Cyclooxygenase-1 is a potential target for prevention and treatment of ovarian epithelial cancer

Abstract

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.

Figure 1

COX-1 is the predominant isoform expressed in OSE cancer cell lines. A, Western blot analysis of COX-1 and COX-2 expression in C1, C2, T1, and T2 OSE cell lines. B, Northern hybridization of Cox-1 and Cox-2 mRNA in C1, C2, T1, and T2 cancer cell line samples. rPL7 is a housekeeping gene.

Figure 2

Tumors generated from OSE cancer cell lines show similar expression of COX-1 and COX-2 as observed for cell lines. A, Western blot analysis of COX-1 and COX-2 expression in tumors. B, Northern hybridization of Cox-1 and Cox-2 in tumor samples. rPL7 is a housekeeping gene. C, in situ hybridization of Cox-1 and Cox-2 in tumor sections. Bar, 500 μm. D, in situ hybridization of Cox-1 and Cox-2 in normal ovaries. Wild-type mice were treated with 5 IU of pregnant mare serum gonadotropin for 48 hours followed by 5 IU of human chorionic gonadotropin to stimulate follicular growth for preparation of ovulation at a defined time point. Ovarian sections from representative mice at 8 hours after hCG treatment. Arrowheads, surface epithelium. Bar, 200 μm.

Figure 3

PGI₂ is the predominant prostaglandin produced in T2 OSE cell lines and is inhibited by a COX-1 selective inhibitor SC-560 in a dose-dependent manner. T2 cells were cultured in 24-well plates and treated with arachidonic acid (AA) with or without COX inhibitors. Prostaglandin (PG) levels were detected by GS/MS. A, prostaglandin synthesis in mouse OSE cancer cells is a function of arachidonic acid concentration. B, effects of COX inhibitors on 6-keto-PGF1α formation as an index of PGI₂ levels. C, effects of COX inhibitors on PGE₂ levels.

Figure 4

PGIS is expressed in OSE cells and tumors. A, Western blotting of PGIS in C1, C2, T1, and T2 OSE cell line samples. B, Western blotting of PGIS in tumor samples. C, in situ hybridization of Pgis in tumor sections. Representative darkfield photomicrographs of tumor sections show hybridization signals. Bar, 500 μm.

Figure 5

COX-1 selective inhibitor SC-560 attenuates OSE tumor growth. Nude mice were allografted with T2 OSE cells and after establishment of the tumors 7 days later they were gavaged orally with the vehicle (0.1 mL), SC-560 (3 mg/kg bd) or celecoxib (50 mg/kg bd) for (A) 18 days (nine mice each for vehicle or SC-560 treated group and six mice for celecoxib treated group; these experiments were repeated at two different times) and (B) for 50 days (5-6 mice per group). Tumor growth was recorded every 2 to 4 days for a period of 59 and 64 days, respectively. Columns, mean; bars, ±SE. *, P < 0.05, significantly different from the vehicle-treated group (unpaired t test).

Figure 6

COX-1 selective inhibitor SC-560 attenuates OSE tumor cell proliferation and accelerates apoptosis. A, cell growth assay. T2 Cells (0.5 × 10⁵) were seeded in 12-well plates and incubated with DMSO (vehicle, 0.1%), SC-560 or celecoxib at different concentrations for indicated days. Cell numbers were counted from triplicate wells. Points, mean of three independent experiments; bars, ±SE. B and C, apoptosis assay. Cells were treated with DMSO, SC-560, or celecoxib at indicated concentrations for 3 days after removal of the serum from the culture media. Number of apoptotic cells was determined by flow cytometry using an Annexin V-FITC kit. Columns, mean of percent apoptotic cells in three separate experiments; bars, ±SE. D, immunostaining of PCNA, Ki67, and TUNEL in tumors. Tumor samples used in this study were derived from nude mice in Fig. 5 legend. Bar, 50 μm.