Expression of 15-Lipoxygenase by Human Colorectal Carcinoma Caco-2 Cells during Apoptosis and Cell Differentiation*

Hideki Kamitani, Mark Geller, and Thomas ElingDagger

From the Eicosanoid Biochemistry Section, Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We studied arachidonic acid metabolism and the expression of cyclooxygenase (Cox) and 15-lipoxygenase (15-LO) in the human colorectal carcinoma cell line, Caco-2, which undergo apoptosis and cell differentiation in the presence of sodium butyrate (NaBT). Caco-2 cells expressed very low levels of Cox-1 but highly expressed Cox-2. NaBT treatment shifted the arachidonic acid metabolites by cell lysates from prostaglandins to 15-hydroxyeicosatetraenoic acid, indicating the presence of a 15-LO. Linoleic acid, an excellent substrate for 15-LO, was metabolized poorly by the Caco-2 cells, but NaBT treatment shifted metabolism to 15-LO metabolite, 13(S)-hydroxyoctadecadienoic acid. Caco-2 cells expressed a 15-LO but only after treatment with NaBT, as determined by Northern blotting. Immunoblotting with anti-human 15-LO antibody detected a 72-kDa band in NaBT-treated Caco-2 cells. Expression of 15-LO mRNA was dependent on the duration of NaBT treatment, with the highest expression observed between 10 and 24 h. Results from expression and metabolism studies with arachidonic and linoleic acid cells indicated Cox-2 was responsible for the lipid metabolism in control cells, whereas 15-LO was the major enzyme responsible after NaBT induction of apoptosis and cell differentiation. The 15-LO in Caco-2 cells was characterized as human reticulocyte 15-LO by reverse transcription-polymerase chain reaction and restriction enzyme analysis. The expression of 15-LO and 15-hydroxyeicosatetraenoic acid or 13(S)-hydroxyoctadecadienoic acid formation correlates with cell differentiation or apoptosis in Caco-2 cells induced by NaBT. The addition of nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly increased NaBT-induced apoptosis, whereas the addition of indomethacin did not alter NaBT-induced apoptosis in the Caco-2 cells. However, indomethacin treatment decreased the expression of Cox-2 in NaBT-treated cells and significantly increased the expression of 15-LO during NaBT treatment. These studies suggest a role for 15-LO, in addition to Cox-2, in modulating NaBT-induced apoptosis and cell differentiation in human colorectal carcinoma cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The first genetic alteration in the multistep process that leads to the development of colon carcinogenesis seems to be the loss of APC gene function. Investigations of APC function and its mutations have provided important clues in understanding colon cancer (1). One of the important changes that results from the loss of the APC gene function is the overexpression of prostaglandin H synthase-2 (cyclooxygenase-2, Cox-2),1 an inducible enzyme that converts arachidonic acid to prostaglandins. The link between APC, Cox-2, and polyp formation was firmly established in studies using APC (ApcDelta 716) and Cox-2 knockout mice (2). Mice carrying the APC mutation overexpress Cox-2 and develop intestinal polyps. When bred to mice with the disrupted Cox-2 gene, the offspring, homozygously deficient for Cox-2, had significantly less polyps than the mice with wild-type Cox-2. These results confirmed Cox-2 as a modulator after APC mutations in colon carcinogenesis and represent a key enzyme in tumor development. The elevated expression of Cox-2 was shown not only in polyps of ApcDelta 716 and Min mice (3) but also in human colorectal tumors (4, 5) and immortalized cell lines of colorectal carcinoma (6). These findings provide an explanation for epidemiological, clinical, animal, and experimental studies which indicate that nonsteroidal anti-inflammatory drugs, which inhibit Cox-2, prevent colorectal cancer.

One possible explanation for the role of Cox-2 in colon carcinogenesis was obtained from studies with rat intestinal epithelial (RIE) cells engineered to overexpress Cox-2 (7). The overexpression of Cox-2 in RIE cells results in a resistance to sodium butyrate (NaBT) or adhesion-induced apoptosis. However, mechanisms that clearly explain how Cox-2 alters or inhibits apoptosis have not been determined. The addition of prostaglandins to cells in culture does not seem to alter cell proliferation and/or apoptosis (8), and other investigations indicate that nonsteroidal anti-inflammatory drug agents may act by a prostaglandin-independent pathway (9). The metabolites of arachidonic acid formed by Cox-2 are considered an important contributor, but we cannot ignore the possible involvement of linoleic acid metabolites, as it is also a substrate for Cox-2 (10). In addition, arachidonic acid and linoleic acid metabolites formed by the enzymatic activity of lipoxygenases may also need to be considered. Our laboratory has shown that both Cox-2 and 15-lipoxygenase (LO) metabolites of arachidonic acid and linoleic acid alter growth factor signaling pathways (11-13). For example, 13(S)-hydroxyoctadecadienoic acid (HODE)/13(S)-hydroperoxyoctadecadienoic acid (HpODE) is formed from linoleic acid in response to epidermal growth factor and contributes to epidermal growth factor-dependent mitogenesis in Syrian hamster fibroblast cells (13). The 13(S)-HpODE augmented the epidermal growth factor receptor signaling pathway by attenuation of receptor dephosphorylation (12). Furthermore, 15-LO metabolites of linoleic acid, 13(S)-HpODE inhibits starvation-induced apoptosis.2 Furthermore, Tang et al. (14) showed that lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, inhibit apoptosis in rat W256 cells. They suggest a role for 12-LO as an inhibitor of apoptosis (15).

