Androgen-independent growth in LNCaP cell lines and steroid uridine diphosphate-glucuronosyltransferase expression

Jiro Kanaya, Mitsuhiro Takashima, Eitetsu Koh, Mikio Namiki
Department of Intrgrated Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan

Asian J Androl 2003 Mar; 5: 9-13             

Keywords: LNCaP; glucuronidation; UGT2B15; antisense cDNA; pcDNA3.1

Abstract

Aim: To investigate the mechanism of androgen-independent growth of prostate cancer after androgen ablation in LNCaP cells and the effect of glucuronidation activity. Methods: To establish androgen-independent growth in prostate cancer LNCaP-SF, continuous passage was performed in androgen-stripped medium and the cells were evaluated for glucuronidation activity. The expression vector of antisense uridine diphosphate glucuronosyl-transferase (UGT) 2B15 cDNA was also constructed and evaluated. Results: LNCaP-SF lead to a higher expression in UGT2B15 and their glucuronidation activity is 2.5 times higher than that of LNCaP cells. Significantly fewer LNCaP and LNCaP-SF than control were transfected with the antisense UGT2B15 cDNA, suggesting that UGT2B15 plays an important part in the glucuronidation activity of androgens in both cells. Conclusion: The alteration of UGT2B15 expression in LNCaP-SF cells is proposed as a biological characteristic involved in the growth of hormone-refractory prostate cancer.

1 Introduction

The prognosis of androgen ablation therapy for advanced prostate cancer is not always satisfactory. Most cases of refractory prostate cancer acquire an androgen-independent phenotype during androgen deprivation.

Recent findings suggest that prostatic cancer cell lines involve various steroidgenic and glucuronidation activities [1, 2]. The uridine diphosphate glucuronosyltrans-ferase (UGT) enzyme catalyzes the transfer of the glucuronyl group from uridine 5'-diphosphoglucuronic acid to active endogenous and exogenous molecules [3, 4]. The glucuronide products become more polar and generally are water-soluble. Glucuronidation of steroids is believed to prevent their interaction with their nuclear receptors and eliminate them from the target cells [5]. Glucuronidation has been demonstrated in the kidney, gut, lung, skin, brain, fat, thymus, prostate and breast but not in the liver. Moreover, Belanger et al [6] have demonstrated high levels of dehydrotestosterone (DHT) glucuronidation in human prostate, breast cyst fluid and ovary follicle fluid.

The purpose of the present study was to examine the molecular biological characteristics of androgen-independent growth of prostate cancer after androgen ablation in LNCaP cells and the effect of glucuronidation activity.

2 Materials and methods

LNCaP FGC (passage 40~45) were purchased from the American Type Culture Collection (Rockville, USA). They were routinely maintained as monolayer cultures and were kept in RPMI-1640 medium supplemented with 10 % (V/V) fetal bovine serum (FBS) and 2 mmol/L of L-glutamine (both from GIBCO BRL, Uxbridge, UK) at 37 in a humidified atmosphere of 5 % CO₂ in air. Subcultures were produced at weekly intervals by using a mixture of 0.05 % trypsin ethylenediaminetetraacetate. For all experiments cells with a low passage range were used.

An androgen-independent LNCaP cell line was established to study the glucuronidation activity. To generate a steroid-free environment, a steroid-reduced medium was used, i.e., a phenol red-free RPMI 1640 medium supplemented with charcoal/dextran-treated FBS to remove the endogenous steroids. An androgen-independent LNCaP cell line was established to study the glucuronidation activity by a long-term (more than six-month) culture of androgen-dependent LNCaP-FGC cells in a RPMI-1640 medium containing the steroid-stripped serum. The surviving cell line was named as LNCaP-SF.

The total RNA from LNCaP and LNCaP-SF was isolated from the cells with an isogen solution (Nippongene, Japan) according to the manufacturer's instructions. UGT2B15 and b-actin mRNAs were measured with the reverse transcription polymerase chain reaction (RT-PCR) using an RT-PCR kit (Gibco Life Technologies, USA). The amplification profile involved denaturation at 95 for 1 min, annealing at 58 for 1 min and extension at 72 for 1 min. This was followed by examination of the relationship between the amount of RT-PCR products and the PCR cycles (21, 24, 27, 30 cycles) in each mRNA. To measure the UGT2B15 mRNA (gene bank accession No. XM_011097), the specific sense primer 5'-CCTTGCCCAGATCCCACAAA-3' and the antisense primer 5'-TATCACAGTTGCCACGCAGG-3' were used. The PCR product size was 535 bp. For the measurement of ?actin, which was used as the control, the sense primer was 5'-GAAAATCTGGCACCACAC-CTT-3' and the antisense primer5'-TTGAAGGTAGTTT CGTGGAT-3'. The PCR product size was 592 bp. The density of each PCR band was analyzed by using a NIH image, and the amount of each UGT2B15 mRNA was expressed as the ratio to b-actin.

