Reagents
PS, Bortezomib and VLX1570 were purchased from Selleck Chemicals, (Houston, Texas, TX, USA). This was dissolved in dimethylsulfoxide and stored at -80°C with being protected from light. We used those at the concentrations up to 200 nM.
Cell lines and culture
A granulosa cell tumor cell line, KGN and COV434 were maintained in DMEM/Ham’s F12 medium supplemented with 10% fetal bovine serum. An ovarian serous adenocarcinoma cell line, SK-OV-3 and a clear cell cancer cell line, RMG-I and OVISE were used in this study.
Cell growth assay and MTT assay
Cell growth was assessed by counting the number of living cells after trypan blue staining. Cell suspensions were plated into 96-well plates in the presence of the drug or solvent alone, incubated as above at 37°C for 1-4 days, and analyzed by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay17.
Apoptosis assay
Apoptosis was examined using an AnnexinV Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) and all samples were analyzed with FACS Calibur flowcytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA)18.
Cell cycle analysis
Cells were fixed with 70% methanol for 30 min and treated with 2 mg/ml ribonuclease A (Nacalai Tesque, Kyoto, Japan) for 30 min at 37°C, then with 50 mg/mL propidium iodide (PI; Sigma, St Louis, MO, USA) for further 20 min at room temperature19.
Immunoblotting analysis
Cell lysates of all five cell lines were prepared in lysis buffer containing 50 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 0.5% TritonX-100, 0.05% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 2 mM phenylmethylsulfonyl fluoride and 1 mM Na3VO4. The lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis was performed as previously described20.
Primary antibodies were obtained from Santa Cruz Biotechnology (bcl2, bcl-XL/Xs; Santa Cruz, CA, USA), Cell Signaling Technology (cleaved-PARP (cPARP), p21, acetyl-H4 (Lys12); Danvers, MA, USA), Sigma-Aldrich (α-tubulin, acetyl-α-tubulin (Lys40); St. Louis, Mo, USA), Horse-radish peroxidase-conjugated mouse and rabbit antibodies were from GE Healthcare Life Sciences (Piscataway, NJ).
Gene expression profiling and gene set enrichment analysis (GSEA)
Gene expression profiling of KGN cells were analyzed in three independent experiments. Treated cells were harvested after 24 h treatment with 100 nM of PS. Total RNA was extracted with RNeasy Mini Kit (Qiagen,Germantown, MD, USA), converted to cDNA and amplified with GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA, USA). The fragmentation, the labeling and the hybridization of cDNA were treated with GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Chips were scanned with a GeneChip Scanner 3000 7G System (Affymetrix).
The gene set enrichment analysis (GSEA; Broad Institute Cambridge, MA, USA) was performed using the gene expression profiling data and by handling the GSEA software. The detail information of these experiments is described in references21. In this study, the whole expression change in the gene sets was defined as statistically significant if both the false discovery rate (FDR) q-values and the familywise error rate (FWER) p-values were less than 0.25.
Statistical analyses
All results are shown as the mean values with ranges. Comparisons between the groups were done using the Dunnett’s and Scheffe’s tests. Differences were considered statistically significant if p-values were less than 0.05. These analyses were carried out using SPSS for Windows version 14.0.