'Combi-targeting' mitozolomide: Conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer

摘要

PURPOSE At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR-overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo. EXPERIMENTAL DESIGN To analyze its mixed EGFR-DNA targeting potential, we performed an enzyme linked immunosorbent assay (ELISA) and western blotting analysis of EGFR phosphorylation in cells stimulated with EGF. DNA damage was analyzed using the comet assay, and apoptosis quantitated by annexin V binding assay. Growth inhibition in vitro was determined by the sulforhodamine B (SRB) assay and in vivo efficacy analyzed in male CD-1 nude mice. RESULTS The results showed that: Under physiological conditions, JDF12 was hydrolyzed to JDF04R and both agents were capable of inhibiting isolated EGFR tyrosine kinase (TK) and EGFR phosphorylation in EGF-stimulated cells. JDF12 significantly damaged DNA, induced apoptosis in DU145 cells and was up to 2-10-fold more potent than equieffective combinations of MTZ and JDF04R or Iressa in a panel that also included LNCaP and its EGFR and ErbB2 transfectants. In vivo, it induced significant antitumor activity in a DU145 xenograft model. CONCLUSIONS The results suggest that the superior cytotoxicity of JDF12 when compared with MTZ and JDF04R may be imputed to its potent EGFR-DNA targeting properties and confirm the ability of this novel strategy to confer EGFR targeting properties to a classical alkylator.