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Carcinogenesis Advance Access originally published online on May 19, 2006
Carcinogenesis 2006 27(11):2308-2315; doi:10.1093/carcin/bgl073
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Alpha-tocopheryl succinate, in contrast to alpha-tocopherol and alpha-tocopheryl acetate, inhibits prostaglandin E2 production in human lung epithelial cells

Eunmyong Lee1,2, Moon-Kyung Choi2, Young-Ju Lee2, Ja-Lok Ku3, Kyung-Hee Kim3, Jin-Sung Choi3 and Soo-Jeong Lim1,2,*

1 Department of Bioscience and Biotechnology, Sejong University Seoul, Korea
2 Research Institute, National Cancer Center Goyang, Gyeonggi, Korea
3 Laboratory of Cell Biology, Cancer Research Center and Cancer Research Institute, Seoul National University College of Medicine Seoul, Korea

*To whom correspondence should be addressed at: Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Korea. Tel: +82 2 3408 3334; Fax: +82 2 3408 3334; Email: sjlim61{at}hotmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The production of prostaglandin E2 (PGE2), a key proinflammatory mediator, is regulated by the availability of its substrate, arachidonic acid (AA), and the activity of the enzyme cyclooxygenase (COX). Increased PGE2 production and COX-2 expression have been observed frequently in specimens from lung cancer patients. Agents that decrease PGE2 production may prevent the initiation and progression of lung cancer. We, therefore, tested the effects of alpha-tocopherol ({alpha}TOL) analogs on PGE2 production in human lung epithelial cells. Alpha-tocopheryl succinate ({alpha}TOS), but not {alpha}TOL or alpha-tocopheryl acetate ({alpha}TOA), inhibited the phorbol 12-myristate 13-acetate (PMA)-stimulated PGE2 production in three human lung epithelial cell lines (BEAS-2B, H460 and A549 cells). The effect of these compounds on PGE2 production was not correlated with their antioxidant activities, since {alpha}TOS alone did not inhibit PMA-induced generation of reactive oxygen species. {alpha}TOS had no effect on PMA-induced AA release or COX-2 expression, although post-incubation with {alpha}TOS inhibited COX activity and prostaglandin (PGE2 and PGF2{alpha}) production in PMA-stimulated cells. {alpha}TOS also blocked the COX activity in A549 cells with endogenous high levels of COX enzymes in the absence of PMA stimulation. In addition, the ability of {alpha}TOS to inhibit COX was affected by AA concentration, suggesting that {alpha}TOS may compete with AA for interaction with COX proteins. These results suggest that {alpha}TOS inhibits COX activity, thereby inhibiting PGE2 production in human lung epithelial cells, despite the lack of antioxidant activity. Administration of {alpha}TOS may block inflammatory responses mediated by PGE2, thereby inhibiting the initiation and progression of lung cancer.

Abbreviations: AA, arachidonic acid; {alpha}TOA, alpha-tocopheryl acetate; {alpha}TOL, alpha-tocopherol; {alpha}TOS, alpha-tocopheryl succinate; COX, cyclooxygenase; DCF, 2', 7'-dichlorofluorescein; DCFH-DA, 2', 7'-dichlorodihydrofluorescein-diacetate; PG, prostaglandin; PGE2/F2{alpha}, prostaglandin E2/F2{alpha}; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Chronic inflammation is a major contributor to the development of degenerative diseases, including cancer, cardiovascular diseases and neurodegenerative disorders (1). Inflammatory mediators such as cytokines and eicosanoids are critical for the initiation and maintenance of cancer cell survival and growth. For example, the concentration of prostaglandin E2 (PGE2) is higher in tumor tissues than in normal tissues (2). In tumor cells, PGE2 has been found to inhibit apoptosis and induce proliferation (3). PGE2 also increases tumor progression by altering cell morphology and by increasing cell motility and migration. Therefore, specific inhibition of PGE2 production may suppress tumor initiation and progression.

