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Introduction

Androgen deprivation therapy is one of the standard treatments for prostate cancer, since the growth and progression of prostate cancer are primarily androgen-dependent (1). However, the progression of an androgen-dependent prostate cancer to an androgen-independent state is a well-established phenomenon (2). Zinc content, which is known to be higher in the prostate compared to any other tissue, has been found to be significantly lower in androgen-independent prostate cancers compared to androgen-dependent ones (3,4). We previously reported that androgen-insensitive AIDL cells, which were established by long-term culture of androgen-sensitive LNCaP cells in a hormone-deprived medium, exhibited a significantly lower zinc content compared to LNCaP cells (5).

Metallothioneins (MTs) and zinc transporters function to maintain cellular zinc homeostasis. MTs are small metal-binding proteins that exist as four isoforms in mammals. Metallothionein (MT)1 and MT2 are major isoforms expressed in the majority of tissues, whereas MT3 and MT4 are minor isoforms normally found in a limited number of tissues (6). The MT3 protein was first isolated as a growth-inhibiting factor from brain neurons and was found to be expressed at high levels in prostate cancer tissues (7). Zinc transporters are classified as the ZnT and ZIP families, belonging to the solute-linked carrier (SLC) gene families SLC30 and SLC39, respectively. SLC30A1 and SLC30A4 expression was reported to be decreased in patients with prostate cancer compared to those with benign prostatic hyperplasia (8,9). SLC39A1 and SLC39A2 expression in prostate tumor tissues obtained from African-American patients was lower compared to that in Caucasian patients (10).

Since zinc levels in the prostate are known to decrease with the progression of prostate cancer during androgen deprivation therapy, we hypothesized that zinc exerts a preventative effect on cancer progression. We previously reported that zinc suppressed the invasiveness of prostate cancer cells and increased the sensitivity of cells to cytotoxic agents (11–13). Therefore, we investigated the effect of androgen on the gene expression of MTs and zinc transporters to elucidate the regulation of zinc levels by androgen in LNCaP cells and demonstrated that MT3 expression is downregulated by androgen.

Materials and methods

Materials

Dihydrotestosterone (DHT) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Bicalutamide was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Charcoal-stripped fetal bovine serum (CS-FBS) was obtained from Invitrogen (Carlsbad, CA, USA). Phenol red-free RPMI-1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.

Cell culture

LNCaP human prostatic carcinoma cells and PC-3 cells were obtained from American Type Culture Collection (Rockville, MD, USA). Androgen-low-sensitive LNCaP-E9 cells have been previously described (14). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37°C.

Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen) and first-strand complementary DNA was synthesized from 5 μg of total RNA using SuperScript III (Invitrogen) as previously described (14). Real-time monitoring of PCR reactions was performed using the Thermal Cycler Dice Real-Time system (Takara, Otsu, Japan) with the SYBR Premix Ex Taq (Takara). At the end of the reaction, a dissociation curve analysis was performed to assess the specificity of the product. PCR was performed under the following conditions: 35 cycles of 30 sec at 95°C, 30 sec at 58°C and 30 sec at 72°C for MT1A, MT2A and MT3; 35 cycles of 30 sec at 95°C, 20 sec at 58°C and 30 sec at 72°C for MT1G and MT1X; 35 cycles of 60 sec at 94°C, 60 sec at 55°C and 60 sec at 72°C for SLC30A1 and SLC39A1; 35 cycles of 30 sec at 95°C, 30 sec at 61°C and 30 sec at 72°C for SLC30A2; and 35 cycles of 10 sec at 95°C and 20 sec at 60°C for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH, a housekeeping gene, was used for normalization of target mRNA expression. The primers used in this study are listed in Table I.

Table I. Primer sequences designed for real-time reverse transcription-polymerase chain reaction.

Table I.

Primer sequences designed for real-time reverse transcription-polymerase chain reaction.

Gene name	Sequence
GAPDH
Sense	5′-CCAGCAAGAGCACAAGAGGA-3′
Antisense	5′-GCAACTGTGAGGAGGGGAGA-3′
SLC30A1
Sense	5′-TGTGAACTTGCCTGCAGAAC-3′
Antisense	5′-GCTTTAGTCCTCCTGGGCTT-3′
SLC30A2
Sense	5′-AGATGCAAGAGGGGAAACCT-3′
Antisense	5′-TGGCAGAGACAGACCTTGTG-3′
SLC39A1
Sense	5′-GTCCTGGTGATGGAGCAGAT-3′
Antisense	5′-CCGATGCCTAGAGGTGTCAT-3′
MT1A
Sense	5′-CTCGAAATGGACCCCAACT-3′
Antisense	5′-ATATCTTCGAGCAGGGCTGTC-3′
MT1G
Sense	5′-GCCAGCTCCTGCAAGTGCAA-3′
Antisense	5′-TCTCCGATGCCCCTTTGCAG-3′
MT1X
Sense	5′-TCTCCTTGCCTCGAAATGGAC-3′
Antisense	5′-GGGGCACACTTGGCACAGC-3′
MT2A
Sense	5′-CCGACTCTAGCCGCCTCTT-3′
Antisense	5′-GTGGAAGTCGCGTTCTTTACA-3′
MT3
Sense	5′-GCTGAGGCAGAAGCAGAGAAG-3′
Antisense	5′-TCATTCCTCCAAGGTCAGCA-3′

Plasmid construction

Genomic DNA from LNCaP cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The 5′-flanking region of the human MT3 gene between −905 and +285 bp was amplified by PCR from genomic DNA using primers containing restriction sites for KpnI and NheI, respectively. The sequences of the primers were as follows: sense, 5′-ACGGTACCTACTGCAGCCTCCTCAACCT-3′; anti-sense, 5′-ACGCTAGCGCTTCTCCAAGCAACTGGAC-3′. The fragment was ligated to a pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI, USA).