Cells from cultures were plated at a density of 110⁶ per well in 6-well plastic plates to reach confluence for the start of the experiment and 24 h was allowed for adhesion. The experiments were performed in fresh medium containing 100 nmol/L of labeled steroid hormones. Radiolabeled (¹⁴C) testosterone was obtained from NEN Life Science Products (Boston, USA).

After 4 h of incubation, labeled androgen metabolite extraction was performed in glass vials using ethyl acetate. The aqueous phase was transferred to separate glass tubes and freeze-dried for 2 h. The dried extracts were then resuspended with the aid of a phosphate buffer (0.1 mol/L, pH 6.5) containing 200 U of b-glucuronidase type VIII (Sigma, USA) and incubated at 37 for 24 h to hydrolyze the steroid conjugates. Following incubation, the steroids were released and extracted again by means of ethyl acetate. The extracted metabolites were separated with thin layer chromatography (TLC) and analyzed with a BAS2000 Radioanalytic imaging system (BAS2000 System Inc., USA) as described in a previous report [7].

2.5 Construction of UGT2B15 antisense RNA eukaryotic expression vector

The gene encoding UGT2B15 was generated with a pGEM-T Easy Vector System (Promega, Madison, WI) according to the manufacturer's instructions. Human prostate QUICK-Clone cDNA was purchased from Clontech (Palo Alto, CA), and the cDNA was amplified by using the primers designed to amplify the sequences encoding UGT2B15 (gene bank accession No. XM_011097). The sense primer was 5'-TAAGACCAG-GATGTCTCTGAAATGGACGTCA-3', and the antisense primer 5'-CCAGGGTTTAATACGTACTTTAGCTGG-3'. The amplification profile involved denaturation at 95 for 1 min, annealing at 60 for 1 min, and extension at 72 for 1 min. The PCR product size was 1773 bp with 35 cycles. The PCR fragment was gel-purified with a QIAEX II gel extraction kit (QIAGEN, Germany) and cloned into the pGEM-T cloning vector (Promega). The recombinant pGEM plasmid was then digested with SpeI and NotI and the 1773 base pair fragment was ligated to the SpeI/NotI-digested pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA). The UGT2B15 antisense RNA eukaryotic expression vector was also constructed, and the orientation of the insert into the vector was confirmed by sequencing according to the dideoxynucleotide chain-termination method of Sanger and by using the sequenase 2.0 version kit (USB, Amersham).

Two µg of the reconstructed pcDNA3.1 plasmid was diluted into 100 µL OPTI-MEM. Four µg lipofect AMINE (both from GIBCO BRL) was diluted into 100 µL OPTI-MEM. Both solutions were incubated for 30 min at room temperature and mixed. After 20 min, 800 µL of cell growth medium without FBS was added. When the cells were at 70 % confluence, the cell layers were washed with serum-free RPMI 1640 medium and the transfection solution was added. After 4 h incubation at 37 , the transfection solution was replaced with a regular cell growth medium. A successful transfection resulted in b-galactosidase expression in a vector that could be easily assayed by using a b-Gal Staining Kit (Invitrogen).

3 Results

We examined the in vitro growth rate of LNCaP-SF in a steroid-stripped medium. The doubling time of LNCaP-SF was approximately 4 days and LNCaP-SF sublines were founded to adapt to androgen withdrawal after more than 60 passages in a steroid-stripped medium. This adaptation was manifested as a higher expression of UGT2B15 in a steroid-stripped medium compared with that of LNCaP in a regular medium (Figure 1ab & c). The steroid-stripped medium in LNCaP-SF resulted in a higher expression in UGT2B15.

Figure 1ab & c. Semiquantification of UGT2B15 expression in LNCaP and LNCaP-SF. A. UGT2B15 expression against b-actin mRNA in LNCaP; B. UGT2B15 expression against b-actin mRNA in LNCaP-SF; C. Level of UGT2B15 mRNA shown as ratio to b-actin. l: LNCaP ; o: LNCaP-SF.