The production of PGE2 begins with the liberation of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2). Subsequently, cyclooxygenase (COX) enzymes convert AA to PGH2 (prostagladin H2), which is converted to various prostaglandins (PGs), including PGE2. Therefore, PGE2 production can be regulated by both substrate availability and COX enzyme activity (4). Of the two isoforms of COX, COX-1 is expressed constitutively in most tissues of the body and acts as a housekeeper enzyme, whereas COX-2 is induced by cytokines, growth factors, carcinogens and tumor promoters (5).

Antioxidant vitamins, which protect against oxidants such as those produced during inflammation, are believed to be important in cancer prevention, primarily by inhibiting inflammatory responses (1). Alpha-tocopherol ({alpha}TOL), one of vitamin E family members, has a potent antioxidant activity and it is the predominant form of vitamin E in vitamin supplements (6). {alpha}TOL also has antithrombotic, anticoagulant, neuroprotective, antiproliferative, immunomodulatory, cell membrane-stabilizing and antiviral actions, which may or may not be associated with its antioxidant activity (7,8). {alpha}TOL has been found to suppress PGE2 production. For example, {alpha}TOL was shown to suppress the oxidized low-density lipoprotein (LDL)-induced PLA2 activity in rat mesangial cells (9) and to attenuate lipopolysaccharide (LPS)-induced COX-2 transcription and synthesis in microglial cells (10). In macrophage cells and aged mice, {alpha}TOL and its analog alpha-tocopheryl acetate ({alpha}TOA) have been found to decrease COX activity but to have no effect on the level of expression of COX mRNA and protein (11,12). Taken together, these findings suggest that supplementation with {alpha}TOL may be beneficial in preventing diseases related to PGE2 mediated inflammatory responses.

Alpha-tocopheryl succinate ({alpha}TOS), which is obtained by the esterification of {alpha}TOL, is a more stable powder form of {alpha}TOL, since the succinate group protects the hydroxyl group of the chromanol ring from oxidation. Despite their similar chemical structures, {alpha}TOS differs in activity from {alpha}TOL. For example, {alpha}TOS is redox-insensitive prior to being cleaved to {alpha}TOL by esterases (13). In contrast to {alpha}TOL, {alpha}TOS induces growth suppression, cell cycle arrest and/or apoptosis in a wide range of cancer cells (14), as well as acting synergistically with the chemopreventive agent selenite to inhibit the growth of prostate cancer cells (15). In addition, {alpha}TOL inhibits protein kinase C (PKC) activity (16), whereas {alpha}TOS activates or inhibits PKC depending on the experimental conditions (17,18).

Lung cancer is one of the leading causes of cancer-related deaths worldwide (19). Since COX-2 overexpression and increased PGE2 production are frequently observed in tissue specimens from lung cancer patients, an agent that decreases PGE2 production at pharmacologically achievable doses may be effective in suppressing the initiation and progression of lung cancer. We therefore tested the effect of {alpha}TOL analogs on PGE2 production in human lung epithelial cells treated with phorbol 12-myristate 13-acetate (PMA), which stimulates AA release (20) and PGE2 production (21). We found that pharmacologically achievable doses of {alpha}TOS, but not of {alpha}TOL or {alpha}TOA, inhibited PGE2 production in human lung epithelial cells despite the lack of antioxidant activity. It seems likely that {alpha}TOS inhibition of PGE2 production is associated with inhibition of COX activity rather than modulation of COX expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines and cultures
The human H460, A549 lung cancer cell lines and BEAS-2B immortalized bronchial epithelial cells were purchased from the ATCC (Manassas, VA). BEAS-2B cells were grown in a Bullet kit (Clonetics, Walkersville, MD) containing serum-free bronchial epithelial cell growth medium, whereas H460 and A549 cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT) and 100 U/ml each of penicillin and streptomycin. Cells were grown in incubators in a humid atmosphere of 95% air and 5% CO2.