These findings suggest a potential involvement of lipoxygenases in the apoptotic process. We have examined arachidonic acid and linoleic acid metabolism in the human colorectal cell line, Caco-2, which undergoes cell differentiation and apoptosis in the presence of NaBT (16). The metabolite profile was dramatically shifted from Cox-2 metabolites to 15-LO-derived metabolites by the NaBT treatment. The NaBT induced a 15-LO that was characterized as the reticulocyte 15-LO previously characterized from human bronchial epithelium (17). In this report, we show the first evidence that a 15-LO is clearly induced in colorectal carcinoma cells. Moreover, the 15-LO may act to modulate NaBT-induced apoptosis and cell differentiation in these cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- The human colorectal carcinoma cell line Caco-2 was obtained from the American Type Culture Collection (ATCC). Caco-2 was grown in Eagle's minimal essential medium, 15% fetal bovine serum (FBS) with 1 mM sodium pyruvate (Life Technologies, Inc.). The media for cells contained gentamicin (1 mg/100 ml, Life Technologies, Inc.). FBS was from Summit, and NaBT was obtained from Sigma. For treating cells with NaBT, cells were cultured in the appropriate media containing serum on 150 cm2 round dishes until nearly confluent. The medium was removed and replaced with FBS medium containing either the solvent or 5 mM NaBT. The NaBT was solibilized by PBS and used. Cells were then harvested at the times stated in the figure legends.

DNA Fragmentation Assay-- The floating cells and the cells attached to the dish were collected separately and sedimented. Washed cell pellets were resuspended in cell lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM EDTA (pH 8.0), 0.5% Triton X-100) and incubated. RNase A (0.5 mg/ml) and proteinase K (0.5 mg/ml) were added, respectively, and incubated for 2 h. DNA was precipitated by ethanol, and 3 µg or 1 × 106 cell numbers of water-diluted sample was run on a 2% agarose gel. Gels were stained with ethidium bromide, and DNA was visualized by UV transilluminater.

Alkaline Phosphatase Activity -- Alkaline phosphatase activity was measured using the p-nitrophenyl phosphate (Sigma) as substrate. The standard assay mixture consisted of 0.5 ml of 2-amino-2-methyl-1-propanol buffer (1.5 mol/liter) (Sigma), 2 mg of substrate, and 100 µl of sonicated protein in a final volume of 1.1 ml. After 15 min at 37 °C, the reaction was terminated by adding 10 ml of 0.05 N NaOH, and the absorbance of the color due to the formation of p-nitrophenol was measured spectrophotometrically at 410 nm. Enzyme activity was expressed in Sigma Unit/0.1 mg protein. The calibration was performed by using p-nitrophenol standard solution (Sigma).

Northern Blot Analysis-- Total RNA from the attached cell was extracted by using TRI Reagent (Sigma) according to the procedure by Chomczynski (18). RNA samples (30 µg per lane) were separated by electrophoresis in a formaldehyde-1% agarose gel. The RNA was transferred in 10 × SCC by capillary action onto nylon membrane (Schleicher & Schuell) and UV-cross-linked with a Stratalinker UV light source (Stratagene). Human 15-LO, Cox-1 and Cox-2 (Oxford Biomedical Research), and human glyceraldehyde-3 phosphate dehydrogenase (CLONTECH) cDNA probes were labeled with [alpha -32P]dCTP (Amersham Pharmacia Biotech) using the Prime-It-II random prime kit (Stratagene). After hybridization and washes, the blots were then exposed to x-ray film (Amersham) for autoradiography.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis-- Cells were washed twice with ice-cold PBS and lysed in protein lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 µg/ml leupeptin and aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). Cells were sonicated four times for 20 s at 50% power for a total protein preparation. Protein content was quantified by the bicinchronic acid method using BCA protein assay reagent (Pierce). Aliquots of the protein preparation were boiled in protein sampling buffer (9% SDS, 15% glycerol, 30 mM Tris-HCl, pH 7.8, 0.05% bromphenol blue, 6% beta -mercaptoethanol) and separated by 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham). Blots were blocked with 10% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and washed. The blots were then incubated in 1% milk in TBS-T with an anti-appropriate specific antibody. CheY-human 15-LO antibody (Ref. 17, provided by Dr. Sigal) and PG27G (Oxford Biomedical Research) antibody were used as the 15-LO and Cox-2 antibodies, respectively. After washing, blots were incubated with anti-rabbit IgG horseradish peroxidase-linked secondary antibody (Amersham). After reacting it by chemiluminescence (the Amersham ECL system), bands were detected after exposure to Hyperfilm-MP (Amersham).

Analysis of Arachidonic and Linoleic Acid Metabolites-- Caco-2 cells cultured in 150-cm2 dishes at each condition were washed with PBS twice. Cells were scraped and collected in 1 ml of the lysis buffer (100 mM Tris-HCl, (pH 8.0), 1 µg/ml leupeptin and pepstatin, 0.5 mM phenylmethylsulfonyl fluoride). Cells were sonicated four times for 20 s at 50% power by the sonic dismembrator for a total protein preparation. Then, protein content was quantified, and 0.8 mg of total cell lysate was used. The sonicates were then diluted with 1 ml of the reaction buffer (100 mM Tris-HCl, 10 mM CaCl2) (2 ml total) and incubated [3H]arachidonic acid (3 µCi, 25 µM) (DuPont NEN) for 30 min or [14C]linoleic acid (3 µCi, 25 µM) (DuPont NEN) for 15 min at 37 °C. Fatty acid compounds were extracted from the incubation buffer by acidification to pH 3.5 with acetic acid and applied to a C18-PrepSep solid phase extraction column (Waters) pretreated with methanol. The samples were then washed with acidified water, eluted with methanol, evaporated to dryness, and reconstituted with high pressure liquid chromatography (HPLC) solvent.

