Skip Navigation


Carcinogenesis Advance Access originally published online on May 16, 2008
Carcinogenesis 2008 29(6):1148-1156; doi:10.1093/carcin/bgn109
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/6/1148    most recent
bgn109v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Seaton, A.
Right arrow Articles by Waugh, D. J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seaton, A.
Right arrow Articles by Waugh, D. J. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

Angela Seaton, Paula Scullin, Pamela J. Maxwell, Catherine Wilson, Johanna Pettigrew, Rebecca Gallagher, Joe M. O'Sullivan, Patrick G. Johnston and David J. J. Waugh*

Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK

* To whom correspondence should be addressed. Tel: +44 2890 972760; Fax: +44 2890 972776; Email: d.waugh{at}qub.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

Abbreviations: AIPC, androgen-independent prostate cancer; AR, androgen receptor; CaP, prostate cancer; Cdk2, cyclin-dependent kinase 2; CXCR, CXC chemokine receptor; DMSO, dimethyl sulphoxide; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; IL, interleukin; JNK, c-jun N-terminal kinase; mRNA, messenger RNA; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PSA, prostate-specific antigen; qPCR, quantitative real-time polymerase chain reaction; rh-IL-8, recombinant human interleukin-8; TBS-T, Tris-buffered saline/0.1% Tween-20


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Prostate cancer (CaP) is the most frequently diagnosed male cancer and the second leading cause of cancer-related deaths in men in Western society. During the initial stages of CaP, growth of the tumor is stimulated by androgen signaling and hence may be successfully controlled using a series of strategies that deplete endogenous androgen expression or interfere with androgen receptor (AR)-mediated signaling (1,2). One such agent is the non-steroidal antiandrogen bicalutamide (Casodex®), which directly antagonizes androgen signaling at the level of the receptor. AR-targeted strategies may be used as a single therapy or, more commonly, in combination with pharmaceutical or surgical castration or ionizing radiation.

CaP cells develop a series of alternative strategies to survive and grow in response to the reduced androgen levels that result from antiandrogen therapy. The development of resistance to antiandrogen therapy results in patient relapse and the emergence of an ablation-resistant or castration-resistant state. The molecular processes underlying this transition are incompletely understood; however, it is increasingly acknowledged that despite their insensitivity to antiandrogen therapy, these tumors frequently remain under the control of AR signaling. Defined mechanisms of resistance include AR gene amplification, AR gene mutations, deregulation of coregulators, ligand-independent activation of the AR and locus-wide chromatin remodeling within AR-regulated genes that permits more efficient transcription (1–8). Ligand-independent activation of the AR has been demonstrated in response to growth factors such as insulin-like growth factor-I (9), neuropeptides like neurotensin (10) and cytokines including interleukin (IL)-6 (11).

IL-8 is a CXC chemokine, the overexpression of which is associated with the angiogenesis, tumorigenicity and lymph node metastasis of androgen-independent prostate cancer (AIPC) in athymic nude mice (12,13). Elevated serum levels of IL-8 have been reported in patients with localized disease and AIPC (14,15). We have demonstrated elevated expression of IL-8 and each of its G-protein-coupled receptors [CXC chemokine receptor (CXCR)-1 and CXCR2] in tumor cells of human prostate biopsy sections compared with normal epithelial cells (16). Expression of IL-8, CXCR1 and CXCR2 all increased with stage of disease: present in prostatic intraepithelial neoplasia but greatest in androgen-independent disease. Furthermore, colorimetric in situ hybridization techniques have detected elevated IL-8 gene expression in prostate biopsy tissue that is associated with both increased Gleason score and pathologic stage of the tumors (17). A previous study has reported IL-8-promoted proliferation of LNCaP cells that was attenuated by pretreatment of the cells with bicalutamide, implying the involvement of the AR in this response (18). In addition, these authors employed a prostate-specific antigen (PSA) promoter-based luciferase reporter construct to indicate that IL-8 signaling increased the AR-mediated transcription and used chromatin immunoprecipitation assays to confirm increased complexing of the AR on the PSA promoter. Collectively, these studies on CaP cell lines and biopsy tissue suggest a potential role for IL-8 in promoting the progression of the disease to the androgen-independent state.

