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The inducible cyclooxygenase-2 (COX-2) gene regulates prostaglandin biosynthesis,is up-regulated in colorectal cancers, and can influence apoptotic susceptibility. We determined whether forced COX-2 expression modulates apoptosis induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of tumor necrosis factor ligand family, and examined determinants of the apoptotic pathway, including membrane death receptors (DR-4 and DR-5). HCT-15 colon cancer cells lacking endogenous COX-2 proteins were stably transfected with the COX-2 cDNA and incubated with TRAIL. Forced COX-2 expression significantly attenuated TRAIL-induced apoptosis and was associated with transcriptional repression of DR-5 and up-regulation of Bcl-2. COX-2 transfectants showed reduced DR-5 mRNA and protein expression as well as reduced caspase-8, caspase-3, and caspase-9 activation relative to parental cells. Sulindac sulfide treatment restored DR-5 expression and, when combined with TRAIL, reduced cell viability to a greater extent than did either drug alone. In summary, modulation of DR-5 and Bcl-2 levels by COX-2 attenuates TRAIL-induced apoptosis and represents a novel mechanism of intrinsic drug resistance in human colon cancer cells.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that converts arachidonic acid to prostaglandins. Overexpression of the COX-2 gene in mammary glands of transgenic mice was sufficient to induce tumorigenesis. We analyzed COX-2 expression in human breast cancers (and breast cancer cell lines) and adjacent ductal carcinoma in situ (DCIS) as well as its association with HER2/neu and clinicopathological variables. Archival primary breast carcinomas (n = 57), adjacent DCIS (n = 14) and DCIS alone (n = 2) were analyzed for COX-2 and HER2 expression by immunohistochemistry using specific monoclonal antibodies. An immunohistochemical scoring system was used. HER2 gene amplification had been analyzed previously by fluorescence in situ hybridization (n = 20). Histology of carcinomas included infiltrating ductal (n = 44), lobular (n = 2), and other (n = 7). Frozen breast cancers and adjacent normal tissue pairs (n = 9) were analyzed for COX-2 mRNA by reverse transcription-PCR. COX-2 and HER2 expression were also analyzed in human breast cancer cell lines (MCF-7, MCF-7/HER2, SK-BR-3, and MDA-MB-231) by immunoblotting. Cytoplasmic COX-2 expression was detected at an intermediate or high level in epithelial cells in 18 of 42 (43%) invasive breast cancers and in 10 of 16 (63%) cases of DCIS. Normal-appearing breast epithelia adjacent to cancer expressed COX-2 in 81% of cases and was generally focal and of similar or decreased intensity relative to adjacent neoplastic epithelia. COX-2 mRNA was detected in all samples analyzed by reverse transcription-PCR and was increased in eight of nine breast cancers relative to paired normal tissue. In archival tumors, no significant correlation was found between COX-2 and HER2 expression/amplification and clinicopathological variables. COX-2 expression was induced in MCF-7 cells stably transfected with HER2, in contrast to parental MCF-7 cells, and was detected in MDA-MB-231, but not SK-BR-3 cells. COX-2 is frequently overexpressed in invasive breast cancers and in adjacent DCIS and, thus, may be an early event in mammary tumorigenesis. Forced HER2 expression in MCF-7 cells was shown to up-regulate COX-2, although no association was found in human tumors. Our results suggest that nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors may be useful in the chemoprevention and therapy of human breast cancer.