Regulation of COX-2 protein expression by Akt in endometrial cancer cells is mediated through NF-κB/IκB pathway
© St-Germain et al; licensee BioMed Central Ltd. 2004
Received: 09 December 2003
Accepted: 11 March 2004
Published: 11 March 2004
Cyclooxygenase-2 (COX-2) has been shown to be highly expressed in a broad series of primary endometrial tumors and its expression may be closely associated with parameters of tumor aggressiveness. In human endometrial cancer, tumor suppressor phosphatase tensin homologue (PTEN) is frequently mutated. In the presence of a mutated PTEN protein, Akt phosphorylation levels increase leading to the activation of this survival pathway. The nuclear transcription factor κB (NF-κB) is a well establish regulator of genes encoding cytokines, cytokine receptors, and cell adhesion molecules that drive immune and inflammatory responses. More recently, NF-κB activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, cell cycle, differentiation, and cell migration. It is known that Akt may act through NF-κB pathway and that COX-2 gene has been shown to be regulated at the promoter level by NF-κB. Recently, we showed that Akt regulates COX-2 gene and protein expressions in phospho-Akt expressing endometrial cancer cells. The present study was undertaken to determine the involvement of NF-κB pathway and IκB (an inhibitor of NF-κB) in the regulation of COX-2 expression and to determine more precisely the downstream targets of Akt involved in this process.
Three different human endometrial cancer cell lines known to have wild type PTEN (HEC 1-A) or a mutated inactive PTEN protein (RL 95-2 and Ishikawa) were used for these studies. Expression IκB and Phospho-IκB were evaluated by Western analysis. The presence of IκB phosphorylation was found in all cell lines studied. There was no difference between cell lines in term of NF-κB abundance. Inhibition of PI 3-K with Wortmannin and LY294002 blocked IκB phosphorylation, reduced NF-κB nuclear activity, reduced COX-2 expression and induced apoptosis. Transfection studies with a dominant negative Akt vector blocked IκB phosphorylation and reduced COX-2 expression. On the opposite, constitutively active Akt transfections resulted in the induction of IκB phosphorylation and up-regulation of COX-2.
These results demonstrate that Akt signals through NF-κB/IκB pathway to induce COX-2 expression in mutated PTEN endometrial cancer cells.
The phosphoinositide 3-kinase (PI 3-kinase) pathway has been implicated in the activation of the proinflammatory transcription factor nuclear factor κB (NF-κB) [1–3]. It has been demonstrated that both the regulatory and the catalytic subunit of phosphatidylinositol 3-kinase (PI 3-K) play a role in NF-κB activation by the tyrosine phosphorylation-dependent pathway . The NF-κB transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes . In addition to its role in inflammation and immune response, NF-κB has also been implicated in the suppression of apoptosis , cellular survival, transformation, and oncogenesis . Predominantly a heterodimeric complex of two polypeptides (p65/RelA and p50), NF-κB lies dormant in the cytoplasm through the binding IκB inhibitory proteins. When phosphorylated on serine 32 and serine 36, IκBα is targeted and degraded by ubiquitin/26 S proteasome pathway liberating the NF-κB heterodimer so that it may translocate to the nucleus and bind DNA. NF-κB binds to cis-acting κB in the promoters and enhancers of key cellular genes. Active, DNA-binding forms of NF-κB are dimeric complexes, composed of various combinations of members of the Rel/NF-κB family of polypeptides (p50, p52, c-Rel, v-Rel, RelA (p65), and RelB). Recently, a large-molecular weight complex was identified that is responsible for phosphorylating IκBα and IκBβ. Two key catalytic sub-units of the IκB kinase (IKK) complex were identified as IKKα and IKKβ . Constitutive NF-κB activation appears to have an important role in tumorigenesis. For example, persistent nuclear NF-κB localization and NF-κB-dependent transcription is detected in breast , ovarian , colon , thyroid  and prostate  tumors. In breast and prostate tumor cells, constitutive NF-κB activity is associated with reduced levels of IκBα that appears related to increased degradation of IκB proteins in these cells .