Here, we report a more direct and extensive characterization of IL-8-promoted AR activation and its role in potentiating androgen-independent outgrowth of human CaP cells. We further demonstrate the novel finding that IL-8 signaling increases AR expression, in addition to studying the dynamics with which this chemokine alters the distribution and transcriptional activation of the AR, resulting in an increased expression of AR-regulated genes. Furthermore, we show that blockade of IL-8 signaling increases the sensitivity of these cells to bicalutamide. Accordingly, we suggest that IL-8-promoted increases in AR expression and activation are consistent with this chemokine promoting the transition to AIPC.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemical and reagents
All chemicals were purchased from the Sigma Chemical Co. (St Louis, MO) unless otherwise stated. BAY11-7082 and c-jun N-terminal kinase (JNK) inhibitor I were both obtained from Merck Chemicals Ltd (Nottinghamshire, UK). AZ10397767 and bicalutamide (Casodex®) were kindly provided under an MTA agreement with AstraZeneca (Alderley Park, UK). BAY11-7082 and JNK inhibitor I were dissolved in dimethyl sulphoxide (DMSO) and used at a final concentration of 5 and 1 µM, respectively. Cells were pretreated with each of these inhibitors for 6 h prior to stimulation with recombinant human interleukin-8 (rh-IL-8) (Peprotech, Rocky Hill, NJ) treatment. Bicalutamide was reconstituted in DMSO and was coadministered with rh-IL-8 or AZ10397767 at a final concentration of 10 µM in LNCaP cells or 1 µM in 22Rv1 cells. Controls for the effect of the DMSO vehicle were conducted in parallel wells.

Cell culture
Human LNCaP cells (kindly provided by Dr Tracy Robson, Queen's University Belfast) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine (Invitrogen Ltd, Paisley, UK) and 1 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). 22Rv1 cells (kindly provided by Dr Antoinette Powell, Trinity College, Dublin) were maintained in RPMI 1640 medium with Glutamax supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Invitrogen). All cultures were maintained in a humidified chamber at 37°C with 5% CO2. Cell culture medium was changed to phenol red-free, serum-free RPMI 1640 medium (Invitrogen Ltd) 24 h prior to any experimental procedure, unless otherwise stated. This was in accordance with previously published experimental procedures (19).

Cell count analysis
Cells were seeded into 24-well plates (2.5 x 104 cells per well) in RPMI 1640 medium and allowed to attach overnight. The medium was replaced with serum-free RPMI prior to the addition of rh-IL-8, AZ10397767 (20 nM) or bicalutamide (1 or 10 µM depending on cell line). Plates were incubated in a humidified chamber at 37°C with 5% CO2 for 48 h and the cells were trypsinized and counted in triplicate using a Coulter Z series particle count and size analyzer (Beckman Coulter, Fullerton, CA). Cell numbers were normalized to control values.

Flow cytometry
Cells were grown on 90 cm2 plates in RPMI 1640 medium and medium was replaced with serum-free RPMI 24 h before addition of rh-IL-8 (3 nM). Following a 24 h exposure to 3 nM rh-IL-8, the cells were scraped into 15 ml tubes, washed twice in phosphate-buffered saline (PBS) containing 0.1% fetal calf serum and resuspended in 1 ml of the same solution. The cells were fixed using 100% ethanol and stored at –20°C overnight. The fixed cells were washed twice in PBS containing 0.1% fetal calf serum, then resuspended in 1 ml of propidium iodide solution (propidium iodide 10 µg/ml and RNase A 25 µg/ml in PBS/0.1% serum) and analyzed by flow cytometry (Beckmann Coulter, Buckinghamshire, UK).

