Tumor-suppressive effects of neutral endopeptidase in androgen-independentprostate cancer cells

Expression of neutral endopeptidase (NEP) 24.11 is diminished in metastatic , androgen-independent prostate cancers (PCs; C, N, Papandreou et al., Nat, Med., 4: 50-57, 1998), To determine the effects on androgen-independent PC cells of overexpressing cell-surface NEP, an inducible tetracycline-regula tory gene expression system was used to stably introduce and express the NE P gene in androgen-independent TSU-Prl cells generating WT-5 cells, which e xpressed high levels of enzymatically active NEP protein when cultured in t he absence of tetracycline, TN12 cells, which contain the identical vectors without the NEP gene and do not express NEP, were used as control. Express ion of NEP in WT-5 cells after removal of tetracycline from the media resul ted in a > 80% inhibition in cell proliferation over a I-meek period (P < 0 .005) compared with control cells. Tumor formation occurred in the prostate glands of orthotopically injected athymic mice killed at 30 days in 4 of 5 mice that mere given injections of 2 x 10(6) WT-5 cells and were fed doxyc ycline (NEP suppressed), and in ail mice that were given injections of TN12 cells and mere fed with or without doxycycline. In contrast, only I of 5 m ouse prostates developed a tumor in mice that were given injections of WT-5 cells and that did not receive doxycycline, Analysis of the mechanisms of NEP-induced growth suppression revealed that NEP expression in WT-5 cells i nduced a 4-fold increase in the number of PC cells undergoing apoptosis, an d increased the expression of p21 tumor suppressor gene protein and the lev el of unphosphorylated retinoblastoma protein as determined by Western blot , Flow cytometric analysis show that induced NEP expression in WT-5 cells r esulted in a G, cell cycle arrest. These data show that NEP can inhibit PC cell growth and tumorigenicity and suggest that NEP has potential as therap y for and rogen-independent PC.