Purpose Troglitazone (TRO) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist. 18 The cells in G1 phase are relatively even more radiosensitive than those in S stage and this could cause radiosensitization. Alternatively TRO stocks a common framework with supplement E that includes a potent antioxidant property [19]. In addition TRO can cause an induction of Cu2+/Zn2+-superoxide dismutase (CuZnSOD) [20 21 and the FG-4592 activated receptor complexes of TRO can induce catalase through binding to its promoter region [22 23 Both SOD and catalase may decrease radiation sensitivity via reactive oxygen species (ROS) scavenging. Although TRO was withdrawn from the market due to idiosyncratic hepatotoxicity [24] there is still much interest about the antitumor effect of TRO [25-27]. Most of antitumor effects was observed at relatively high concentrations (20-50 μM) of TRO while the clinically achievable concentrations are around 2-5 μM [28 29 In addition although combining low-dose PPARγ agonists with other drugs [27 30 is usually highly effective combining with radiation therapy has not been reported yet. From the above we were interested in what FG-4592 will be the net result of combining low dose TRO and radiation by the contradictory effect of TRO; SOD or catalase induction and increased G1 cells. PPARγ agonist mediated growth inhibition is usually PPARγ-dependent or -impartial [31 32 Therefore we investigated the combining effects of TRO and radiation in cervix cancer cell lines with different level of PPARγ expression. Materials and Methods 1 Cells and culture conditions The cell lines were purchased from the Korean Cell Line Lender (Seoul Korea). Cells were produced in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Recreation area Memorial Institute (RPMI) made up of 10% fetal bovine serum (FBS) supplemented with 100 IU/mL penicillin 100 μg/mL streptomycin. The cells were kept in a humidified atmosphere made up of 5% CO2 at 37℃ and passaged by trypsinization. TRO was purchased from Cayman Chemical (Ann Arbor MI USA) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 0.1% DMSO in the culture medium. All standard culture reagents were from Invitrogen (Carlsbad CA USA). 2 Protein extraction for western blot and catalase activity assay Cell proteins was obtained by rinsing the cells with phosphate-buffered saline (PBS) pH 7.2 three times scraping the cells from the culture flasks with a rubber policeman. The cells were washed with PBS by centrifugation two times. Pellets were FG-4592 kept frozen at -80℃ until use. At the time of analysis the cell pellets were resuspended in one volume of 50 mM potassium phosphate buffer FG-4592 (pH 7.85) and sonicated on ice 3 times for 10 seconds using a sonicator (Branson 1510R-DTH Danbury CT USA). Protein concentration was decided using the FG-4592 Bradford method using bovine serum albumin as a standard [33]. 3 Western blot analysis Proteins was denatured in 1 volume of sample buffer made up of 62.5 mM Tris-HCl (pH 6.8) 10 glycerol 2 sodium dodecyl sulfate (SDS) 5 β-mercaptoethanol (v/v) and 2-3 drops of saturated bromophenol blue answer at 100℃ for 3 minites. The proteins were separated in a 12.5% denaturing polyacrylamide gel by electrophoresis and then transferred onto nitrocellulose membranes at 100 V for 1 hour on ice. The blots were then blocked in 4% dry milk in Tween-Tris buffered saline (TTBS 0.02 M Tris buffer [pH 7.0] and 0.5% Tween 20) at room temperature for 2 hours and incubated with primary antibody (1:1 0 in TTBS at 4℃ overnight. After washing three times with TTBS 5 minutes each the blots were incubated with secondary antibody (1:10 0 After cleaning 3 x the blots had been visualized using chemiluminescence (Intron Biotechnology Seongnam Korea). The antibodies for Mouse monoclonal to PROZ CuZnSOD MnSOD and catalase had been bought from AbFrontier (Seoul Korea). 4 Catalase activity assay Cells had been treated with 2-10 μM of TRO every day and night. After getting cell pellets as described protein concentration was determined using Bradford assay previously. Catalase activity was quantitated following decomposition of H2O2 in 240 nm [34] spectrophotometrically. The catalase activity was portrayed as U/mg proteins. 5 RNA isolation and quantitative real-time polymerase string response RNA was isolated in the cells using TRIzol reagent based on the manufacturer’s process (Invitrogen Carlsbad CA USA). cDNA synthesized using iScript invert transcriptase reagent (Bio-Rad Hercules CA USA) from 1 μg of RNA. For real-time quantitative change.