Reagents and antibodies
AA and antibodies to human COX-2 were obtained from Cayman Chemical (Ann Arbor, MI). {alpha}TOS, {alpha}TOA, {alpha}TOL and PMA were purchased from Sigma (St Louis, MO). 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR). Antibodies to human COX-1 and cPLA-2 were purchased from Santa Cruz Biotechnology (Santa cruz, CA) and antibodies to ß-actin were purchased from Sigma (St. Louis, MO). All other chemicals were of reagent grade and used without further purification. {alpha}TOL analogs were dissolved in dimethyl sulfoxide (DMSO) at 50 mM and then diluted with culture media. The final concentration of DMSO in all samples did not exceed 0.1%.

Immunoblotting
Treated cells were scraped from the culture, washed twice with PBS and incubated for 15–30 min on ice in lysis buffer containing 150 mM NaCl, 10 mM Tris, 0.2% Triton X-100, 0.3% NP-40, 0.2 mM Na3VO4 and protease inhibitors (pH 7.4) (Roche). After centrifugation at 13 200x g for 15 min at 4°C, supernatants were collected and the protein concentration in each was measured by the Bradford method. Aliquots of supernatants containing equal amounts of protein were boiled in SDS-reducing buffer for 5 min, electrophoresed on SDS–polyacrylamide gels and transferred on to nitrocellulose membranes. Membranes were blocked with 4% non-fat dry milk and probed with specific primary antibodies, followed by incubation with appropriate peroxidase-conjugated secondary antibodies. Blots were developed with ECL reagent (Amercham, Arlington Heights, IL) according to the manufacturer's protocol.

PGE2, PGF2{alpha} release and COX activity assay
Cells (5 x 104 cells/ml) plated in 24-well tissue culture plates in RPMI 1640 medium containing 5% FBS were treated with various reagent. After incubation, the supernatant conditioned medium was harvested and then assayed for PGE2 or PGF2{alpha} levels using a specific enzyme immunoassay (EIA) kit according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). Medium alone without cells was incubated under the same conditions and used as blank control for the EIA. Levels of PGE2 or PGF2{alpha} were normalized to the number of cells.

In separate experiments designed to determine the activity of the COX enzyme, COX activity was quantified by providing cells with exogenous AA, the substrate for COX and measuring its conversion to PGE2. Briefly, cells were treated, after which cells were washed with phosphate-buffered saline (PBS; pH 7.4), and fresh medium containing AA was added for 20 min at 37°C; then the medium was collected and subjected for the PGE2 EIA.

AA release
Cells in 24-well plates were labeled with [3H]AA (0.1 µCi/ml/well) (Perkin-Elmer Life Science, Boston, MA) for 12–16 h at 37°C in serum-free RPMI media. After labeling, cells were washed three times with PBS and incubated in 0.5% bovine serum albumin (BSA)-containing serum-free medium containing various reagents as indicated for the specific experiments. At the end of the incubation, the medium was removed and centrifuged at 3000x g for 10 min to remove floating cells. The radioactivity in the supernatant was then measured by liquid scintillation spectrometry.

Reactive oxygen species (ROS)-generation assay
Non-fluorescent DCFH-DA, hydrolyzed to DCFH inside cells, yields highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of intracellular H2O2 and related peroxides. Therefore, the DCF fluorescence intensity has been reliably used to evaluate the total generation of ROS including superoxide, hydrogen peroxide and peroxynitrite formed intracellularly (22). After treatment, the cells were harvested, washed twice with PBS, resuspended in serum-free medium, and incubated with 5 µM DCFH-DA for 15 min at 37°C. The cells were washed with ice-cold PBS and placed on ice, and cell fluorescence was measured by flow cytometry (FACSCalibur, BD Biosciences). As a positive control, cells were treated with H2O2 and processed for ROS detection.

Cell growth and viability assay
Cells were seeded in 96-well plates in 0.1 ml of media supplemented with 5% FBS. On the following day cells were treated with {alpha}TOS. Control and treated cultures received the same amount of DMSO. Cell growth and viability were measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The formation of formazan crystals by active mitochondrial respiration in cells was determined using a microplate spectrophotometer (BioTek, Winooski, VT) after dissolving the crystals in DMSO.

