Activation of cpla 2 is required for leukotriene D 4 -induced proliferation in colon cancer cells

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1 Carcinogenesis vol.26 no.11 pp , 2005 doi: /carcin/bgi159 Advance Access publication June 23, 2005 Activation of cpla 2 is required for leukotriene D 4 -induced proliferation in colon cancer cells Ladan Parhamifar, Bengt Jeppsson 1 and Anita Sjölander Experimental Pathology, The Department of Laboratory Medicine and 1 Surgery, The Department of Clinical Sciences, Lund University, Malmö University Hospital, SE Malmö, Sweden To whom correspondence should be addressed. Tel: þ ; Fax: þ ; Anita.Sjolander@med.lu.se It is well documented that prolonged inflammatory conditions, particularly those relating to the colon, have been shown to induce cancer. We have previously demonstrated that the pro-inflammatory mediator leukotriene D 4 (LTD 4 ) induces survival and proliferation in intestinal cells and that its receptor, CysLT 1, is upregulated in human colon cancer tissue. Here we demonstrate, for the first time that in both Int 407 (a non-transformed human intestinal epithelial cell line) and Caco-2 cells (a human colorectal carcinoma cell line), cytosolic phospholipase A 2 a (cpla 2 a)is activated and translocates to the nucleus upon LTD 4 stimulation via a calcium-dependent mechanism that involves activation of protein kinase C (PKC), and the mitogenactivated protein kinases ERK1/2 and p38. We also show with a cpla 2 a promoter luciferase assay, that LTD 4 induces an increase in the transcriptional activity of cpla 2 a via activation of cpla 2 a and the transcription factor NFkB. Interestingly we demonstrate here that both the basal and the LTD 4 -induced cpla 2 a activity is elevated 3-fold in Caco-2 colon cancer cells compared with Int 407 cells. The difference in basal activity was confirmed in human colon tumor samples by the finding of a similar increase in cpla 2 a activity when compared with normal colon tissue. A functional role of the increased cpla 2 a activity in tumor cells was revealed by our findings that inhibition of this enzyme reduced both basal and LTD 4 - induced proliferation, the effects being most pronounced in Caco-2 tumor cells. The present data reveal that cpla 2 a, an important intracellular signal activated by inflammatory mediators, is an important regulator of colon tumor growth. Introduction Cysteinyl leukotrienes (LTC 4, LTD 4, LTE 4 ) are derived from arachidonic acid (AA) through the activation of 5-lipoxygenase (5-LO), and mediate their signals through either of two seven-transmembrane G-protein coupled receptors, CysLT 1 and Abbreviations: 5-LO, 5-lipoxygenase; AA, arachidonic acid; Caco-2 cells, a human colorectal carcinoma cell line; cpla 2 a, cytosolic phospholipase A 2 a; COX, cyclooxygenase; IBD, inflammatory bowel diseases; Int 407, a non-transformed human intestinal epithelial cell line; LTD 4, leukotriene D 4 ; MAPK, mitogen-activated protein kinases; NSAID, non-steroidal antiinflammatory drug; NFkB, nuclear factor kappa B; PKC, protein kinase C; PTX, pertussis toxin. CysLT 2 (1,2). Leukotrienes are important pro-inflammatory mediators, and their involvement is implicated in many inflammatory conditions (3,4). In previous experiments using non-transformed intestinal epithelial cells we have shown that LTD 4, through the CysLT 1 receptor, upregulates several proteins related to carcinogenesis, such as cyclooxygenase (COX-2), 5-LO, b-catenin and the survival protein Bcl-2 (5). In addition, we have also shown that LTD 4 mediates survival and proliferation in these cells through the CysLT 1 receptor, and that the same receptor is upregulated in colon cancer tissue (6,7). Most recently we have also demonstrated an increased accumulation of the CysLT 1 receptor in the nuclei in colon cancer tissue and in the colon cancer cell line Caco-2 (8). Our data suggest that LTD 4, through its receptor CysLT 1 could in part be responsible for the observed relationship between intestinal inflammation and a subsequent development of cancer. A connection between cancer and inflammation was made in 1863 by Rudolf Virchow, who suggested that the origin of cancer was at sites of chronic inflammation (9). Today, it is believed that the tumor micro-environment equates to 15% of all global cancer cases, for example via infectious agents and the chronic inflammatory response they elicit (10). The most obvious and widely accepted connection is the one between inflammatory bowel diseases (IBD) and colon cancer. Patients with chronic inflammation in their intestinal tract lasting longer than 8 years, have a 30 50% increased risk of developing colon cancer (11). Pathways that can contribute to inflammation-induced cancer include factors involved in cell proliferation, such as COXs and prostaglandin formation, enhanced levels of lipoxygenases and thereby leukotrienes as mentioned above, and nuclear factor kappa B (NFkB) that is believed to function as a tumor promoter in inflammation based cancer (12). In this context it should be mentioned that a phospholipase A 2 has been suggested to regulate the DNA-binding ability of NFkB (13,14). As already mentioned, all cysteinyl leukotrienes are derived from AA that has been released from internal membrane lipids via the action of phospholipase A 2 s. These enzymes can be divided into three major groups based on size, ability to be secreted and calcium dependency (15). The three groups consist of the low molecular weight secretory phospholipase A 2 s (spla 2, kda), the calcium-independent phospholipase A 2 s (ipla 2, 80 kda) and the high molecular weight cytosolic phospholipase A 2 s (cpla 2 s, 85 kda) (15). The cpla 2 group consists of 3 isoforms, cpla 2 a, cpla 2 b and cpla 2 g, where cpla 2 a is the prototypic isoform (16). When activated by phosphorylation, via an increase in the cytosolic concentration of calcium, cpla 2 a translocates from the cytosol to either the nuclear membrane, endoplasmatic reticulum, Golgi or plasma membrane depending on cell type and stimulus (17 20). At these sites it catalyses the hydrolysis of membrane glycerophospholipids at the Sn-2 position to liberate AA (15). The expression and activity of PLA 2 has in several studies been related to cancer. For example, an increase in cpla 2 a Carcinogenesis vol.26 no.11 # Oxford University Press 2005; all rights reserved. 1988

