Cancer Prevention Research The Cyclooxygenase Inhibitor Sulindac Sulfide Inhibits EP4 Expression and Suppresses the Growth of Glioblastoma Cells

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal antiinflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal–regulated kinase kinase-1/extracellular signal–regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide–induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity. Glioblastomas are the most common primary malignant brain tumors. Despite advancements in cancer therapies including surgery, radiation therapy, and chemotherapy, the median survival time is 12 to 15 months for patients newly diagnosed with glioblastoma (1). Shono et al. (2) reported that high cyclooxygenase-2 (COX-2) expression is associated with clinically more aggressive gliomas and is a strong predictor of poor survival. Thus, the activation of EP2 and EP4 is a potential modulator of glioblastoma progression. The overexpression of COX-2 is observed in several human cancers and increased COX-2 expression is associated with a poor prognosis in colon (3), breast (4), ovarian (5), and pancreatic cancers (6). The overproduction of prostaglandin E2 (PGE2) promotes tumor growth by binding to receptors designated EP1, EP2, EP3, and EP4. The EP receptor signaling pathways control cell proliferation, invasion, apoptosis, and angiogenesis. Deletion of the EP2 and EP4 receptors in mice causes a reduction of tumor growth in colorectal and breast cancers (7). EP2 and/ or EP4 expression is upregulated compared with normal tissues in colorectal (7) and breast (8) cancers, and EP4 mRNA expression is upregulated in human astrocytoma cells (9). Raza et al. (10) reported that the EP4 expression in surgically resected glioblastoma tissues is also upregulated compared with the expression in tissues from anaplastic astrocytomas. Nonsteroidal anti-inflammatory drugs (NSAID) exert their effects by inhibiting COX activity, thereby reducing the levels of prostaglandins. Some NSAIDs have chemopreventive effects against the development of human cancers, including glioblastomas (11–13). COX inhibitors may be useful, but the recently discovered toxic side effects of the selective COX-2 inhibitors seem to eliminate their clinical use. Sulindac, the prodrug for sulindac sulfide, a nonselective COX inhibitor, is well documented as an effective drug in preventing the development of intestinal polyps in experimental animals and of tumors in humans, but little information is available of prevention of glioblastoma by sulindac. The chemopreventive effect of sulindac sulfide and other NSAIDs, in general, seems to be complex and not exclusively dependent on inhibition of COX activity. Drugs such as sulindac sulfide, as well as other chemopreventive chemicals, increase the expression of Authors' Affiliations: Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina; Division of Neurosurgery, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan; and Department of Pathobiology, College of Veterinary Medicine, the University of Tennessee, Knoxville, Tennessee Received 7/1/09; revised 8/12/09; accepted 9/1/09; published OnlineFirst 11/24/09. Grant support: NIH, National Institute of Environmental Health Sciences Intramural Research Program. Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). Requests for reprints: Thomas E. Eling, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, 111 TW Alexander Drive, Research Triangle Park, NC 27709. Phone: 919-541-3911; Fax: 919-541-0146; E-mail: [email protected]. ©2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-09-014