Zinc and Metallothionein Levels and Expression of Zinc Transporters in Androgen-Independent Subline of LNCaP Cells
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《男科医学杂志》
the Laboratory of Pharmaceutics, Gifu Pharmaceutical University, Mitahora-higashi, Gifu, Japan; Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee; and the Department of Urology, Mie University School of Medicine, Edobashi, Tsu, Mie, Japan.
Abstract
Zinc levels in the prostate have been reported to be associated with the development and progression of malignant prostate cells. To investigate the reason why the zinc content decreases during the progression of prostate cancer to an androgen-independent state, we compared the expression levels of metallothionein and zinc transporters between androgen-responsive LNCaP cells and its androgen-independent subline, AIDL cells. AIDL cells showed lower zinc levels than LNCaP cells and comparable levels of androgen receptor expression to LNCaP cells, consistent with some clinical aspects of androgen-independent prostatic cancer. AIDL cells exhibited a lower expression of zinc transporter 1 (ZnT1) and higher expression of ZnT3 than LNCaP cells. The content of metallothionein, which is a major zinc-binding protein, was significantly lower in AIDL cells than in LNCaP cells. Furthermore, the expression of ZnT3 mRNA was decreased by incubating LNCaP cells in medium containing hormone-stripped fetal calf serum and increased by addition of synthetic androgen R1881 to the medium, whereas the intracellular zinc levels were not affected under these conditions. These findings suggest that factors such as ZnT1 and metallothioneins other than ZnT3 are associated with the low intracellular zinc content in AIDL cells.
Key words: Hormone-refractory, metallothionein, ZnT1, ZnT3
High concentrations of zinc, an essential nutrient for most cells and tissues, are known to be retained in the prostate gland and secreted into the seminal plasma (Mawson and Fischer, 1951, 1952, 1953). A deficiency of zinc leads to male hypogonadism accompanied by inhibition of sperm formation and a lack of secondary sex characteristics (Prasad, 1983, 1985). Zinc has also been suggested to have an antibacterial function in seminal plasma (Fair et al, 1976). Moreover, prostatic zinc has been found to control prostatic epithelial cell growth through inhibition of mitochondrial aconitase activities, cell cycle arrest, and induction of cell detachment (Costello and Franklin, 1981; Costello et al, 1997; Iguchi et al, 1998; Liang et al, 1999). Recently, zinc in prostate cancer has been reported to regulate the metastasis through the inhibition of the enzymatic activity of aminopeptidase N and urokinase-type plasminogen activator (Ishii et al, 2001). Thus the physiological functions of zinc in the prostate have been gradually revealed.
Possible relations between changes of zinc content in the prostate and prostatic diseases have been investigated. The zinc level in prostate cancer was observed to decrease to that detected in nonprostate tissues (Gyorkey et al, 1967; Ogunlewe and Osegbe, 1989). Zinc levels in seminal plasma of patients with bacterial prostatitis or sterility were also reported to be consistently lower than those in healthy subjects (Fair et al, 1976; Fair and Parrish, 1981). High levels of zinc have been found in the prostate grand with benign prostatic hyperplasia compared with the normal prostate (Gyorkey et al, 1967; Ogunlewe and Osegbe, 1989). Although functional disorder of zinc homeostasis seems to be associated with prostatic disease, it is unclear whether diseases cause the disruption of zinc homeostasis or vice versa.
Since the growth and progression of prostate cancer are initially androgen-dependent, androgen ablation therapies have been the standard treatment for prostate cancer (Huggins and Hodges, 1941). Hormone ablation therapy only causes a temporary regression of prostate cancer; however, the progression of an androgen-dependent prostate cancer to an androgen-independent state is a well-established phenomenon (Emmett et al, 1960). In androgen-independent prostate cancer, zinc levels have been found to be much lower than those in an androgen-dependent state (Shiina et al, 1996a,b), but little information is available as to why zinc content decreases during the progression of prostate cancer to a hormone-independent state after androgen withdrawal therapy.
Many transporters and metal-binding proteins regulating zinc homeostasis have been identified in mammals. Natural resistance-associated macrophage protein 2 (Nramp2) is a metal ion transporter that imports a variety of metal ions such as Fe, Zn, Cd, and Cu from the extracellular environment into cells (Gunshin et al, 1997). Zinc transporter 1 (ZnT1) is a zinc transporter responsible for zinc efflux across the plasma membranes to prevent zinc toxicity (Palmiter and Findley, 1995). Zinc transporter 2 (ZnT2) is involved in zinc uptake into vesicles (endosome/lysosome compartment) in intestine, kidney, and testis (Palmiter et al, 1996a). Zinc transporter 3 (ZnT3) is expressed in the brain and is responsible for the accumulation of zinc in synaptic vesicles (Palmiter et al, 1996b). Zinc transporter 4 (ZnT4) is associated with zinc efflux or compartmentalization in the mammary gland, and is essential for regulating the zinc content of milk (Huang and Gitschier, 1997; Murgia et al, 1999). Zn-regulated transporter/Fe-regulated transporterlike proteins (ZIP) are suggested to be involved in zinc influx across the plasma membranes because forced expression of these transporters in mammalian cells increased their zinc uptake (Gaither and Eide, 2000). Nramp2, ZnT1, and ZnT4 are ubiquitously expressed in most tissues, whereas ZnT2 and ZnT3 display tissue-restricted expression in mammals. Metallothioneins are a major family of zinc-binding proteins and are well known to play an important role in zinc uptake, distribution, storage, and release (Cousins, 1985).