Apoptosis enzyme-linked immunosorbent assay (ELISA)
Histone-associated DNA fragments were quantified using a photometric enzyme immunoassay using Cell Death Detection ELISAplus (Roche Applied Bioscience) following the manufacturer's protocol.

Statistical analysis
Statistically significant differences between values obtained under different experimental conditions were determined using two-tailed unpaired Student's t-tests.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
{alpha}TOS, but not {alpha}TOL or {alpha}TOA, decreased PMA-stimulated PGE2 production in lung epithelial cells
We assayed the effects of {alpha}TOL analog pretreatment on PMA-stimulated PGE2 production in several human lung epithelial cell lines with different basal expression of COX-1 and COX-2 proteins (Figure 1A). In H460 cells, 50 ng/ml PMA increased PGE2 production 20-fold compared with vehicle-treated cells, whereas pretreatment with 10 and 20 µM {alpha}TOS resulted in PGE2 production that was 35.1 and 13.0% of that in cells treated with PMA alone, respectively (Figure 1B). In BEAS-2B cells, PGE2 production was increased 2-fold by PMA, whereas pretreatment with 20 µM {alpha}TOS caused PGE2 production to revert to basal levels (Figure 1C). In A549 cells, PGE2 production was increased 6.8-fold by PMA, whereas pretreatment with 20 µM {alpha}TOS resulted in PGE2 production that was 46.0% of that in cells treated with PMA alone (Figure 1D). We found that at concentrations of 20 and 100 µM, of both {alpha}TOL and {alpha}TOA had no effect on PMA-stimulated PGE2 production in these cell lines (Figure 1B–D). Thus, of the {alpha}TOL analogs, only {alpha}TOS could inhibit PMA-stimulated PGE2 production in lung epithelial cell lines.


Figure 1
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  		Fig. 1 Effects of {alpha}TOL analogs on PMA-induced PGE2 production in human lung epithelial cells. (A) COX-1 and COX-2 protein expression in lung epithelial cell lines. Cell lysates were obtained from exponentially growing cells and subjected to immunoblotting with appropriate antibodies. Immunoblotting with an antibody to ß-actin was used to ensure equal loading of proteins in each lane (lower panel). (B) H460, (C) BEAS-2B and (D) A549 lung epithelial cells were plated at 5 x 104 cells/well density in duplicates for each experiment. Cells were pretreated with indicated doses of {alpha}TOS, {alpha}TOL, {alpha}TOA and the corresponding amount of DMSO in controls for 4 h before the treatment with 50 ng/ml of PMA. After overnight incubation, conditioned medium was collected and the PGE2 concentration in the conditioned media was determined by an ELISA and normalized to cell numbers as described in the text. Results are for duplicate assays in each of at least two independent experiments (mean ± SD). **, P < 0.005; ***, P < 0.001 by unpaired t-test.

 
Utilizing HPLC analysis to determine the time-dependent uptake of {alpha}TOS, {alpha}TOL and {alpha}TOA into H460 cells (23), we found that all three were taken up at comparable rates (data not shown). This was consistent with earlier findings (24) and indicated that differential effects of {alpha}TOL analogs on PGE2 production were not due to differences in cellular uptake.

{alpha}TOL and {alpha}TOA, but not {alpha}TOS, inhibited PMA-induced ROS generation in lung epithelial cells
Previous findings have indicated that vitamin E analogs inhibit PGE2 production via the regulation of enzymes in the PGE2 synthesis pathway, and that this inhibition is associated with antioxidant activity (12). Although {alpha}TOS is redox-insensitive, it may be rapidly changed to a metabolite with antioxidant activity even more potent than that of {alpha}TOL. To compare the antioxidant activities of the three {alpha}TOL analogs, we stimulated A549 cells with PMA, which generates ROS (20), and measured ROS generation by the increase in DCF fluorescence. We found that exposure of A549 cells for 2 h to 1 µg/ml PMA increased DCF fluorescence 1.4-fold (Figure 2). Pretreatment of A549 cells with {alpha}TOS had no effect on ROS generation, whereas pretreatment with {alpha}TOL or {alpha}TOA completely blocked the PMA-induced increase in ROS (Figure 2). These findings suggest that neither {alpha}TOS nor a possible intracellular metabolite acts as an antioxidant in lung epithelial cells and that {alpha}TOS appears to inhibit PGE2 production despite its lack of antioxidant activity.


