Journal of Andrology
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Published-Ahead-of-Print October 3, 2007, DOI:10.2164/jandrol.107.003608
Journal of Andrology, Vol. 29, No. 2, March/April 2008
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.107.003608

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Downregulation of Thymosin β4 Expression by Androgen in Prostate Cancer LNCaP Cells

KAZUHIRO IGUCHI*, MAI ITO*, SHIGEYUKI USUI*, ATSUSHI MIZOKAMI{dagger}, MIKIO NAMIKI{dagger} AND KAZUYUKI HIRANO*

From the * Laboratory of Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan; and {dagger} Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Sciences, Ishikawa, Japan.

Correspondence to: Dr Kazuyuki Hirano, Laboratory of Pharmaceutics, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan (e-mail: hirano{at}gifu-pu.ac.jp).
Received for publication July 6, 2007; accepted for publication September 13, 2007.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Androgen ablation therapy is an effective treatment for advanced prostate cancer, but the tumor often progresses toward a more aggressive phenotype. We determined the changes in genes associated with the malignant progression and found increased thymosin β4, involved in tumor metastasis, in androgen-sensitive LNCaP cells grown in the medium with androgen-deficient, charcoal-stripped fetal calf serum. The mRNA expression of thymosin β4 was determined by real-time polymerase chain reaction analysis. The transcriptional activity of thymosin β4 was measured by luciferase assay using reporter plasmid containing 5'-flanking region of thymosin β4. Thymosin β4 mRNA expression was increased in LNCaP cells in the androgen-deficient condition and decreased by dihydrotestosterone treatment. Androgen receptor antagonist bicalutamide inhibited thymosin β4 expression in a dose-dependent manner. In androgen receptor–negative PC-3 cells, no significant effects on thymosin β4 gene expression were observed. The regulation of thymosin β4 mRNA expression by androgen is due to the transcriptional activation. Deletion analysis revealed that the region between –83 bp and –46 bp of the thymosin β4 gene is responsible for the regulation of the transcriptional activity by androgen. Thymosin β4 expression is negatively controlled at the transcriptional level by androgen.

     Key words: Androgen receptor, androgen ablation therapy


Prostate cancer is the most common cancer in men and the second leading cancer-related cause of death in the Western countries (Jemal et al, 2007). Most patients with advanced prostate cancer are currently treated with an androgen ablation therapy because the growth and progression of prostate cancer are initially androgen dependent (Huggins and Hodges, 1941; Isaacs, 2005). The androgen ablation therapy causes tumor regression in more than 80% of cases, but the prostate cancer frequently progresses from an androgen-dependent to an aggressive androgen-independent state after the therapy (Emmett et al, 1960; Isaacs, 2005). The prostate cancer in this state is often difficult to cure, and the development of effective treatment strategies is under consideration.

Many reasons have been proposed as to why prostate cancer cells acquire aggressive phenotypes, such as rapid growth, increased invasive and metastatic potentials, and resistance to apoptosis following androgen ablation therapy (Pienta and Bradley, 2006). These include changes in the expression of androgen-regulated genes after the therapy. For instance, Gleave et al (1999) have demonstrated that the increased Bcl-2 expression after androgen withdrawal contributes to the resistance to apoptotic induction by androgen ablation and chemotherapeutic agents. Xing and Rabbani (1999) have revealed that the expressions of urokinase-type plasminogen activator involved in tumor metastasis are regulated by androgen.

Thymosin β4 is a small, acidic, 4.9-kDa protein and functions as a major G-actin sequestering factor in mammalian cells (Low et al, 1981; Safer et al, 1991). The physiologic role of thymosin β4 also includes involvement in wound healing, cell differentiation, tumor metastasis, and angiogenesis (Malinda et al, 1999; Kobayashi et al, 2002; Cha et al, 2003; Philp et al, 2003; Bock-Marquette et al, 2004; Smart et al, 2007). In tumor cells, increased thymosin β4 expression is associated with changes in the expression of various genes involved in malignancy, such as vascular endothelial growth factor, E-cadherin, and survivin (Cha et al, 2003; Wang et al, 2003, 2004; Hsiao et al, 2006). Clinically, thymosin β4 expression has been reported to increase in several metastatic tumor cells, such as metastatic colorectal and tongue squamous cell carcinoma (Yamamoto et al, 1993; Vigneswaran et al, 2005). Thymosin β4 is suggested to be a molecular target for antitumor strategies (Goldstein, 2003).