2 Role of activation of cpla 2 in colon cancer cells expression has been shown in a variety of tumors, including colon cancer (21,22). Interestingly enough, the non-steroidal anti-inflammatory drug (NSAID) acetylsalicylic acid that is known to reduce the risk for colorectal cancer by as much as 40%, also inhibits the mrna expression of cpla 2 (23,24). Furthermore, the well-known role of COX-2 and its generation of prostaglandins in the pathogenesis of colorectal cancer (25) are also related to cpla 2 a since this enzyme is the predominant mediator releasing AA for COX-2 in the intestine. Correspondingly it has been demonstrated that cpla 2 a is involved in COX-2 mediated PGE 2 production and proliferation in human cholangiocarcinoma cells (26). Furthermore, deletion of cpla 2 in APC min/ mice results in an 83% reduction in tumor numbers in the small intestine (27). In the current study, a possible signaling relationship between LTD 4 and cpla 2 a activation has been investigated in intestinal epithelial cells and related to tumor cell growth. We have in the present study investigated the possibility that CysLT 1 receptor-induced signaling is involved in cpla 2 a activation, an incident that would initiate an additional production of pro-inflammatory leukotrienes. The main findings in our study is the demonstration of an essential role for cpla 2 a in LTD 4 induced proliferation thereby indicating its potential as a target in cancer therapy (28). Materials and methods Materials The inhibitors PD98059 and SB were purchased from New England Biolabs, Inc. (Beverly, MA). Antibodies against ERK 1/2 (p42/44) and phospho-erk1/2 (p42/44), cpla 2 a and phospho-cpla 2 a were purchased from Cell Signaling Technology (Beverly, MA). LTD 4 was obtained from Cayman Chemical (Ann Arbor, MI). The cpla 2 promoter construct was a kind gift from Dr R Nemenoff (Denver, CO). Methyl-[ 3 H] thymidine, ECL western blot detection reagents and Hyperfilm were from Amersham International (Buckinghamshire, UK). GF109203X, MAPT/AM, Bayu and pyrrolidin-1 were from Calbiochem (San Diego, CA). Pertussis toxin (PTX) was obtained from Speywood Pharma (Maidenhead, UK). Peroxidase-linked goat anti-rabbit antibody and fluorescence mounting medium originated from Dako A/S (Copenhagen, Denmark). The Dual Luciferase Reporter Assay Kit was purchased from Promega (Madison, WI), and Lipofectamine was from Invitrogen (Carlsbad, CA). ZM198,615 (ICI-198,615) was a kind gift from Astra Zeneca (Lund, Sweden). Ionophore A23187 was obtained from ICN Biomedicals (Temecula, CA). All cell culture medium was purchased from Invitrogen Life Technologies. Alexa 488 was obtained from Molecular Probes (Leiden, Netherlands) and the COX-2 antibody, ab15191, was purchased from Abcam (Cambridge, UK). AACOCF3 and all other chemicals were of analytical grade and purchased from Sigma Chemical (St Louis, MO). Cell culture and human colon tissue samples Non-transformed human embryonic intestinal epithelial cells, Int 407 cells (29), exhibiting typical epithelial growth and morphology were cultured in 75 cm 2 flasks as a monolayer to 80% confluence for 5 days in Eagle s Basal medium, supplemented with 15% newborn calf serum, 55 mg/ml streptomycin and 55 U/ml penicillin. Caco-2 cells derived from human colon cancer (30), and exhibiting an adherent epithelial growth pattern, were cultured to 80% confluence for 5 days in DMEM supplemented with 10% fetal bovine serum (FBS), 55 mg/ml streptomycin, and 55 U/ml penicillin. Fresh biopsies of human colon cancer tissue, paired with a corresponding normal tissue sample, taken for analysis of their content of COX-2 and active/phosphorylated cpla 2 were obtained. Upon removal the samples were immediately frozen by immersing them in liquid nitrogen. The local ethical committee at Lund University (LU-52-99) has approved the part of this study that relates to the analysis of the collected clinical material from colon cancer patients. Cell lysis and fractionation Caco-2 cells were left in serum-free medium for 2 h, treated with or without the ionophore A23187 (5 mm, 5 min), or pre-incubated with inhibitors in the absence or presence of LTD 4 (80 nm, 10 min, 15 min or 20 min). cpla 2 a inhibitors AACOCF3 (10 mm, 1 h) and pyrrolidin-1 (4 mm, 30 min), PTX (500 ng/ml, 2 h), Ca 2þ inhibitor MAPT/AM (10 mm, 1h), ERK1/2 inhibitor PD98059 (50 mm, 30 min), p38 inhibitor SB (20 mm, 30 min), CysLT 1 receptor inhibitor ZM198,615 (40 mm, 30 min), a NFkB inhibitor BAYu (20 mm, 60 min) and PKC inhibitor GF109203X (2 mm, 15 min) were added prior to LTD 4 addition. The stimulations were terminated by the addition of ice-cold lysis buffer A [20 mm Na HEPES (ph 8.0), 2 mm MgCl 2,1 mm EDTA, 5 mm orthovandate, 60 mg/ml phenylmethylsulfonyl fluoride (PMSF) and 4 mg/ml leupeptin] and the cells were placed on ice. The cells were scraped from the flasks, homogenized and used as whole cell lysates, or subjected to N 2 -decompression at 1000 p.s.i. for 10 min, using a cell disruption bomb (Parr Instrument, Moline). The intact nuclei were collected by centrifugation at 200 g and washed twice in buffer A. The supernatant was centrifuged at g for 10 min, and the resulting supernatant was fractioned into cytosol and plasma membrane fractions by centrifugation at g for 1 h. Gel electrophoresis and immunoblotting Sections from pairs of fresh normal and cancerous colon tissues, or cell lysates were solubilized by boiling at 100 C for 5 min in a sample buffer [62 mm Tris (ph 6.8), 1.0% SDS, 10% glycerol, 15 mg/ml dithiothreitol and 0.05% bromphenol blue]. Equal amounts of protein (30 50 mg protein/well) were loaded and subjected to electrophoresis on 8% homogeneous polyacrylamide gels in the presence of SDS. The separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were washed once for 5 min in TTBS (TBS with 0.1% Tween-20) and then blocked (1 TBS, 0.1% Tween-20 and 5% w/v non-fat dry milk) for 1 h at room temperature. The membranes were incubated overnight at 4 C with specific antibodies against phospho-cpla 2, total cpla 2, phospho-p38 MAPK, total p38 MAPK or COX-2 (diluted 1:250), all others diluted 1:1000, or 1:500 for tissue samples, in