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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*
Open AccessPublished:December 28, 2001DOI:https://doi.org/10.1074/jbc.M107829200
      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.
      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
      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 (
      • Taketo M.M.
      ,
      • Simmons D.L.
      • Levy D.B.
      • Yannoni Y.
      • Erikson R.L.
      ,
      • Dubois R.N.
      • Abramson S.B.
      • Crofford L.
      • Gupta R.A.
      • Simon L.S.
      • Van De Putte L.B.
      • Lipsky P.E.
      ,
      • Smith W.L.
      • Garavito R.M.
      • DeWitt D.L.
      ). 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 (
      • Sano H.
      • Kawahito Y.
      • Wilder R.L.
      • Hashiramoto A.
      • Mukai S.
      • Asai K.
      • Kimura S.
      • Kato H.
      • Kondo M.
      • Hla T.
      ,
      • Ristimaki A.
      • Honkanen N.
      • Jankala H.
      • Sipponen P.
      • Harkonen M.
      ,
      • Zimmermann K.C.
      • Sarbia M.
      • Weber A.A.
      • Borchard F.
      • Gabbert H.E.
      • Schror K.
      ,
      • Hida T.
      • Yatabe Y.
      • Achiwa H.
      • Muramatsu H.
      • Kozaki K.
      • Nakamura S.
      • Ogawa M.
      • Mitsudomi T.
      • Sugiura T.
      • Takahashi T.
      ). 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 (
      • Rioux N.
      • Castonguay A.
      ). Clinical observations from two independent reports (
      • Hida T.
      • Yatabe Y.
      • Achiwa H.
      • Muramatsu H.
      • Kozaki K.
      • Nakamura S.
      • Ogawa M.
      • Mitsudomi T.
      • Sugiura T.
      • Takahashi T.
      ,
      • Wolff H.
      • Saukkonen K.
      • Anttila S.
      • Karjalainen A.
      • Vainio H.
      • Ristimaki A.
      ) 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 (
      • Nicolson G.L.
      ,
      • Noel A.
      • Gilles C.
      • Bajou K.
      • Devy L.
      • Kebers F.
      • Lewalle J.M.
      • Maquoi E.
      • Munaut C.
      • Remacle A.
      • Foidart J.M.
      ). Increasing tumorigenic potential by COX-2 overexpression has been suggested to be associated with resistance to apoptosis (
      • Tsujii M.
      • DuBois R.N.
      ). Exposing HCA-7 colon cancer cells to a COX-2 inhibitor, SC-58125, inhibited growth and increased apoptotic cells, which PGE2 stimulation reversed (
      • Sheng H.
      • Shao J.
      • Morrow J.D.
      • Beauchamp R.D.
      • DuBois R.N.
      ). 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 (
      • Hao C.M.
      • Komhoff M.
      • Guan Y.
      • Redha R.
      • Breyer M.D.
      ). 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.

      DISCUSSION

      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 (
      • Tsujii M.
      • DuBois R.N.
      ) or PGE2 exposure (
      • Sheng H.
      • Shao J.
      • Morrow J.D.
      • Beauchamp R.D.
      • DuBois R.N.
      ) 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 (
      • Kozopas K.M.
      • Yang T.
      • Buchan H.L.
      • Zhou P.
      • Craig R.W.
      ). 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 (
      • Kuo M.L.
      • Chuang S.E.
      • Lin M.T.
      • Yang S.Y.
      ,
      • Chao J.R.
      • Wang J.M.
      • Lee S.F.
      • Peng H.W.
      • Lin Y.H.
      • Chou C.H.
      • Li J.C.
      • Huang H.M.
      • Chou C.K.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ,
      • Reynolds J.E.
      • Yang T.
      • Qian L.
      • Jenkinson J.D.
      • Zhou P.
      • Eastman A.
      • Craig R.W.
      ). 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 (
      • Moulding D.A.
      • Quayle J.A.
      • Hart C.A.
      • Edwards S.W.
      ), 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 (
      • McGinty A.
      • Chang Y.W.
      • Sorokin A.
      • Bokemeyer D.
      • Dunn M.J.
      ). A different study found that an apoptosis-related gene, dynein light chain (DLC), was up-regulated in PC12 cells by COX-2 expression or PGE2treatment (
      • Chang Y.W.
      • Jakobi R.
      • McGinty A.
      • Foschi M.
      • Dunn M.J.
      • Sorokin A.
      ). DLC expression prevented apoptosis of PC12 cells by reducing neuron nitric-oxide synthase activity. Themcl-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-2gene family (
      • Kelley T.J.
      • Cotton C.U.
      • Drumm M.L.
      ,
      • Kim B.C.
      • Lee M.N.
      • Kim J.Y.
      • Lee S.S.
      • Chang J.D.
      • Kim S.S.
      • Lee S.Y.
      • Kim J.H.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). 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 (
      • Hsu A.L.
      • Ching T.T.
      • Wang D.S.
      • Song X.
      • Rangnekar V.M.
      • Chen C.S.
      ). 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 (
      • Kuo M.L.
      • Chuang S.E.
      • Lin M.T.
      • Yang S.Y.
      ,
      • Wang J.M.
      • Chao J.R.
      • Chen W.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ) have pointed out that the anti-apoptotic mcl-1gene, 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 (
      • Kuo M.L.
      • Chuang S.E.
      • Lin M.T.
      • Yang S.Y.
      ). 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 (
      • Sheng H.
      • Shao J.
      • Washington M.K.
      • DuBois R.N.
      ).
      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. PPARα, PPARδ, PPARγ), which acts directly as a transcription factor upon ligand binding (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      ). Many studies have demonstrated that the receptor-coupled G protein can transduce the signal to the PI3K (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ). 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.

      Acknowledgments

      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.

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