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J. Biol. Chem., Vol. 276, Issue 52, 48997-49002, December 28, 2001

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Cyclooxygenase-2 Inducing Mcl-1-dependent Survival Mechanism in Human Lung Adenocarcinoma CL1.0 Cells

INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE/Akt PATHWAY^*

Ming-Tsan Lin, Rung-Chi Lee^§, Pan-Chyr Yang^¶, Feng-Ming Ho, and Min-Liang Kuo^§**

From the  Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan, ^§ Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ^¶ Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan, and  Tao-Yuan General Hospital, Taoyuan 330, Taiwan

Received for publication, August 15, 2001, and in revised form, October 3, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclooxygenase 2 (COX-2) has been reported to be commonly expressed in advanced stages of human lung adenocarcinoma. In this study, the COX-2 constitutive expression vector was transfected into a human lung adenocarcinoma cell line CL1.0 and several clones were obtained which stably expressed COX-2. These COX-2-overexpressed clones demonstrated remarkable resistance to apoptosis induced by Ultraviolet B (UVB) irradiation, vinblastine B (VBL) cell lymphoma-2 (Bcl-2), or other anti-cancer drugs. To understand how COX-2 prevents apoptosis, the investigators examined the expression level of Bcl-2 family members. Mcl-1, but not other Bcl-2 members, was significantly up-regulated by COX-2 transfection or prostaglandin E₂ (PGE₂) treatment. Treatment of COX-2-overexpressed cells (cox-2/cl.4) with two specific COX-2 inhibitors, NS-398 and celecoxib, caused an effective reduction of the increased level of Mcl-1. These data suggest that the expression level of Mcl-1 is tightly regulated by COX-2. Moreover, transfection of cox-2/cl.4 cells with antisense Mcl-1 enhanced apoptosis induced by UVB irradiation, revealing that Mcl-1 plays a crucial role in cell survival activity mediated by COX-2. Furthermore, COX-2 transfection or PGE₂ treatment evidently activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Inhibition of the PI3K pathway by LY294002 or wortmannin effectively attenuated the increased level of Mcl-1 induced by COX-2 or PGE₂. Blocking the PI3K activity with a dominant-negative vector, DN-p85, also greatly diminished the level of Mcl-1 and enhanced UVB-elicited cell death in cells transfected by COX-2. In a similar way, LY294002 inhibited cell survival and Mcl-1 level in PGE₂-treated CL1.0 cells. These findings suggest that COX-2 promotes cell survival by up-regulating the level of Mcl-1 by activating the PI3K/Akt-dependent pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclooxygenases (COX)¹ are the key enzyme that mediates the production of prostaglandins (PGs) from arachidonic acid. Two COX isoforms have been identified, COX-1 and COX-2. COX-1 is expressed constitutively, whereas COX-2 is induced by growth factors, tumor promoters, and cytokines (1-4). The increased expression of COX-2 has been reported to correlate with the malignant changes observed in a variety of human cancers, including colorectal, gastric, esophageal, brain, and lung tumors (5-8). In lung tumorigenesis, administrating a specific COX-2 inhibitor, NS-398, clearly prevented carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors in A/J mice (9). Clinical observations from two independent reports (8, 10) detected a markedly higher COX-2 expression in human lung adenocarcinomas but a lower expression in squamous cell carcinomas or small cell lung cancers. These results indicate that incremental COX-2 expression is critical for pathogenic alteration during the development of human lung cancer.

Inappropriate induction of apoptosis has been associated with organ injury, whereas a failure to undergo apoptosis may cause abnormal cell growth and lead to certain malignant phenotypes, e.g. tumor invasion and metastasis (11, 12). Increasing tumorigenic potential by COX-2 overexpression has been suggested to be associated with resistance to apoptosis (13). Exposing HCA-7 colon cancer cells to a COX-2 inhibitor, SC-58125, inhibited growth and increased apoptotic cells, which PGE₂ stimulation reversed (14). Inhibiting COX-2 activity by SC-58236 or down-regulation of the COX-2 protein by antisense expression in medullary interstitial cells apparently causes an apoptosis (15). Therefore, the above data suggest that COX-2 may function as a survival factor by protecting cells from apoptosis. However, no researchers have discovered the detailed mechanisms underlying how COX-2 induces certain downstream effector genes to prevent apoptosis.