Figure 2
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  		Fig. 2 Effect of {alpha}TOL analogs on the PMA generation of ROS in A549 cells. Cells were treated with vehicle alone or with the indicated concentrations of {alpha}TOL analogs. After 4 h incubation, 1 µg/ml of PMA was added. Cells incubated with PMA for 2 h were subjected to ROS determination. ROS data are expressed as the increase in channel fluorescence of treated cells relative to vehicle-treated cells. Results are for duplicate assays in each of two independent experiments (mean ± SD). **, P < 0.005 by unpaired t-test.

 
{alpha}TOS did not block PMA-induced AA release
To gain insight into the mechanism by which {alpha}TOS regulates PGE2 production in lung epithelial cells, we tested whether {alpha}TOS regulates the process leading to the release of AA. We found that PMA had no effect over time on the expression of cPLA2, the enzyme primarily responsible for the production of AA, in H460, A549 and BEAS-2B cells (Figure 3A). In contrast, AA release was time-dependently increased by incubation with PMA (Figure 3B), and this increase was not blocked by {alpha}TOS pretreatment (Figure 3C). Furthermore, {alpha}TOS inhibition of PGE2 production was observed in the presence of exogenously supplied AA (data not shown). These data suggest that {alpha}TOS inhibited PMA-induced PGE2 release without affecting AA release.


Figure 3
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  		Fig. 3 Effect of {alpha}TOS on the PMA-induced AA release in human lung epithelial cells. (A) PMA did not cause upregulation of cPLA2 expression. cPLA2 expression was assayed by immunoblotting, in cells harvested after incubation with PMA for indicated times. Immunoblotting with an antibody to ß-actin was used to control for equal loading of proteins per lane. (B) PMA time-dependently caused the induction of AA release. [3H]AA-labeled H460 cells were incubated with PMA (50 ng/ml). At the time indicated, the amount of [3H]AA released into the media was determined and expressed as a percentage of the radioactivity of media treated with vehicle alone. (C) {alpha}TOS pretreatment did not reverse the PMA-induced AA release. [3H]AA-labeled H460 cells were treated with indicated dose of {alpha}TOS for 4 h, and then incubated with PMA (50 ng/ml). At 9 h post-incubation, [3H]AA release was determined as described in the text. The results shown are the average of two different experiments assayed in triplicate.

 
{alpha}TOS did not affect PMA upregulation of COX-2
We next investigated whether {alpha}TOS inhibited PGE2 production by inhibiting PMA-induced upregulation of COX. Incubation of lung epithelial cells with PMA increased COX-2 protein expression in a time-dependent manner, whereas COX-1 expression remained unchanged (Figure 4A). The vehicle, DMSO, had no effect on PMA-induced COX-2 protein expression. COX-2 mRNA expression was also upregulated by PMA (data not shown). Pretreatment of cells with {alpha}TOS did not significantly inhibit PMA upregulation of COX-2 (Figure 4B), suggesting that {alpha}TOS does not interfere with the process leading to COX-2 upregulation.


Figure 4
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  		Fig. 4 Effect of {alpha}TOS on the expression and activity of COX induced by PMA. (A) PMA caused COX-2 upregulation, while retaining COX-1 expression. COX-1 and COX-2 expression was assayed by immunoblotting, in H460, BEAS-2B and A549 cells harvested after incubation with PMA for indicated times. Immunoblotting with an antibody to ß-actin was used to control for equal loading of proteins per lane. (B) {alpha}TOS did not affect PMA-induced COX-2 upregulation in lung epithelial cells. COX-1 and COX-2 expression was assayed by immunoblotting, in H460 cells harvested after incubation with PMA in the presence of 20 µM of {alpha}TOS.