In the present study, to clarify the role of zinc in the progression of prostate cancer to an androgen-independent state, we investigated expression levels of zinc transporters and metallothionein in androgen-independent LNCaP (AIDL) cells and native LNCaP cells. Elucidation of the zinc retention system in the prostate will be useful for investigation of prostatic diseases.
Materials and Methods
Cell Culture
Androgen-responsive LNCaP cells were purchased from American Type Culture Collection (Rockville, Md), and androgen-independent LNCaP cells (AIDL) were established by Dr. T. Ohnishi at Mie University Faculty of Medicine. The AIDL cells were established from LNCaP cells by maintaining in phenol red-free RPMI-1640 medium (Sigma, St. Louis, Mo) with 2% charcoal-stripped fetal calf serum (CS-FCS) over 2 years (Kokontis et al, 1994; Onishi et al, 2000, 2001). LNCaP and AIDL cells were cultured in phenol red-free RPMI-1640 medium with 10% FCS (LNCaP) or 2% CS-FCS (AIDL), respectively.
Cell Proliferation
Cell proliferation was evaluated by measurement of the fluorescence intensity in the presence of alamar blue (Wako, Osaka, Japan) (Pagé et al, 1993). Cells were seeded in 96-well multidishes (Sumilon, Tokyo, Japan) at a density of 1 x 104 cells/well in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS, and after 1 day, androgen agonist R1881 (NEN Life Science, Boston, Mass) was added to the culture medium. After 72 h, alamar blue solution was added to the medium, and the plates were incubated for 4 hours. The fluorescence intensity was measured using a Cytofluor 2350 with excitation and emission wavelengths at 530 nm and 590 nm, respectively.
Zinc Assay
Zinc concentration was measured using 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol disodium salt dihydrate (5-Br-PAPS, Dojindo Laboratory, Kumamoto, Japan) (Yamashita et al, 1996). Briefly, cells were cultured in 90-mm dishes (Sumilon) in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS for 3 days. After being washed with phosphate-buffered saline, the cells were pelleted, treated with 10% trichloroacetic acid on ice for 15 minutes, and centrifuged at 4°C for 10 minutes. The resulting supernatant was incubated with 0.08 nM 5-Br-PAPS and 29 mM salicyladoxime for 10 minutes at room temperature. The absorbance of the mixture was measured at 570 nm with a microplate reader.
Metallothionein Assay
The total amount of metallothionein was determined by the cadmium-saturation assay (Onosaka et al, 1978). Briefly, cells were incubated in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS for 3 days. The cells were collected, resuspended in 10 mM Tris-HCl (pH 7.4), and sonicated. The cell lysate was then centrifuged at 105 000 x g at 4°C for 60 minutes. The resulting supernatant was incubated with 0.2 μg of Cd2+ for 10 minutes at room temprature. After removal of unbound Cd2+ by using hemoglobin as a chelator, Cd2+ bound to metallothionein was measured with an atomic adsorption spectrophotometer (Shimadzu AA-6500).
Semiquantitative Reverse Transcriptase Polymerase Chain Reaction
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, Cal) according to the manufacturer's instructions. Extracted RNA was dissolved in diethylpyrocarbonate-treated water and quantified by measuring the absorbance at 260 nm. Aliquots of 5 μg of total RNA were used to synthesize the first-strand complementary DNA with SuperScript II (Invitrogen) and subjected to polymerase chain reaction (PCR) amplification with the oligonucleotide primers listed in Table 1 using a thermal cycler. The optimal PCR conditions were determined as the amount of amplification product in proportion to that of input RNA. PCR was performed under the following conditions: 26 cycles of 1 minute at 94°C, 1 minute at 57°C, and 1 minute at 72°C for androgen receptor (AR); 24 cycles of 1 minute at 94°C, 1 minute at 54°C, and 1 minute at 72°C for prostate-specific antigen (PSA); 26 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C for ZnT1 and ZnT4; 28 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C for ZnT3 and ZIP1; 25 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C for Nramp2; 28 cycles of 1 minute at 94°C, 1 minute at 62°C, and 1 minute at 72°C for ZIP3; 23 cycles of 45 seconds at 94°C, 45 seconds at 60°C, and 2 minutes at 72°C for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). G3PDH served as an internal RNA control to allow comparison of RNA levels among different specimens. After PCR, the reaction products were resolved on 1.75 % agarose gels and visualized with ethidium bromide.