Because COX-2 is selectively expressed in human lung adenocarcinomas, this study investigated whether COX-2 would alter the cellular sensitivity to apoptosis and the possible action mechanism in a human lung adenocarcinoma CL1.0 cell line. To address this issue, the COX-2 expression vector was transfected into CL1.0 cells, and several stable clones overexpressing COX-2 were obtained. These stable clones displayed a substantial level of Mcl-1 protein and exhibited a remarkable resistance to apoptosis. Interestingly, exposure of CL1.0 cells with PGE₂ also caused an up-regulation of the Mcl-1 protein as well as an increase in anti-apoptotic activity. Transient transfection of antisense Mcl-1 into COX-2 overexpressed cells caused them to be more susceptible to cytotoxicity induced by UV irradiation. Further experiments examined the role of the PI3K/Akt pathway in COX-2- or PGE₂-mediated Mcl-1 up-regulation and anti-apoptotic activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection-- Human lung adenocarcinoma cell line CL1.0 was cultured as previously described (16). CL1.0 cells accurately expressing the COX-2 were established by transfection with a COX-2 constitutive expression vector, pSG5-Cox-2, employing the TransFast^TM transfection reagent (Promega, Madison, WI). After 48 h of transfection, cells were trypsinized and replated in RPMI 1640 with 10% fetal calf serum and 300 µg/ml hygromycin B. Hygromycin B-resistant clones were selected and expanded. The level of COX-2 and COX-1 was determined by Western blotting.

PGE₂ Assay-- 5 × 10⁴ cells/well of different stable COX-2-overexpressed clones (cox-2/cl.2, cox-2/cl.4, and cox-2/cl.17) were plated in 6-well dishes and grown to a 60-70% confluence in growth medium by 24 h. Then the culture medium was collected with an enzyme immunoassay to verify amounts of PGE₂ secreted by these stable clones. The production of PGE₂ was standardized to protein concentrations.

Transient Transfection with Antisense Mcl-1-- COX-2-overexpressing CL1.0 cells (cox-2/cl.4) were plated 24 h before transfection at a density of 5 × 10⁴ cells onto a clover glass. Cells were transfected with 5 µg of antisense Mcl-1 plasmid (pcDNA3-mcl-1-AS) or control plasmid (pcDNA3) utilizing TransFast^TM transfection reagent. Transfections were performed in triplicate. Twenty-four hours after transfection, transfected cells were changed to a serum-free medium for a further 12 h and were then treated with UVB irradiation (100 mJ/cm²) or none. After treatment, the extent of apoptosis was determined by TUNEL assays with the In Situ Death Detection Kit, Fluorescein (Roche Molecular Biochemicals) according to the manufacturer's instructions. Apoptotic cells were visualized by fluorescence microscopy.

DNA Fragmentation Assay-- The DNA fragmentation on agarose gel electrophoresis was detected as described previously (17).

Immunoblotting-- The cellular lysates were prepared as described previously (18). A 50-100-µg sample of each lysate was subjected to electrophoresis on 10% SDS-polyacrylamide gels. The samples were then electroblotted on nitrocellulose paper. After blocking, blots were incubated with anti-Mcl-1, anti-Bcl-2, anti-Bcl-x_L, anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA), anti-COX-1, anti-COX-2 (Transduction Laboratories, Lexington, KY), anti-Akt, and anti-phospho-Akt (New England BioLabs) antibodies in PBST (phosphate-buffered saline within 0.1% Tween 20) for 1 h followed by two washes (15 min each) in PBST. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) were employed to depict the protein bands on membranes.