Previous reports indicate that the transcription factor NF-κB can function upstream of cyclooxygenase-2 (COX-2) to control transcription of this gene through the IKK pathway activation . Cyclooxygenase (COX) is the rate-limiting enzyme involved in the biosynthesis of prostaglandins (PG) and exists in two isoforms: COX-1 (constitutively expressed) and COX-2 (the regulated isoform). Cyclooxygenase-2 (COX-2) up-regulation has been found in several type of cancers such as colon carcinomas , cervix , head and neck , bladder , pancreas , stomach , prostate  and breast . It is believed that COX-2 and PGs, particularly PGE2, may be key elements in the evolution of tumor transformation and malignancy. Epidemiological studies showed that nonsteroidal anti-inflammatory drugs (NSAIDs) can be used for cancer prevention . It has been shown that COX-2 expression in colorectal carcinoma cells provides a growth and survival advantage and increases tumor cell invasiveness (see  for a review). Additionally, more evidences suggest that COX-2 is highly express in a broad series of primary endometrial tumors and its expression may closely be associated with parameters of tumor aggressiveness .
Akt is a serine/threonine protein kinase also known as protein kinase B or Rac [25–27]. Akt is an inactive cytosolic protein recruited to the plasma membrane, and activated by phosphorylation at threonine 308 and serine 473 in response to growth factors or cytokines [28–30] via the product of PI 3-K, phosphatidylinositol 3,4,5-triphosphate (PIP3). Upon phosphorylation, Akt has been shown to phosphorylate and to block the action of several pro-apoptotic proteins such as Bad . Akt also blocks cytochrome C release from the mitochondria through the regulation of Bcl-2  and regulates expression of cIAP-1 . Inhibition of Akt has been shown to induce apoptosis [33–35] and was shown to be a downstream target of NF-κB . Recently, it has been demonstrated that Akt in involved in IKK phosphorylation resulting in NF-κB activation . In a number of different cancers, the tumor suppressor phosphatase tensin homologue (PTEN, a lipid phosphatase) is frequently mutated. PTEN mutations have been found in several types of endometrial cancer [38–41]. PTEN dephosphorylates PIP3 into inactive PIP2, which blocks Akt activation. Moreover, we have previously shown that Akt regulates COX-2 gene and protein expressions.
We have demonstrated recently that Akt directly regulates COX-2 gene and protein expression in endometrial cancer cells . The present study was undertaken to determine the involvement of NF-κB pathway and IκB in the regulation of COX-2 expression and to determine more precisely the downstream targets of Akt involved in this process. We hypothesized that PTEN mutation increase Akt activity which may, in turn, be involved in the activation of NF-κB. Our results demonstrate that activity of NF-κB is up-regulated in human endometrial cancer cells expressing phospho-Akt and is responsible for the increase of COX-2 gene expression.
Inhibition of PI 3-K induces apoptosis
Inhibition of the PI 3-kinase/Akt signaling pathway reduces phosphorylation of IκB and activates NF-κB translocation into the nucleus
Constitutively active Akt transfections resulted in the induction of IκB phosphorylation and up-regulation of COX-2 expression
Dominant negative Akt vector blocked IkB phosphorylation, which leads to the activation of apoptosis
The ability of NF-κB to promote cell proliferation, to suppress apoptosis, to promote cell migration, and to suppress differentiation apparently have been co-opted by cellular and viral oncoproteins to promote oncogenesis. It is known that Akt may act through NF-κB pathway  and that COX-2 gene has been shown to be regulated at the promoter level by NF-κB . The activity of NF-κB is tightly controlled by inhibitory IκB proteins that bind to NF-κB complexes and thus sequester NF-κB in the cytoplasm. Stimuli such as cytokines promote the serine phosphorylation of IκB and its polyubiquitination and proteosome-mediated degradation and thereby induce NF-κB translocation to the nucleus. Since Akt phosphorylation has been shown to activate NF-κB in other systems, a similar sequence of events might be involved in phospho-Akt expressing RL 95-2 and Ishikawa human endometrial cancer cell lines used in the present study. PTEN is a crucial phosphatase involved in the regulation of Akt phosphorylation: the presence of an active PTEN protein blocks Akt phosphorylation by the dephosphorylation of PI 3-K product, PIP3 . In the presence of a mutated-PTEN protein, activation of Akt generally occurs constitutively. We have demonstrated previously that Akt is constitutively phosphorylated/activated in two mutated-PTEN human endometrial cancer cell lines that have been used in the present study (RL 95-2 and Ishikawa) [32, 42]. Whereas, phosphorylation of Akt was absent in one wild-type PTEN cell line (HEC 1-A).