Sequences of oligonucleotide primers for PCR
Protein Assay
The protein concentration was determined by Lowry assay using bovine serum albumin as a standard (Lowry et al, 1951).
Results
Characterization of AIDL and LNCaP Cells
AIDL cells, which can grow well in the absence of androgen, are an LNCaP subline derived by continuous passaging in hormone-depleted medium. The viability of LNCaP cells incubated for 72 hours in CS-FCS-containing medium was approximately half of that in FCS-containing medium, whereas the viability of AIDL cells incubated in CS-FCS-containing medium was 1.3-fold of that in FCS-containing medium (data not shown). A previous study showed that the activities of mitogen-activated protein kinases were higher in AIDL cells than in LNCaP cells (Onishi et al, 2001). Moreover, to ascertain that AIDL cells exhibit features of androgen-independent prostate cancer, androgen responsiveness of cell proliferation, AR and PSA expressions, and intracellular zinc levels were investigated. Androgen responsiveness of these cells was assessed by measuring the effect of synthetic androgen R1881 on cell growth. As shown in Figure 1A, the proliferation of LNCaP cells was stimulated by R1881 in a dose-dependent manner but no significant effect on the growth of AIDL cells was observed. The expression of AR in AIDL cells showed approximately the same as that in LNCaP cells; however, PSA expression was not detected in AIDL cells (Figure 1B). Therefore, we next assessed the transcriptional activity of AR in AIDL cells by measuring induction of PSA mRNA in response to R1881 treatment. As shown in Figure 1C, increased expression of PSA was observed after 8-16 days of incubation in R1881-stimulated AIDL cells, whereas the same treatment increased the expression after 1 day in LNCaP cells. These results suggest that the androgen-independent behavior in AIDL cells possibly involves an alteration of AR function or an abnormality in AR signal transduction pathway. We also determined zinc levels in AIDL and LNCaP cells. As shown in Figure 2, the zinc levels in AIDL cells were significantly lower than those in LNCaP cells. Thus, AIDL cells were judged to retain some features observed in the androgen-independent prostate cancer.
Expression of Zinc Transporters and Metallothionein in AIDL and LNCaP Cells
Zinc homeostasis in eukaryotic cells is believed to be controlled by zinc uptake, elimination, and intracellular sequestration, which are regulated by the expression levels of various zinc transporters and the major zinc-binding protein metallothionein (Beyersmann and Haase, 2001). To determine factors involved in the decrease of zinc content in androgen-independent prostatic cancer, we next tried to examine the expression of zinc transporters and metallothionein contents in LNCaP and AIDL cells. As shown in Figure 3, the levels of ZnT1 and ZnT3 mRNAs were approximately 1.5-fold and 4-fold higher in AIDL cells than LNCaP cells. The expressions of ZnT4, Nramp2, ZIP1, and ZIP3 showed no significant differences between them. Moreover, as shown in Figure 4, the levels of metallothionein in AIDL cells were approximately threefold lower than those in LNCaP cells.
Effect of R1881 on Intracellular Zinc and Metallothionein Levels, and Expressions of Zinc Transporters
Since AIDL cells were generated by long-term culture of androgen-responsive LNCaP cells in the medium containing hormone-stripped serum, the decrease in intracellular zinc and metallothionein and the increase in ZnT1 and ZnT3 expressions in AIDL cells may be mediated by androngenic regulation. Therefore, we next tried to assess the effects of androgen-sufficient or -deficient conditions on zinc accumulation, metallothionein levels, and ZnTs expressions in LNCaP cells. As shown in Figure 5, the expression of ZnT3 mRNA in LNCaP cells was decreased by the treatment with 100 nM R1881, whereas slightly increased by hormone ablation, suggesting that androgen negatively regulated ZnT3 expression. The altered expressions of ZnT3 were seen in androgen-responsive LNCaP cells but not in androgen-independent AIDL cells, supporting the idea that the expression of ZnT3 was regulated by androgen (Figure 5C). The expressions of the other zinc transporters including ZnT1 were not observed to be androgen regulated. The zinc and metallothionein levels were also not affected by treatment of LNCaP cells with either R1881 or androgen ablation (Figure 6).
Figure 6. Effects of androgen on zinc and metallothionein levels in LNCaP cells. LNCaP cells were treated under the same conditions as described in Figure 5 (A). After 4 d of incubation, intracellular zinc (A) and metallothionein (B) levels were determined. Values represent the means ± SD from 3 incubations.