PI3K Assay-- PI3-kinase activities were assayed as described previously (19). Briefly, 10⁷ cells received different treatments and were washed twice with ice cold phosphate-buffered saline and lysed with 1 mM lysis buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, 20 mM Tris, pH 8.0, 2 mM orthovanadate). Cell extracts were incubated with 1 µg of anti-phosphotyrosine antibody overnight at 4 °C. The immunocomplex was precipitated with 50 µl of protein A-Sepharose for 1 h at 4 °C and washed three times with lysis buffer, twice with LiCl buffer (0.5 M LiCl, 100 mM Tris, pH 7.6), and twice with TNE buffer (10 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA). The immunocomplex was preincubated with 10 µl of 20 mM Hepes (pH 7.4), containing 2 mg/ml propidium iodide (Sigma) on ice for 10 min. Kinase reaction was performed by adding 40 µl of reaction buffer (10 µCi of [-³²P]ATP, 20 mM Hepes, pH 7.4, 20 µM ATP, 5 mM MgCl₂) at room temperature for 15 min. Adding 100 µl of 1 M HCl extracted with 200 µl of a 1:1 mixture of chloroform and methanol stopped the reaction. The radiolabeled lipids were separated by thin-layer chromatography and visualized by phosphorimaging.

Transient Transfection and LacZ Cell Death Assay-- CL1.0 cells were plated 24 h before transfection at a density of 1 × 10⁴ cells/well in a 6-well plate. Cells were co-transfected with pCMV--gal plasmid (1 µg), which expressed -galactosidase, and plasmids (4 µg) containing the COX-2 (pSG5-Cox-2), DN-p85, or pcDNA3 control vector utilizing the TransFast^TM transfection reagent. Transfections were performed in triplicate. Twenty-four hours after transfection, the cell medium was replaced with a fresh serum-free medium for 12 h and then exposed to 100 mJ/cm² UVB or none. Cells were washed after treatment and fixed in PBS containing 2% formaldehyde and 0.2% glutaraldehyde. These cells were washed twice more with PBS, resuspended in staining solution containing PBS (pH 7.4), 1 mM MgCl₂, 10 mM K₄Fe (CN)₆, 10 mM K₃He(CN)₆, and 1 mM 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal; added just before use) for 4-24 h, and washed twice with PBS. The -galactosidase-positive cells (blue living cells) in each well were counted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cox-2 Expression Causes Resistance to Apoptosis in Human Lung Adenocarcinoma CL1.0 Cells-- The human COX-2 constitutive expression plasmid, pSG5-Cox-2, and the control vector were transfected into human adenocarcinoma CL1.0 cells, which have a relatively low level of COX-2 protein. This action was aimed at determining whether alteration of the COX-2 level would change the cellular sensitivity to apoptosis. After transfection, cells were cultured in a medium containing 300 µg/ml hygromycin B. Each colony that grew after hygromycin selection was picked and expended. Western blot analysis revealed that three stable colonies (cox-2/cl.2, cox-2/cl.4, and cox-2/cl.17) were randomly selected and expressed a 4-6-fold increase of COX-2 protein compared with the vector control cells (Fig. 1A). The level of COX-1 protein in these transfectants and control cells remained unaltered (Fig. 1A). Elevated levels of PGE₂ production, assessed by enzyme-linked immunosorbent assay (Fig. 1B), paralleled the increased expression of COX-2 protein in the human lung cancer CL1.0 cells. This implies that the exogenously overexpressed COX-2 displayed enzymatic activities in CL1.0 cells.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   A, determination of the protein levels of COX-2 and COX-1 in COX-2-transfected CL1.0 cells. The COX-2-overexpressed cells (cox-2/cl.2, cox-2/cl.2, and cox-2/cl.17) and the vector control cells were obtained as described under "Experimental Procedures." Equal aliquots of protein extracted from these cells were electrophoresed, and the proteins were transferred to the nitrocellulose filter. The nitrocellulose filter was probed with their specific antibodies as indicated. B, PGE2 production increases in COX-2 transfected CL1.0 cells. The postculture medium was collected for 24 h, as described under "Experimental Procedures," and assayed for PGE2 by enzyme immunoassay. Columns, mean; bars, S.D.; n = 6.