primary antibody solution [TBS with 0.1% Tween-20 and 5% bovine serum albumin (BSA)], followed by three washes, each for 5 min in TTBS. The anti-actin antibody was diluted 1:200 in 2% BSA/phosphate buffered saline (PBS). The membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:2000 in blocking buffer or 1:3000 in 1% BSA/PBS for actin, and the membranes were washed extensively. Thereafter the membranes were incubated with ECL western blot detection reagents, and exposed to Hyperfilm-ECL to visualize immunoreactive proteins. Transient transfection and dual-luciferase Ò reporter assays Int 407 and Caco-2 cells were cultured for 5 days to 50 60% confluency in 24-well plates. The cpla 2 promoter construct was transiently transfected at a final concentration of 1 mg/ml, and the control Renilla luciferase vector was present at all times at 0.2 mg/ml to normalize for transfection efficiency. The luciferase assays were carried out as described in the Dual Luciferase methodology according to Promega. Briefly, vector DNA was allowed to form complexes with Lipofectamine, which was used at 0.003% v/v (final concentration). Cells were washed once in serum-free medium and the DNAlipofectamine mixture containing the cpla 2 promoter plasmid and the Renilla plasmid, was added. The transfection continued at 37 C for 6 h, after which the medium was changed to normal growth medium and the cells were allowed to grow for another 48 h. During the last 2 h the cells were kept in serum-free medium and treated with or without A23187 (5 mm, 30 min) or pre-treated with the following inhibitors, cpla 2 a inhibitors AACOCF3 (10 mm, 1 h) and pyrrolidin-1 (4 mm, 30 min), PTX (500 ng/ml, 2 h), Ca 2þ chelator MAPT/AM (10 mm, 1h), ERK1/2 inhibitor PD98059 (50 mm, 30 min), p38 inhibitor SB (20 mm, 30 min), CysLT 1 receptor inhibitor ZM198,615 (40 mm, 30 min), PKC inhibitor GF109203X (2 mm, 15 min), 5-LO inhibitor MK866 (5 mm, 30 min) or NFkB inhibitor Bayu (20 mm, 1 h), in the absence or presence of LTD 4 (80 nm, 1.5, 4 or 18 h). Cells were then washed in PBS and lysed with dual luciferase reporter (DLR; provided by Promega) and passive lysis buffer (250 ml/well for 15 min at room temperature). Lysed samples were collected, a 20 ml portion of lysate was transferred to a luminometer test tube containing 100 ml Luciferase Assay Buffer II, and the luciferase reaction was recorded using a Berthold MiniLumat LB9506 luminometer (Berthold Technologies, Bad Wildbad, Germany). The control Renilla luciferase signal was recorded after the addition of 100 ml Stop and Glo buffer, and the ratio between the firefly and Renilla luciferase was calculated. Immunofluorescent staining Cells were grown on cover slips to 50 60% confluence, pre-treated with cpla 2 inhibitors AACOCF3 (10 mm, 1 h) or pyrrolidin (4 mm, 30 min), and treated with or without LTD 4 (80 nm, 15 min). Cells were washed three times with cold PBS, fixed for 15 min in 4% ice-cold paraformaldehyde in PBS. This was followed by blocking in a 3% BSA and PBS solution for 30 min. Thereafter, the cells were incubated at room temperature with primary antibody against phospho-cpla 2 (1:50) in a 1% BSA and PBS solution for 30 min. Cells were washed five times in PBS and incubated with a conjugated secondary 1989

3 L.Parhamifar, B.Jeppsson and A.Sjölander antibody (goat anti-rabbit IgG Alexa 488) in a 1% BSA and PBS solution for 1 h. Following five washes with PBS, the cover slips were mounted on glass slides with a fluorescence-mounting medium. The mounted slides were examined in a Nikon TE300 microscope ( plan Apochromat oil immersion objective), integrated in deconvolution fluorescence microscopy. Thymidine incorporation and cell counting Both cell lines were cultured for 5 days in 24-well plates and incubated in the absence or presence of the cpla 2 a inhibitors, AACOCF3 (10 mm) or pyrrolidin (4 mm) for 1 h prior to LTD 4 (80 nm) and also during the subsequent stimulation for 18, 24 or 48 h. Cellular DNA synthesis was assayed by adding 3 H-labeled thymidine (0.5 mci) during the last 24 h of treatment. The experiments were terminated and the cells were washed twice with PBS, treated with 10% trichloracetic acid for 30 min and lysed in 1 M NaOH. The level of radioactivity indicating the incorporation of [ 3 H]thymidine into DNA was measured in a ß-liquid scintillation counter (LKB rack Beta, Wallace). For the cell counting experiment the cells were serum starved and treated with the cpla 2 inhibitors AACOCF3 and pyrrolidin, and LTD 4 as indicated above. During the course of the experiment, fresh media, LTD 4 and inhibitors were replaced every day. To determine the number of viable cells, counting was done in the presence of 0.2% trypan blue. Hoeschst staining Cells were cultured on glass cover slips and exposed to cpla 2 a inhibitors AACOCF3 (10 mm) and pyrrolidin (4 mm) for 1 h. The cells were subsequently washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min at 37 C and then incubated for 15 min with the Hoechst stain (10 mg/ml) in PBS. The cover slips were then washed and mounted on glass slides. Samples were examined and photographed with a Nikon Eclipse 800 microscope, using a Plan-Apo 60 objective. Results Activation and nuclear translocation of cpla 2 a upon LTD 4 treatment The effect of LTD 4 on activity and cellular localization of cpla 2 a in epithelial cells has not previously been shown, but it is established that the calcium ionophore A23187 causes activation and cellular translocation of cpla 2 a in this cell type (31). In the experiments outlined in Figure 1, the activation and translocation of cpla 2 a was investigated in colon cancer cells (Caco-2) upon LTD 4 treatment using ionophore A23187 treatment as a positive control. First, Caco-2 cells were serum starved for 2 h, pre-treated or not with cpla 2 a inhibitor AACOCF3 (10 mm, 1 h) and then stimulated with LTD 4 (80 nm, 15 min) or A23187 (5 mm, 5 min) as indicated in Figure 1A. To evaluate the activity and distribution of cpla 2 a, the cells were immunostained with a phosphocpla 2 a antibody. The first image reveals the basal level of phospho-cpla 2 in untreated control cells (Figure 1A, left panel). With ionophore or LTD 4 treatment the amount of phosphorylated cpla 2 is profoundly increased in the