Indeed, our results demonstrate that the presence of IκB phosphorylation was found in all cell lines studied. There was no difference between cell lines in term of NF-κB abundance indicating that NF-κB expression is not involved in the regulation of COX-2 gene expression. However, NF-κB was shown to be activated and present in the nucleus of the two mutated-PTEN endometrial cancer cells (RL 95-2 and Ishikawa) expressing phospho-Akt. Thus, the presence of a wild-type PTEN protein results in the reduction of Akt activity/phosphorylation leading to the inhibition of IκB phosphorylation and the sequestration of NF-κB. On the opposite, the presence of a mutated PTEN protein enables Akt phosphorylation, which in turn may phosphorylate IκB allowing NF-κB to be translocated to the nucleus to induce transcription of genes involved in cell survival. These results demonstrate that the ability of PTEN to negatively regulate the PI 3-K/Akt/NF-κB pathway may be important to its role of tumor suppressor protein.
In the present study, we have investigated the role of PI 3-kinase in NF-κB activation. Various PI 3-kinase inhibitors such as Wortmannin, LY294002 and dominant-negative Akt expression vector were used to fully prove the involvement of PI 3-K/Akt pathway in the regulation of NF-κB activity. Inhibition of PI 3-K with Wortmannin and LY294002 blocked Akt and IκB phosphorylation and reduced COX-2 expression in RL 95-2 and Ishikawa cells. However, PI 3-K inhibitors had no effect in HEC 1-A cells (a cell line with non detected Akt phosphorylation and no detectable level of COX-2) confirming that COX-2 is a targeted gene downstream of Akt. Transfection studies with a dominant negative Akt vector in mutated-PTEN RL 95-2 cells blocked IκB phosphorylation, increased IκB expression and leaded to the activation of apoptosis. These PI 3-K/Akt inhibition studies demonstrate that PI 3-K and Akt are required for NF-κB activation.
To further confirm the involvement of Akt and NF-κB /IκB pathway in the control of COX-2 expression, transfections with a constitutively active (CA) Akt expression vector were carried out using RL 95-2 cells. As hypothesized, CA-Akt transfection induced COX-2 expression and confirmed the results obtained with DN-Akt inhibition and PI 3-K inhibition studies. Moreover, these transfections resulted in the induction of IκB phosphorylation. The subsequent degradation of IκB allows the release and translocation of NF-κB to the nucleus. Recent evidences also suggest that CCAAT/enhancer-binding protein beta (C/EBPβ), a transcription factor, is involved downstream Akt activation pathway [46–48]. Studies have demonstrated that C/EBPβ is an essential transcription factor for COX-2 gene regulation [49, 50] indicating that activation of C/EBPβ by Akt may be in part responsible for COX-2 gene expression. Another study showed that inactivation of GSK-3β through activation of Akt plays an important role in the UVB induction of COX-2 transcription . Thereby, C/EBPβ and GSK-3β may be other different targets following Akt phosphorylation to activate cell survival through COX-2 gene expression and PGE2 secretion.