A time-course study of glucuronidation metabolism in these cell lines over 4 h indicated that metabolism was linear throughout this period (data not shown). Figure 2 shows the formation and identification of androgen glucuronides when 100 nmol/L of testosterone was used as the substrate. Treatment of the polar phase with b-glucuronidase resulted in a major part of the radioactivity being converted to the organic phase, which was consisted entirely of testosterone and DHT (Figure 2B) as identified by TLC. This result implies the conversion of testosterone and DHT to a glucuronide conjugate.

3.3 Glucuronidation activity in LNCaP and LNCaP-SF cell lines

The antisense cDNA-mediated decrease in endogenous UGT2B15 resulted in a reduction in the glucuroni-dation activity in LNCaP and LNCaP-SF cells in vitro. LNCaP-SF cells showed a 2.5 times higher glucuroni-dation activity than did LNCaP cells (Figure 3, control). Because UGT2B15 is thought to degrade androgens, an increase in UGT2B15 could result in increasing degradation and hence a decrease in the bioactivity of androgens.

Figure 3. Effect of glucuronidation transfected with anti-UGT2B15 DNA. Testosterone glucuronide of LNCaP and LNCaP-SF determined after transfection of anti-UGT2B15 cDNA. Data in meanSEM were from three separate experiments. ^bP<0.05, compared with control. AU: Arbitrary Unit.

3.4 Transfection assay

b-galactosidase expression in a vector was used to evaluate the rate of transfection, which was founded to be more than 60 % (Figure 4). LNCaP and LNCaP-SF transfected with the antisense UGT2B15 cDNA were signicantly lower than that of the control, suggesting that UGT2B15 played an important part in the glucuroni-dation activity of testosterone in both LNCaP and LNCaP-SF (Figure 3). These results seem to support the hypothesis that glucuronidation may act as a regulator of androgen metabolism.

Figure 4. b-galactosidase expression in pcDNA3.1 transfected with lipofectin. A successful transfection resulted in b-galactosidase expression in pcDNA3.1 as assayed by b-Gal Staining Kit. Efficiency of transfection was more than 60 %.

Development of androgen-independent growth is a major obstacle in the treatment of human prostate cancer. As certain aspects of the growth of androgen-independent clones remain obscure, we investigated the molecular biological characteristics of the transition from androgen-dependent to androgen-independent growth in human prostate cancer LNCaP cells.

The androgen-independent cell line LNCaP-SF was established from LNCaP cells through prolonged androgen deprivation culture. The LNCaP cells remained proliferative despite the androgen-free condition. A recent study has reported that several sublines of LNCaP showed androgen-independent growth, indicating that some biological changes occur in LNCaP cells with androgen deprivation [8-12].

On the other hand, previous in vitro findings have shown that androgens are converted into a glucuronide conjugate in LNCaP but not in androgen-insensitive cell line PC-3 or DU145 [7], while no sulfate conjugation is seen in any of these cell lines [1]. Glucuronidation implies the presence of UGT activity in LNCaP. UGT enzymes have been classified into several sub-families [13] and the nucleotide sequence of UGT2B enzymes is about 80 to 95 % identical with each other [3, 13]. In humans, UGT2B enzymes are widely expressed in extra-hepatic tissues and the presence of steroid UGT activities is found in several peripheral tissues, namely, the prostate, testis, skin, breast, kidney, brain and ovary [4]. It has been proposed that glucuronidation inactivates steroid hormones in peripheral steroid target tissues including the prostate [14]. Moreover, a specific transcript of UGT2B15 gene [15, 16] has been identified in prostate LNCaP [7].

The androgen-independent subcellline LNCaP-SF showed a loss of androgen dependency and the proliferation of cells in a steroid-free medium. Therefore, androgen-independent subcell line LNCaP-SF is thought to be an important feature of the hormone-refractory cancer. Our study raised some interesting findings concerning UGT activity. LNCaP-SF showed a higher glucuroni-dation activity than that of the parental LNCaP cell, and glucuronidation activities were reduced by anti-UGT2B15 transfection in both LNCaP and LNCaP-SF. Since UGT2B15 is thought to contribute to glucuronide conjugation, an increase in glucuronidation activity could result in increasing degradation and reducing bioactivity of androgens in androgen independent LNCaP.