In the present study, we investigated the mechanisms underlying the transition to an aggressive phenotype after androgen ablation therapy by examining the effect of androgen withdrawal on gene expressions related to tumor malignancy in androgen-sensitive prostate cancer LNCaP cells. Here, we report that thymosin β4 expression was increased in LNCaP cells maintained under an androgen-deprived culture condition.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials

Dihydrotestosterone was purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan). Bicalutamide was kindly supplied by AstraZeneca K.K. (Osaka, Japan). All other chemicals were of analytical grade.

Cell Culture

Human prostatic carcinoma LNCaP cells (androgen sensitive) and PC-3 and DU-145 cells (androgen insensitive) were from American Type Culture Collection (Rockville, Md). LNCaP-E9 cells (low androgen sensitive) were obtained by the limiting dilution method (Iguchi et al, 2007). The cells were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS) under a humidified atmosphere with 5% CO2 at 37°C. For steroid-free conditions, Phenol red–free RPMI-1640 medium was used with charcoal-stripped FCS (CS-FCS).

Real-Time Reverse Transcription–Polymerase Chain Reaction

To study the effect of steroid hormones, LNCaP, LNCaP-E9, and PC-3 cells were cultured in phenol-red free RPMI-1640 medium containing either 5% CS-FCS or 5% FCS for 1, 2, and 3 days. To study the effect of dihydrotestosterone, LNCaP and PC-3 cells were incubated in the medium containing 5% CS-FCS for 36 hours. Then, the cells were seeded and treated with indicated concentrations of dihydrotestosterone for 48 hours. In the case of studying the effect of bicalutamide, LNCaP and PC-3 cells were incubated in RPMI-1640 medium+10% FCS and treated with the indicated concentrations of bicalutamide for 48 hours. After incubation, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif), and then first-strand complementary DNA was synthesized from 5 µg total RNA using SuperScript III (Invitrogen), as described previously (Iguchi et al, 2006). Real-time monitoring of reactions was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, Calif) with the SYBR Premix Ex Taq (Takara Bio Inc, Otsu, Japan). At the end of the PCR, a dissociation curve analysis was performed to examine the specificity of the product. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization of thymosin β4 mRNA expression level. The PCR was performed under the following conditions: 35 cycles of 15 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C for thymosin β4, and 35 cycles of 10 seconds at 95°C and 20 seconds at 60°C for GAPDH. The primers used in this study were 5'-CAGACCAGACTTCGCTCGTA-3' and 5'-GCTTCTCCTGTTCAATCGT-3' for thymosin β4, and 5'-CCAGCAAGAGCACAAGAGGA-3' and 5'-GCAACTGTGAGGAGGGGAGA-3' for GAPDH.

Plasmid Construction

Genomic DNA from LNCaP cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The 5'-flanking region of human thymosin β4 gene between –2650 and +44 bp was amplified by PCR from the genomic DNA using primers containing restriction sites for KpnI and NheI, respectively. The sequences of the primers were: sense, 5'-GAGGTACCGGAAAGCACAACTGCCTAGC-3'; antisense, 5'-TAGCTAGCGTACGAGCGAAGTCTGGTCTG-3'. The fragment was ligated to a pGL3-basic firefly luciferase reporter gene (Promega, Madison, Wis) and was designated as –2650pGL3. The various lengths of the 5'-flanking regions of thymosin β4 gene (between –695 and +44, –403 and +44, –193 and +44, –83 and +44, and –46 and +44) were amplified by PCR from –2650pGL3 using the same antisense primer and different sense primers containing restriction sites for KpnI. The fragments were ligated to a pGL3-basic firefly luciferase reporter gene and were designated as –695pGL3, –403pGL3, –193pGL3, –83pGL3, and –46pGL3, respectively. The sense primers were as follows: 5'-GGGGTACCTGCTAAGAGGGAGGTGTTT-3' for –695pGL3, 5'-GGGGTACCCGCCCTTGTGTGGAGATGT-3' for –403pGL3, 5'-GGGGTACCTTCGCCATCGTTGTGGTTAG-3' for –193pGL3, and 5'-GGGGTACCGAAGGAGTTAAGC-3' for –46pGL3.