COX-2 has been shown to contribute to tumorigenesis and the malignant phenotype of tumor cells by different mechanisms, including: (1) inhibition of apoptosis; (2) increased angiogenesis; (3) increased invasiveness; (4) modulation of inflammation/immuno-suppression; and (5) conversion of procarcinogens to carcinogens (see  for a review). An evident correlation between COX-2 expression and inhibition of apoptosis has been established, associated with increased PGE2 levels resulting in modulation of pro- and anti-apoptotic factors such as Bcl-2 . We have showed previously that COX-2 inhibition with NS-398 in RL 95-2 and Ishikawa cells results in the inhibition of Akt phosphorylation and induction of apoptosis suggesting that the Akt/NF-κB/COX-2 pathway is an important point of control of cell survival.
In summary, the present study demonstrates a crucial role for Akt in the regulation of NF-κB expression through the phosphorylation of IκB in human endometrial cancer cells. The results demonstrate that Akt signals through NF-κB/IκB pathway to induce COX-2 gene and protein expression. There is compelling evidence that NF-κB is deregulated in many forms of cancer and its inhibition is a logical therapy for certain cancers and for adjuvant approaches to cancer therapy. Indeed, this study shows that NF-κB/IκB pathway could be a good target for gene therapy in endometrial cancers. Further studies on other signaling factors/transcription factors such as GSK-3β and C/EBPβ activation/phosphorylation will provide more insight into the complex mechanisms by which Akt regulates COX-2 gene expression in human endometrial cancer cells.
Wortmannin, LY294002, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and Hoechst 33258 were obtained from Sigma (St. Louis, MO). DMEM-F12, Mc Coy's 5A medieum, FBS serum and PCR primers were purchased from Invitrogen (Burlington, ON). Anti-human PhosphoPlus Akt (Ser473), Akt, IκBα and Phospho-IκBα antibodies were obtained from Cell Signaling Technology and anti-human COX-2 were obtained from Cedarlane Laboratories (Hornby, ON). Secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody was purchased from BioRad (Mississauga, ON). Dominant negative (DN) and constitutively active (CA) Akt vectors were generously provided by Dr Zhenguo Wu, Hong Kong University of Science and Technology.
Human endometrial cancer cells (HEC 1-A and RL 95-2) were obtained from ATCC. Ishikawa cells were generously provided by Dr Sylvie Mader, Université de Montréal, Canada. Cells were cultured in 75 cm2 bottles at 37°C in an atmosphere of 5% CO2. Ishikawa cells were maintained in DMEM-F12 supplemented with 2.438 g/L of NaHCO3, FBS (10%) and gentamycin (50 μg/ml). HEC 1-A cells were grown in Mc Coy's 5A medium supplemented with 2.2 g/L of NaHCO3, FBS (10%) and gentamycin (50 μg/ml). RL 95-2 were cultured in DMEM-F12 supplemented with 1.75 g/L of NaHCO3, HEPES (5 μM), insulin (2.5 μg/ml), FBS (10%) and gentamycin (50 μg/ml). 1 × 106 cells were plated in log growth phase into 6 wells plates for 24 hrs in the above culture medium prior to initiation of treatment. Wortmannin dose (50 μg/ml) and 24 hours time were chosen following dose-responses and time-course preliminary studies.
Cells were plated at a density of 4 × 105 cells/well in six-well plates 24 hours before transfection. RL 95-2 cells were transfected with DN-Akt and CA-Akt vectors. Transient transfection of the cells was carried out with 1 μg of DNA/well using Effectene (Qiagen, Mississauga, ON), according to the protocol suggested by the manufacturer. Empty vector was used as the transfection control. Transfection efficiencies were determined by Western analysis using an anti-Akt antibody.
NF-κB cheluminescent assay
The BIOXYTECH NF-κB Chemiluminescent Assay (Medicorp, Montreal, QC) employs an oligonucleotide containing the DNA binding NF-κB consensus sequence bound to a 96-well plate. NF-κB present in the sample binds specifically to the oligonucleotide coated on the plate. The DNA bound NF-κB is selectively recognized by the primary antibody (p50 and p105 specific). A secondary antibody-alkaline phosphatase conjugate binds to the primary antibody. Then, the Relative Light Units (RLU) is measured using a chemiluminescence detector after addition of the alkaline phosphatase substrate.