To investigate a possible role of COX-2 in cell survival, these COX-2-overexpressed cells were treated with different apoptosis-inducing agents. Fig. 2A reveals that all the COX-2-overexpressed cells (cox-2/cl.2, cox-2/cl.4, and cox-2/cl.17) displayed a remarkable resistance to vinblastine (VBL)-induced apoptosis, as agarose gel electrophoresis of DNA laddering demonstrated. In contrast, vector control cells were highly susceptible to VBL treatment. The apoptosis-resistant characteristic of these COX-2-overexpressed cells was also noted after treatment with other anti-cancer drugs such as paclitaxel or topotecan (not displayed). Apoptotic cells were stained with Hoechst 33258 fluorescent dye (i.e. chromatin condensation and nuclear segmentation) as an additional apoptosis assessment. Fig. 2B demonstrates that the cox-2/cl.4 cells had fewer apoptotic cells than the vector control cells when exposed to UVB irradiation (100 mJ/cm²). Few if any apoptotic cells could be detected in cox-2/cl.4 or control cells without UVB exposure (data not displayed). Exposure of parental CL1.0 cells to 1-4 µg/ml PGE₂ consistently resulted in a dose-dependent decrease in apoptotic cells induced by UVB irradiation compared with cells in the absence of PGE₂, as determined by the TUNEL assay (Fig. 2C). These findings indicate that overexpression of COX-2 or its elevated product, PGE₂, renders cells more resistant to apoptosis stresses.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   COX-2-overexpressed or PGE2-treated cells confer resistance upon apoptosis. A, COX-2 transfectant-resisting VBL-induced DNA fragmentation. Each cell clone was treated with 3 µM VBL for 16 h, and the cellular DNA was extracted as described under "Experimental Procedures." The DNA laddering was analyzed with 1.8-2% agarose gel. B, morphological examination of cox-2/cl.4 and vector control cells after treatment with 100 mJ/cm2 UVB. The apoptotic characteristics such as nuclear fragmentation and chromatin condensation were determined by staining with Hoechst 33258 fluorescent dye. C, PGE2 prevents UVB-induced apoptosis in parental CL1.0 cells. Parental human lung adenocarcinoma CL1.0 cells were pre-exposed as indicated to 0-4 µg/ml PGE2 for 1 h and then treated with 100 mJ/cm2 UVB. After treatment, apoptotic cells were detected and quantified employing the TUNEL method with the In Situ Death Detection Kit, Fluorescein (Roche Molecular Biochemicals) according to the manufacturer's instructions. The labeled cells were examined with a fluorescence microscope. Data are the means of triplicate determinations; error bars, S.D.

Cox-2 Stimulation Up-regulates Mcl-1 but Not Other Bcl-2 Family Members-- The expression level of the Bcl-2 family proteins was first examined to identify the possible downstream gene(s) regulated by COX-2 or PGE₂. Fig. 3A reveals that the level of Mcl-1 protein was significantly increased by ~3-4-fold in cox-2/cl.2 and cl.4 stable clones compared with the vector control cells. In contrast, the levels of other Bcl-2 family members such as Bcl-x_L and Bcl-2 were only slightly affected either in COX-2 stable clones or control cells. We further examined whether the level of Mcl-1 protein in cox-2/cl.4 cells would be altered by treatment with COX-2 inhibitors. Fig. 3B shows, under the noncytotoxic dose, that both celecoxib (10 µM) and NS-398 (25 µM) strongly diminished the endogenous level of Mcl-1 in the COX-2 stable clone. Under the same circumstance, both inhibitors could inhibit the level of PGE₂, an indicator for COX-2 activity, in COX-2-overexpressed cells by 70-80% (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A, immunoblot analysis of Mcl-1, Bcl-2, and Bcl-xL in COX-2-overexpressed cell lines. Cell extracts were prepared and immunoblotting performed as described under "Experimental Procedures." B, COX-2 inhibitors attenuated the increased level of Mcl-1 in COX-2-overexpressed cells. Cox-2/cl.4 cells were plated at a density of 1-3 × 106 cells/100-mm dish. The cultures were treated with 10 µM celecoxib or 25 µM NS-398 for 16 h; then the treated cells were collected and cell lysates were prepared for immunoblotting. Lane 1, vector control cells; lane 2, cox-2/cl.4 cells; lane 3, cox-2/cl.4 cells treated with 10 µM celecoxib; lane 4, cox-2/cl.4 cells treated with 25 µM NS-398. C, transient transfection with COX-2 vector increased Mcl-1 protein level. CL1.0 cells were transiently transfected with 0, 2, or 4 µg of pSG5-Cox-2 or equal amounts of control vector pSG5 as described under "Experimental Procedures." After transfection, each cell lysate was obtained and subjected to electrophoresis and immunoblotting with anti-Mcl-1, anti-Cox-2, and anti-Bcl-2 antibodies. D, PGE2 elevated the level of Mcl-1 protein in CL1.0 cells. Human CL1.0 cells were serum-free for 24 h and then were treated with 4 µg/ml of PGE2 for various periods of time as indicated. Cells were lysed, and the supernatants were subjected to immunoblotting with anti-Mcl-1, anti-Bax, and anti-Bcl-2 antibodies.

