Enterolactone glucuronide and beta-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment

摘要

Evidence from preclinical and animal studies demonstrated an anticancer effect of flaxseed lignans, particularly enterolactone (ENL), against prostate cancer. However, extensive first-pass metabolism following oral lignan consumption results in their systemic availability primarily as glucuronic acid conjugates (ENL-Gluc) and their modest in vivo effects. To overcome the unfavorable pharmacokinetics and improve their effectiveness in prostate cancer, antibody-directed enzyme prodrug therapy (ADEPT) might offer a novel strategy to allow for restricted activation of ENL from circulating ENL-Gluc within the tumor environment. The anti-prostate-specific membrane antigen (PSMA) antibody D7 was fused with human beta-glucuronidase (h beta G) via a flexible linker. The binding property of the fusion construct, D7-h beta G, against purified or cell surface PSMA was determined by flow cytometry and Octet Red 384 system, respectively, with a binding rate constant, K-d,K- of 2.5 nM. The enzymatic activity of D7-h beta G was first tested using the probe, 4-methylumbelliferone glucuronide. A 3.8-fold greater fluorescence intensity was observed at pH 4.5 at 2 h compared with pH 7.4. The ability of D7-h beta G to activate ENL from ENL-Gluc was tested and detected using LC-MS/MS. Enhanced generation of ENL was observed with increasing ENL-Gluc concentrations and reached 3613.2 ng/mL following incubation with 100 mu M ENL-Gluc at pH 4.5 for 0.5 h. D7-h beta G also decreased docetaxel IC50 value from 23 nM to 14.9 nM in C4-2 cells. These results confirmed the binding and activity of D7-h beta G and additional in vitro investigation is needed to support the future possibility of introducing this ADEPT system to animal models.