suggested these differences in esterase activities could be exploited to develop prodrugs that selectively target cancer cells. The esterases observed selleck chemical by Yamazaki et al. were, however, never identified. The primary focus of the work presented here was to identify the specific esterases differentially expressed in tumorigenic human prostate cancer cells and in non tumorigenic prostate epithelial cells. We compared the esterase activity profiles of RWPE 2, LNCaP, DU 145, and PC 3 tumorigenic prostate cell lines to RWPE 1 nontu morigenic prostate epithelial cells using the naphthyl acetate substrate and the chiral naphthyl ester substrates naphthyl N acetyl S alaninate and naphthyl N acetyl R alaninate. These substrates were previously used by Yamazaki et al. Figure 2 shows the structures of the various substrates.

In addition, we have advanced the Yamazaki method of detecting esterases by using a native electroblot method that markedly in creases the sensitivity for detecting esterase activity bands compared to that observed in n PAGE gels. We identified oxidized protein hydrolase, also called N acylaminoacyl peptide hydrolase, as a key esterase that is overexpressed in the tumorigenic LNCaP cell line. OPH is a serine esterase protease that has a well characterized esterase activity towards naphthyl butyrate and an exopeptidase activity for removing the N terminally acetylated amino acid residues from peptides proteins. Immunohistochemistry of primary pros tate tumor sections indicate that OPH is highly expressed in some prostate tumors, suggesting that OPH could have potential as a drug target in prostate cancer.

The overexpression of OPH in some prostate cancers suggests that chemotherapeutic prodrugs esters modeled after known ester substrates of OPH have potential in treating some prostate cancers. Methods Materials Porcine liver esterase, digitonin, naphthyl acetate, fast blue RR salt, goat anti rabbit HRP conjugate polyclonal antibody, and diisopropyl fluorophosphate were purchased from Sigma Chemical Company. Novex Tris glycine native sample buffer, NuPAGE LDS sample buffer, Novex Tris glycine gels, NativeMark unstained protein standards, Protein A agarose beads, penicillin streptomycin solution, and geneticin were purchased from Invitrogen.

Precision plus protein standards were purchased from Bio Rad, the BCA kit and the In gel tryptic digestion kit were purchased from Pierce, ZipTipU C18 tips were purchased from Millipore, 3,3,5,5 tetramethylbenzidine was purchased from Promega, rabbit polyclonal anti AARE antibody was purchased from Abcam, superose 12 column was purchased from GE Healthcare, pCDNA3. promotion information 1 vector encoding OPH Flag was a kind gift from Dr. M. Hayakawa. Substrates R and S isomers of ANAA were synthesized and purified as previously described and stored at 20 C. Stock solutions of 100 mM naphthyl acetate were prepared in DMSO and stored at 20 C.