Discussion
To mimic androgen ablation therapy for the treatment of prostate cancer, we chronically cultured androgen-responsive LNCaP cells in androgen-reduced conditions, generating an androgen-independent derivative AIDL, which retained limited hormone-responsive proliferation (Onishi et al, 2000, 2001). Although AIDL cells retained a similar level of AR mRNA expression, transcriptional activity of AR in AIDL cells was significantly lower than that in LNCaP cells, suggesting that AIDL cells exhibit an AR abnormality such as AR gene mutation. These findings confirm some clinical observations of hormone-refractory prostate cancer. Moreover, AIDL cells showed much lower zinc levels compared with native LNCaP cells, consistent with the clinical evidence that zinc levels in androgen-independent prostate cancer was much lower than those in an androgen-dependent state (Shiina et al, 1996a,b). Thus, AIDL cells seem to be useful for investigating the zinc retention system in hormone-refractory prostate cancer.
In the present study, we analyzed the expression of zinc transporters and metallothionein content in LNCaP and AIDL cells to obtain clues about the reason why the zinc content decreases during the progression of prostatic cancer. Our experiments revealed that ZnT1 and ZnT3 mRNA expressions were higher and metallothionein levels were lower in AIDL cells than in native LNCaP cells. Since ZnT3 was found abundantly and has been believed to play a crucial role in the regulation of zinc transport into vesicles in mammalian brain (Cole et al, 1999; Lee et al, 2000), the increased expression of ZnT3 would affect the cellular localization of zinc but not affect the total amount of intracellular zinc. In fact, the R1881-treated LNCaP cells showed no significant changes of zinc content, although the expression of ZnT3 mRNA was decreased in the cells. These results suggest that the decrease in the expression level of ZnT3 mRNA does not reflect the change in the intracellular zinc content in LNCaP cells.
ZnT-1 transports zinc out of cells and has been suggested to play a key role in cellular zinc homeostasis (Palmiter et al, 1995). Increased cellular zinc content induces ZnT1 gene expression to efflux zinc from cells (Palmiter et al, 1995; McMahon and Cousins, 1998). The regulation of ZnT1 mRNA expression was found to be mediated by binding of metal response element-binding transcription factor-1 to metal response elements (MREs) sequence present in the ZnT1 promoter region (Langmade et al, 2000; Andrews, 2001). These findings led us to speculate that decreased cellular zinc content in AIDL cells may lead to a decrease in ZnT1 expression. However, the expression was observed to be higher than in LNCaP cells. The reason why the expression of ZnT1 increased in AIDL cells is unknown, but from the viewpoint of its function as effluxing zinc, the increased ZnT1 expression may result in the low intracellular zinc content in the cells.
Since metallothionein acts as a zinc store in the cell and its gene is also transcriptionally regulated by intracellular zinc thorough cis-acting MREs (Andrews, 2001), the low level of zinc in AIDL cells could be closely related to the low expression of metallothionein observed in these cells. However, the reason for the decreased expression of metallothionein in AIDL cells is probably more complex since its expression is also regulated by various stress factors, including glucocorticoids, cytokines, and reactive oxygen species (Palmiter, 1998). Therefore, the low level of metallothionein in AIDL cells, which is generated to be cultured in the medium containing 2% hormone-stripped FCS, may partially result from its culture condition.
The high-level expression of ZnT3 was observed in LNCaP cells cultured in hormone-reduced medium. It is not clear from our experiments whether the increased expression of ZnT3 is mediated directly by hormone ablation or indirectly through alterations of environmental androgen. Although an androgen response element was not found in the 5' promoter region of ZnT3 gene [data not shown; the sequence was described in the GenBank database (accession number NT_022184)], since cis-element sequence that is required to mediate a negative regulation by androgen is almost unknown, the unidentified androgen response element may exist in the upstream region of the ZnT3 gene. We are going to further investigate the transcriptional regulation of the ZnT3 gene.
Expression of metallothionein in rat prostate is known to be regulated by androgen, but the degree of the androgenic effect is different among lobes of prostate (Ghatak et al, 1996; Tohyama et al, 1996; Yamashita et al, 1996). The rat prostate consists of at least 3 anatomically independent lobes, designated as the ventral, lateral, and dorsal prostate. In ventral prostate, metallothionein expression was not detectable and castration appeared to induce metallothionein gene expression, but testosterone administration after castration had no effect on the expression (Tohyama et al, 1996). In dorsolateral prostate, metallothionein was present and testosterone caused a reversal of the effect of castration on metallothionein induction (Ghatak et al, 1996; Tohyama et al, 1996). In lateral prostate, metallothionein expression was not regulated by androgen (Yamashita et al, 1996). These findings suggest that metallothionein gene expression is regulated by androgen in the dorsal prostate, but not lateral and ventral prostate. LNCaP cells have been reported to exhibit the characteristics of lateral prostate cells (Costello et al, 1999); therefore, our observation of no effect on metallothionein expression in LNCaP cells by androgen supports the previous studies.
In conclusion, we first showed that the expression of ZnT3 mRNA underwent an androgenic regulation in LNCaP cells and the high-level expression of ZnT3 mRNA was detected in the androgen-independent subline derived from LNCaP cells. In addition, high levels of ZnT1 and low levels of metallothionein were detected in AIDL cells. The elucidation of the relation between the expressions of ZnT1, ZnT3, and metallothionein and the progression of prostate cancer to the hormone-refractory state might help in understanding the physiology of prostate cancer.