nuclei (Figure 1A, middle panels). LTD 4 did not cause an activation or translocation in cells pre-treated with the cpla 2 a inhibitor AACOCF3 (Figure 1A, right panel). Cells were then treated with LTD 4 or ionophore, as above, after which the cells were disintegrated and preparation of nuclei and cytosol fractions were performed. Figure 1B shows anti-phospho-cpla 2 a and anti-total-cpla 2 a antibody blots of these fractions. Clearly, LTD 4 stimulation for 15 min increased the level of phosphorylated cpla 2 a in the nucleus (140%), while decreasing it in the cytosol (70%), and while the levels of total cpla 2 were unchanged. The control experiments with ionophore revealed, as in Figure 1A, an increase in phosphorylated cpla 2 a in both the cytosol and the nuclei (Figure 1B). In all experiments, we detected a low basal content of both active and inactive cpla 2 a in the nuclei, a phenomenon previously observed by others (32). The reason(s) for the basal nuclear 1990 Fig. 1. Activation and nuclear translocation of cpla 2 upon LTD 4 treatment in Caco-2 cells. (A) Caco-2 cells were grown on cover slips, pre-incubated or not with the cpla 2 inhibitor AACOCF3 (10 mm, 1 h), and then stimulated with 10 mm A23817 (5 min) or 80 nm LTD 4 (15 min). Thereafter the cells were fixed, permeabilized and stained with an anti-phospho-cpla 2 antibody and an Alexa 488-conjugated secondary antibody. Shown are representative images of, from left to right, untreated control cells, A23817 stimulated cells, LTD 4 -stimulated cells, and cells pre-treated with AACOCF3 and then stimulated with LTD 4.(B) Representative western blots of cytosolic and nuclear fractions, as described in Materials and methods, of Caco-2 cells treated with A21387 or LTD 4 as described above and indicated in the figure. The membranes were immunoblotted with antibodies against phospho-cpla 2 (upper panels) or total cpla 2 (lower panels). (C) Accumulated densitometric analysis of phospho-cpla 2 levels in cytosolic and nuclear fractions in the blots obtained in (B). The data are given as percent of control and represent means SEM of more than three separate experiments and the statistical analysis was performed with Student s t-test. P location of this protein is, however, yet unclear. These representative findings were confirmed by densitometric analysis of the amounts of phosphorylated-cpla 2 a in the fractions under different conditions (Figure 1C). Intracellular signals participating in LTD 4 -induced activation of cpla 2 a The intracellular signals involved in LTD 4 -induced cpla 2 a activation in intestinal epithelial cells were subsequently characterized. The variations in phospho-cpla 2 protein levels in unstimulated control cells are possibly due to a certain variation in the basal activity of cpla 2 in the cells or, perhaps, more likely to the well known difficulty in comparing protein levels on different blots since the conditions during the devel-

4 Role of activation of cpla 2 in colon cancer cells Fig. 2. LTD 4 -induced activation of cpla 2 a and its regulation in Caco-2 cells. Caco-2 cells were pre-incubated with either a receptor antagonist, an inhibitor or a calcium chelator, as described in Materials and methods and indicated in the figure, and then stimulated with or without LTD 4 (80 nm) for 15 min. The subsequent western blot analyses on total cell lysates were, for determination of activation levels, done with antibodies against phospho-cpla 2 a, phospho-erk1/2 or phospho-p38 and, for loading controls, with total cpla 2 a, total p38 or total ERK1/2. (A) Representative blots of cells pre-incubated with ZM198,615 (40 mm, 30 min) or PTX (500 ng/ml, 2 h), followed by treatment with LTD 4 for 15 min. (B) Representative blots of cells pre-incubated with GF109203X (2 mm, 15 min) or MAPT/AM (10 mm, 1 h), (C, E G) PD98059 (50 mm, 30 min) or (C, F and G) SB (20 mm, 30 min), and then followed by treatment with LTD 4 for 15 min. Representative blots of cells pre-incubated or not with PD98059 or SB203580, and then stimulated LTD 4 (80 nm) or A23187 (10 mm) for the indicated periods of time. (D) Representative blots of cells pre-incubated with AACOCF3 (10 mm, 1 h) or pyrrolidin-1 (4 mm, 30 min), and then stimulated with LTD 4 for 15 min. The blots shown are representative of three separate experiments. opment of two different western blots can never be identical. Figure 2A shows that both the CysLT 1 receptor inhibitor ZM198,615 and PTX abolished the LTD 4 -induced increase in cpla 2 a activity, indicating that the signal is mediated via the CysLT 1 receptor and a Ga i -protein. In Figure 2B the cells were treated with the PKC inhibitor GF109203X, and the intracellular calcium chelator MAPT/AM, and again the LTD 4 -induced activation of cpla 2 a was completely inhibited by GF109203X and partially inhibited by MAPT/AM. Different MAPKs have, by other agonists and in other cell types, been shown to activate cpla 2 a by phosphorylation at Ser-505 (33), and we have previously shown that LTD 4 can activate these MAPKs via a PKC-dependent pathway (34). Therefore we pre-treated the cells with PD98059 or SB203580, and as seen in Figure 2C this revealed that the LTD 4 -induced activation of cpla 2 requires both ERK1/2 and p38 signaling activities. These results are in accordance with our previous observations that stimulation of intestinal cells with LTD 4 triggers, within minutes, a calcium signal, activation of PKC and ERK1/2 (35,36) and, in the present study, also activation of p38 was seen at 10 min (Figure 2E). Interestingly, the LTD 4 -induced activation of p38 was blocked by pre-treatment with PD98059 (Figure 2F), but the activation of ERK1/2 was only partially inhibited by pre-treatment with SB and GF109203X (Figure 2G). These data suggest that activation of PKC is located upstream of ERK1/2, that is, located upstream of p38 in the LTD 4 -induced signaling pathway responsible for the activation of cpla 2 a. As a control for previous (Figure 1) and subsequent experiments, we also confirmed that the cpla 2 a inhibitors AACOCF3 and pyrrolidin both abolished the cpla 2 a activation induced by LTD 4 (Figure 2D). LTD 4 regulates cpla 2 a promoter activity Transient transfections were performed to determine whether the cpla 2 promoter could