Objective The goal of this research was to research whether selective cyclooxygenase (COX) inhibitors promote paclitaxel-induced apoptosis in taxane-resistant ovarian tumor cells by suppressing gene expression. Selective CBL-SL COX inhibitors considerably promote paclitaxel-induced cell loss of life in taxane-resistant ovarian tumor cells within Flubendazole (Flutelmium) a prostaglandin-independent manner. COX inhibitors could be potent therapeutic tools to promote paclitaxel sensitization of taxane-resistant ovarian cancers by suppressing gene acts as a cell membrane pump extruding drugs to the extracellular space thereby reducing drug accumulation in cells. As reported previously numerous toxins and chemotherapeutic drugs including taxanes and anthracyclines are substrates [7]. Cyclooxygenase-2 (COX-2) is usually a key enzyme in transforming arachidonic acid to prostaglandins and it has been accepted as an unfavorable prognostic factor in numerous tumors including ovarian malignancy [8]. To overcome drug resistance by COX-2 COX-2 inhibitors have been studied in various cancers such as leukemia colon cancer and hepatocellular carcinoma [9-12]. Selective COX-2 inhibitors have been recognized for their anticancer effects in addition to their effects in treating rheumatoid arthritis and managing pain [13]. Moreover selective COX-2 inhibitors sensitize MDR malignancy cells to chemotherapeutic drugs in either COX-2-dependent or COX-2-impartial mechanisms [14]. The aim of the present study was to demonstrate the usefulness of selective COX inhibitors in promoting paclitaxel-induced apoptosis in taxane-resistant ovarian malignancy cells and to understand the mechanisms involved in the selective COX inhibitors suppressing gene expression. MATERIALS AND METHODS 1 Reagents and antibodies NS-398 and SC-560 selective COX inhibitors were purchased from Cayman Chemical Co. (Ann Arbor MI USA). Paclitaxel was purchased from your Cheil General Hospital Pharmacy (Seoul Korea). LY294002 a selective PI3K inhibitor was purchased from Cell Signaling Technology (Beverly MA USA). Prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich (St Louis MO USA). Antibodies against COX-1 COX-2 and cleaved poly ADP ribose polymerase Flubendazole (Flutelmium) (PARP) were supplied by Cell Signaling Technology (Beverly MA USA). The anti-antibody was obtained from Abcam (Cambridge UK). The anti-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz CA USA). The anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology. 2 Cell culture Human ovarian carcinoma cell lines SKOV3ip1 HeyA8 (taxane-sensitive) SKOV3ip2-TR HeyA8-MDR (taxane-resistant) were provided by Dr. AK Flubendazole (Flutelmium) Sood (Texas MD Flubendazole (Flutelmium) Anderson Malignancy Center TX USA). SKOV3ip1 and HeyA8 cells were produced in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 0.5% gentamicin. SKOV3ip2-TR and HeyA8-MDR cells were produced in RPMI 1640 supplemented with 10% FBS and 0.5% gentamicin and with 300 ng/mL paclitaxel. 3 Immunoblot analysis Cells (5×105) were lysed within a lysis buffer (10 mM Tris-HCl [pH 8.0] 150 mM NaCl 1 NP-40 1 sodium deoxycholate 0.1% sodium dodecyl sulphate [SDS]) protein were resolved by SDS-polyacrylamide gel electrophoresis (Web page) and transferred onto nitrocellulose membranes (Millipore Bedford MA USA). Membranes had been obstructed with 5% skim dairy in tris-buffered saline with tween 20 (TBS-T) buffer (10 mM Tris-HCl [pH 7.5] 150 mM 0 NaCl.1% Tween-20) for one hour and incubated with relevant antibodies for 18 hours at 4℃. Membranes had been cleaned with TBS-T buffer and incubated for one hour at area temperatures (RT) with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG at a dilution of just one 1:5 0 Proteins bands had been visualized using improved chemiluminescence (Amersham-Pharmacia Piscataway NJ USA). 4 MTT assay Cells (3×103) had been seeded in 96-well microplates treated with COX inhibitors PGE2 or paclitaxel as indicated. At 72 hours after incubation 100 μL/well of 2 mg/mL 3-(4 5 5 bromide (MTT) option was put into the microplates. Two hours after MTT treatment the moderate was taken out and formazan crystals had been dissolved with the addition of 100 μL dimethylsulfoxide per well. Cell viability was examined by calculating the absorbance at 590 nm using an enzyme-linked immunosorbent assay audience. 5 Total RNA isolation and invert transcription polymerase string response Total RNA was extracted using TRIzol reagent (Invitrogen Carlsbad CA USA). One microgram from the isolated.