Luciferase assay

LNCaP cells were seeded at a density of 2×105 cells/well into a 24-well culture plate (Nalge Nunc International, Rochester, NY, USA). After 24 h, the medium was changed to phenol red-free RPMI-1640 supplemented with DHT and/or bicalutamide. Cells were then cotransfected with 0.7 μg of pGL3-MT3 (−905 to +285) firefly luciferase reporter plasmid and 0.1 μg of Renilla luciferase plasmid pRL-CMV using Lipofectamine® 2000 (Invitrogen), according to the manufacturer's instructions. Forty-eight hours after transfection, cell lysates were prepared and luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.

Statistical analysis

Significance was assessed by a one-way ANOVA followed by Dunnett's test using Prism4 software (GraphPad Software, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of androgen on zinc transporter and metallothionein expression in prostate cancer cells

The effect of DHT on the mRNA expression of zinc transporters and MTs in prostate cancer cells was first assessed by real-time RT-PCR analysis. As shown in Fig. 1, DHT decreased the expression of MT3 in androgen-sensitive LNCaP cells in a dose-dependent manner, but did not affect SLC30A1, SLC30A2, SLC39A1, MT1A, MT1G, MT1X or MT2A expression. In PC-3 cells lacking androgen receptors, DHT exerted no effect on the expression of MT3 (Fig. 2B). In addition, MT3 expression in LNCaP cells was markedly higher with the medium containing androgen-deficient CS-FBS compared to the normal FBS (Fig. 2A). The MT3 expression in androgen-low-sensitive LNCaP-E9 cells was significantly higher in the medium containing CS-FBS compared to the normal FBS; however, the expression was less pronounced compared to that in LNCaP cells. There were no differences in MT3 expression between PC-3 cells in the medium containing FBS and those in CS-FBS. The effect of bicalutamide, an androgen receptor antagonist, on MT3 expression was then examined. Bicalutamide increased the mRNA expression in LNCaP cells, whereas it exerted no effect on PC-3 cells (Fig. 2C). Moreover, the suppression of MT3 expression triggered by DHT was inhibited in the presence of bicalutamide (Fig. 2D). These results suggest that MT3 expression is downregulated by androgen.

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.

Androgen regulates MT3 transcriptional activity in LNCaP cells

The luciferase reporter assay was then performed using MT3 reporter constructs, to determine whether the downregulation of MT3 expression by androgen is due to transcriptional regulation. As shown in Fig. 3, DHT decreased luciferase activity in LNCaP cells and this decrease was reversed by bicalutamide. Taken together, these results suggest that MT3 expression may be regulated by androgen transcriptional control in LNCaP cells.

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.

Discussion

In this study, we demonstrated that MT3 expression was regulated by androgen in prostate cancer cells. Although DHT decreased MT3 expression in androgen-sensitive LNCaP cells, bicalutamide exerted the opposite effect. The induction of MT3 expression by androgen withdrawal was lower in androgen-low-sensitive LNCaP-E9 cells compared to that in LNCaP cells. In addition, no change was observed in MT3 expression following androgen withdrawal or treatment with DHT or bicalutamide in PC-3 cells lacking androgen receptors. These results suggest that the expression of MT3 in LNCaP cells is downregulated by androgen. The androgen receptor binds to specific DNA motifs known as androgen-response elements (AREs) in the regulatory regions of target genes. AREs comprise two 6-bp elements separated by a 3-bp spacer, 5′-AGAACAnnnTGTTCT-3′ (15). The sequence (5′-TCTTGTnnnACAGGC-3′), which is similar to the reverse sequence of ARE, is found upstream in the MT3 gene at the position −871 to −857.

Previous studies suggested that the regulation of MT3 expression appears to be complicated due to cell-type specificity and differences between species (7,16–18). Specifically, MT3 expression was observed to be upregulated by androgen in rodent tissues (17,19). In a previous study, we reported that MT3 expression in the lateral lobe of the rat prostate was decreased following castration (20), a finding inconsistent with our present results, according to which MT3 mRNA expression in LNCaP cells is downregulated by androgen. Therefore, the 5′-flanking region of the MT3 gene was compared between rats and humans to investigate the opposite regulation of MT3 expression in these species. ARE and the reverse sequence of ARE were not found within 3 kb of the 5′-upstream region of the MT3 gene in rats, although the sequence exists in this 3-kb region in humans. The existence of ARE and the reverse sequence of ARE may be responsible for the differences in androgen-regulated MT3 expression between the two species.

The mechanisms underlying the transition to the aggressive phenotype following androgen deprivation therapy have yet to be elucidated. However, one of the reasons considered is the altered gene expression associated with alterations in the tumor malignant potential triggered by androgen withdrawal. In this study, we demonstrated that MT3 expression was downregulated by androgen in LNCaP cells. MT3 has been shown to be involved in growth inhibition, prevention of hypoxic damage and chemotherapy resistance (21–23). These results suggest that the androgenic regulation of MT3 expression may be the mechanism underlying the progression of prostate cancer.

References