 
{alpha}TOS inhibition of COX activity
Since {alpha}TOS had no effect on PMA-induced AA release or COX expression, we tested the effect of {alpha}TOS on COX activity in intact cells. We found that exposure of PMA-prestimulated H460 cells to {alpha}TOS resulted in a decrease in COX activity (Figure 5A). {alpha}TOS inhibition of COX activity was also observed in other cell lines prestimulated with PMA (data not shown). Under these experimental conditions, COX-2 protein levels were not affected by treatment with {alpha}TOS in any cell lines (data not shown). These data suggest that {alpha}TOS inhibited COX activity in intact lung epithelial cells.


Figure 5
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  		Fig. 5 {alpha}TOS inhibited COX activity in lung epithelial cells. (A) Inhibition of COX activity by post-incubation with {alpha}TOS in PMA-stimulated H460 cells. Cells were pretreated with PMA. After 6 h incubation, cells were washed and incubated with fresh medium containing vehicle or {alpha}TOS for 16 h. Medium was then removed and replaced with a fresh serum-free media containing 10 µM AA for 20 min. PGE2 in the medium was measured. COX activity (%) was expressed as the ratio of PGE2 produced in cells treated with PMA in the presence of {alpha}TOS to that in cells treated with PMA alone. The results shown are the average of two different experiments assayed in duplicate. *, P < 0.05;**, P < 0.005 by unpaired t-test, compared with cells treated with PMA alone (B) COX inhibitory effect of {alpha}TOS in A549 cells. The COX activity of A549 cells was determined after overnight incubation with {alpha}TOS or {alpha}TOL at indicated concentrations. **, P < 0.005 by unpaired t-test, compared with control cells. (C) Immunoblot analysis of COX-1 and COX-2 expression in A549 cells treated with {alpha}TOS for 16 h. (D) Effect of AA on the {alpha}TOS-induced decreases in COX activity. A549 cells were incubated with 20 µM of {alpha}TOS for 16 h. After removing supernatant, fresh media containing indicated concentrations of AA was added and incubated for another 20 min. PGE2 in the medium was measured, and the COX activity (%) was expressed as the ratio of PGE2 produced in cells treated with {alpha}TOS to that in cells treated with DMSO at the corresponding AA concentrations. The results shown are the average of two different experiments assayed in duplicate.

 
To determine if {alpha}TOS inhibits COX activity in cells with endogenous levels of these COX enzymes in the absence of PMA stimulation, we tested the effects of {alpha}TOS on COX activity in A549 cells, which produce high endogenous levels of PGE2 due to the relatively high expression of COX-2 (Figure 1A). We found that {alpha}TOS dose-dependently inhibited COX activation (Figure 5B) and PGE2 production (data not shown), without changing the levels of COX-1 and COX-2 expression (Figure 5C). The ability of {alpha}TOS to inhibit COX activity decreased with increasing AA concentration (Figure 5D), suggesting that {alpha}TOS may act as a competitive inhibitor of AA.

In addition to PGE2, COX can produce other prostaglandins such as PGF2{alpha}. To investigate whether COX inhibition by {alpha}TOS also similarly affected the production of other prostaglandins, we checked the {alpha}TOS-induced changes in the PGF2{alpha} production in lung epithelial cells. Regardless of PMA stimulation, the PGF2{alpha} production was too low to detect any changes in both H460 and BEAS-2B cells (data not shown). In A549 cells, PMA stimulation greatly increased the PGF2{alpha} production, which was dose-dependently blocked by {alpha}TOS treatment (Figure 6). Considered together, these data suggest {alpha}TOS inhibition of COX activity as one mechanism by which it blocks the production of prostaglandins in lung epithelial cells


Figure 6
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  		Fig. 6 {alpha}TOS blocked the PGF2{alpha} production in PMA-stimulated lung epithelial cells. A549 cells were pretreated with PMA. After 6 h incubation, cells were washed and incubated with fresh medium containing vehicle or {alpha}TOS for 16 h. After overnight incubation, conditioned medium was collected and the PGF2{alpha} concentration in the conditioned media was determined by an ELISA and normalized to cell numbers as described in the text. Results are for duplicate assays in each of at least two independent experiments (mean ± SD). *, P < 0.05 by unpaired t-test.