High Pressure Liquid Chromatography-- Reverse-phase HPLC analysis was performed using an Ultrasphere ODS column (5 µm; 4.6 × 250 mm; Beckman). The solvent system consisted of a methanol/water gradient at flow rate of 1.1 ml/min as described previously (19). Radioactivity was monitored using a Flow Scintillation Analyzer (Packard) with EcoLume (ICN Biochemicals) as the liquid scintillation mixture. To separate 9-HODE and 13-HODE, straight-phase HPLC was used. The straight-phase HPLC used a Waters µPorasil column (10 µm; 3.9 × 300 mm) with a flow rate of 2 ml/min and eluted with hexane:diethylether:acetic acid (1000:200:1) for 60 min. UV analysis was performed by monitoring absorbance at 235 nm with a Waters 990 photodioarray detector. Authentic standards of 9(R)-HODE, 13(S)-HODE, 15(S)-HETE, prostaglandin E2 (PGE2), and prostaglandin F2alpha were obtained from Cayman Chemical and used.

RT-PCR and Restriction Enzyme Analysis-- First-strand complementary DNA (cDNA) was generated using 1 µg of total RNA as template. Oligo(dT)18 primer (20 pmol) was used to prime a standard reverse-transcription (RT) reaction. AdvantageTM RT-for-polymerase chain reaction (PCR) kit was purchased from CLONTECH and used for this cDNA synthesis. As a control for possible contamination of genomic DNA, the same RNA was also prepared to react without reverse transcriptase. The reaction solution was diluted to a final volume of 100 µl by DEPC-treated water, and the 5 µl was used as PCR. PCR mixture consisted of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 2.0 units of Taq DNA polymerase (Pharmacia), and 0.4 µM each of the following primers: P1, 5'-GAGTTGACTTTGAGGTTTCGC-3'; P2, 5'-GCCACGTCTGTCTTATAGTGG-3'; P3, 5'-CCCTGTGGATGAGCGATTTC-3'; and P4, 5'-AAGTGTCCCCTCAGAAGATG-3' in a total volume of 50 µl. Thermocycling was performed according to the following profile: 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min, repeated 35 times followed by a final extension at 72 °C for 7 min. cDNA fragments generated by RT-PCR using P1 and P2 primers were digested with PstI or HindIII (Boehinger Mannheim). Analysis of the fragments was performed on a 1.8% agarose gel electrophoresis and then photographed.

Inhibition of 15-LO by Nordihydroguaiaretic Acid (NDGA) and Apoptosis-- To inhibit the activity of 15-LO in Caco-2 cells treated with NaBT, NDGA (Sigma), a inhibitor of lipoxygenase, was added in addition to NaBT. Before assessing the effect of attenuating the 15-LO on NaBT-induced apoptosis, the optimal concentration of NDGA for inhibiting 15-LO without affecting Cox activity was determined by studying arachidonic acid metabolism. The procedure for metabolism study and HPLC analysis were described above. As described previously, the floating Caco-2 cells were apoptotic, and thus the population of cells floating can be used as a measure of apoptosis. Cells were cultured on 6-well dishes (10 cm2) in FBS containing medium until subconfluent. Then, the media were replaced by the following four conditions: serum-containing medium (FBS), 5 mM of NaBT in FBS, NDGA (10 µM) in FBS, and NDGA (10 µM) containing 5 mM NaBT in FBS. NDGA was added to the medium every 24 h. NDGA was dissolved in ethanol, and the final concentration of ethanol in medium was less than 0.01%. The numbers of attached and floating cells were respectively counted with a hemocytometer in each condition at every 24 h until 96 h. Five dishes were performed in each condition. The ratio of the numbers of floating cells per total cells was calculated as the indicator of apoptosis. Also, the attached cells and the floating cells were harvested, and genomic DNA was prepared and assayed for the fragmentation of DNA.

Inhibition of Cox-2 by Indomethacin and Apoptosis -- To inhibit the Cox activity of Caco-2 cells, indomethacin (Sigma) was added to the cells during NaBT treatment. Cells were suspended and cultured on 6-well dishes (10 cm2) until subconfluent. Then, the media were replaced with media containing FBS under the following conditions: control, 5 mM NaBT, indomethacin (1 µM), and 5 mM NaBT and indomethacin (1 µM). Indomethacin was administered at every 12 h. The numbers of attached and floating cells were counted with a hemocytometer in each condition at every 24 h until 96 h. Cells were also lysed at 24, 48, and 72 h after treatment for immunoblot analysis.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Apoptosis and Cell Differentiation in Caco-2 Cells during NaBT Treatment-- During culture of colorectal cell lines, some cells detach from the dish and float in the medium. Evidence indicates the floating cells have undergone apoptosis (20). NaBT is reported to induce apoptosis and cell differentiation in various human colorectal carcinoma cells in culture (16, 21-23) including Caco-2 cells. In our experiments, we observed, microscopically, Caco-2 cells floating in the media with increasing time of treatment with NaBT. Two procedures were performed to evaluate the cell status during treatment with NaBT of Caco-2 cells. Fragmentation of DNA, an index of apoptosis, was determined by an examination of DNA ladder formation in the floating and attached cells. Cell differentiation was measured by alkaline phosphatase activity. The characteristic apoptotic DNA ladders were detected in the floating cells after treatment with NaBT at 72 and 96 h as shown in Fig. 1A, and we could not clearly observe DNA ladders in the attached cells. Thus, NaBT induced apoptosis in the Caco-2 cell characterized by the detachment of cells from the dishes and the presence of cells floating in the media. These observations and conclusions are consistent with the previous reports (16, 21-23). The alkaline phosphatase activity, a marker of cell differentiation (24), was clearly increased in the attached cells by the NaBT treatment (Fig. 1B). At 24 h, FBS-treated cells showed means 8.7 ± 1.4 S.E. units/0.1 mg protein, and NaBT-treated cells showed 16.2 ± 2.8 S.E. units/0.1 mg protein. The difference of alkaline phosphatase activity was clearer at 48 h between FBS and NaBT-conditioned cells, 10.4 ± 0.8 S.E. units/0.1 mg protein and 133.6 ± 13.4 S.E. units/0.1 mg protein, respectively. Alkaline phosphatase activity was increased with increasing time of exposure to NaBT, indicating an increase in cell differentiation. Thus, NaBT induced both cell differentiation and apoptosis in Caco-2 cells.


