Suppression of metallothionein 3 gene expression by androgen in LNCaP prostate cancer cells

Abstract

Androgen deprivation therapy is the standard treatment for prostate cancer. However, tumors often progress towards a more aggressive phenotype despite treatment. Prostate tissue has a high zinc concentration, which may correlate with prostate cancer progression. Therefore, we investigated the effect of dihydrotestosterone (DHT) on the gene expression of metallothioneins (MTs) and zinc transporters in prostate cancer with quantitative real-time polymerase chain reaction (PCR). The MT3 gene expression in LNCaP cells was suppressed by DHT in a dose-dependent manner. However, it increased in a culture medium containing androgen-deficient charcoal-stripped fetal bovine serum (FBS). Bicalutamide, an androgen receptor antagonist, increased the gene expression of MT3 and partially reversed the suppression of MT3 gene expression induced by DHT. In PC-3 cells lacking androgen receptors, DHT and bicalutamide exerted no effect on MT3 gene expression. The reporter gene assay with a luciferase reporter plasmid containing the 5'-flanking region of MT3 demonstrated a decrease in luciferase activity caused by DHT that was reversed by bicalutamide. These results suggest that MT3 gene expression is downregulated by androgen.

Keywords: LNCaP; androgen; metallothionein 3; prostate.

Figure 1.

Effect of dihydrotestosterone (DHT) on zinc transporter and metallothionein mRNA expression. LNCaP cells were cultured in phenol red-free RPMI-1640 medium supplemented with 2% charcoal-stripped fetal bovine serum for 1 day and then treated with the indicated concentrations of DHT for 48 h. Total RNA was isolated from cells and subjected to real-time reverse transcription-polymerase chain reaction analysis. ^**P<0.01 vs. untreated LNCaP cells.

Figure 2.

Androgen regulates MT3 mRNA expression in LNCaP cells. (A) Cells were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (white bars) or phenol red-free RPMI-1640 medium supplemented with 5% charcoal-stripped fetal bovine serum (CS-FBS) (black bars) for 1, 2 and 3 days. (B) PC-3 cells were cultured in phenol red-free RPMI-1640 medium supplemented with 2% CS-FBS for 1 day and then treated with the indicated concentrations of dihydrotestosterone (DHT) for 48 h. (C) Cells were treated with the indicated concentrations of bicalutamide for 48 h. (D) LNCaP cells were treated with 10 nM DHT and the indicated concentrations of bicalutamide for 48 h. After incubation, total RNA was isolated from cells and subjected to real-time reverse transcription-polymerase chain reaction analysis. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. control.

Figure 3.

Androgen regulates MT3 transcriptional activity in LNCaP cells. LNCaP cells were transfected with pGL3-basic vector containing the MT3 upstream region (−905 to +285) and phRL-CMV. Cells were then treated with the indicated concentration of (A) dihydrotestosterone (DHT) or (B) DHT plus bicalutamide. After 48 h, cell lysates were prepared. Firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay system and normalized with Renilla luciferase activity. *P<0.05, ^**P<0.01 vs. untreated LNCaP cells.