COX-2 with another selective COX-2 antagonist, SC58125, impeded cell growth and proliferation and increased apoptosis. 13 Polyps from Min mice, which carry a nonsense mutation in the APC gene, have been found to contain significantly elevated levels of PGE2 and LTB4.48 In this report, we show that NS-398 treatment of intestinal epithelial cells counteracted the LTD4-induced increase in [3-catenin after 1 hour (Figure 6A). Treatment of Min

mice with sulindac decreased ~-catenin levels and resulted in temporary tumor regression. However, focally heightened expression of Bcl-2 and unchanged LT levels conferred resistance to sulindac treatment within weeks.48, 44 Until now, it has been assumed that the increased cell turnover observed in inflamed intestinal mucosa can cause neoplasia. 49 LT levels are significantly elevated in both intestinal inflammatory lesions and tumors; hence it is noteworthy that these proinflammatory mediators, among them LTD4 and LTB4, which act via G p r o t e i n - c o u p l e d receptors, can induce changes that are characteristic of transformed cells. More specifically, such changes include increased expression of COX-2, [3-catenin, and Bcl-2 and/or translocation of these proteins to the membrane fraction. These alterations are associated with decreased apoptotic ability and appear to be partially mediated by prostaglandins, as in the case of [3-catenin. The LTD 4induced formation of prostaglandins does not account for all of the observed effects; the Bcl-2 protein level was still increased in the absence of prostaglandin production. This effect on Bcl-2 is a likely explanation for the rescuing capacity of LTD4 in NS-398 treated cells. Further understanding of LT signaling is needed to map the location of the events we observed in our experiments. In conclusion, our data indicate the existence of a novel process governed by mediators that are common in both inflammation and neoplasia, and they elucidate the interaction of these 2 states in conditions such as ulcerative colitis. References

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Received November 8, 1999. Accepted May 26, 2000. Address requests for reprints to: Anita Sj61ander, Ph.D., Division of Experimental Pathology, Department of Laboratory Medicine, Lund University, U-MAS Entrance 78, S-205 02 Maim6, Sweden. e-mail: [email protected]; fax: (46) 40-337353. Supported by grants from the Swedish Medical Research Council (project 10356; to A.S.), Inga and John Hains' Foundation, the Ake Wiberg Foundation, the Crafoord Foundation, and Foundations at Maim6 University Hospital; and by grants from the Royal Physiographic Society in Lund (to J.F.O.). The authors thank Maria Juhas for excellent technical assistance and Patty Ohdman for linguistic revision of the manuscript.