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Fig. 1.   Induction of apoptosis and cell differentiation in Caco-2 cells after treatment with NaBT. A, DNA laddering produced by Caco-2 cells after treatment with NaBT. The electrophoretic pattern of DNA isolated from Caco-2 cells is shown. DNA was extracted from the attached and floating cells cultured in 5 mM NaBT for 72 and 96 h. The left lane shows DNA size marker. Each lane contains 3 µg of the extracted DNA and was analyzed on 2% agarose gel. B, induction of cellular differentiation in the attached Caco-2 cells after treatment with NaBT. The near confluent Caco-2 cells cultured in serum-containing medium (FBS) on 10-cm2 dishes were washed by PBS. Then, the media were replaced by FBS or 5 mM NaBT containing FBS, and the attached cells were collected as cell lysates at 4, 24, 48, 72, and 96 h after treatment. Three independent dishes were examined in each condition, and results are plotted as the mean ± S.E. Enzyme activity is measured in Sigma unit per 0.1 mg of protein

Cox-1, Cox-2, and 15-LO Expression in Caco-2 Cells-- The expression of Cox-l, Cox-2, and 15-LO was determined during NaBT-induced apoptosis and cell differentiation. RNA was prepared from both attached and floating cells. However, because the floating cells had undergone apoptosis, RNA was degraded; thus, expression could only be determined in intact attached cells. Total RNA (30 µg) was prepared from FBS-cultured cells as the normal culture condition and from cells cultured with 5 mM NaBT for 72 h. The results for the expression of three oxygenases by Northern analysis are shown in Fig. 2. Little or no expression of Cox-1 and 15-LO was observed, but high expression of Cox-2 was detected, which is in agreement with a previous report by Kutchera et al. (6). NaBT treatment did not increase Cox-1 expression. In Caco-2 cells, NaBT down-regulated the expression of Cox-2 also seen in Fig. 2. Treatment of the cells with NaBT resulted in the expression of a 15-LO. Although we investigated the expression of 15-LO in the other four different colorectal carcinoma cell lines, the expression of 15-LO was not observed in any cells cultured in FBS (data not shown). The RNA obtained from normal human tracheobronchial epithelial cells treated with retinoic acid was used as positive control for expression of 15-LO (25).


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Fig. 2.   Expression of Cox-1, Cox -2, and 15-LO in Caco-2 cells before and after treatment with NaBT. Northern hybridization blots comparing levels of mRNA expression of Cox-1, Cox-2, and 15-LO in pair of the FBS-starved cells and the 5 mM NaBT-treated cells for 72 h. Each lane contains 30 µg of total RNA. Equal loading was observed by equivalent density of hybridization to the human glyceraldehyde-3 phosphate dehydrogenase (G3PDH) cDNA. Normal human tracheobronchial epithelial (NHTBE) cells (25) are used as the positive control for the expression of 15-LO. The mRNA for Cox-1, Cox-2, and 15-LO is 2.7, 4.5, and 2.7 kb, respectively.

Time Course for the Expression of Cox-2 and 15-LO in Caco-2 Cells Treated with NaBT-- Northern and immunoblot analysis was used to evaluate the time course for Cox-2 and 15-LO expression during NaBT induction of apoptosis and cell differentiation. RNAs were extracted and assessed from cells attached to the dish at 11 points of time during treatment. Surviving cells, cells treated with NaBT for 96 h then fed for about 7 days until confluent with FBS containing media, were included. NaBT-treated cells continued to express Cox-2 with a decreasing level of expression observed from 10 h to 48 h of exposure (Fig. 3A). No expression of 15-LO at the earliest times of NaBT treatment was observed, but at 6 h a band of 2.7 kb, which hybridized to the human 15-LO probe, was detected. The 15-LO expression increased until 96 h. However, the surviving cells, which were again cultured in serum after 96 h of NaBT treatment, showed no expression of 15-LO. The cultured cells under identical conditions except for NaBT showed Cox-2 expression but no expression of 15-LO at all times (data not shown).


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Fig. 3.   Time course for the expression of Cox-2 and 15-LO during NaBT-induced apoptosis in Caco-2 cells. A, Northern blot analysis. The near confluent Caco-2 cells cultured in FBS were treated with 5 mM NaBT containing fresh medium and then incubated for the stated time points. Total RNA was extracted from the cells at times indicated after treatment and analyzed by Northern hybridization. Surviving cells in the right lane are the cells cultured in FBS for 7 days after 96 h of exposure by 5 mM NaBT. Each lane contains 30 µg of total RNA, and equal loading was observed by equivalent density of hybridization to the human glyceraldehyde-3 phosphate dehydrogenase (G3PDH) cDNA. The mRNA for Cox-2 and 15-LO is 4.5 and 2.7 kb, respectively. The blot was hybridized, stripped, and then hybridized with the next probe. B, immunoblot analysis. The near confluent Caco-2 cells cultured in FBS were treated with 5 mM NaBT or the fresh FBS and then incubated for the stated time points. Protein was extracted from the cells at the times indicated after treatment, and surviving cells were separated on 8% SDS-polyacrylamide gels. Each lane contains 15 µg of the total cell lysates. Cellular expression of Cox-2 and 15-LO were estimated using anti-human Cox-2 and anti-human 15-LO antibody.

Immunoblot analysis was used to confirm the expression of Cox-2 and 15-LO in control and NaBT-treated cells (Fig. 3B). Protein expression could only be determined in attached cells; the protein was degraded in the apoptotic floating cells. Cox-2 protein was detected in the FBS-cultured cells at all time points examined, and the surviving cells also expressed similar amounts of Cox-2. Furthermore, Cox-2 expression was decreased by NaBT induction, a result that is consistent with the Northern data. We did not observe any protein that reacts to the human 15-LO antibody in the cells grown in FBS. In contrast, treatment with NaBT increased the expression of 15-LO protein. We began to detect expression of 15-LO between 6 and 14 h after treatment with NaBT. After 24-48 h, significant expression of 15-LO was observed. Taken together, these results support the conclusion for the expression of 15-LO during NaBT-induced cell differentiation and apoptosis in Caco-2 cells.