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Abstract
Zinc levels in the prostate have been reported to be associated with the development and progression of malignant prostate cells. To investigate the reason why the zinc content decreases during the progression of prostate cancer to an androgen-independent state, we compared the expression levels of metallothionein and zinc transporters between androgen-responsive LNCaP cells and its androgen-independent subline, AIDL cells. AIDL cells showed lower zinc levels than LNCaP cells and comparable levels of androgen receptor expression to LNCaP cells, consistent with some clinical aspects of androgen-independent prostatic cancer. AIDL cells exhibited a lower expression of zinc transporter 1 (ZnT1) and higher expression of ZnT3 than LNCaP cells. The content of metallothionein, which is a major zinc-binding protein, was significantly lower in AIDL cells than in LNCaP cells. Furthermore, the expression of ZnT3 mRNA was decreased by incubating LNCaP cells in medium containing hormone-stripped fetal calf serum and increased by addition of synthetic androgen R1881 to the medium, whereas the intracellular zinc levels were not affected under these conditions. These findings suggest that factors such as ZnT1 and metallothioneins other than ZnT3 are associated with the low intracellular zinc content in AIDL cells.
Key words: Hormone-refractory, metallothionein, ZnT1, ZnT3
High concentrations of zinc, an essential nutrient for most cells and tissues, are known to be retained in the prostate gland and secreted into the seminal plasma (Mawson and Fischer, 1951, 1952, 1953). A deficiency of zinc leads to male hypogonadism accompanied by inhibition of sperm formation and a lack of secondary sex characteristics (Prasad, 1983, 1985). Zinc has also been suggested to have an antibacterial function in seminal plasma (Fair et al, 1976). Moreover, prostatic zinc has been found to control prostatic epithelial cell growth through inhibition of mitochondrial aconitase activities, cell cycle arrest, and induction of cell detachment (Costello and Franklin, 1981; Costello et al, 1997; Iguchi et al, 1998; Liang et al, 1999). Recently, zinc in prostate cancer has been reported to regulate the metastasis through the inhibition of the enzymatic activity of aminopeptidase N and urokinase-type plasminogen activator (Ishii et al, 2001). Thus the physiological functions of zinc in the prostate have been gradually revealed.
Possible relations between changes of zinc content in the prostate and prostatic diseases have been investigated. The zinc level in prostate cancer was observed to decrease to that detected in nonprostate tissues (Gyorkey et al, 1967; Ogunlewe and Osegbe, 1989). Zinc levels in seminal plasma of patients with bacterial prostatitis or sterility were also reported to be consistently lower than those in healthy subjects (Fair et al, 1976; Fair and Parrish, 1981). High levels of zinc have been found in the prostate grand with benign prostatic hyperplasia compared with the normal prostate (Gyorkey et al, 1967; Ogunlewe and Osegbe, 1989). Although functional disorder of zinc homeostasis seems to be associated with prostatic disease, it is unclear whether diseases cause the disruption of zinc homeostasis or vice versa.
Since the growth and progression of prostate cancer are initially androgen-dependent, androgen ablation therapies have been the standard treatment for prostate cancer (Huggins and Hodges, 1941). Hormone ablation therapy only causes a temporary regression of prostate cancer; however, the progression of an androgen-dependent prostate cancer to an androgen-independent state is a well-established phenomenon (Emmett et al, 1960). In androgen-independent prostate cancer, zinc levels have been found to be much lower than those in an androgen-dependent state (Shiina et al, 1996a,b), but little information is available as to why zinc content decreases during the progression of prostate cancer to a hormone-independent state after androgen withdrawal therapy.
Many transporters and metal-binding proteins regulating zinc homeostasis have been identified in mammals. Natural resistance-associated macrophage protein 2 (Nramp2) is a metal ion transporter that imports a variety of metal ions such as Fe, Zn, Cd, and Cu from the extracellular environment into cells (Gunshin et al, 1997). Zinc transporter 1 (ZnT1) is a zinc transporter responsible for zinc efflux across the plasma membranes to prevent zinc toxicity (Palmiter and Findley, 1995). Zinc transporter 2 (ZnT2) is involved in zinc uptake into vesicles (endosome/lysosome compartment) in intestine, kidney, and testis (Palmiter et al, 1996a). Zinc transporter 3 (ZnT3) is expressed in the brain and is responsible for the accumulation of zinc in synaptic vesicles (Palmiter et al, 1996b). Zinc transporter 4 (ZnT4) is associated with zinc efflux or compartmentalization in the mammary gland, and is essential for regulating the zinc content of milk (Huang and Gitschier, 1997; Murgia et al, 1999). Zn-regulated transporter/Fe-regulated transporterlike proteins (ZIP) are suggested to be involved in zinc influx across the plasma membranes because forced expression of these transporters in mammalian cells increased their zinc uptake (Gaither and Eide, 2000). Nramp2, ZnT1, and ZnT4 are ubiquitously expressed in most tissues, whereas ZnT2 and ZnT3 display tissue-restricted expression in mammals. Metallothioneins are a major family of zinc-binding proteins and are well known to play an important role in zinc uptake, distribution, storage, and release (Cousins, 1985).
