P450arom is the rate-limiting enzyme that catalyzes the final step in the conversion pathway from androgen to estrogen. The quantity and activity
of P450arom can directly affect the levels of estrogen in normal or abnormal tissues, in order to maintain estrogen-related physiologic functions in normal tissues. Meanwhile, P450arom play a role in the pathogenesis and prognosis of estrogen-dependent diseases. The HSP inhibitor activity of P450arom see more is regulated by prostaglandin E2 (PGE2), which is affected by cyclooxygenase-2 (COX-2). We hypothesize that COX-2/PGE2/P450arom might be a signaling pathway in estrogen-dependent diseases to regulate the autocrine activity of estrogen in cancerous tissues. Previous reports indicated that HER-2/neu regulated the expression of COX-2 as the upstream molecular of COX-2-mediated signal pathways [2, 3]. In the present paper, our results demonstrated that transfection with HER-2/neu in endometrial cells induced the activation of COX-2/PGE2/P450arom signal, resulting in the increase of autocrine estrogen from endometrial cells. Materials and methods Cell culture The Ishikawa cell line was kindly supplied by the Department of Pathophysiology,
Beijing University. Cells were cultured in RIPM1640 with 10% fetal TPX-0005 ic50 bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in an incubator maintained at 37°C and 5% CO2. Celecoxib, a selective COX-2 inhibitor, was purchased from Santa Cruz Biotechnology and dissolved in DMSO to generate a 100 mM stock solution that was stored at −20°C. For inhibition experiment,
confluence cells were starved by serum deprivation overnight. Then, cells were treated with 80 μM celecoxib and incubated for 48 h. Construction of pcDNA3.1-HER2/neu Upstream (5′-TGGGAGCCTGGCATTTCTG-3′) and downstream (5′-TCCGGCC ATGCTGAGATGTA-3′) 2-hydroxyphytanoyl-CoA lyase primers were designed based on HER-2/neu cDNA sequence obtained from GenBank. For cloning, HindIII/XbaI restriction endonuclease sites were inserted flanking the target gene primers. Primers were synthesized by TaKaRa Biotechnology Co., Ltd. Total RNA was isolated from Ishikawa cells using TRIzol reagent (TaKaRa, China) according to the manufacturer’s instructions. HER-2/neu cDNA was reverse-transcribed using the One Step RNA PCR Kit (TaKaRa) according to the manufacturer’s recommendations. PCR conditions included denaturation at 94°C for 5 min, 25 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C for 6 min, with a final extension at 72°C for 10 min. PCR products were separated on 1% agarose gel and eluted. The PCR product was sent to TaKaRa for sequencing. PcDNA3.1 plasmid and HER2 cDNA were digested with HindIII/XbaI double endonucleases. The digested products were separated by agarose gel electrophoresis and purified. Pure HER2 cDNA and vector were mixed at a 4:1 ratio and were ligated at 16°C for 20 h.