Inhibition of cyclooxygenase-1 and -2 expression by targeting the endothelin a receptor in human ovarian carcinoma cells.

New therapies against cancer are based on targeting cyclooxygenase (COX)-2. Activation of the endothelin A receptor (ET(A)R) by endothelin (ET)-1 is biologically relevant in several malignancies, including ovarian carcinoma. In this tumor, the ET-1/ET(A)R autocrine pathway promotes mitogenesis, apoptosis protection, invasion, and neoangiogenesis. Because COX-1 and COX-2 are involved in ovarian carcinoma progression, we investigated whether ET-1 induced COX-1 and COX-2 expression through the ET(A)R at the mRNA and protein level in HEY and OVCA 433 ovarian carcinoma cell lines by Northern blot, reverse transcription-PCR, Western blot, and immunohistochemistry; we also investigated the activity of the COX-2 promoter by luciferase assay and the release of prostaglandin (PG) E(2) by ELISA. ET-1 significantly increases the expression of COX-1 and COX-2, COX-2 promoter activity, and PGE(2) production. These effects depend on ET(A)R activation and involve multiple mitogen-activated protein kinase (MAPK) signaling pathways, including p42/44 MAPK, p38 MAPK, and transactivation of the epidermal growth factor receptor. COX-2 inhibitors and, in part, COX-1 inhibitor blocked ET-1-induced PGE(2) and vascular endothelial growth factor release, indicating that both enzymes participate in PGE(2) production to a different extent. Moreover, inhibition of human ovarian tumor growth in nude mice after treatment with the potent ET(A)R-selective antagonist ABT-627 is associated with reduced COX-2 and vascular endothelial growth factor expression. These results indicate that impairing COX-1 and COX-2 and their downstream effect by targeting ET(A)R can be therapeutically advantageous in ovarian carcinoma treatment. Pharmacological blockade of the ET(A)R is an attractive strategy to control COX-2 induction, which has been associated with ovarian carcinoma progression and chemoresistance.

Spinella F ，Rosanò L ，Di Castro V ，Nicotra MR ，Natali PG ，Bagnato A ... -  《CLINICAL CANCER RESEARCH》

Endothelin-1 and endothelin-3 promote invasive behavior via hypoxia-inducible factor-1alpha in human melanoma cells.

Endothelin (ET) B receptor (ET(B)R), which is overexpressed in human cutaneous melanomas, promotes tumorigenesis upon activation by ET-1 or ET-3, thus representing a potential novel therapeutic target. Hypoxia-inducible factor-1alpha (HIF-1alpha) is the transcriptional factor that conveys signaling elicited by hypoxia and growth factor receptors. Here, we investigated the interplay between ET axis and hypoxia in primary and metastatic melanoma cell lines. We report that under normoxic conditions, ET(B)R activation by ET-1/ET-3 enhances vascular endothelial growth factor (VEGF) up-regulation, cyclooxygenase (COX)-1/COX-2 protein expression and COX-2 promoter activity, prostaglandin E(2) (PGE(2)) production, and do so to a greater extent under hypoxia. Moreover, COX-1/COX-2 inhibitors block ET-induced PGE(2) and VEGF secretion, matrix metalloproteinase (MMP) activation, and cell invasion, indicating that both enzymes function as downstream mediators of ET-induced invasive properties. The ET(B)R selective antagonist BQ788 or transfection with ET(B)R small interfering RNA (siRNA) block the ET-mediated effects. ETs also increase HIF-1alpha expression under both normoxic and hypoxic conditions and its silencing by siRNA desensitizes COX-2 transcriptional activity, PGE(2) and VEGF production, and MMP activation in response to ET-3, implicating, for the first time, HIF-1alpha/COX as downstream targets of ET(B)R signaling leading to invasiveness. In melanoma xenografts, specific ET(B)R antagonist suppresses tumor growth, neovascularization, and invasiveness-related factors. Collectively, these results identify a new mechanism whereby ET-1/ET-3/ET(B)R axis can promote and interact with the HIF-1alpha-dependent machinery to amplify the COX-mediated invasive behavior of melanoma. New therapeutic strategies using specific ET(B)R antagonist could provide an improved approach to the treatment of melanoma by inhibiting tumor growth and progression.

Spinella F ，Rosanò L ，Di Castro V ，Decandia S ，Nicotra MR ，Natali PG ，Bagnato A ... -  《CANCER RESEARCH》

Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells.