be regulated by LTD 4. Briefly, cells were grown in 12-well plates for 3 days, transfected with the cpla 2 promoter, for the gene activity, in the absence or presence of LTD 4 and different inhibitors, and monitored using a luciferase assay. In Figure 3 cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. The transcriptional activity of the cpla 2 promoter increased with 50% after LTD 4 treatment for 90 min (1.5 h), 35% after 120 min (2 h), 55% after 240 min (4 h) and almost no increase was seen after 18 h (P ; Figure 3A). In summary, the transcriptional activity reached its maximal level after 90 min (1.5 h), temporarily declined around 120 min (2 h), regained its maximal level around 240 min (4 h) and finally diminished and almost returned to its basal level, after 18 h. The 5-LO inhibitor MK866 had no effect on the basal transcriptional level, but reduced (P ) the effect of LTD 4 stimulation (Figure 3A). The ionophore A23187, used as a positive control, induced a 75% increase in the transcriptional activity (P ; Figure 3C). In order to study the intracellular signals involved in the LTD 4 -induced cpla 2 a transcriptional pathway, cells were treated with different inhibitors. Figure 3B shows the luminescence activity of cells pre-treated with the CysLT 1 receptor antagonist ZM198,615 or PTX, in the absence or presence of LTD 4 (90 min). The pre-treatments clearly abolished the LTD 4 -induced increase in cpla 2 promoter activity. We subsequently impaired the activity of PKC with GF109203X, and abolished a rise in cytosolic free Ca 2þ with MAPT/AM. The results show that both the inhibitor and the Ca 2þ chelator inhibited the LTD 4 -induced increase in transcription (Figure 3C). Furthermore, the MAPK inhibitors and the NFkB inhibitor Bayu produced similar results (Figure 3D). It has been shown that cpla 2 regulates the DNA binding activity of NFkB (13,14). We therefore studied the transcriptional activity when inhibiting cpla 2 a. The results show that the level of LTD 4 -induced cpla 2 transcription was totally abolished in the presence of a cpla 2 inhibitor (Figure 3D). Curiously the CysLT 1 receptor antagonist ZM198,615 and PTX both impaired the basal transcription of cpla 2, although the involvement of downstream signals are 1991

5 L.Parhamifar, B.Jeppsson and A.Sjölander Fig. 3. Intracellular signals involved in LTD 4 -induced gene activation of cpla 2 in Caco-2 cells. Luciferase assays of cpla 2 transcription in Caco-2 cells transiently transfected with a cpla 2 promoter construct and a Renilla plasmid. The Caco-2 cells were subsequently incubated in the absence or presence of 80 nm LTD 4 (1.5, 2, 4 or 18 h), pre-treated or not with a receptor antagonist, a calcium chelator or the indicated different inhibitors (MK866, ZM198,615, PTX, GF109203X, MAPT/AM, PD98059, SB or AACOCF3) as described in Materials and methods and in Figure 2, and then stimulated or not with either LTD 4 (80 nm, for 1.5 h) or A23187 (5 mm, 30 min, positive control). In addition, we also pretreated the cells with, a NFkB inhibitor (BAYu , 20 mm, 60 min) before they were stimulated or not with LTD 4 (80 nm, 90 min). Each obtained cpla 2 luciferase value was measured and normalized against its corresponding Renilla value. The cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. The data are given as percent of control and represent means SEM of at least three separate experiments, and the statistical analyses were performed with Student s t-test. P and P harder to evaluate than those mediating the LTD 4 -induced increase in cpla 2 promoter activity (Figure 3). The cpla 2 a inhibitor AACOCF3, which abolished the LTD 4 -induced activation but not the basal activity of cpla 2 (Figure 2D), also abolished the LTD 4 -induced but not the basal transcriptional cpla 2 promoter activity (Figure 3E). Analysis of a non-transformed intestinal epithelial cell line, Int 407, also revealed a LTD 4 -induced phosphorylation of cpla 2 a and a subsequent translocation of active cpla 2 a to the nuclei (Figure 4A). In accordance with this, both LTD 4 and the A23187 ionophore increased the cpla 2 promoter activity in Int 407 cells with a similar time kinetics as in Caco-2 cells. The signals involved in this agonist-induced cpla 2 transcription in Int 407 cells (Figure 4B F) were the same as in Caco-2 cells. In Figure 4B F, cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. Another interesting observation in the luciferase assays was that the basal level of transcription was 410-fold higher in Caco-2 tumor cells ( arbitrary units) when compared with the Int 407 cells ( arbitrary units). Although the CysLT 1 receptor antagonist ZM198,615 had a minute effect on the basal transcription of cpla 2 in 1992 Int 407 cells, the basal activity in these cells was generally much less sensitive to the different signaling inhibitors (Figure 4B F) than the Caco-2 tumor cells. As in Caco-2 cells, the cpla 2 a inhibitor AACOCF3 abolished the LTD 4 - induced but not the basal transcriptional cpla 2 promoter activity (Figure 4F). In support of this we performed western blot assays that clearly revealed an increased cpla 2 a protein content in both cell-lines upon LTD 4 stimulation (Figure 5A and B). The importance of the previously indicated intracellular signals (Figures 3 and 4) were also confirmed by the analysis of cpla 2 a protein content in both cell-lines (Figure 5A and B). As seen in Figure 5A, the cpla 2 a protein level in both cell lines was increased by exposure to LTD 4 between 1.5 and 4 h but had returned to basal level after 18 h. LTD 4 -induced activation of cpla 2 a is part of the proliferative response in both Caco-2 and Int 407 cells We know that LTD 4 increases the rate of proliferation in intestinal epithelial cells via an ERK1/2-dependent signaling pathway (6), and therefore we evaluated the role of LTD 4 - induced activation of cpla 2 a in the regulation process of proliferation. The growth rates of Int 407 cells and Caco-2 cells were evaluated, and the results reveal that LTD 4 induces an increase in both cell lines (Figure 6A and B). The insets in Figure 6A and B reveal that stimulation with LTD 4 causes significant increases in cell growth in both cell lines even after