Once prostate cancer becomes androgen-indepen-dent, the cancer cells are resistant to growth suppression by secondary endocrine therapy and over-expression of UGT may result in the removal of all other steroid compounds.

In conclusion, our findings suggest that excess UGT2B15 enzyme activity and expression is proposed as one biological characteristic involved in the growth of hormone-refractory prostate cancer.

References

[1] Guillemette C. Glucuronosyltransferase activity in human cancer cell line LNCaP. Mol Cell Endocrinol 1995; 107: 131-9.
[2] Hum DW, Belanger A, Levesque E, Barbier O, Beaulieu M, Albert C, et al. Characterization of UDP-glucuronosyl-transferases active on steroid hormones. J Steroid Biochem Mol Biol 1999; 69: 413-23.
[3] Levesque E, Beaulieu M, Guillemette C, Hum DW, Belanger A. Effect of interleukins on UGT2B15 and UGT2B17 steroid uridine diphosphate-glucuronosyltransferase expression and activity in the LNCaP cell line. Endocrinology 1998; 139: 2375-81.
[4] Belanger A, Hum DW, Beaulieu M, Levesque E, Guillemette C, Tchernof A, et al. Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues. J Steroid Biochem Mol Biol 1998; 65: 301-10.
[5] Guillemette C, Hum DW, Belanger A. Specificity of glucuro-nosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least. J Steroid Biochem Mol Biol 1995; 55: 355-62.
[6] Belanger A, Guillemette C, Hum DW. Evidence for a role of glucuronosyltransferase in the regulation of androgen action in the human prostatic cancer cell line LNCaP. J Steroid Biochem Mol Biol 1996; 57: 225-31.
[7] Koh E, Kanaya J, Namiki M. Adrenal steroids in human prostatic cancer cell lines. Arch Androl. 2001; 46: 117-25.
[8] Kokontis JM, Hay N, Liao S. Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol Endocrinol 1998; 12: 941-53.
[9] Gao M, Ossowski L, Ferrari AC. Activation of Rb and decline in androgen receptor protein precede retinoic acid-induced apoptosis in androgen-dependent LNCaP cells and their androgen-independent derivative. J Cell Physiol 1999; 179: 336-46.
[10] Culig Z, Hoffmann J, Erdel M, Eder IE, Hobisch A, et al. Switch from antagonist to agonist of the androgen receptor bicalutamide is associated with prostate tumour progression in a new model system. Br J Cancer 1999; 81: 242-51.
[11] Lu S, Tsai SY, Tsai MJ. Molecular mechanisms of androgen-independent growth of human prostate cancer LNCaP-AI cells. Endocrinology 1999; 140: 5054-9.
[12] Igawa T, Lin FF, Lee MS, Karan D, Batra SK, Lin MF. Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model. Prostate. 2002; 50: 222-35.
[13] Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Belanger A, et al. The UDP glycosyltransferase gene super-family: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 1997; 7: 255-69.
[14] Belanger G, Beaulieu M, Marcotte B, Levesque E, Guillemette C, Hum DW, et al. Expression of transcripts encoding steroid UDP-glucuronosyltransferases in human prostate hyperplastic tissue and the LNCaP cell line. Mol Cell Endocrinol 1995; 113: 165-73.
[15] Guillemette C, Hum DW, Belanger A. A Regulation of steroid glucuronosyltransferase activities and transcripts by androgen in the human prostatic cancer LNCaP cell line. Endocrinolog 1996; 137: 2872-9.
[16] Guillemette C, Levesque E, Beaulieu M, Turgeon D, Hum DW, Belanger A. Differential regulation of two uridine diphospho-glucuronosyltransferases, UGT2B15 and UGT2B17, in human prostate LNCaP cells. Endocrinology 1997; 138: 2998-300.

home

Correspondence to: Eitetsu Koh, MD, PhD, Department of Urology, Kanazawa University Graduate School of medical Science, 15-1 Takarama-machi Kanazawa, 920-8641 Japan.
Tel: +81-76-265 2393, Fax: +81-76-222 6726
E-mail: kohei@med.kanazawa-u.ac.jp
Received 2002-11-04      Accepted 2003-01-14

This article has been cited by other articles:

•  Hajdinjak T, Zagradisnik B.  Prostate cancer and polymorphism D85Y in gene for dihydrotestosterone degrading enzyme UGT2B15: Frequency of DD homozygotes increases with Gleason score. PROSTATE 59 (4): 436-439 JUN 1 2004  [Abstract] [Full Text]
•