Quantitative real-time polymerase chain reaction
Total RNA was isolated using RNAStat60 (Biogenesis, Oxford, UK) according to the manufacturer's instructions. For real-time polymerase chain reaction (PCR), 1 µg of total RNA was oligo(dT) reverse transcribed using MMLV-RT (Invitrogen) according to the manufacturer's instructions. The complementary DNA (50 ng) was mixed with primers (2 nM), sterile water and SYBR Green PCR mastermix (Finnzymes, Espoo, Finland). The primer sequences were as follows—18s: forward, 5'-CATTCGTATTGCGCCGCT-3' and reverse, 5'-CGACGGTATCTGATCGTC-3'; AR: forward, 5'-CGGAAGCTGAAGAAACTTGG-3' and reverse, 5'-CGTGTCCAGCACACACTACA-3'; PSA: forward, 5'-TGAGCCTCCTGAAGAATCGA-3' and reverse, 5'-TTGCGCACACACGTCATT-3' and cyclin-dependent kinase 2 (Cdk2): forward, 5'-ATGGAGAACTTCCAAAAGGTGGA-3' and reverse, 5'-CAGGCGGATTTTCTTAAGCG-3'. Real-time PCR was carried out in a 96-well plate using an Opticon 2 Continuous Fluorescence Detector (Bio-Rad, Hertfordshire, UK). Amplification was 95°C for 15 min and 45 cycles at 95°C for 15 s, 55°C for 30 s and 72°C for 60 s. The threshold cycle (CT), which indicates the relative abundance of a particular transcript, was calculated for each reaction by the Opticon 2 system. Unknown expression levels were determined from standard curve dilutions and normalized against 18s.

Cell lysate and nuclear extract preparation
For cell lysate preparation, cells were washed twice with 1 x PBS, harvested and resuspended in RIPA buffer [150 mM NaCl, 1% Triton X-100, 10 mM Tris–HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate and one Complete Mini protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN) per 10 ml RIPA buffer]. Cells were lysed by passing through a needle (21 gauge) 10 times and centrifuged at 10 000g for 5 min to clear. To prepare nuclear extracts, cells were washed twice with 1 x PBS, harvested and resuspended in hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and one Complete Mini protease inhibitor tablet/10 ml hypotonic buffer], pelleted by centrifugation at 10 000g at 4°C for 10 min, and supernatant discarded. The pelleted cells were then lysed in hypotonic buffer containing 0.1% (vol/vol) Nonidet P-40. Samples were centrifuged for 10 min at 10 000g at 4°C to pellet the nuclei. The nuclear pellet was then resuspended in nuclear lysis buffer (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid, 25% glycerol and one Complete Mini protease inhibitor cocktail tablet/10 ml nuclear lysis buffer), incubated on ice for 30 min and centrifuged at 10 000g at 4°C for 10 min. The supernatant containing the nuclear extract was removed and added to 50 µl of storage buffer [10 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM ethylenediaminetetraacetic acid, 10% glycerol and one Complete Mini protease inhibitor cocktail tablet/10 ml buffer]. Protein concentrations for cell and nuclear lysates were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL).

Western blotting
Cell lysates (50 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences, Buckinghamshire, UK). Membranes were washed in Tris-buffered saline/0.1% Tween-20 (TBS-T) then blocked for 1 h at room temperature in 5% bovine serum albumin/TBS-T. For detection of the AR, primary antibody (Millipore, Billerica, MA) was used at 1:400 in 5% bovine serum albumin/TBS-T. The membranes were washed three times in TBS-T and then incubated with a rabbit horseradish peroxidase-labeled secondary antibody (GE Healthcare UK Ltd, Buckinghamshire, UK). Following three washes in TBS-T, the bands were detected using enhanced chemiluminescence (ECL plus reagents, Amersham Biosciences). Membranes were reprobed to ensure equal loading with glyceraldehyde 3-phosphate dehydrogenase antibody (Biogenesis, Dorset, UK) in the case of cell lysates and TATA box-binding protein antibody (Abcam, Cambridgeshire, UK) in the case of the nuclear extracts.

