We have previously characterized the expression of OPH in LNCaP,

We have previously characterized the expression of OPH in LNCaP, RWPE 1, COS 7 and COS 7 OPH cell lines. Moreover, Kumar et al. have Oligomycin A IC50 characterized the degree of Akt activation in RWPE 1, LNCaP, DU145 and PC3 cells as well as the basal levels of oxidative stress. We found that S NPAA was the most effective prodrug in its ability to deplete GSH, cause oxidative stress, induce apoptosis, and de crease cell viability, particularly in cell lines overex pressing OPH. Methods Materials Reduced glutathione, digitonin, dimethyl sulfoxide, 2,2,2 trichloroacetic acid, 2,4 dinitro phenylhydrazine, 5,5 dithiobionitrobenzoic acid and diisopropyl fluorophosphate were purchased from Sigma Chemical Company.

DMEM, KSFM and growth factors, and RPMI 1640 cell medium, penicillin streptomycin solution, and genet icin and KB plus DNA ladder, Celltracker Inhibitors,Modulators,Libraries blue, 10kD spin columns, and EnzChek Caspase 3 assay kit were purchased from Invitrogen. BCA kit and the anti DYKDDDDK antibody were purchased from Pierce. Celltiter Inhibitors,Modulators,Libraries 96 AQueous One MTS kit, described as the MTS viability assay in experiments, was purchased from Promega and contained CellTiter96 Aqueous One Solution composed of a tetrazolium compound phenyl N acetyl L alaninate was synthesized as previously described. R NPAA, S NQM, and R NQM were synthesized with the fol lowing modifications. R enantiomers were synthesized using N acetyl D alanine in place of N acetyl L alanine. The naphthyl core of NQM Inhibitors,Modulators,Libraries prodrugs were synthesized by re placing 4 phenol with 4 1 naphthol.

Cell culture and lysates Tumorigenic cell lines LNCaP, DU 145, and PC 3 and the non tumorigenic cell line RWPE 1, and COS 7 cells were purchased from American Type Culture Collection, cultured according to ATCCs in structions and supplemented with 100 U ml penicillin and 100 mg ml streptomycin. Inhibitors,Modulators,Libraries Cells were detached from the 75 cm3 cell culture flasks after reaching 80% confluence by washing the cells with PBS followed by the addition of 0. 25% trypsin. The detached cells were centrifuged at Inhibitors,Modulators,Libraries 500 g for 5 min and washed with PBS to remove trypsin. Cells were centrifuged a second time and pellets stored at ?80 C. Cell pellets of each cell line were lysed using 2% digitonin in PBS on ice with vortexing every two min. After 10 min of incubation on ice, the ly selleck compound sates were centrifuged at 18,000 g for 5 min at 4 C and the supernatant collected. Protein concentrations were determined with the BCA kit using the manufac turers instructions. Semi purified OPH from rat liver OPH was semi purified from 100 g of rat liver using the method described by Stone et al. The pooled semi purified rOPH was analyzed by mass spectroscopy as described by Stone et al. to verify that no other esterases or proteases were present.

We have previously characterized the expression of OPH in LNCaP,