Luciferase Assay

LNCaP and DU-145 cells were seeded at a density of 5 x 104 cells/well (LNCaP) or 2.5 x 104 cells/well (DU-145) into a 24-well culture plate (Nalge Nunc, Rochester, NY). After 24 hours, the medium was changed to Phenol red–free RPMI-1640 medium containing 5% FCS, 5% CS-FCS, 5% CS-FCS + 1 nM dihydrotestosterone, or 5% FCS + 10 µM bicaltamide without antibiotics. Then, the cells were cotransfected with 0.22 µg of each firefly luciferase reporter plasmid and 0.7 ng Renilla luciferase plasmid pRL-CMV using FuGene6 reagent (Roche Diagnostics, Indianapolis, Ind) according to the manufacturer's instructions. For experiments with DU-145 cells, the cells were cotransfected with full-length human androgen receptor expression plasmid pSGAR2 (0.044 µg) along with 0.22 µg firefly luciferase reporter plasmid and 1.45 ng phRL-CMV. Seventy-two hours after transfection, the cell lysates were prepared, and luciferase activities were measured using the Dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized with Renilla luciferase activity.

Statistical Analysis

Variation in the results obtained between 2 groups was calculated by Student's t test, and the significance of differences between multiple groups were assessed by 1-way analysis of variance followed by the Dunnet test.


Figure 1
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  		Figure 1. The mRNA expression of thymosin β4 in prostate cancer cells. (A) LNCaP, (B) LNCaP-E9, and (C) PC-3 cells were incubated in the medium containing either 5% FCS (white bars) or 5% CS-FCS (black bars) for 1, 2, and 3 days, and the mRNA expression was quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). The mRNA level of the cells incubated in medium containing FCS for 1 day was taken as 100%. Values represent the mean ± SD from at least 2 independent experiments, each performed in triplicate. * indicates P < .05; **, P < .01; ***, P < .001.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
First, we examined the effect of steroid hormones on thymosin β4 expression in prostate cancer cells. Real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression of thymosin β4 in androgen-sensitive LNCaP cells was definitely higher in the medium containing CS-FCS than FCS after the first, second, and third days of incubation (Figure 1A). In the case of low androgen–sensitive LNCaP-E9 cells, the thymosin β4 mRNA expression was significantly increased after 3 days of incubation in the medium containing CS-FCS compared with FCS (Figure 1B). Androgen receptor–negative PC-3 cells showed no differences in thymosin β4 mRNA expression level between FCS and CS-FCS (Figure 1C).

Second, to examine whether the observed increase in thymosin β4 mRNA expression is mediated by an androgen receptor, we tested the effect of dihydrotestosterone and androgen receptor antagonist bicalutamide on thymosin β4 mRNA expression. The treatment of LNCaP cells with dihydrotestosterone significantly reduced thymosin β4 mRNA expression (Figure 2A). Moreover, bicalutamide treatment increased thymosin β4 mRNA expression in a dose-dependent manner (Figure 2B). In PC-3 cells treated with dihydrotestosterone or bicalutamide, no change in thymosin β4 mRNA expression was observed (Figure 2C and D).


Figure 2
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  		Figure 2. Androgenic regulation of thymosin β4 mRNA expression in LNCaP cells. (A, B) LNCaP and (C, D) PC-3 cells were treated with indicated concentrations of either (A, C) dihydrotestosterone or (B, D) bicalutamide for 48 hours, respectively. The mRNA expression was quantified using real-time RT-PCR. Values represent the mean ± SD from at least 2 independent experiments, each performed in triplicate. * indicates P < .05; **, P < .01; ***P < .001 vs control.

 
Third, to determine whether the downregulation of thymosin β4 mRNA expression by androgen is due to transcriptional regulation, the luciferase reporter assay was performed using thymosin β4 reporter constructs. It can be seen from Figure 3A that an approximately 3.5-fold increase in luciferase activity was observed in LNCaP cells cultured in the medium containing CS-FCS than FCS. The luciferase activity of CS-FCS was decreased to the same level as that of FCS by the addition of 1 nM dihydrotestosterone. Moreover, treatment with an androgen receptor antagonist bicalutamide significantly increased the luciferase activity in LNCaP cells in the medium containing FCS.


Figure 3
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  		Figure 3. Androgenic regulation of luciferase activity of thymosin β4 reporter plasmid. LNCaP (A) and DU-145/AR (B) cells were transfected with 2650pGL3 and pRL-CMV as described in "Materials and Methods." The luciferase activity in cells incubated in the medium containing FCS (A) or CS-FCS (B) was taken as 100%. Data represented are the mean ± SD from at least 2 independent experiments, each performed in triplicate. ** indicates P < .01; ***, P < .001. DHT indicates dihydrotestosterone; BIC, bicalutamide.

