Cancer cell esterases are often overexpressed and some have chiral specificities different from those of corresponding normal cells. Carboxylesterases in particular are known to be overexpressed in several cancers. Additionally, cancer cells often exhibit high levels of intrinsic oxidative stress that is required for survival and an aggressive phenotype. We hypothesized that these 2 characteristics of cancer cells could be exploited to aid in the detection and treatment of prostate cancer. We have developed a fluorogenic ester probe that is activated by carboxylesterase to help distinguish tumorigenic cells from nontumorigenic prostate cells. Ester prodrugs have the same activation mechanism and have been thought to be a promising approach in cancer therapy. Prodrugs are inactive drugs that can be selectively activated by a specific enzyme. We have developed a chiral ester prodrug strategy using native polyacrylamide gel electrophoresis (n-PAGE) and proteomic methods to compare and identify the esterase profiles of several tumorigenic and nontumorigenic prostate cell lines. Our results showed that cell lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines exhibit differential esterase activity compared with non-tumorigenic RWPE-1 prostate cell lysates when incubated with α- naphthyl acetate or α-naphthyl N-acetyl-alaninate ester substrates and a diazonium salt. We have identified oxidized protein hydrolase (OPH), a serine esterase/protease that catalyzes the removal of N-acylated residues from proteins, to be differentially expressed between some tumorigenic and nontumorigenic prostate cell lines. OPH was found to have high hydrolytic activity towards the S-isomer of α-naphthyl N-acetylalaninate (S-ANAA) chiral ester. LNCaP lysates incubated with N-acetyl-alanyl-p-nitroanilide, a known OPH substrate, had twofold higher OPH activity compared with RWPE-1 lysates. We have also developed and tested novel glutathione depleting prodrugs modeled after S-ANAA that increase oxidative stress and induced apoptosis in tumorigenic prostate cells with little effect on nontumorigenic RWPE-1 cells. These results suggest that ester molecular beacon probes and ester prodrugs may be effective in identifying and treating prostate cancer tumors that overexpress esterases with little effect on normal prostate cells.

Cancer cell esterases are often overexpressed and can have chiral specificities different from that of the corresponding normal cells and can, therefore, be useful targets for activating chemotherapeutic prodrug esters. Prodrug esters are inactive compounds that can be preferentially activated by esterase enzymes. Moreover, cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species (ROS) and a decreased expression of some enzymatic antioxidants. Prodrugs designed to induce additional oxidative stress can selectively induce apoptosis in cancer cells already exhibiting a high level of intrinsic oxidative stress. This study focused on the in vitro evaluation of four novel prodrug esters: the R- and S- chiral esters of 4-[(nitrooxy)methyl]phenyl N-acetylalaninate (R- and S-NPAA) and the R- and S- chiral esters of 4-[(nitrooxy)methyl]naphth-1-yl N-acetylalaninate (R- and S-NQM), which are activated, to varying extents, by oxidized protein hydrolase (OPH, EC 3.4.19.1) yielding a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. OPH is a serine esterase/protease that is overexpressed in some human tumors and cancer cell lines.Methods: To evaluate the chiral ester prodrugs, we monitored cellular GSH depletion, cellular protein carbonyl levels (an oxidative stress biomarker) and cell viability in tumorigenic and nontumorigenic prostate cancer cell lines.Results: We found that the prodrugs were activated by OPH and subsequently depleted GSH. The S-chiral ester of NPAA (S-NPAA) was two-fold more effective than the R-chiral ester (R-NPAA) in depleting GSH, increasing oxidative stress, inducing apoptosis, and decreasing cell viability in tumorigenic prostate LNCaP cells but had little effect on non-tumorigenic RWPE-1 cells. In addition, we found that that S-NPAA induced apoptosis and decreased cell viability in tumorigenic DU145 and PC3 prostate cell lines. Similar results were found in a COS-7 model that overexpressed active human OPH (COS-7-OPH).Conclusions: Our results suggest that prostate tumors overexpressing OPH and/or exhibiting a high level of intrinsic oxidative stress may be susceptible to QM generating prodrug esters that are targeted to OPH with little effect on non-tumorigenic prostate cells.