Chemopreventive action of Green Tea Catechins (GTCs) was proven effective in several experimental models. Clusterin (CLU) gene is potently up-regulated in the regressing rat prostate following androgen ablation. We reported its down-regulation in human CaP, suggesting that it might be a tumor-suppressor gene in the prostate. Here, normal (NPC), SV40-immortalized (PNT2, PNT1a), tumorigenic androgen-independent (PC3, DU145) human prostate epithelial cells and spontaneously developing CaP transgenic mice (TRAMP model) were treated with GTCs to define their molecular action by studying the expression of CLU during both progression and chemoprevention of CaP. GTCs were extracted from green tea leaves and content was assessed by HPLC (EGC 5,5%; EC 12,24%; EGCG 51,88%; ECG 6,12%; total GTCs 75,7%). EGCG was purchased from Sigma. Pure EGCG or GTCs were given to NPC, PNT2, PNT1a, PC-3 and DU145 cells. TRAMP mice received 0.3% GTCs daily in drinking water soon after weaning. CLU expression was assessed by Northern and Western blot in comparison with histone H3 mRNA (marker of the S-phase of cell proliferation) and GAPDH mRNA (housekeeping). EGCG induced dose-response cell death in PNT2, PNT1a, PC3 and DU145 (IC50: 31.8, 83.6, 99.4 and 202.3 mg/ml, respectively) without any significant effect on NPC cells. GTCs were as effective as EGCG. Both EGCG and GTCs treatment induced casp-8 and –3 activation and CLU expression. In the TRAMP model, CLU mRNA and protein were potently down-regulated in parallel to casp-9 activation when CaP progressed at 17 and 22 weeks of age. Histone H3 was potently up-regulated instead, as expected (SV40 expression induces proliferation of prostate cells in TRAMP mice). Oral GTCs administration reduced CaP onset from 100% to 20% without any side effect. GTCs treatment induced CLU mRNA and protein in parallel to casp-9 expression and activation until 24 weeks of age, also causing strong repression of histone H3 mRNA. Changes in CLU expression in the TRAMP model were fully validated by immunohistochemistry analysis using both polyclonal anti-mouse clusterin antibody from Santa Cruz, USA, and a new affinity-purified polyclonal anti-mouse clusterin antibody that we have recently developed. EGCG and GTCs showed CaP chemopreventive activity both in vitro and in vivo. This effect involved CLU over-expression, apoptosis activation and decreased prostate cell proliferation. These data support the hypothesis that CLU is a new anti-oncogene for CaP, suggesting that GTCs chemopreventive action is directly mediated by sustained CLU gene over-expression in the prostate.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]