Metabolites of Arachidonic Acid in NaBT-treated Caco-2 Cells-- To study arachidonic acid metabolism, NaBT-treated cells and FBS-cultured cells were sonicated, and proteins were estimated. The lysates, 0.8 mg of protein, were then incubated with exogenous [3H]arachidonic acid (3 µCi; 25 µM) for 30 min. Reverse-phase HPLC analysis of the extracts is shown in Fig. 4. For FBS-treated cells, PGE2 (retention time 20~23 min) and prostaglandin F2alpha (retention time 23~29 min) were the major metabolites, and HETE (retention time 72~78 min) was the minor metabolite (Fig. 4A). The formation of the metabolites was inhibited by indomethacin (5 µM), indicating metabolism by Cox-2 activity (Fig. 4B). In contrast, NaBT-treated cells showed a significant increase in the formation of 15-HETE (retention time 73~76 min) and moderate formation of PGE2 (Fig. 4C). Moreover, the formation of the PGE2 was inhibited by 5 µM of indomethacin, but the formation of 15-HETE peak was not inhibited (Fig. 4D), indicating metabolism by both Cox-2 and 15-LO, respectively. The 15-HETE metabolite also coeluted with standard on reverse-phase HPLC and had a UV absorption at 234 nm (data not shown). These results confirm the expression of an active 15-LO and expression of the level of transcription and translation.


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Fig. 4.   Analysis of metabolites of [3H]arachidonic acid in NaBT-treated Caco-2 cells. The (0.8 mg) protein from total cell lysates was reacted in 2 ml of reaction buffer containing 25 µM [3H]arachidonic acid (3 µCi) and incubated for 30 min at 37 °C. Radiolabeled products were separated by reverse-phase HPLC as described under "Experimental Procedures." A, products formed in the incubation of arachidonic acid with the lysates of Caco-2 cells cultured in serum containing medium (FBS) for 48 h. B, products formed in the incubation of arachidonic acid and indomethacin (5 µM) with the lysates of cells cultured in FBS. C, products formed in the incubation of arachidonic acid with the lysates of cells cultured in 5 mM NaBT containing FBS for 48 h. D, products formed in the incubation of arachidonic acid and indomethacin (5 µM) with the lysates of cells cultured in 5 mM NaBT containing FBS.

Metabolites of Linoleic Acid in NaBT-treated Caco-2 Cells-- Linoleic acid is an excellent substrate for the human reticulocyte 15-LO (10) but a poor substrate for 15(S)-LO (or 15-LO-2). Cox-2 converts linoleic acid to 9(R)-HODE (26), whereas 15-LO forms 13(S)-HODE, and thus linoleic acid metabolism is an excellent indicator of a metabolism shift from Cox-2 to 15-LO. Also, our studies with fibroblasts indicate 13(S)-HpODE and 13(S)-HODE as potent modulators of signaling pathways, which control cell growth, apoptosis, and other biological process (11, 12). Thus, [14C]linoleic acid metabolism studies were performed on cell sonicates (0.8 mg of total protein) prepared from FBS- and NaBT-treated cells. The metabolites of linoleic acid are shown in Fig. 5. In NaBT-treated cells, the 10~12% of linoleic acid was converted to a HODE metabolite(s) at 73 min retention time by reverse-phase HPLC (Fig. 5B). These metabolites coeluted with 9/13-HODE standard and had a UV absorption at 234 nm because 13-HODE and 9-HODE have the same retention time by our reverse-phase HPLC system. Moreover, this peak was not suppressed by addition of indomethacin (5 µM) (data not shown). FBS-treated cells converted only 2~4% of linoleic acid to metabolite peaks at 73 min retention time, and several metabolites around the predicted 9/13-HODE peak could be seen (Fig. 5A). To identify 9-HODE and 13-HODE from FBS- and NaBT-treated cells, straight-phase HPLC was performed. Metabolite peaks were collected from reverse-phase HPLC and then analyzed by straight-phase HPLC with 9(R)- and 13(S)-HODE standards. 9-HODE (retention time 11 min) is the dominant metabolite formed in FBS-treated cells (Fig. 5C), whereas 13-HODE (retention time 7 min) is the major metabolite in NaBT-treated cells (Fig. 5D). These results are consistent with the conclusion that Cox-2 is responsible for the metabolism in FBS-treated cells, whereas 15-LO is responsible for the metabolism in Caco-2 cells during NaBT-induced apoptosis. The data also support the conclusion that a 15-LO is induced by the NaBT treatment. Moreover, these results indicated that linoleic acid is a better substrate than arachidonic acid for this inducible 15-LO.


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Fig. 5.   Analysis of metabolites of [14C]linoleic acid in NaBT-treated Caco-2 cells. The 0.8-mg protein from total cell lysates was reacted in 2 ml of reaction buffer containing 25 µM [14C]linoleic acid (3 µCi) and incubated for 15 min at 37 °C. Radiolabeled products were separated by reverse-phase HPLC. The metabolites with retention times from 70 to 80 min were collected in four separate sets, pooled, evaporated, and then separated by straight-phase HPLC. A, incubation of linoleic acid with lysates of Caco-2 cells cultured in FBS for 48 h separated by reverse-phase HPLC. B, incubation of linoleic acid with lysates of cells cultured in 5 mM NaBT containing FBS for 48 h separated by reverse-phase HPLC. C, separation of 9- and 13-HODE isomers from incubation A by straight-phase HPLC. D, separation of 9- and 13-HODE isomers from incubation B by straight-phase HPLC.

