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Cancer Research

in 10% charcoal-striped FBS RPMI 1640 either with or without dihydrotestosterone (1 108 mol/L) and either with capsaicin

(2 104

mol/L) or diluent control for 24 hours. Luciferase activity

was measured and the fold induction calculated relative to that measured in control cells (Fig. 2). Dihydrotestosterone increased reporter activity >60-fold compared with control LNCaP cells. Capsaicin completely inhibited this effect. These results indicate that capsaicin inhibited the ability of androgen to transactivate the PSA promoter/enhancer. Additional experiments used the lucifer- ase-reporter construct containing either a 496-bp fragment of the PSA enhancer, containing six AREs or ARE4-E4Lux (multimerized four consensus AREs from the PSA promoter; ref. 23); luciferase activity was stimulated by dihydrotestosterone (1 108 mol/L), and capsaicin again completely inhibited this stimulation for each construct (data not shown).

Capsaicin reduces androgen-induced PSA and AR expres- sion. Protein levels of PSA were examined in the androgen- sensitive prostate cancer cell line, LNCaP. Cells were incubated with 109 mol/L dihydrotestosterone either with or without capsaicin (0.5, 1, or 2 104 mol/L, 24 hours). As expected, PSA

expression was induced by dihydrotestosterone, which was blocked by capsaicin (104 mol/L; Fig. 3A). The expression of AR was also induced by dihydrotestosterone, and capsaicin (104 mol/L) inhibited this effect with levels of AR protein becoming undetect- able in the presence of 2 104 mol/L capsaicin.

To determine whether capsaicin affected the transcription of AR, RNA expression levels of AR were measured by real-time reverse transcription-PCR. LNCaP cells were incubated with 109 mol/L dihydrotestosterone either with or without capsaicin (2 104 mol/L) for either 12 or 24 hours. Capsaicin inhibited dihydrotestosterone-induced RNA expression of AR within 12 hours (Fig. 3B).

To determine whether the inhibitory effect of capsaicin on PSA transcriptional activity in LNCaP cells was solely a result of down- regulation of AR protein levels, AR cDNA expression vector (pCMV-AR) was placed in LNCaP cells (Fig. 3C). These cells were also transfected with PSA P/E-Luc and cultured either with or without dihydrotestosterone (108 mol/L) and either with or without capsaicin (104 mol/L) for 24 hours. Cells were harvested and Western blot analysis and Luciferase assay were done.

Figure 1. LNCaP and PC-3 prostate cancer cells: effect of capsaicin on their clonal proliferation, cell cycle, apoptosis, and related proteins. A, cells were cultured in soft agar with increasing amounts of capsaicin, colonies were counted (day 14) and expressed as a percentage of colonies relative to untreated control plates. Points, means of three independent experiments done in triplicate plates; bars, FSD. B, PC-3 cells were exposed to increasing amounts of capsaicin for 24 hours, and cell cycle analysis was done. C, cells were cultured for 24 hours with increasing amounts of capsaicin. Apoptosis was measured by TUNEL assay and expressed as a percentage of apoptotic cells relative to untreated control PC-3 and LNCaP cells. Columns, means of three independent experiments; bars, FSD. D, LNCaP cells were incubated with capsaicin (2 104 mol/L) for indicated times. Lysates were analyzed by Western blot for levels of p53, Bax, p21 Waf1, and GAPDH.

Cancer Res 2006; 66: (6). March 15, 2006

3224

www.aacrjournals.org

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