In the present study, to clarify the role of zinc in the progression of prostate cancer to an androgen-independent state, we investigated expression levels of zinc transporters and metallothionein in androgen-independent LNCaP (AIDL) cells and native LNCaP cells. Elucidation of the zinc retention system in the prostate will be useful for investigation of prostatic diseases.
Materials and Methods
Cell Culture
Androgen-responsive LNCaP cells were purchased from American Type Culture Collection (Rockville, Md), and androgen-independent LNCaP cells (AIDL) were established by Dr. T. Ohnishi at Mie University Faculty of Medicine. The AIDL cells were established from LNCaP cells by maintaining in phenol red-free RPMI-1640 medium (Sigma, St. Louis, Mo) with 2% charcoal-stripped fetal calf serum (CS-FCS) over 2 years (Kokontis et al, 1994; Onishi et al, 2000, 2001). LNCaP and AIDL cells were cultured in phenol red-free RPMI-1640 medium with 10% FCS (LNCaP) or 2% CS-FCS (AIDL), respectively.
Cell Proliferation
Cell proliferation was evaluated by measurement of the fluorescence intensity in the presence of alamar blue (Wako, Osaka, Japan) (Pagé et al, 1993). Cells were seeded in 96-well multidishes (Sumilon, Tokyo, Japan) at a density of 1 x 104 cells/well in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS, and after 1 day, androgen agonist R1881 (NEN Life Science, Boston, Mass) was added to the culture medium. After 72 h, alamar blue solution was added to the medium, and the plates were incubated for 4 hours. The fluorescence intensity was measured using a Cytofluor 2350 with excitation and emission wavelengths at 530 nm and 590 nm, respectively.
Zinc Assay
Zinc concentration was measured using 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol disodium salt dihydrate (5-Br-PAPS, Dojindo Laboratory, Kumamoto, Japan) (Yamashita et al, 1996). Briefly, cells were cultured in 90-mm dishes (Sumilon) in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS for 3 days. After being washed with phosphate-buffered saline, the cells were pelleted, treated with 10% trichloroacetic acid on ice for 15 minutes, and centrifuged at 4°C for 10 minutes. The resulting supernatant was incubated with 0.08 nM 5-Br-PAPS and 29 mM salicyladoxime for 10 minutes at room temperature. The absorbance of the mixture was measured at 570 nm with a microplate reader.
Metallothionein Assay
The total amount of metallothionein was determined by the cadmium-saturation assay (Onosaka et al, 1978). Briefly, cells were incubated in phenol red-free RPMI-1640 medium supplemented with 2% CS-FCS for 3 days. The cells were collected, resuspended in 10 mM Tris-HCl (pH 7.4), and sonicated. The cell lysate was then centrifuged at 105 000 x g at 4°C for 60 minutes. The resulting supernatant was incubated with 0.2 μg of Cd2+ for 10 minutes at room temprature. After removal of unbound Cd2+ by using hemoglobin as a chelator, Cd2+ bound to metallothionein was measured with an atomic adsorption spectrophotometer (Shimadzu AA-6500).
Semiquantitative Reverse Transcriptase Polymerase Chain Reaction
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, Cal) according to the manufacturer's instructions. Extracted RNA was dissolved in diethylpyrocarbonate-treated water and quantified by measuring the absorbance at 260 nm. Aliquots of 5 μg of total RNA were used to synthesize the first-strand complementary DNA with SuperScript II (Invitrogen) and subjected to polymerase chain reaction (PCR) amplification with the oligonucleotide primers listed in Table 1 using a thermal cycler. The optimal PCR conditions were determined as the amount of amplification product in proportion to that of input RNA. PCR was performed under the following conditions: 26 cycles of 1 minute at 94°C, 1 minute at 57°C, and 1 minute at 72°C for androgen receptor (AR); 24 cycles of 1 minute at 94°C, 1 minute at 54°C, and 1 minute at 72°C for prostate-specific antigen (PSA); 26 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C for ZnT1 and ZnT4; 28 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C for ZnT3 and ZIP1; 25 cycles of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C for Nramp2; 28 cycles of 1 minute at 94°C, 1 minute at 62°C, and 1 minute at 72°C for ZIP3; 23 cycles of 45 seconds at 94°C, 45 seconds at 60°C, and 2 minutes at 72°C for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). G3PDH served as an internal RNA control to allow comparison of RNA levels among different specimens. After PCR, the reaction products were resolved on 1.75 % agarose gels and visualized with ethidium bromide.