6 Role of activation of cpla 2 in colon cancer cells Fig. 4. LTD 4 -induced activation and nuclear translocation of cpla 2 and activation of the cpla 2 promoter in Int 407 cells. (A) Int 407 cells were grown on cover slips, pre-incubated or not with the cpla 2 inhibitor AACOCF3 (10 mm, 1 h), and then stimulated with 10 mm A23817 (5 min) or 80 nm LTD 4 (15 min). Thereafter the cells were fixed, permeabilized and stained with an anti-phospho-cpla 2 antibody and an Alexa 488-conjugated secondary antibody. Shown from left to right, are representative images of untreated control cells, A23817 stimulated cells, LTD 4 -stimulated cells and cells pre-treated with AACOCF3, and then stimulated with LTD 4.(B) Luciferase assays of cpla 2 transcription in Caco-2 cells transiently transfected with a cpla 2 promoter construct and a Renilla plasmid. The Int 407 cells were subsequently incubated in the absence or presence of 80 nm LTD 4 (1.5, 2, 4 or 18 h), pre-treated or not with, a receptor antagonist, a calcium chelator or the indicated different inhibitors (MK866, ZM198,615, PTX, GF109203X, MAPT/AM, PD98059, SB or AACOCF3) as described in Materials and methods and in Figure 2, and then stimulated or not with either LTD 4 (80 nm, for 1.5 h) or A23187 (5 mm, 30 min, positive control). In addition, we also pretreated cells with, a NFkB inhibitor (BAYu , 20 mm, 60 min) before they were stimulated or not with LTD 4 (80 nm, 90 min). Each obtained cpla 2 luciferase value was measured and normalized against its corresponding Renilla value. The cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. The data are given as percent of control and represent means SEM of at least three separate experiments, and the statistical analyses were performed with Student s t-test. P and P day. Furthermore, when either Caco-2 or Int 407 cells were pre-treated with a cpla 2 inhibitor (AACOCF3 or pyrrolidin) they both exhibited an abolished LTD 4 -induced growth rate (Figure 6A and B). Similar results were obtained when the growth rates of these two cell lines were assayed by thymidine incorporation for 18, 24 or 48 h (Figure 6A and B). In both Caco-2 and Int 407 cells two structurally different cpla 2 inhibitors, AACOCF3 and pyrrolidin, abolished the LTD 4 - induced thymidine incorporation after 18 h (Figure 6A and B). In Figure 6, cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. To verify that the effects were indeed due to a decrease in proliferation and not to an effect on apoptosis, as previously reported (37), both cell lines were treated with AACOCF3 or pyrrolidin for 5 days under the same conditions as in Figure 6, stained with Hoechst and then examined in a microscope. No increase in the number of apoptotic cells was observed in either Caco-2 or Int 407 cells (data not shown). Increased levels of cpla 2 in Caco-2 colon cancer cells and human colon tumor tissue Based on the observation that cpla 2 transcription was more elevated in Caco-2 colon cancer cells than in non-transformed intestinal Int 407 cells, we decided to determine the activity and levels of the protein in both cell lines. Representative western blots and the accumulated densitometric results are shown in Figure 7A. In each experiment, equal amounts of total protein from unstimulated and LTD 4 stimulated Int 407 and Caco-2 cells were loaded and analyzed on the same gel and PVDF membrane. We observed a 3-fold higher protein content of active/phosphorylated cpla 2 a in unstimulated Caco-2 colon cancer cells compared with unstimulated non- 1993

7 L.Parhamifar, B.Jeppsson and A.Sjölander Fig. 5. LTD 4 effect on the total protein levels of cpla 2 a in Int 407 and Caco-2 cells. (A) Representative western blots of total cell lysates, as described in Materials and methods, of Int 407 and Caco-2 cells without and with 80 nm LTD 4 stimulation for 1.5, 4 or 18 h. The membranes were immunoblotted with antibodies against total cpla 2 (upper panels; densitometric analysis of total cpla 2 levels of the blots are shown) or actin (lower panels). In (B) cells were pre-treated with the indicated different inhibitors (ZM198,615, PTX, GF109203X, MAPT/AM, SB203580, PD98059 or BAYu ) as described in Materials and methods, and then stimulated with LTD 4 (80 nm, 1.5 h). Representative western blots of Int 407 and Caco-2 cells are shown. transformed Int 407 cells. Accordingly the level of active/ phosphorylated cpla 2 a after LTD 4 stimulation was also 3-fold higher in Caco-2 colon cancer cells compared with Int 407 cells. The total amount of cpla 2 a in Caco-2 cells was also significantly higher than that in Int 407 cells. These results led us to analyze a series of paired samples from fresh normal and cancerous colon tissues, with respect to the protein content of phospho-cpla 2 a, COX-2 (tumor control) and actin (loading control). Figure 7B shows the result of the analysis of four pairs of normal and tumor tissue samples. Equal amounts of total protein from the four pairs of normal and tumor tissue were loaded and analyzed on the same gel and PVDF membrane. In three of the four pairs the tumor sample has a higher level of phospho-cpla 2 than the corresponding normal sample (Figure 7B). The first pair of control and tumor tissues shows no cpla 2 a activity. We do not have an explanation for this. However, it could reflect a heterogeneity among the tumors or within a specific tumor. Similar results were obtained when the samples were analyzed for their content of COX-2, used here as a tumor control (Figure 7B). In the present study we frequently also detected the inducible form of COX, COX-2, which is normal colon tissue. A finding in good agreement with the results presented in other studies of COX-2 (7,38). It is possible that the lack of a difference between normal and tumor colon cancer tissue in the first pair is due to an unequal loading as indicated by the difference in actin content. Regardless of this, the accumulated densitometric results show a statistically significant increased level of active/phosphorylated cpla 2 a in human colon cancer tissue compared with normal human colonic tissue Discussion Chronic inflammatory conditions are intimately linked to an increased risk for subsequent development of cancer, including colon cancer. In agreement, NSAIDs have been shown to reduce the occurrence of colon