Following treatment, both floating and attached cells were resuspended in 10% formalin containing Hoechst 33258 for 24 hours at 4°C. Hoechst nuclear staining was viewed and photographed using a Olympus BX60 fluorescence microscope and a Coolsnap-Pro CF digital Camera (Carsen Group, ON). Cells with typical apoptotic nuclear morphology (nuclear shrinkage, condensation and fragmentation) were identified and counted, using randomly selected fields on numbered photographic slides, of which the "counter" was not aware of the treatment, so as to avoid experimental bias. A minimum of 200 cells per treatment group was counted in each experiment and results are presented as a percentage of apoptotic cells/non-apoptotic cells.
Terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)
Cells (floating and attached) were pooled, placed on a positively charged microscope slide, dried and rinsed with PBS. Slides were incubated with proteinase K (20 μg/ml) for 30 min at room temperature. Slides were washed twice with PBS and endogenous peroxidase was inactivated with 0.3 % hydrogen peroxide in methanol for 30 min. Slides were rinsed with buffer and incubated with 10 mM citrate solution for two minutes on ice. Then, tissue sections were rinsed with PBS and incubated with TdT labelling reaction (In Situ Cell Death Detection, POD, Roche) for 30 min at 37 °C in humidified environment. Slides were washed three times in PBS and tissue sections were blocked with BSA 3% for 20 min at room temperature. Converter-POD solution was added and incubated 30 min at 37°C in humidified environment. Slides were washed 5 min in PBS and color development was achieved by incubation using DAB substrate. Cells were finally counterstained with hematoxylin. Negative control was performed using the same protocol without TdT enzyme. TUNEL positive cells were counted as described with the Hoechst nuclear staining assay.
Protein extraction and Western analysis
Cells (both floating and attached) were trypsinized, lysed in lysis buffer (PBS 1 × pH 7.4; 1% Nonidet P-40; 0.5% Sodium deoxycholate; 0.1% SDS; Protease Inhibitor Cocktail Tablets (Roche)), frozen and thawed three times, and centrifuged (13000 × g, 20 min at 4°C) to remove insoluble material. Supernatant was recovered and stored at -20°C pending analysis. Protein content was determined with the Bio-Rad DC Protein Assay according to manufacturer instructions. Protein extracts (50 μg) were heated (95°C, 3 min), resolved by 10% SDS-Polyacrylamide gel electrophoresis (PAGE) and electro-transferred to nitrocellulose membranes (15 V, 30 min) using a semi-dry transfer (Bio-Rad, Mississauga, ON). Membranes were then blocked (2 hrs, RT) with PBS containing 5% milk powder + 0.05% Tween 20, then incubated with anti-COX-2 (1:1000), anti-Akt (1:1000), anti-Phospho-PKB/Akt (1:250), anti-IκBα (1:1000), anti-Phospho-IκBα (1:500) (overnight, 4°C), and subsequently with Horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:3000; RT, 45 min). Peroxidase activity was visualized with the ECL kit (Amersham, Arlington Heights, IL), according to the manufacturer's instructions.
All experiments were repeated at least three times. Data were subjected to one-way ANOVA or student t test (PRISM software version 4.0; GraphPad, San Diego, CA). Differences between experimental groups were determined by the Tukey's test.
This work has been supported by grants from the Fond de la Recherche en Santé du Québec (FRSQ; 23112-1777 and 23502-2725) and the Canadian Institute of Health Research (CIHR; IGO-66069 and MOP-66987). Eric Asselin is a recipient of a fellowship from FRSQ. Veronique Gagnon is a recipient of a FRSQ scholarship.
