Chemotherapeutic approaches to glioblastoma which are highly
Chemotherapeutic approaches to glioblastoma, which are highly invasive, are still largely unsuccessful and have only a minimal impact on patients’ survival rate . New agents that target the specific molecular aberrations in the tumors are presently being investigated for their efficacy against this malignancy . Mitochondria play an essential role in apoptosis by releasing apoptogenic molecules such as cytochrome c and apoptosis-inducing factor, and the voltage dependent anion channel (VDAC) and mitochondrial translocator protein (TSPO) are taking part in activation of the mitochondrial apoptosis pathway . TSPO and VDAC are involved in the generation of reactive oxidative species to induce the mitochondrial apoptosis pathway and has become a target for drug development directed to find treatment for neurodegenerative diseases and cancer. In the field of anti-cancer gene therapy, the activation of anti-apoptotic genes in tumors is a compelling molecular target , and the identification of novel anti-apoptotic genes that are expressed in tumor cells is expected to lead to new anti-cancer gene therapeutics. Using a yeast-based system, previously established for the functional screening of anti-apoptotic mammalian genes from a tumor cell-derived cDNA library , we identified FPPS as a novel inhibitor of paclitaxel-induced cell death in U87MG glioblastoma cells. In the present study, we show that FPPS inhibits paclitaxel-induced apoptosis through mevalonate metabolism and the c-Jun N-terminal kinase (JNK)-mediated apoptotic signal pathway. Paclitaxel, pamidronate and farnesyl diphosphate (FPP) were purchased from Sigma–Aldrich Co. (St. Louis, MO). Monoclonal and polyclonal Pam3CSK4 Biotin receptor against Bax, Bcl-X, caspase-3, cytochrome c, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other polyclonal antibodies against p53, JNK, phospho-JNK, p38, phospho-p38, extracellular signal regulated kinase (ERK), phospho-ERK, Akt, phospho-Akt, and Flag were purchased from Cell Signaling (Beverly, MA). Other reagents used were of the highest grade available. Human glioblastoma U87MG cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U/ml penicillin G, and 100μg/ml streptomycin at 37°C and 5% CO. Stable transfectants were generated by transfection of U87MG cells with the vector Flag-pcDNA3.1 or Flag-pcDNA3.1-FPPS, which was constructed by the insertion of human FPPS cDNA digested with BamHI/XbaI into the corresponding sites of Flag-pcDNA3.1. Following transfection, cells were cultured in medium containing 100μg/ml of hygromycin (Sigma–Aldrich Co., St. Louis, MO). Several independent clones were isolated, and positive clones were identified by Western blot analysis of cell lysates using an anti-Flag antibody. Cells expressing Flag or Flag-FPPS were seeded onto 24-well plates at a density of 1×10 cells per well. After incubation overnight, the cells were incubated with the indicated reagents for 48h, and then cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously . For the analysis of cell cycle profiles, cells treated with the concentrations of paclitaxel indicated were stained with propidium iodide, and cellular DNA was measured as described previously . Analysis of apoptosis by means of an Annexin-V-Fluos Staining Kit (Roche Applied Science, Indianapolis, IN) and fluorescence-activating cell sorting was performed using a FACS Calibur system (Becton Dickinson Biosciences, Franklin Lakes, NJ) as described previously . Cells were treated with specific reagents for the time periods indicated and were washed twice in ice-cold PBS and then resuspended in S-100 buffer [20mM HEPES (pH 7.5), 10mM KCl, 1.9mM MgCl, 1mM EGTA, 1mM EDTA, and a mixture of protease inhibitors]. The cell homogenate was centrifuged at 1,000× for 5min, and the resulting supernatants were re-centrifuged at 14,000× for 30min to collect the mitochondria-rich (pellet) and the cytoplasmic (supernatant) fractions. For the isolation of whole cell extracts, cells were lysed in a PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) for 90min at −20°C. The cell lysates were centrifuged at 10,000× for 20min and the supernatants (whole cell extracts) were collected. For detection of immunoreactive proteins by Western blotting, aliquots of mitochondrial, cytoplasmic, and whole cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences, Uppsala, Sweden). Membranes, blocked with 5% non-fat milk, were reacted with the antibodies indicated overnight at 4°C, and immunoreactive bands were detected using the ECL reagent (Bionote, Gyeonggi-do, Korea).