Sequences of oligonucleotide primers for PCR
Protein Assay
The protein concentration was determined by Lowry assay using bovine serum albumin as a standard (Lowry et al, 1951).
Results
Characterization of AIDL and LNCaP Cells
AIDL cells, which can grow well in the absence of androgen, are an LNCaP subline derived by continuous passaging in hormone-depleted medium. The viability of LNCaP cells incubated for 72 hours in CS-FCS-containing medium was approximately half of that in FCS-containing medium, whereas the viability of AIDL cells incubated in CS-FCS-containing medium was 1.3-fold of that in FCS-containing medium (data not shown). A previous study showed that the activities of mitogen-activated protein kinases were higher in AIDL cells than in LNCaP cells (Onishi et al, 2001). Moreover, to ascertain that AIDL cells exhibit features of androgen-independent prostate cancer, androgen responsiveness of cell proliferation, AR and PSA expressions, and intracellular zinc levels were investigated. Androgen responsiveness of these cells was assessed by measuring the effect of synthetic androgen R1881 on cell growth. As shown in Figure 1A, the proliferation of LNCaP cells was stimulated by R1881 in a dose-dependent manner but no significant effect on the growth of AIDL cells was observed. The expression of AR in AIDL cells showed approximately the same as that in LNCaP cells; however, PSA expression was not detected in AIDL cells (Figure 1B). Therefore, we next assessed the transcriptional activity of AR in AIDL cells by measuring induction of PSA mRNA in response to R1881 treatment. As shown in Figure 1C, increased expression of PSA was observed after 8-16 days of incubation in R1881-stimulated AIDL cells, whereas the same treatment increased the expression after 1 day in LNCaP cells. These results suggest that the androgen-independent behavior in AIDL cells possibly involves an alteration of AR function or an abnormality in AR signal transduction pathway. We also determined zinc levels in AIDL and LNCaP cells. As shown in Figure 2, the zinc levels in AIDL cells were significantly lower than those in LNCaP cells. Thus, AIDL cells were judged to retain some features observed in the androgen-independent prostate cancer.
Expression of Zinc Transporters and Metallothionein in AIDL and LNCaP Cells
Zinc homeostasis in eukaryotic cells is believed to be controlled by zinc uptake, elimination, and intracellular sequestration, which are regulated by the expression levels of various zinc transporters and the major zinc-binding protein metallothionein (Beyersmann and Haase, 2001). To determine factors involved in the decrease of zinc content in androgen-independent prostatic cancer, we next tried to examine the expression of zinc transporters and metallothionein contents in LNCaP and AIDL cells. As shown in Figure 3, the levels of ZnT1 and ZnT3 mRNAs were approximately 1.5-fold and 4-fold higher in AIDL cells than LNCaP cells. The expressions of ZnT4, Nramp2, ZIP1, and ZIP3 showed no significant differences between them. Moreover, as shown in Figure 4, the levels of metallothionein in AIDL cells were approximately threefold lower than those in LNCaP cells.
Effect of R1881 on Intracellular Zinc and Metallothionein Levels, and Expressions of Zinc Transporters
Since AIDL cells were generated by long-term culture of androgen-responsive LNCaP cells in the medium containing hormone-stripped serum, the decrease in intracellular zinc and metallothionein and the increase in ZnT1 and ZnT3 expressions in AIDL cells may be mediated by androngenic regulation. Therefore, we next tried to assess the effects of androgen-sufficient or -deficient conditions on zinc accumulation, metallothionein levels, and ZnTs expressions in LNCaP cells. As shown in Figure 5, the expression of ZnT3 mRNA in LNCaP cells was decreased by the treatment with 100 nM R1881, whereas slightly increased by hormone ablation, suggesting that androgen negatively regulated ZnT3 expression. The altered expressions of ZnT3 were seen in androgen-responsive LNCaP cells but not in androgen-independent AIDL cells, supporting the idea that the expression of ZnT3 was regulated by androgen (Figure 5C). The expressions of the other zinc transporters including ZnT1 were not observed to be androgen regulated. The zinc and metallothionein levels were also not affected by treatment of LNCaP cells with either R1881 or androgen ablation (Figure 6).
Figure 6. Effects of androgen on zinc and metallothionein levels in LNCaP cells. LNCaP cells were treated under the same conditions as described in Figure 5 (A). After 4 d of incubation, intracellular zinc (A) and metallothionein (B) levels were determined. Values represent the means ± SD from 3 incubations.
Discussion
To mimic androgen ablation therapy for the treatment of prostate cancer, we chronically cultured androgen-responsive LNCaP cells in androgen-reduced conditions, generating an androgen-independent derivative AIDL, which retained limited hormone-responsive proliferation (Onishi et al, 2000, 2001). Although AIDL cells retained a similar level of AR mRNA expression, transcriptional activity of AR in AIDL cells was significantly lower than that in LNCaP cells, suggesting that AIDL cells exhibit an AR abnormality such as AR gene mutation. These findings confirm some clinical observations of hormone-refractory prostate cancer. Moreover, AIDL cells showed much lower zinc levels compared with native LNCaP cells, consistent with the clinical evidence that zinc levels in androgen-independent prostate cancer was much lower than those in an androgen-dependent state (Shiina et al, 1996a,b). Thus, AIDL cells seem to be useful for investigating the zinc retention system in hormone-refractory prostate cancer.