 
Apoptosis induced by TOS was not mediated by PGE2 suppression
Earlier studies including ours showed that {alpha}TOS, but not {alpha}TOL or {alpha}TOA, inhibited growth and induced apoptosis in human lung cancer cells (23), raising one possibility that {alpha}TOS may influence the lung cancer cell growth and death via suppressing PGE2 production through modulation of COX activity. To investigate this, we checked whether incubation with {alpha}TOS at doses inhibiting PGE2 production (10 and 20 µM) up to 72 h induced apoptosis in A549 cells. We found no significant changes in the growth and apoptosis in {alpha}TOS-treated cells (data not shown). Apoptosis was observed only when A549 cells were treated with {alpha}TOS at doses >40 µM, and the addition of exogenous PGE2 to the medium did not reverse the growth inhibition and apoptosis induced by 40 µM {alpha}TOS (Figure 7A). Furthermore, growth inhibition and apoptosis induced by {alpha}TOS was not affected by forced COX-2 expression in H460 cells (25) (Figure 7B). These data suggest that {alpha}TOS ability to induce growth inhibition and apoptosis in lung cancer cells is not directly associated with its ability to suppress PGE2 production.


Figure 7
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  		Fig. 7 {alpha}TOS ability to induce growth inhibition and apoptosis in lung cancer cells is not directly associated with its ability to suppress PGE2 production. (A) Effect of addition of exogenous PGE2 to the medium on the growth inhibition and apoptosis induction after {alpha}TOS treatment in A549 cells. Cells were plated for 24 h and incubated with 40 µM doses of TOS in the presence or absence of PGE2. Apoptosis ELISA assay and MTT assay were performed after 24 and 48 h treatment. Results are representative data of at least two independent experiments. Growth data are expressed as percentage growth (mean ± SD) relative to control (CTL, vehicle-treated) cells. (B) Effect of {alpha}TOS on the growth and apoptosis of H460 cells stably transfected with COX-2 or control vector (25). Cells were plated for 24 h and incubated with increasing (MTT assay) or fixed (40 µM) (apoptosis assay) doses of {alpha}TOS. Apoptosis ELISA assay and MTT assay were performed after 24 and 48 h treatment with {alpha}TOS. Results are representative data of at least two independent experiments. Growth data are expressed as percentage growth (mean ± SD) relative to control (CTL, vehicle-treated).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
PGE2 plays a key role in inflammation and its associated diseases, such as cancer and cardiovascular disease. PGE2 production is regulated by the activity of its synthesizing enzyme, COX, and the availability of the enzyme substrate, AA. The release of AA from the sn-2 position of membrane phospholipid is a highly regulated process that occurs through the action of members of PLA2 complex family. The 85 kDa cPLA2 is considered the enzyme primarily responsible for the production of AA in a wide range of cells, including epithelial cells (26,27).

PMA has been found to induce AA release (4,28,29), either by increasing the level of cPLA2 protein (29,30) or by enzymatic activation (20,31). We have shown here that PMA-induced AA release in lung epithelial cells. Under our experimental conditions, however, PMA did not significantly alter cPLA2 expression, suggesting that PMA induces AA release through mechanisms other than regulation of cPLA2 expression. For example, PMA may increase AA release by affecting the activity of cPLA2 in lung epithelial cells. In agreement with previous results (32,33), we also found that PMA upregulated the expression of COX-2, without affecting expression of COX-1, in human lung epithelial cells. Taken together, we found that the PMA-induced increase in PGE2 production was associated with an increase in substrate availability as well as increased COX-2 enzyme expression.

