In ovarian cancer, very limited number of studies has directly examined the effect of altering CLU expression on cell death and survival. Thus, prognostic significance of CLU expression in ovarian Selleckchem SAHA HDAC cancer patients remains controversial [26–29]. To establish the clinical significance of CLU as a potential molecular target to predict survival in ovarian cancer patients, we conducted this study. Methods
Cell line Human ovarian cancer cell line, KF, was provided as a generous gift by Dr. Yoshihiro Kikuchi, National Defence Medical College, Saitama, Japan. Another ovarian adenocarcinoma cell lines, SKOV-3 and OVK-18 cells, were purchased from ATCC, and clear cell carcinoma cell lines, KOC-7c and TU-OC-1, were provided as a generous gift by Dr. Junzo Kigawa, Tottori
University, Japan. All cell lines were maintained in RPMI-1640 supplemented with 2 mM L-glutamine, and 10% FCS (Sigma, St. Louis, MO, USA) OVK18 cells, maintained in DMEM supplemented with 2 mM L-glutamine and 10% FCS (Sigma). Both KF-TX and SKOV-3-TX clone were established from parental cell lines KF and find more SKOV-3, respectively by maintaining each clone in increasing sublethal concentration of TX (up to 10 nM for KF-TX and 2 nM for SKOV-3-TX) for more than ten months then IC50 of each clone was determined by the viability assay after three days treatment. Antibodies and reagents Mouse anti-human CLU (clone 41 D, Upstate Biotechnology, Lake Placid, NY, USA) was used at 1:1,000 dilution for western blotting. Immunoblotting detection was done with anti-mouse secondary horseradish-peroxidase-conjugated antibodies (Dako) diluted 1:2,000. TX was supplied by Bristol-Myers Co. Ltd. (Japan). We then prepared Selleckchem C59 stock solution by diluting TX in the media
at a final concentration of 4 μM and further working dilutions were carried out to reach the desired concentration. Antisense oligodeoxynucleotide against CLU (OGX-011) was provided by Oncogenex (Canada). Transient transfection of KF-TX cells with si-RNA or OGX-011 To knock down the expression of CLU, siRNA or OGX-011 was used in this study. Validated siRNA oligomers directed against the s-CLU mRNA leader endoplasmic reticulum signal peptide (s-CLU-siRNA) [30] and a control sequence which does not match any gene sequence (Cont-siRNA) were synthesized by Ambion (USA): s-CLU-siRNA, 5-GCG UGC AAA GAC UCC AGAAdTdT-3 and 3-dTdTCGC ACG UUU CUG AGG UCU U-5; Cont-siRNA, 5-GCG CGC UUU GUA GGA UUC GdTdT-3 and 3-dTdTCGCGCG AAA CAU CCU AAG C-5. s-CLU-siRNA or cont-siRNA were transfected into ovarian cancer cells (105 cells/60-mm dish) using SiPORT Neofex (Ambion; USA) at a final concentration of 200 nM. KF-TX cells were cultured to 50% confluence. Transfection of OGX-011 was done twice using Effectine (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.