Characterization of 15-LO cDNA in NaBT-treated Caco-2 Cells-- The expression and activity of 15-LO in NaBT-treated Caco-2 cells were confirmed by Northern, immunoblot, and HPLC analysis. We wanted to further characterize the inducible 15-LO in Caco-2 cells. Two human 15-LO have been characterized, the reticulocyte 15-LO, which was isolated by Sigal et al. (27) and highly expressed in pulmonary epithelial cells, and the 15(S)-LO (or 15-LO-2), which was discovered in epithelial tissue by Brash et al. (28). We isolated RNA and made cDNA from the NaBT-treated Caco-2 cells and characterized the 15-LO by restriction enzyme analysis. Two different pairs of primers were employed based on the cDNA sequence of human reticulocyte 15-LO. As shown in Fig. 6A, P1 and P2 primers yield a 952-bp fragment and P3/P4 yield a 619-bp fragment. Moreover, P1/P2 fragment has two PstI sites and a HindIII site. Thus, three fragments (390, 291, and 271 bp) are predicted by PstI digestion, and two fragments (648 and 304 bp) are predicted by HindIII digestion. After synthesis of first strand cDNA from total RNA of NaBT-treated Caco-2 cells, PCR was performed with the above primers. At the same time, the RNA samples without reverse transcriptase were amplified to neglect the contamination of the genomic DNA. Two PCR fragments and restriction analysis gave a restriction map that agreed with the predicted size as shown in Fig. 6B. This experiment was repeated twice with the same findings. These results confirm the presence of the reticulocyte type 15-LO expression in human colorectal carcinoma cell line Caco-2 by treatment with NaBT.


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Fig. 6.   Characterization of 15-LO cDNA induced by NaBT in Caco-2 cells. A, a part of restriction map in the human reticulocyte 15-LO cDNA (27). The predicted sizes of RT-PCR products by using Primer 1, 2 and Primer 3, 4 (represented by an arrow) are illustrated. Also, the digestion pattern by PstI and HindIII are illustrated. The sequences of these primers are described under "Experimental Procedures." B, restriction enzyme analysis of 15-LO cDNA fragment generated by RT-PCR in NaBT-treated Caco-2 cells. Lane 1, size marker; lane 2, PCR product (952 bp) generated by primer 1 and 2; lane 3, PstI digestion pattern (390, 291, and 271 bp) for the PCR product by primer 1 and 2; lane 4, HindIII digestion pattern (648 and 304 bp) for the PCR product by primer 1 and 2; lane 5, PCR product (619 bp) generated by primer 3 and 4; lane 6, negative control. DNA without reverse transcriptase was generated by RT-PCR using primer 1 and 2. DNA separation is carried out by 1.8% agarose gel electrophoresis.

Inhibition of 15-LO in NaBT-treated Caco-2 Cells-- The next question is whether the inducible 15-LO plays a role in the NaBT-induced apoptosis. To address this question, we treated cells with an inhibitor of 15-LO, NDGA. NDGA is well known as the inhibitor of lipoxygenases, but it can inhibit cyclooxygenase at high concentrations (29). The optimum concentration of NDGA for inhibition of the lipoxygenase without inhibiting Cox-2 activity was determined by HPLC analysis of arachidonic acid metabolism. NDGA, 10 µM, which inhibited lipoxygenase activity without significant effects on Cox activity (data not shown), was used for the following apoptosis experiment. To examine if the 15-LO modulated NaBT-induced apoptosis, we measured the number of floating (apoptotic) and attached cells during the course of NaBT treatment in the presence of the 15-LO inhibition NDGA. This method has been extensively used to estimate apoptosis in colorectal cells (30). Apoptosis of the floating cells was also confirmed by DNA fragmentation analysis. The ratio of apoptosis to attached cells in NaBT-treated Caco-2 cells gradually increased with duration of NaBT treatment. NDGA treatment significantly enhanced the NaBT-induced apoptosis compared with that of NaBT-treated Caco-2 cells without NDGA at each time point from 48 to 96 h (apoptotic ratio: at 96 h, 0.51 ± 0.025 S.E. in NaBT, 0.85 ± 0.009 S.E. in NaBT plus NDGA) (Fig. 7A). Treatment with NDGA alone did not enhance apoptosis in the Caco-2 cells. Moreover, the nucleosomal DNA ladder formation could only be seen in floating cells from the Caco-2 cells treated with NaBT or NaBT with NDGA (Fig. 7B). The data support the conclusion that inhibition of 15-LO by NDGA enhances apoptosis in NaBT-treated Caco-2 cells.


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Fig. 7.   Effect of NDGA for NaBT-induced apoptosis in Caco-2 cells. A, enhanced apoptosis by 10 µM NDGA in NaBT-treated Caco-2 cells. The 60% confluent cells were cultured in the various conditioned media on 10-cm2 dishes. Cell counts of attached cells and floating cells were determined at 24, 48, 72, and 96 h, and the ratio of floating cells to total cells are plotted. These results are mean ± S.E. of five separate dishes. bullet , FBS; black-triangle, 5 mM NaBT; open circle , 10 µM NDGA in FBS; triangle , 10 µM NDGA in NaBT. B, DNA from NaBT-cultured cells with and without NDGA for 96 h was prepared and analyzed by 2% agarose gel electrophoresis. Each lane contains genomic DNA extracted from 1 × 106 cells. lane 1, attached cells of NaBT treatment; lane 2, attached cells of NaBT and 10 µM NDGA treatment; lane 3, floating cells of NaBT treatment; lane 4, floating cells of NaBT and 10 µM NDGA.