To exclude the possibility that an artificial drug selection enhanced anti-apoptotic Mcl-1 protein in these COX-2 stable clones, investigators transfected CL1.0 cells with COX-2 expression vector or treated them with various concentrations of PGE₂ and then examined the expression of Mcl-1 protein. Western blot analysis revealed that CL1.0 cells displayed an apparent dose-dependent increase in Mcl-1 protein (~4-8-fold) when transfection occurred with the 2 or 4 µg of pSG5-Cox-2 vector but not with the control vector, pSG5 (Fig. 3C). A commonly used liposomal transfection method achieved a high transfection efficiency of >40% in the human adenocarcinoma CL1.0 cells (not displayed). CL1.0 cells were treated next with 4 µg/ml PGE₂ for various periods of time, and then the change of Mcl-1 protein level was detected by Western blotting. Fig. 3D clearly demonstrates that the level of Mcl-1 protein rapidly increased 1 h after PGE₂ treatment, peaked at 3-6 h, and then maintained a high level of Mcl-1 over 12 h. In contrast, the level of Bcl-2 protein was little affected during the PGE₂ treatment. These results suggest that COX-2 overexpression or PGE₂ elevated the Mcl-1level in CL1.0 cells but not other Bcl-2 family members.

Mcl-1 Protein Involved in the Anti-apoptotic Effect of Cox-2-- The COX-2-overexpressed cells (cox-2/cl.4) were transfected with a constitutive antisense Mcl-1 expression vector to further investigate the role of Mcl-1 in COX-2-mediated anti-apoptotic activity. However, following several rounds of stable clone selection, none of the clones that survived could express antisense Mcl-1. This probably occurred because of the severely cytotoxic effect of the constitutive antisense Mcl-1 expression. A transient transfection death assay was therefore conducted to investigate this phenomenon. Cox-2/cl.4 cells were initially transfected with empty vectors or antisense Mcl-1 vectors. Twenty-four hours after transfection, transfected cells were changed to a serum-free medium for a further 12 h and were then treated with UVB irradiation (100 mJ/cm²) or none. After treatment, the extent of apoptosis was defined as the number of cells positively stained with TUNEL and identified under a fluorescence microscope. Fig. 4A reveals that cox-2/cl.4 cells displayed a resistant phenotype to UVB-induced apoptosis when transfected with a control vector or none. Transfection of cox-2/cl.4 cells with antisense Mcl-1enhanced apoptotic cell death induced by UVB irradiation. Under the same circumstances, the antisense Mcl-1 vector effectively attenuated the endogenous level of Mcl-1 in cox-2/cl.4 cells by transfection (Fig. 4B). The above results strongly suggest that the mcl-1 gene is critically involved in the COX-2-mediated anti-apoptotic effect in human adenocarcinoma CL1.0 cells.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   A, transfection with antisense Mcl-1 attenuated the anti-apoptotic activity of COX-2-overexpressed cells. Cox-2/cl.4 cells were plated at a density of 5 × 104 onto a coverglass. Cox-2/cl.4 cells were transfected with 5 µg of antisense mcl-1 vector (pcDNA3-mcl-1-AS) or control vector (pcDNA3). Transfection was performed in triplicate. 24 h after transfection, cells were changed to a serum-free medium for a further 12 h and were then exposed to 100 mJ/cm2 UVB. The apoptotic cells were determined using the TUNEL assay. Results displayed are the mean ± S.D. from three independent experiments. B, transfection with antisense Mcl-1 significantly reduced the endogenous level of Mcl-1 protein in cox-2/cl.4 cells. Immunoblotting determined the level of Mcl-1 protein.