In the present study, we analyzed the expression of zinc transporters and metallothionein content in LNCaP and AIDL cells to obtain clues about the reason why the zinc content decreases during the progression of prostatic cancer. Our experiments revealed that ZnT1 and ZnT3 mRNA expressions were higher and metallothionein levels were lower in AIDL cells than in native LNCaP cells. Since ZnT3 was found abundantly and has been believed to play a crucial role in the regulation of zinc transport into vesicles in mammalian brain (Cole et al, 1999; Lee et al, 2000), the increased expression of ZnT3 would affect the cellular localization of zinc but not affect the total amount of intracellular zinc. In fact, the R1881-treated LNCaP cells showed no significant changes of zinc content, although the expression of ZnT3 mRNA was decreased in the cells. These results suggest that the decrease in the expression level of ZnT3 mRNA does not reflect the change in the intracellular zinc content in LNCaP cells.
ZnT-1 transports zinc out of cells and has been suggested to play a key role in cellular zinc homeostasis (Palmiter et al, 1995). Increased cellular zinc content induces ZnT1 gene expression to efflux zinc from cells (Palmiter et al, 1995; McMahon and Cousins, 1998). The regulation of ZnT1 mRNA expression was found to be mediated by binding of metal response element-binding transcription factor-1 to metal response elements (MREs) sequence present in the ZnT1 promoter region (Langmade et al, 2000; Andrews, 2001). These findings led us to speculate that decreased cellular zinc content in AIDL cells may lead to a decrease in ZnT1 expression. However, the expression was observed to be higher than in LNCaP cells. The reason why the expression of ZnT1 increased in AIDL cells is unknown, but from the viewpoint of its function as effluxing zinc, the increased ZnT1 expression may result in the low intracellular zinc content in the cells.
Since metallothionein acts as a zinc store in the cell and its gene is also transcriptionally regulated by intracellular zinc thorough cis-acting MREs (Andrews, 2001), the low level of zinc in AIDL cells could be closely related to the low expression of metallothionein observed in these cells. However, the reason for the decreased expression of metallothionein in AIDL cells is probably more complex since its expression is also regulated by various stress factors, including glucocorticoids, cytokines, and reactive oxygen species (Palmiter, 1998). Therefore, the low level of metallothionein in AIDL cells, which is generated to be cultured in the medium containing 2% hormone-stripped FCS, may partially result from its culture condition.
The high-level expression of ZnT3 was observed in LNCaP cells cultured in hormone-reduced medium. It is not clear from our experiments whether the increased expression of ZnT3 is mediated directly by hormone ablation or indirectly through alterations of environmental androgen. Although an androgen response element was not found in the 5' promoter region of ZnT3 gene [data not shown; the sequence was described in the GenBank database (accession number NT_022184)], since cis-element sequence that is required to mediate a negative regulation by androgen is almost unknown, the unidentified androgen response element may exist in the upstream region of the ZnT3 gene. We are going to further investigate the transcriptional regulation of the ZnT3 gene.
Expression of metallothionein in rat prostate is known to be regulated by androgen, but the degree of the androgenic effect is different among lobes of prostate (Ghatak et al, 1996; Tohyama et al, 1996; Yamashita et al, 1996). The rat prostate consists of at least 3 anatomically independent lobes, designated as the ventral, lateral, and dorsal prostate. In ventral prostate, metallothionein expression was not detectable and castration appeared to induce metallothionein gene expression, but testosterone administration after castration had no effect on the expression (Tohyama et al, 1996). In dorsolateral prostate, metallothionein was present and testosterone caused a reversal of the effect of castration on metallothionein induction (Ghatak et al, 1996; Tohyama et al, 1996). In lateral prostate, metallothionein expression was not regulated by androgen (Yamashita et al, 1996). These findings suggest that metallothionein gene expression is regulated by androgen in the dorsal prostate, but not lateral and ventral prostate. LNCaP cells have been reported to exhibit the characteristics of lateral prostate cells (Costello et al, 1999); therefore, our observation of no effect on metallothionein expression in LNCaP cells by androgen supports the previous studies.
In conclusion, we first showed that the expression of ZnT3 mRNA underwent an androgenic regulation in LNCaP cells and the high-level expression of ZnT3 mRNA was detected in the androgen-independent subline derived from LNCaP cells. In addition, high levels of ZnT1 and low levels of metallothionein were detected in AIDL cells. The elucidation of the relation between the expressions of ZnT1, ZnT3, and metallothionein and the progression of prostate cancer to the hormone-refractory state might help in understanding the physiology of prostate cancer.
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