- Lilienbaum A, Israel A: From calcium to NF-kappa B signaling pathways in neurons. Mol Cell Biol. 2003, 23: 2680-2698.PubMed CentralView ArticlePubMedGoogle Scholar
- Kane LP, Shapiro VS, Stokoe D, Weiss A: Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol. 1999, 9: 601-604.View ArticlePubMedGoogle Scholar
- Beraud C, Henzel WJ, Baeuerle PA: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation. Proc Natl Acad Sci U S A. 1999, 96: 429-434.PubMed CentralView ArticlePubMedGoogle Scholar
- Lenardo MJ, Baltimore D: NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989, 58: 227-229.View ArticlePubMedGoogle Scholar
- Wang CY, Mayo MW, Baldwin A.S., Jr.: TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science. 1996, 274: 784-787.View ArticlePubMedGoogle Scholar
- Mercurio F, Manning AM: Multiple signals converging on NF-kappaB. Curr Opin Cell Biol. 1999, 11: 226-232.View ArticlePubMedGoogle Scholar
- Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M: The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell. 1997, 91: 243-252.View ArticlePubMedGoogle Scholar
- Rayet B, Gelinas C: Aberrant rel/nfkb genes and activity in human cancer. Oncogene. 1999, 18: 6938-6947.View ArticlePubMedGoogle Scholar
- Bours V, Dejardin E, Goujon-Letawe F, Merville MP, Castronovo V: The NF-kappa B transcription factor and cancer: high expression of NF-kappa B- and I kappa B-related proteins in tumor cell lines. Biochem Pharmacol. 1994, 47: 145-149.View ArticlePubMedGoogle Scholar
- Dejardin E, Deregowski V, Chapelier M, Jacobs N, Gielen J, Merville MP, Bours V: Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells. Oncogene. 1999, 18: 2567-2577.View ArticlePubMedGoogle Scholar
- Visconti R, Cerutti J, Battista S, Fedele M, Trapasso F, Zeki K, Miano MP, de Nigris F, Casalino L, Curcio F, Santoro M, Fusco A: Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression. Oncogene. 1997, 15: 1987-1994.View ArticlePubMedGoogle Scholar
- Herrmann JL, Beham AW, Sarkiss M, Chiao PJ, Rands MT, Bruckheimer EM, Brisbay S, McDonnell TJ: Bcl-2 suppresses apoptosis resulting from disruption of the NF-kappa B survival pathway. Exp Cell Res. 1997, 237: 101-109.View ArticlePubMedGoogle Scholar
- Gasparian AV, Yao YJ, Kowalczyk D, Lyakh LA, Karseladze A, Slaga TJ, Budunova IV: The role of IKK in constitutive activation of NF-kappaB transcription factor in prostate carcinoma cells. J Cell Sci. 2002, 115: 141-151.PubMedGoogle Scholar
- Plummer SM, Holloway KA, Manson MM, Munks RJ, Kaptein A, Farrow S, Howells L: Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-kappaB activation via the NIK/IKK signalling complex. Oncogene. 1999, 18: 6013-6020.View ArticlePubMedGoogle Scholar
- Shao J, Sheng H, Inoue H, Morrow JD, DuBois RN: Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells. J Biol Chem. 2000, 275: 33951-33956.View ArticlePubMedGoogle Scholar
- Kulkarni S, Rader JS, Zhang F, Liapis H, Koki AT, Masferrer JL, Subbaramaiah K, Dannenberg AJ: Cyclooxygenase-2 is overexpressed in human cervical cancer. Clin Cancer Res. 2001, 7: 429-434.PubMedGoogle Scholar
- Dannenberg AJ, Altorki NK, Boyle JO, Lin DT, Subbaramaiah K: Inhibition of cyclooxygenase-2: an approach to preventing cancer of the upper aerodigestive tract. Ann N Y Acad Sci. 2001, 952: 109-115.View ArticlePubMedGoogle Scholar
- Bostrom PJ, Aaltonen V, Soderstrom KO, Uotila P, Laato M: Expression of cyclooxygenase-1 and -2 in urinary bladder carcinomas in vivo and in vitro and prostaglandin E2 synthesis in cultured bladder cancer cells. Pathology. 2001, 33: 469-474.View ArticlePubMedGoogle Scholar