cancer (39). These data suggest that inflammatory mediators can be part of colon cancer development. In previous studies, we have shown that LTD 4 via its receptor CysLT 1 signals proliferation and survival in intestinal cells (6), and that the CysLT 1 receptor is upregulated in human colon cancer tissue (7). Here we show for the first time that the presence of LTD 4 triggers activation of cpla 2 a and its translocation to the nucleus in an intestinal tumor cell line, Caco-2 and in a non-transformed intestinal epithelial cell line, Int 407. These data are in agreement with previous studies in a human endothelial cell line, EA.HY.926, where cpla 2 was shown to translocate to the nucleus and there to be involved in proliferative signaling (32). In this context we have previously also shown that LTD 4 stimulation causes the CysLT 1 receptor, COX-2 and 5-LO to translocate to the nucleus in intestinal cells (8). Together these findings increase the chances of a nuclear production of prostaglandins and leukotrienes and a subsequent local activation and proliferative signaling of the CysLT 1 receptor in intact intestinal cells, as recently demonstrated in an isolated intact nuclei from these cells (8). In the two cell lines we also investigated the signaling pathway(s) involved in LTD 4 -induced cpla 2 a activation and translocation. Our results demonstrate that a CysLT 1 receptorinduced activation of a PTX-sensitive G-protein, is the initial activation event in both Caco-2 and Int 407 cells. The pathway from the CysLT 1 receptor to the activation of cpla 2 a also included an increase in cytosolic calcium and activation of

8 Role of activation of cpla 2 in colon cancer cells A Number of cells x Day Int 407 LTD4 Control AAC+ LT AAC Pyrr + LT Pyrr days B Number of cells x Day Caco-2 5 LTD4 Control AAC + LT AAC Pyrr+ LT Pyrr days [3H] Thymidine incorp % of control LTD 4 AACOCF3 Pyrrolidin PKC. The fact that MAPT/AM only partially inhibits the LTD 4 -induced activation of cpla 2 a can be explained by the presence of calcium-independent activation mechanisms of cpla 2 a, for example through phosphatidylinositol biphosphate (40). These results correspond to previous data from our group demonstrating that LTD 4 can activate a calciumdependent PKC (35,36). More interesting was the finding that both the ERK1/2 and the p38 MAPKs were crucial for LTD 4 - induced activation and translocation of cpla 2 a. These results are in agreement with previous studies showing that nuclear translocation of cpla2 is regulated by both ERK1/2 and p38 MAPKs in endothelial cells (32), and that both these MAPKs can phosphorylate cpla 2 a at the most important residues in terms of regulation of its enzymatic activity, Ser-505 and Ser-727 (28). The fact that inhibition of ERK1/2 abolished LTD 4 -induced activation of p38, but inhibition of p38 did not significantly affect the LTD 4 -induced activation of ERK1/2, indicates that activation of ERK1/2 is upstream of p38, similar to that previously demonstrated in other situations (41) (h) LTD (h) AACOCF Pyrrolidin Fig. 6. Activation of cpla 2 is required for LTD 4 -induced proliferation in both Caco-2 and Int 407 cells. (A and B) The cells were incubated in the absence or presence of the indicated cpla 2 a inhibitor (AACOCF3 or pyrrolidin), and in the absence or presence of LTD 4 (80 nm) as indicated for up to 5 days. The proliferative responses were determined each day by counting the cells in the presence of 0.05% trypan blue. In all groups the medium and, if present, the indicated cpla 2 a inhibitor and LTD 4 were replaced with fresh medium, inhibitor and LTD 4 every 24 h. In both insets (A and B) the white bars indicate the controls whereas the black bars in both panels specify from the left to the right cell exposed to: LTD 4, AACOCF3, AACOCF3 þ LTD 4, pyrrolidin, or pyrrolidin þ LTD 4. The results shown are based on three separate experiments and are given as means. (A and B) Thymidine uptake analysis of Caco-2 and In 407 cells was performed after the cells were pretreated or not with the indicated cpla 2 a inhibitor (AACOCF3 or pyrrolidin) for 15 min and then, still in the presence of the inhibitor, stimulated or not with LTD 4 for 18, 24 or 48 h. In the experiments outlined in the lower panel in Figure 5A and B methyl-[ 3 H]thymidine (0.5 mci/well) was added for the last 24 h. The experiments were terminated by lysing the cells and mixed the lysates with scintillation liquid, after which the radioactivity was measured in a LKB Wallace 1209 RackNBeta counter. The cells stimulated with LTD 4 in the absence of any inhibitor and cells incubated with only an inhibitor (all marked x ) were statistically compared with the controls (open bars). However, cells incubated with an inhibitor and stimulated with LTD 4 (unmarked) were statistically compared with cells stimulated with LTD 4 alone. The results shown are based on three separate experiments and are given as means SEM. The statistical analyses were performed with Student s t-test. P and P See online Supplementary material for a colour version of this figure. [3H] Thymidine incorp % of control Another interesting aspect revealed by our present study is the fact that the basal cpla 2 a activity was impaired, in particular in the tumor cell line, in the presence of a CysLT 1 receptor antagonist. There are at least two possible explanations for this observation; the first being that these cells have an endogenous release of LTD 4. However, this has to the best of our knowledge not been shown in epithelial cells. Another alternative explanation could be that the CysLT 1 receptor exhibits spontaneous activity in the absence of its ligand (42). Several G-protein coupled receptors in different cells have been well documented to exhibit such activities (43). Furthermore, specific receptor antagonists have been shown to abolish the spontaneous activities of such receptors (42,44). A spontaneous activity of the CysLT 1 receptor, or an endogenous formation of LTD 4, further underlines the importance of the demonstrated increased expression of this receptor in human colon cancer tissue (7). We have also investigated, in both Caco-2 and Int 407 cells, whether addition of LTD 4 could activate the transcriptional 1995