Inhibition of Cox-2 in NaBT-treated Caco-2 Cells-- Several questions arise from these data. Is NaBT-induced apoptosis regulated by Cox-2 expression in Caco-2 cells as is reported for overexpressing Cox-2 in RIE cells (7), and is the expression of 15-LO linked to Cox-2 in these cells? Cox-2 in Caco-2 cells was decreased by NaBT treatment as indicated by Northern and immunoblot analysis at different times after NaBT treatment. In contrast, 15-LO expression increased during the NaBT treatment (Fig. 3). To address these questions, indomethacin was added to the cells to inhibit Cox-2 activity, and based upon reports in the literature (7), we expected Cox-2 inhibition to result in an increase in apoptosis in the NaBT-treated cells. However, the addition of indomethacin (1 µM) at every 12 h did not alter the number of apoptotic floating cells in the NaBT-treated cells or FBS-treated cells at any time point examined, as shown in Fig. 8A. This result seems to suggest the decreasing Cox-2 is not modulating NaBT-induced apoptosis in Caco-2 cells. To further examine this issue, we measured the expression of Cox-2 and 15-LO in the attached cells under these conditions. Protein expression could not be estimated in apoptotic cells because of degradation. Fig. 8B shows the comparative expression of Cox-2 and 15-LO by immunoblot analysis. NaBT treatment stimulated the expression of 15-LO as expected, but the addition of indomethacin (1 µM) further enhanced the expression of the 15-LO in the NaBT-treated cells at 24, 48, and 72 h after treatment. Cox-2 expression was decreased by NaBT treatment. The addition of indomethacin (1 µM) to NaBT-treated cells further decreased the Cox-2 expression as compared with cells treated with NaBT alone. These data indicate that the expression of this inducible 15-LO is linked to the Cox-2 expression during the induction of apoptosis. Suppression of Cox-2 expression by NaBT with or without indomethacin is compensated by an expression of 15-LO in Caco-2 cells.


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Fig. 8.   Effect of indomethacin for NaBT-induced apoptosis in Caco-2 cells. A, the effect of 1 µM indomethacin for NaBT-induced apoptosis in Caco-2 cells. The 60% confluent cells were cultured in the various conditioned medium on 10-cm2 dishes. Cell counts of attached cells and floating cells were determined at 24, 48, 72, and 96 h, and ratio of floating cells to total cells are plotted. These results are mean ± S.E. of five separate dishes. bullet , FBS; black-triangle, 5 mM NaBT; open circle , 1 µM indomethacin in FBS; triangle , 1 µM indomethacin in NaBT. B, comparative expression of Cox-2 and 15-LO by NaBT-treated cells incubated with and without indomethacin (1 µM, Indo). The near confluent Caco-2 cells cultured in FBS were treated with 5 mM NaBT or the fresh FBS with or without indomethacin (1 µM). Protein was extracted from the attached cells at 24, 48, and 72 h after treatment and separated on 8% SDS-polyacrylamide gel. Each lane contains 15 µg of the total cell lysates. Expression of Cox-2 and 15-LO were estimated by using anti-human Cox-2 and human 15-LO antibody.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Colorectal tumor formation results from the accumulation of specific genetic changes. The homeostasis of the colorectal epithelial cells, which give rise to the tumor, depends not only on the rate of cell proliferation but also on apoptosis. Mutation in the APC gene is an early event in the development of colorectal cancer and is associated with a decrease or inhibition in apoptosis (30). Studies with mice clearly link the loss of APC gene function with the induction of Cox-2 and polyp growth (3). In addition, other studies using mice with the disrupted Cox-2 gene provide evidence to support the hypothesis that Cox-2 expression is an early rate-limiting step in the development of adenoma formation (2). The most compelling evidence linking the overexpression of Cox-2 to altered apoptosis and cell differentiation was provided by studies with RIE cells (7). The overexpression of Cox-2 in RIE cells prevented NaBT and adhesion-induced apoptosis and cell differentiation. Apoptosis was restored by treatment with a Cox inhibitor (7). Human colorectal cell lines have been useful in investigations of the cellular mechanisms associated with Cox-2 expression and altered apoptosis. Apoptosis in human colorectal carcinoma cell lines can be induced by inhibitors of Cox-2 by both Cox-2 dependent (31, 32) and independent (8, 9) pathways. However, the addition of exogenous prostaglandins failed to reverse inhibitor-induced apoptosis (8).

We have examined unsaturated fatty acid metabolism and the expression of the enzymes responsible for lipid metabolism during NaBT-induced apoptosis and cell differentiation in human colorectal carcinoma Caco-2 cells. These cells have been frequently used, undergo apoptosis and cell differentiation in response to NaBT, and have a mutated APC gene expressing the truncated APC protein product (30, 33). The arachidonic acid metabolism by Caco-2 cells was dramatically shifted during the NaBT treatment from prostaglandins to metabolites of 15-LO, mainly 15-HETE. Likewise, the metabolism of linoleic acid, a predominate unsaturated fatty acid present in cells and the preferred substrate for the reticulocyte 15-LO, was dramatically shifted from 9-HODE to 13-HODE. Furthermore, the NaBT treatment resulted in a significant enhancement of overall cis-unsaturated fatty acid metabolism with the lipoxygenase-derived metabolites predominating. Caco-2 cells poorly expressed Cox-1, but high expression of Cox-2 was observed. Treatment with NaBT modestly attenuated the expression of Cox-2 and significantly increased the expression of a 15-LO. The NaBT-induced 15-LO in Caco-2 cells was confirmed by RT-PCR and restriction enzyme analysis as the human reticulocyte 15-LO isolated by Sigal et al. (27). Ample evidence is presented in this report to support the conclusion that during NaBT-induced apoptosis and cell differentiation in Caco-2 cells, significant expression of the human reticulocyte 15-LO occurs, which shifts the metabolism from prostaglandin to lipoxygenase-catalyzed metabolites. This is the first report demonstrating the expression and enzymatic activity of human reticulocyte 15-LO in a human colorectal carcinoma cell line. Whether 15-LO expression also occurs in human colon tumors is currently under investigation.