The peroxisome proliferator-activated receptor-gamma (PPARgamma), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the control of cyclooxygenase (COX) 2 expression in some tissue, although the exact mechanism(s) of this activity has not been elucidated. In this study we explored the possible mechanism(s) of control of COX-2 gene expression through PPARgamma signaling in human cervical cancer. Using primary human cervical tissues and the CaSki human cervical cancer cell line, we assayed for PPARgamma and COX-2 mRNA expression by reverse transcription-PCR. Nuclear protein binding activities to three response elements located in the COX-2 promoter [nuclear factor kappaB (NFkappaB), cyclic AMP response element, and activator protein (AP)-2] were measured by gel mobility shift assays. We used transient transfection assays with COX-2 promoter reporter gene constructs to determine the regulatory sites in this promoter, which mediates PPARgamma regulation of COX-2 activity. We showed, for the first time, that primary human cervical cancer tissues express PPARgamma. Using CaSki cells, we demonstrated that COX-2 and PPARgamma mRNA levels were inversely regulated by PPARgamma ligands in that these compounds up-regulated PPARgamma but down-regulated COX-2. In contrast, epidermal growth factor (EGF), a potent activator of COX-2, decreased PPARgamma mRNA levels. This down-regulation of PPARgamma mRNA by EGF was blocked in the presence of NS-398, a selective COX-2 inhibitor. PPARgamma ligands suppressed the binding activities of AP-1 (binding to CRE) and NFkappaB but not AP-2. Transient transfection results indicated that EGF stimulated whereas PPARgamma ligands inhibited COX-2 promoter (-327/+59) activity. This effect by PPARgamma ligands on the COX-2 promoter was blocked when the CRE, but not the NFkappaB, binding site was mutagenized. Cervical cancer cells express readily detectable levels of PPARgamma. There is reciprocal negative regulation between COX-2 and PPARgamma signaling in human cervical cancer cells. The ability of PPARgamma ligands to inhibit COX-2 appears to be mediated predominantly through inhibition of AP-1 protein binding to the CRE site in the COX-2 promoter.

Han S ，Inoue H ，Flowers LC ，Sidell N ... -  《CLINICAL CANCER RESEARCH》

IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells.

Nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer and this effect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/ cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE2 levels are higher in proliferating cells compared with post-confluent differentiated cells and in cells that constitutively overexpress IGF-II. Up-regulation of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfection of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative effect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events.

Di Popolo A ，Memoli A ，Apicella A ，Tuccillo C ，di Palma A ，Ricchi P ，Acquaviva AM ，Zarrilli R ... -  《ONCOGENE》

Gastrin-induced cyclooxygenase-2 expression in Barrett's carcinogenesis.

Cyclooxygenase (COX)-2 has been causally implicated in carcinogenesis. The evidence for increased COX-2 in the malignant progression of Barrett's esophagus is contradictory. We hypothesize that COX-2 expression may be causally affected by the gastrin status via the cholecystokinin 2 (CCK(2)) receptor. COX-2 and prostaglandin E(2) expression were evaluated by Western blotting and enzyme-linked immune assay in samples of squamous esophagus, Barrett's esophagus with varying degrees of dysplasia to adenocarcinoma, and normal duodenum. Differentiation status was evaluated by histopathology and villin expression. A longitudinal case-control study compared COX-2 in patients who progressed to adenocarcinoma with nonprogressors matched for age and length of follow-up. Messenger RNA levels of gastrin and CCK(2) receptor in biopsies and cell lines were evaluated by reverse transcription-PCR, and in vitro gastrin stimulation was conducted with and without inhibitors for CCK(2) (YM022) and COX-2 (NS-398). Cell proliferation was evaluated using minichromosome maintenance protein 2 (Mcm2) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. COX-2 expression is significantly increased in Barrett's esophagus before dysplasia development. Expression is highly variable within Barrett's dysplasia and adenocarcinoma samples independent of differentiation status. In a longitudinal case-control study, the expression levels within patients increased over time, regardless of the degree of malignant progression. Biopsies from nondysplastic Barrett's esophagus expressed increased gastrin mRNA levels compared with other biopsies. Gastrin significantly induced COX-2, prostaglandin E(2), and cell proliferation in biopsies and cell lines. Gastrin-induced proliferation can be inhibited by YM022 and NS-398. COX-2 is up-regulated early in the Barrett's metaplasia sequence. During carcinogenesis, gastrin is a significant determinant of COX-2 activity levels via the CCK(2) receptor.

Abdalla SI ，Lao-Sirieix P ，Novelli MR ，Lovat LB ，Sanderson IR ，Fitzgerald RC ... -  《CLINICAL CANCER RESEARCH》