Function of PI3K/Akt Pathway in COX-2-mediated Anti-apoptotic Effect and Mcl-1 Up-regulation-- Because the PI3K/Akt pathway plays a central role in integrating a diverse survival signal triggered by numerous growth factors (20-22), COX-2 was tested to ascertain whether it could activate this pathway. To evaluate this, the endogenous Akt activity in cox-2/cl.2 and cox-2/cl.4 cells was examined by determining the serine-phosphorylated status of Akt, employing an anti-phospho-Akt antibody. The Akt activity correlates well with the phosphorylation of Akt molecules on serine 473 (23). Interestingly, these COX-2-overexpressed cells displayed a significantly increased Akt phosphorylation over the vector control cells (Fig. 5A). Furthermore, treatment with a PI3K inhibitor, LY294002 (10 µM), greatly reduced the phosphorylation of Akt in these COX-2 expressed cells (Fig. 5A). This indicates that PI3K functions upstream of Akt in response to the COX-2-elicited survival signal. The authentic PI3K activity of CL1.0 cells after treatment with PGE₂ was subsequently determined. Immunoprecipitates with the anti-phosphotyrosine antibody revealed a substantial increase in PI3K activity 15-60 min after exposure to 4 µg/ml PGE₂, and 10 µM LY294002 could completely inhibit this increase (Fig. 5B). The above results suggest that the PI3K/Akt pathway is indeed activated by COX-2 or its product, PGE₂.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   The PI3K/Akt pathway was activated by COX-2 and PGE2. A, phosphorylation of Akt at Ser473 was increased in COX-2-overexpressed cell lines. COX-2-overexpressed cells, cox-2/cl.2 (cl.2) and cox-2/cl.4 (cl.4), and vector control cells (Vector) were treated with or without 10 µM LY294002 (LY) for 6 h, and then each cellular lysate was prepared to perform electrophoresis and immunoblotting as described under "Experimental Procedures" employing a specific anti-phospho-Akt antibody or anti-Akt antibody. B, activation of PI3K activity by PGE2 in CL1.0 cells. Cells were treated as indicated, and lysates with equal amounts of protein were subjected to immunoprecipitations with anti-phosphotyrosine antibody. The immunocomplex was employed for PI3K activity assays as described under "Experimental Procedures."

The next aim of the investigation was to ascertain whether PI3K activity inhibition might affect the anti-apoptotic activity and Mcl-1 up-regulation induced by COX-2 or PGE₂. To address this issue, a COX-2 vector and a dominant-negative form of the regulatory subunit of PI3K, DN-p85, were concomitantly transfected into CL1.0 cells. Western blot analysis revealed that DN-p85 completely attenuated COX-2-mediated Mcl-1 up-regulation (Fig. 6A). Utilizing an anti-hemagglutinin antibody (data not shown) or detecting the serine phosphorylation of Akt (Fig. 6B) confirmed the successful expression of the DN-p85 in transfected cells. To determine the effect of DN-p85 on COX-2-mediated cell survival, a transient transfection death assay was conducted employing the -galactosidase expression plasmid (pCMV--gal) as a survival marker. The transfection results revealed that the DN-p85 transfection, but not the control pcDNA3, abolished the COX-2-induced cell survival activity when exposed to UVB irradiation, as determined by the decrease of blue surviving cells expressing -galactosidase activity (Fig. 6C).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Inhibiting PI3K activity by transfection with DN-p85 reduced COX-2-induced Mcl-1 up-regulation and cell survival. Transfection with DN-p85 effectively attenuated COX-2-mediated Mcl-1 up-regulation (A) and Akt phosphorylation (B). CL1.0 cells were transfected with pSG5-Cox-2 (4 µg/well) and DN-p85 (4 µg/well) or control pcDNA3 vector (4 µg/well). After 48 h of transfection, cells lysates were obtained and subjected to electrophoresis and immunoblotting with anti-Mcl-1 (A) or anti-phospho-Akt (B) antibodies. C, DN-p85 transfection abolished COX-2-induced anti-cell death activity. CL1.0 cells were transfected with pSG5-Cox-2 (4 µg/well), pCMV--gal (1 µg/well), and either DN-p85 (4 µg/well) or an empty vector (4 µg/well). After 24 h of transfection, the cell medium was replaced with a fresh serum-free medium for 12 h, and then the cells were exposed to 100 mJ/cm2 UVB or none. After treatment, cells were washed and fixed to determine -galactosidase activity as described under "Experimental Procedures." The surviving cells were counted as the number of blue cells expressing -galactosidase activity.