The relationship between Cox-2 expression, which is modulated by the APC gene alteration, and 15-LO expression during apoptosis is apparent. During the course of NaBT treatment, there seems to be an inverse relationship between Cox-2 expression and 15-LO expression. At times during the apoptosis and cell differentiation process where the lowest expression of Cox-2 was detected, the highest 15-LO expression was observed. In addition, treatment of the cells with an inhibitor of Cox-1 and -2, indomethacin, attenuates the expression of Cox-2 in NaBT-treated cells and enhances the expression of 15-LO by NaBT at times during the course of apoptosis. These findings suggest Cox-2 and 15-LO are working in parallel to modulate the process of apoptosis and cell differentiation in the Caco-2 cells. The regulation of Cox-2 has been extensively studied, and several studies have indicated either the up-regulation (34, 35) or down-regulation (36, 37) by the stable metabolites formed from arachidonic acid by this enzyme. Studies suggest a regulation via the SP-1, NFkappa B, NF-IL6, AP-1, AP-2, and CRE sites present in the Cox-2 gene (38, 39), but regulation by NaBT has not been reported. The regulation of the human 15-LO is poorly understood. The inflammatory cytokines IL-4 and IL-13 enhance the expression of 15-LO in the human lung carcinoma A-459 (40), human monocytes (41-43), and human bronchial epithelial cells (25). The expression of the 15-LO is most notable in human airway epithelial cells (44, 45) but has also been detected in monocytes and skin (46). There is no report that NaBT, the inducer of cell differentiation or apoptosis by the mechanism of histone hyperacetylation (47), regulates the expression of 15-LO.

The importance of the 15-LO expression in the process of NaBT-induced apoptosis and cell differentiation is not clear. Inhibition of the lipoxygenase activity by NDGA at concentrations determined to attenuate only the 15-LO activity in these cells enhanced the NaBT-induced apoptosis, which supports the hypothesis for 15-LO metabolites acting as inhibitors of apoptosis. Several reports in the literature (14, 15) and in this laboratory2 also suggest that lipoxygenase metabolites including 15-LO metabolites can act as inhibitors of apoptosis. However, NDGA is a general inhibitor of lipoxygenases and can influence the redox state of cells (48, 49), so this conclusion must be viewed with caution. We considered directly testing the 15-LO metabolites of arachidonic acid and linoleic acid, but the high binding of the metabolites to proteins and other materials in the tissue culture system severely compromises this approach. An alternate approach is to prepare Caco-2 cells, which highly express 15-LO, as a tool to study the importance of the lipoxygenase in apoptosis. These experiments are currently in progress. Indomethacin at concentrations that inhibit Cox-2 did not attenuate NaBT-induced apoptosis, which was an unexpected result in view of other reports in the literature that clearly indicate inhibition of Cox-2 enhances apoptosis in colorectal carcinoma cells (50). An explanation for this observation was provided by analysis of the expression of Cox-2 and 15-LO under these conditions. Indomethacin enhanced the expression of 15-LO, which should increase the formation of 15-LO-derived metabolites. These metabolites would act to replace the prostaglandins and attenuate the apoptosis. Thus, the net response to indomethacin treatment is lack of effect on NaBT-induced apoptosis. Thus, Cox-2 and 15-LO may act in concert to attenuate apoptosis in the Caco-2 cells. The inverse relationship between Cox-2 and 15-LO expression (note Fig. 3) is further support for this notion.

Linoleic acid and not arachidonic acid is the preferred substrate of the reticulocyte 15-LO, and thus linoleic acid metabolites must be considered as potentially biologically active. Considerable 13-HODE was produced from the cell lysates from NaBT-treated cells during cell differentiation and apoptosis. Because NaBT induced cell differentiation as well as apoptosis, we must also consider if these lipids are modulators of cell differentiation. In the human airway epithelium, the expression of 15-LO is observed during retinoid-induced cell differentiation (25). Furthermore, increased expression of rabbit reticulocyte 15-LO is observed during reticulocyte maturation (51). The overexpression of peroxisome proliferator-activated receptor (PPAR)gamma in rat intestinal tumors was recently reported, and Caco-2 cells were found to express the highest levels of PPARgamma of all the human colorectal cell lines tested (52). Furthermore, 13(S)-HODE and 9(S)-HODE bind to and activate the PPARgamma (53). These lipid metabolites induce monocyte maturation and regulate gene expression. The induction of 15-LO during NaBT treatment of Caco-2 cells may be a response to rescue the cells from apoptosis. One can speculate that the 15-LO acts as an anti-apoptotic signal that directs the cell from apoptosis to a newly differentiated cell. The overexpression of Cox-2 observed after APC mutations acts as a more prolonged or constitutive signal to attenuate apoptosis. Thus, both Cox-2 and 15-LO may act to modulate apoptosis and cell differentiation in colorectal carcinoma cells.

    ACKNOWLEDGEMENTS

We thank Julie Angerman-Stewart for technical assistance, Dr. Elliott Sigal for kindly providing 15-LO antibody, and Dr. Raymond N. DuBois for helpful discussions throughout these studies.

    FOOTNOTES

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

Dagger To whom correspondence should be addressed: Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3911; Fax: 919-541-0146; E-mail: Eling{at}niehs.nih.gov.

The abbreviations used are: Cox, cyclooxygenase; NaBT, sodium butyrate; HETE, hydroxyeicosatetraenoic acid; LO, lipoxygenase; HODE, hydroxyoctadecadienoic acid; RT-PCR, reverse transcription-polymerase chain reaction; RIE, rat intestinal epithelial; HpODE, hydroperoxyoctadecadienoic acid; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; PG, prostaglandin; PPAR, peroxisome proliferator-activated receptor; kb, kilobase(s); bp, base pair(s); NDGA, nordihydroguaiaretic acid.

2 W. C. Glasgow, A. L. Everhart, and Eling, T. E., unpublished observation.

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Abstract
Introduction
Procedures
Results
Discussion
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