- Kokawa A, Kondo H, Gotoda T, Ono H, Saito D, Nakadaira S, Kosuge T, Yoshida S: Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by cyclooxygenase inhibitors. Cancer. 2001, 91: 333-338.View ArticlePubMedGoogle Scholar
- Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M: Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res. 1997, 57: 1276-1280.PubMedGoogle Scholar
- Gupta S, Srivastava M, Ahmad N, Bostwick DG, Mukhtar H: Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate. 2000, 42: 73-78.View ArticlePubMedGoogle Scholar
- Howe LR, Subbaramaiah K, Brown AM, Dannenberg AJ: Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr Relat Cancer. 2001, 8: 97-114.View ArticlePubMedGoogle Scholar
- Sandler RS: Epidemiology and risk factors for colorectal cancer. Gastroenterol Clin North Am. 1996, 25: 717-735.View ArticlePubMedGoogle Scholar
- Ferrandina G, Legge F, Ranelletti FO, Zannoni GF, Maggiano N, Evangelisti A, Mancuso S, Scambia G, Lauriola L: Cyclooxygenase-2 expression in endometrial carcinoma: correlation with clinicopathologic parameters and clinical outcome. Cancer. 2002, 95: 801-807.View ArticlePubMedGoogle Scholar
- Bellacosa A, Testa JR, Staal SP, Tsichlis PN: A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science. 1991, 254: 274-277.View ArticlePubMedGoogle Scholar
- Coffer PJ, Woodgett JR: Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991, 201: 475-481.View ArticlePubMedGoogle Scholar
- Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA: Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A. 1991, 88: 4171-4175.PubMed CentralView ArticlePubMedGoogle Scholar
- Stephens L, Anderson K, Stokoe D, Erdjument-Bromage H, Painter GF, Holmes AB, Gaffney PR, Reese CB, McCormick F, Tempst P, Coadwell J, Hawkins PT: Protein kinase B kinases that mediate phosphatidylinositol 3, 4, 5-trisphosphate-dependent activation of protein kinase B. Science. 1998, 279: 710-714.View ArticlePubMedGoogle Scholar
- Hayakawa J, Ohmichi M, Kurachi H, Kanda Y, Hisamoto K, Nishio Y, Adachi K, Tasaka K, Kanzaki T, Murata Y: Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res. 2000, 60: 5988-5994.PubMedGoogle Scholar
- Suzuki Y, Nakabayashi Y, Takahashi R: Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci U S A. 2001, 98: 8662-8667.PubMed CentralView ArticlePubMedGoogle Scholar
- Davies MA, Koul D, Dhesi H, Berman R, McDonnell TJ, McConkey D, Yung WK, Steck PA: Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN. Cancer Res. 1999, 59: 2551-2556.PubMedGoogle Scholar
- Gagnon V, St-Germain ME, Parent S, Asselin E: Akt activity in endometrial cancer cells: regulation of cell survival through cIAP-1. Int J Oncol. 2003, 23: 803-810.PubMedGoogle Scholar
- Asselin E, Wang Y, Tsang BK: X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat granulosa cells during follicular development. Endocrinology. 2001, 142: 2451-2457.PubMedGoogle Scholar
- Asselin E, Mills GB, Tsang BK: XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells. Cancer Res. 2001, 61: 1862-1868.PubMedGoogle Scholar
- Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C: PI3K/Akt and apoptosis: size matters. Oncogene. 2003, 22: 8983-8998.View ArticlePubMedGoogle Scholar
- Meng F, Liu L, Chin PC, D'Mello SR: Akt is a downstream target of NF-kappa B. J Biol Chem. 2002, 277: 29674-29680.View ArticlePubMedGoogle Scholar