 
To confirm that the changes of the luciferase activity are mediated by androgen receptor, DU-145 cells were transfected with androgen receptor expression plasmid pSGAR2, and we examined the effect of dihydrotestosterone and bicalutamide on the luciferase activity of thymosin β4 reporter plasmid. As shown in Figure 3B, the luciferase activity was decreased by dihydrotestosterone treatment, and the inhibitory effect of dihydrotestosterone on luciferase activity was suppressed by addition of bicalutamide.

Subsequently, to define the regulatory elements of thymosin β4 gene responsive to androgen, various deletion constructs of the thymosin β4 gene 5'-flanking region were constructed, and luciferase assay was performed. Figure 4 shows that increased luciferase activity in LNCaP cells cultured in the medium containing CS-FCS was detected when the cells were transfected with the deletion constructs –2650pGL3, –695pGL3, –403pGL3, –193pGL3, or –83pGL3. No significant changes in luciferase activity were observed in LNCaP cells transfected with –46pGL3 under CS-FCS conditions and with dihydrotestosterone.


Figure 4
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  		Figure 4. Luciferase activity of the deletion constructs of the 5'-flanking region of thymosin β4 in LNCaP cells. LNCaP cells were transfected with 2650pGL3, 695pGL3, 403pGL3, 193pGL3, 83pGL3, or 46pGL3 and pRL-CMV as described in "Materials and Methods." After 72 hours, the cell lysates were prepared, and luciferase activities were measured. The luciferase activity in cells incubated in the medium containing 5% FCS was taken as 100%. Data represented are the mean ± SD from at least 2 independent experiments, each performed in triplicate. * indicates P < .05; **, P < .01; ***, P < .001.

 


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we present evidence that thymosin β4 expression was regulated by androgen in prostate cancer cells. Although dihydrotestosterone decreases the gene expression of thymosin β4 in androgen-sensitive LNCaP cells, bicalutamide exhibits the opposing effect. Furthermore, the decrease in thymosin β4 expression was observed in LNCaP cells treated with synthetic androgen R1881 (data not shown). The induction of thymosin β4 gene expression by androgen withdrawal was reduced in androgen low-sensitive LNCaP-E9 cells compared with that in LNCaP cells. In addition, there was no change in thymosin β4 gene expression after androgen withdrawal, dihydrotestosterone or bicalutamide treatment in androgen receptor–negative PC-3 cells. These results strongly suggest that the expression of thymosin β4 is negatively affected by androgen. Evidence for the modulation of thymosin β4 expression by endogenous and exogenous factors has been published. For example, the expression of thymosin β4 has been shown to be positively controlled by growth hormone in rat heart (Yoshioka et al, 2006). Antitumor drugs also affect the intracellular concentration of thymosin β4 in prostate and breast cancer cells (Iguchi et al, 1999; Otto et al, 2002). Nonsteroidal anti-inflammatory drugs have been reported to induce the expression of thymosin β4 in human colon cancer cells (Jain et al, 2004).

The downregulation of thymosin β4 expression by androgen occurs at the transcriptional level. Thus, either androgen withdrawal or bicalutamide increases the transcriptional activity of thymosin β4 in LNCaP cells, but dihydrotestosterone treatment decreases the transcriptional activity of thymosin β4. Furthermore, in DU-145 cells transfected with wild-type androgen receptor, the transcriptional activity of thymosin β4 was inhibited by dihydrotestosterone, and the inhibition was reversed by the addition of bicalutamide. However, there is no consensus sequence for steroid response elements (SREs: 5'-TGACGTC-3'; Nelson et al, 1999) within 2650 bp upstream of the thymosin β4 gene, but the region responsible for androgen has been revealed to be localized between –83 bp and –46 bp of the 5'-flanking region of thymosin β4 gene. This region contains a consensus sequence for a cyclic AMP response element (CRE: 5'-TGACGTC-3'; Bokar et al, 1988) and, interestingly, dihydrotestosterone has been found to cause the phosphorylation of CRE binding protein (CREB) in LNCaP cells (Unni et al, 2004). Moreover, CREB has been identified to be an androgen receptor coactivator (Fronsdal et al, 1998), and the expression has been shown to be downregulated by androgen (Comuzzi et al, 2004). Taken together, the androgenic regulation of thymosin β4 gene expression may be involved in the activation of CREB by androgen.

In conclusion, we first found that the expression of thymosin β4, which is involved in tumor progression, increases in prostate cancer LNCaP cells after androgen withdrawal. Although it is necessary to clarify that thymosin β4 is expressed at increased levels in prostate cancer tissues from patients treated with androgen ablation therapy, based on the findings of this study, we infer that androgenic regulation of thymosin β4 expression is one possible cause for tumor progression following this therapy.


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