A similar observation was made in CL1.0 cells treated with PGE₂. Fig. 7A reveals that PGE₂-induced Mcl-1 up-regulation was almost completely attenuated in the presence of LY294002 or wortmannin. Also, LY294002 treatment greatly sensitized PGE₂-treated cells to UVB irradiation-elicited apoptosis as determined by a TUNEL assay (Fig. 7B). The above data clearly reveal that COX-2-mediated cell survival activity requires activation of the PI3K/Akt-dependent pathway and its subsequent downstream gene, mcl-1.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   PI3K inhibitors reduced PGE2-induced Mcl-1 up-regulation and anti-apoptotic activity. A, PI3K inhibitors blocked the PGE2-induced Mcl-1 up-regulation. CL1.0 cells were serum-starved for 24 h and were then treated with 4 µg/ml PGE2 with or without 10 µM LY294002 (LY) or 50 nM wortmannin (Wort) for 6 h. After treatment, cells were lysed, and the supernatants were subjected to immunoblotting with anti-Mcl-1 and anti-Bcl-2 antibodies. B, LY294002 abolished the anti-apoptotic activity in CL1.0 cells induced by PGE2. Human lung adenocarcinoma CL1.0 cells were pre-exposed to 4 µg/ml PGE2 with or without 10 µM LY294002 for 6 h and were then further treated with 100 mJ/cm2 UVB. After treatment, apoptotic cells were detected and quantified using the TUNEL method with the In Situ Death Detection Kit, Fluorescein (Roche Molecular Biochemicals) according to the manufacturer's instructions. The labeled cells were examined with a fluorescence microscope. Data are the means of triplicate determinations; bars, S.D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Accumulating evidence has suggested that cancer cells expressing higher levels of COX-2 may obtain a survival advantage that eventually facilitates the tumor development and progression. Although these studies have established a direct relationship between COX-2 expression and cell survival in different cell systems, the precise mechanism by which COX-2 prevents cell death has seldom been investigated and remains elusive. This study demonstrated that COX-2 overexpression or PGE₂ treatment induced an increase in a novel anti-apoptotic protein Mcl-1 through the PI3K/Akt-dependent pathway in human adenocarcinoma cells. An antisense Mcl-1 transfection assay ensured a crucial role for Mcl-1 in the COX-2-mediated anti-apoptotic effect in lung adenocarcinoma CL1.0 cells. Other researchers distinctly observed that either forced expression of COX-2 in intestinal cells (13) or PGE₂ exposure (14) to colon cancer cells caused up-regulation of the Bcl-2 protein. The level of Bcl-2 protein, however, was unchanged in our cell system, suggesting induction of certain members of the Bcl-2 family by COX-2, depending upon the cell context.

The mcl-1 gene, one of the Bcl-2 family members, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells (24). Overexpression of Mcl-1 delays apoptosis induced by a broad array of agents such as c-Myc overexpression, growth factor withdrawal and other cytotoxic agents (17, 25, 26). These findings correspond to our current data, suggesting that certain types of cancer cells require Mcl-1 to survive. Many cytokines and growth factors have already been reported as able to induce Mcl-1 expression (27), but this is first time it has been demonstrated that the COX-2-derived prostanoids can do so. COX-2 has been reported to inhibit nerve growth factor withdrawal apoptosis in differentiated PC12 cells (28). A different study found that an apoptosis-related gene, dynein light chain (DLC), was up-regulated in PC12 cells by COX-2 expression or PGE₂ treatment (29). DLC expression prevented apoptosis of PC12 cells by reducing neuron nitric-oxide synthase activity. The mcl-1 and DLC genes are the only two downstream effectors responsible for COX-2-mediated anti-apoptotic signal identified thus far.