9 L.Parhamifar, B.Jeppsson and A.Sjölander Fig. 7. Elevated levels of active cpla 2 a in human colon cancer cells. (A) This panel shows a representative western blot in which the levels of phospho-cpla 2 a and total cpla 2 a in Caco-2 and Int 407 cells have been analyzed in cells stimulated or not with 80 nm LTD 4 for 15 min. The diagram outlines the accumulated densitometric analysis of phospho-cpla 2 levels from the blots. The data are calculated as fold increase of untreated Int 407 cells, and are given as means SEM of at least three separate experiments. The statistical analysis were performed with Student s t-test. P and P (B) Pairs of fresh normal and cancerous colon tissue from four different patients were analyzed by western blotting for their contents of active/phosphorylated cpla 2 a, COX-2 (tumor control) and actin (loading control). The diagram outlines the accumulated densitometric analysis of phospho-cpla 2 and COX-2 levels. The data have in each case been calculated as percent of its own normal control tissue and are given as means SEM from four separate pairs. The statistical analysis were performed with Student s t-test. P and P activity of the cpla 2 a gene. With a luciferase assay we could demonstrate that LTD 4 increases the transcriptional activity of the cpla 2 a promoter, and that this transcription is dependent on activation of the CysLT 1 receptor, PKC, ERK1/2, and p38. In good agreement, it has been shown that nerve growth factorinduced cpla 2 expression in PC12 cells is mediated via activation of PKC, ERK1/2 and p38 (45). The demonstration that cpla 2 a can participate in the regulation of the DNAbinding activity of NFkB (13), lead us to examine if NFkB activation is necessary for LTD 4 -induced transcriptional activity of the cpla 2 a promoter. Our results indicated that the LTD 4 -induced transcriptional activation of cpla 2 is dependent on NFkB which is in line with the finding that the cpla 2 promoter contains NF-kB binding sites (46). These results are particularly interesting since NSAIDs have been shown to reduce colitis associated cancer by inhibition of the NF-kB signaling pathway in a mouse model (12). We can, however, at this point not exclude or conclude that LTD 4 has a direct effect on NF-kB activity. Obviously this needs to be further elucidated. The fact that the same signaling molecules, activated by LTD 4, mediate both activation and the transcriptional activity of the cpla 2 promoter suggest the existence of a positive loop whereby activation of cpla 2 increases its own expression level and thus an additional elevation of cpla 2 activity. The present finding that two structurally different cpla 2 a inhibitors, AACOCF3 and pyrrolidin, both inhibited the LTD 4 -induced transcriptional activity of the cpla 2 a promoter indicates that such a cpla 2 a driven positive loop is present in intestinal cells. Control experiments revealed that both 1996 LTD 4 -induced cpla 2 a phosphorylation and translocation are almost completely abolished by these cpla 2 a inhibitors. Intriguingly, the level of cpla 2 transcription activity in nontransformed Int 407 cells is lower than in colon cancer Caco-2 cells. In agreement with this, other groups have demonstrated high levels of cpla 2 in gastrointestinal cancer (47). Furthermore, we show that the colon cancer cell line Caco-2 also has higher levels of active cpla 2 a than a non-transformed cell line. These findings in human cell lines were supported by the present demonstration of higher levels of phosphorylated cpla 2 a in human colon tumor specimens compared with normal tissue from the same patient. In contrast, it has been shown that the level of cpla 2 mrna in mouse colon tumors is reduced as is its enzymatic activity (48). Clearly there exists an important difference in cpla 2 regulation between human colon cancer cells and mouse colon cancer cells. To gain further support for our finding of an increased activity of cpla 2 in human colon cancer cells, we investigated a possible functional role for active cpla 2 a in human colon cancer cells. In our previous studies, we have shown that LTD 4 can signal for increased proliferation, a hallmark of cancer cells. Our present findings show that treatment with either one of two different cpla 2 a inhibitors reduces LTD 4 -induced proliferation in both non-transformed human intestinal cells and in human colon cancer cells. However, inhibition of cpla 2 a did not affect the rate of apoptosis in these cells, a phenomenon sometimes associated with inhibition of cell proliferation (49). Somewhat different results were obtained by Longo et al. (49), who demonstrated that in a mouse colon cancer cell line (WB-2054-M4) thymidine incorporation is reduced by a spla 2 inhibitor but not by a cpla 2 inhibitor, whereas in a nonmalignant rat intestinal cell line (IEC-18) both inhibitors reduced the thymidine incorporation. There appear to exist important differences in the functional role of cpla 2 in human and mouse cancer cells. The fact that cpla 2 a activation is involved in regulation of human intestinal cell proliferation is indirectly supported by our observation that colon cancer cells exhibit increased cpla 2 a activity. The present finding that CysLT 1 receptor-induced cpla 2 a activity is an important signaling event in the regulation of human colon cancer growth provides an additional link between inflammation and cancer. Acknowledgements The authors are grateful to Dr R.Nemenoff (Denver, USA) for the cpla 2 promoter construct. This work was supported by grants to A.S. from the Swedish Cancer Foundation, the Swedish Medical Research Council, the Foundations at Malmö University Hospital, the Ruth and Richard Julins Foundation, Magnus Bergvalls Foundation, Gunnar Nilssons Foundation, and the Österlund Foundation. Conflict of Interest Statement: None declared. References 1. Lynch,K.R., O Neill,G.P., Liu,Q. et al. (1999) Characterization of the human cysteinyl leukotriene CysLT 1 receptor. Nature, 399, Heise,C.E., O Dowd,B.F., Figueroa,D.J. et al. (2000) Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem., 275, Funk,C.D. (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294, Serhan,C.N., Haeggström,J.Z. and Leslie,C.C. (1996) Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J., 10,