Vitamin E analogs such as {alpha}TOL have been found to inhibit PGE2 production in macrophage cells (12). In lung epithelial cells, however, {alpha}TOL was not effective (11), suggesting that the potency of {alpha}TOL depends on cell type. In the present study, we found, however, that {alpha}TOS, a succinyl analog of {alpha}TOL, dose-dependently inhibited PGE2 production in lung epithelial cells. {alpha}TOS did not inhibit PMA-stimulated AA release, while the ability of {alpha}TOS to reduce PGE2 production was observed in the presence of exogenous AA. These data suggest that {alpha}TOS-induced reduction in PGE2 was not caused by regulation of PMA-induced AA release. We found, however, that the effect of {alpha}TOS on PGE2 production in intact lung epithelial cells was primarily due to its inhibition of COX activity. This is supported by our findings showing that {alpha}TOS inhibited PMA-stimulated PGE2 synthesis without altering expression of COX-1 and COX-2; that post-incubation with {alpha}TOS inhibited COX activity and prostaglandin (PGE2 and PGF2{alpha}) production in cells prestimulated with PMA; and that {alpha}TOS inhibited COX activity in cells with endogenously high expression of COX-2.

Since vitamin E is one of the most potent natural scavengers for ROS, its benefits to human health have been thought to be due to its antioxidant properties. For example, vitamin E analogs such as {alpha}TOL have been found to attenuate COX activity by scavenging the oxidant hydroperoxide, which is necessary for COX activation (12,34). While {alpha}TOS, but not {alpha}TOL or {alpha}TOA, significantly inhibited COX activity and PGE2 production in human lung epithelial cells, both {alpha}TOL and {alpha}TOA completely abrogated PMA-induced ROS generation whereas {alpha}TOS did not, suggesting that neither {alpha}TOS nor a possible intracellular metabolite has antioxidant activities inside cells. These results indicate that the ability of {alpha}TOS to inhibit COX activity does not result from the antioxidative properties of the former. Vitamin E is thought to act via non-antioxidant mechanisms in cell signaling, interfering with enzymatic activity, apoptosis and modulating gene expression. For example, {gamma}TOL, a less potent antioxidant than {alpha}TOL, inhibited COX activity and PGE2 generation in lung epithelial cells, whereas {alpha}TOL did not (11), suggesting that certain analogs of vitamin E may inhibit COX activation and subsequent PGE2 production via a non-antioxidant mechanism. Our results suggest that the ability of certain vitamin E analogs such as {alpha}TOS to reduce PGE2 production is due to the interaction between these analogs and the enzymes and proteins involved in PGE2 production. Since the ability of {alpha}TOS to inhibit COX activity decreased with increasing AA concentration, {alpha}TOS may compete with AA in interactions with COX proteins. With our current data, we cannot determine whether {alpha}TOS equally or preferentially inhibited COX-1 and/or COX-2. In addition, the precise mechanism by which {alpha}TOS inhibits COX activity in intact cells remains to be determined, since we failed to show that {alpha}TOS inhibits COX activity of purified enzymes (data not shown).

An inducible microsomal prostaglandin E synthetase (mPGES) was characterized recently as an enzyme to convert COX-derived PGH2 to PGE2 (35). Upregulation of both COX-2 and mPGES was observed to contribute to enhanced production of PGE2 in lung cancer (36). Therefore, it will be of interest to investigate whether {alpha}TOS also modulates the expression or activity of mPGE2 to block PGE2 production in lung epithelial cells.

In conclusion, we have shown that pharmacologically relevant concentrations (18) of {alpha}TOS inhibited COX activity and PGE2 synthesis in human lung epithelial cell lines. These findings suggest that administration of {alpha}TOS may provide an effective way to inhibit inflammatory responses, thereby inhibiting the initiation and progression of lung cancer. In addition, it will be of interest to compare mixed supplementation with {alpha}TOL and {alpha}TOS with that of each alone on the inflammatory response in epithelial cells and macrophages. The use of {alpha}TOL analogs may contribute to the design of better strategies against inflammatory diseases and cancer.


   Acknowledgments
 
This work was supported by a grant from the National Cancer Center (No. 0310010-3) to S.J.L.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 9, 2005; revised April 26, 2006; accepted May 5, 2006.


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