This investigation also revealed that the PI3K/Akt signaling pathway could be activated and involved in Mcl-1 up-regulation by COX-2 expression or PGE₂. Emerging evidence has demonstrated that the PI3K/Akt signaling pathway promotes growth-mediated cell survival and restricts apoptosis by modifying the anti-apoptotic and pro-apoptotic activities of members of the bcl-2 gene family (20-22). Interestingly, celecoxib, a specific COX-2 inhibitor, has been found to promote apoptosis in human prostatic cancer cells by attenuating the Akt activity but not the level of Bcl-2 protein (30). This implies that the Akt-related pathway is important for COX-2-induced anti-apoptotic activity but that it might be dissociated from the expression of Bcl-2 protein. Our previous studies (17, 31) have pointed out that the anti-apoptotic mcl-1 gene, but not bcl-2, is a direct downstream target of the PI3K/Akt signaling pathway induced by interleukin-6 or -3. The cells overexpressing an activated Akt, Myr-Akt, also augmented the expression of Mcl-1 but not Bcl-2 (17). A recent study has also demonstrated that PGE₂ treatment activates the PI3K/Akt pathway, which is required to increase growth and motility in colon carcinoma cells (32).

How do COX-2-derived prostanoids, e.g. PGE₂, activate the PI3K? Two classes of prostaglandin receptors transduce signals upon binding of the ligand, the G-coupled cytoplasmic receptor class (i.e. EP1-4 for PGE₂) and the nuclear PPAR receptor class (i.e. PPAR, PPAR, PPAR), which acts directly as a transcription factor upon ligand binding (33). Many studies have demonstrated that the receptor-coupled G protein can transduce the signal to the PI3K (34). Logically, the cytoplasmic EP receptors are the preferred mediator for the prostanoid-induced PI3K/Akt-dependent cell survival effect. Further investigation will be needed to determine whether the EP receptor or PPAR receptor is involved in the anti-apoptotic activity by COX-2-derived prostanoids.

Although COX-2 expression is thought to be crucial for the development of certain human cancers, the downstream signal that mediates the neoplastic effects is poorly understood. The current investigation has revealed that either overexpression of COX-2 or exposure to PGE₂ can increase the apoptosis threshold in human lung adenocarcinoma cells by up-regulating the mcl-1 gene. It also found the PI3K/Akt signaling pathway to be involved in regulating Mcl-1 expression. This work verifies a new downstream agent of COX-2. That verification will help researchers to understand better the precise mechanism of the COX-2-mediated carcinogenic process.

	ACKNOWLEDGEMENTS

We thank Dr. H.-F. Yang-Yen for helpful instructions on the PI3K assay and Dr. R.-H. Chen for providing dominant-negative Akt and p85. We also acknowledge Ted Knoy for his critical editing of the manuscript.

	FOOTNOTES

^* This research was supported by the National Science Council of the Republic of China, Taiwan under Contracts NSC-89-2316-B-002-031 and NSC 89-2320-B-002-259.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.

^** To whom correspondence should be addressed: Laboratory of Molecular & Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University No. 1, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan. Fax: 886-2-2341-0217; E-mail: toxkml@ha.mc.ntu.edu.tw.

Published, JBC Papers in Press, October 3, 2001, DOI 10.1074/jbc.M107829200

	ABBREVIATIONS

The abbreviations used are: COX, cyclooxygenase; PGE₂, prostaglandin E₂; PI3K, phosphatidylinositol 3-kinase; VBL, vinblastine; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; PPAR, peroxisome proliferator activator receptor; Bcl, B cell lymphoma; Mcl, myeloid cell leukemia.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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