- Wang Y, Chang J, Li YC, Li YS, Shyy JY, Chien S: Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway. Am J Physiol Heart Circ Physiol. 2004, 286: H685-H692.View ArticlePubMedGoogle Scholar
- Risinger JI, Hayes AK, Berchuck A, Barrett JC: PTEN/MMAC1 mutations in endometrial cancers. Cancer Res. 1997, 57: 4736-4738.PubMedGoogle Scholar
- Risinger JI, Hayes K, Maxwell GL, Carney ME, Dodge RK, Barrett JC, Berchuck A: PTEN mutation in endometrial cancers is associated with favorable clinical and pathologic characteristics. Clin Cancer Res. 1998, 4: 3005-3010.PubMedGoogle Scholar
- Tashiro H, Blazes MS, Wu R, Cho KR, Bose S, Wang SI, Li J, Parsons R, Ellenson LH: Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res. 1997, 57: 3935-3940.PubMedGoogle Scholar
- Marsh DJ, Dahia PL, Caron S, Kum JB, Frayling IM, Tomlinson IP, Hughes KS, Eeles RA, Hodgson SV, Murday VA, Houlston R, Eng C: Germline PTEN mutations in Cowden syndrome-like families. J Med Genet. 1998, 35: 881-885.PubMed CentralView ArticlePubMedGoogle Scholar
- St-Germain ME, Gagnon V, Mathieu I, Parent S, Asselin E: Akt regulates COX-2 mRNA and protein expression in mutated-PTEN human endometrial cancer cells. Int J Oncol. 2004, 24: 1311-1324.PubMedGoogle Scholar
- Kane LP, Shapiro VS, Stokoe D, Weiss A: Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol. 1999, 9: 601-604.View ArticlePubMedGoogle Scholar
- Schmedtje J.F., Jr., Ji YS, Liu WL, DuBois RN, Runge MS: Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem. 1997, 272: 601-608.View ArticlePubMedGoogle Scholar
- Maehama T, Dixon JE: The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3, 4, 5-trisphosphate. J Biol Chem. 1998, 273: 13375-13378.View ArticlePubMedGoogle Scholar
- Piwien-Pilipuk G, Van Mater D, Ross SE, MacDougald OA, Schwartz J: Growth hormone regulates phosphorylation and function of CCAAT/enhancer-binding protein beta by modulating Akt and glycogen synthase kinase-3. J Biol Chem. 2001, 276: 19664-19671.View ArticlePubMedGoogle Scholar
- Guo S, Cichy SB, He X, Yang Q, Ragland M, Ghosh AK, Johnson PF, Unterman TG: Insulin suppresses transactivation by CAAT/enhancer-binding proteins beta (C/EBPbeta). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major activation domain of C/EBPbeta. J Biol Chem. 2001, 276: 8516-8523.View ArticlePubMedGoogle Scholar
- Wang L, Shao J, Muhlenkamp P, Liu S, Klepcyk P, Ren J, Friedman JE: Increased insulin receptor substrate-1 and enhanced skeletal muscle insulin sensitivity in mice lacking CCAAT/enhancer-binding protein beta. J Biol Chem. 2000, 275: 14173-14181.View ArticlePubMedGoogle Scholar
- Gorgoni B, Caivano M, Arizmendi C, Poli V: The transcription factor C/EBPbeta is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts. J Biol Chem. 2001, 276: 40769-40777.View ArticlePubMedGoogle Scholar
- Reddy ST, Wadleigh DJ, Herschman HR: Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J Biol Chem. 2000, 275: 3107-3113.View ArticlePubMedGoogle Scholar
- Tang Q, Gonzales M, Inoue H, Bowden GT: Roles of Akt and glycogen synthase kinase 3beta in the ultraviolet B induction of cyclooxygenase-2 transcription in human keratinocytes. Cancer Res. 2001, 61: 4329-4332.PubMedGoogle Scholar
- Dempke W, Rie C, Grothey A, Schmoll HJ: Cyclooxygenase-2: a novel target for cancer chemotherapy?. J Cancer Res Clin Oncol. 2001, 127: 411-417.View ArticlePubMedGoogle Scholar
- Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN: Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res. 1998, 58: 362-366.PubMedGoogle Scholar
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