Transfection and luciferase assay
Cells were plated in six-well plates (2.5 x 104 cells per well) in RPMI 1640 medium and incubated for 48 h at 37°C with 5% CO2. Transfection of the cells with 4 µg pGL3 basic vector (Promega, Madison, WI) or with 4 µg of an ARE-Elb-LUC plasmid (kind gift from Prof. Nancy Weigel, Baylor College of Medicine, Houston, TX) was carried out using GeneJuice transfection reagent (Merck Chemicals) according to their protocol. Cells were also cotransfected with 0.08 µg of a Renilla luciferase plasmid as a transfection control. Plates were returned to the incubator and 24 h prior to IL-8 addition medium was replaced with serum-free phenol red-free RPMI medium. rh-IL-8 (3 nM) was added for the desired time and the samples were analyzed by luciferase assay using the Promega Dual Luciferase assay kit (Promega) according to the manufacturer's protocol. The AR transcriptional activity in each sample was determined by adjusting for transfection efficiency using the Renilla readout followed by normalization to sample matched pGL3 values.

siRNA treatment
Commercially synthesized oligonucleotide pools (Dharmacon, Chicago, IL) to specifically target the AR were transfected into the cells according to the manufacturer's protocol. A commercially available scrambled oligonucleotide (Dharmacon) was also used in these experiments as a control. The AR targeted siRNA and the scrambled control was each used at a concentration of 100 nM and successful depletion of the target was confirmed by quantitative real-time polymerase chain reaction (qPCR) and immunoblotting. Following transfection, the cells were incubated for 24 h and the medium changed to serum-free, phenol red-free RPMI medium for a further 24 h. rh-IL-8 was added to the cells and 24 h later, the cells were collected for analysis of AR-dependent gene expression by qPCR.

PSA protein analysis
Cells were plated in 24-well plates (5 x 104 cells per well) in RPMI 1640 medium. Following an overnight incubation, the medium was replaced with serum-free, phenol red-free RPMI 1640 and stimulated with 3 nM rh-IL-8 for the required time. A time-matched control with no rh-IL-8 was also conducted. The supernatant was removed from the cells and centrifuged at 1000g for 5 min to remove any cell debris and the supernatant stored at –70°C until assayed. The cell number in each well was determined by parallel cell count analysis. The PSA concentration in each sample was determined using the ARCHITECT total PSA assay according to the manufacturer's instructions. This assay is a two-step immunoassay to determine the presence of total PSA (both free PSA and PSA complexed to alpha-1-antichymotrypsin), using chemiluminescent microparticle immunoassay technology with flexible assay protocols, referred to as Chemiflex®. The PSA quantity in each sample was correlated to cell number and compared with a time-matched unstimulated sample.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
Cells were seeded into 96-well plates (3 x 103 cells per well) in RPMI 1640 medium and allowed to attach overnight. Serial dilutions of bicalutamide were added to the cells alone or in combination with AZ10397767 (20 nM). Plates were incubated in a humidified chamber at 37°C with 5% CO2 for 72 h, then 50 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (2 mg/ml) was added and the plates were returned to the incubator for 4 h. Medium and any non-metabolized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were aspirated from the wells and the formazan crystals dissolved in 100 µl DMSO. Absorbance was read at 570 nm using a microplate reader (Molecular Devices, Wokingham, UK).

Statistical analysis
Two-tailed Student's t-test analysis was used to compare means where appropriate using GraphPad Prizm software.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
IL-8 signaling induces androgen-independent growth of LNCaP and 22Rv1 cells
The LNCaP cell line was originally derived from a human lymph node lesion and characterized to be responsive to androgen stimulation. This cell line contains a point mutation in the AR that alters the ligand specificity of the receptor, making it additionally responsive to adrenal androgens, estrogens and progesterones (20). Hence, our experiments were performed under serum-free conditions in phenol red-free medium to remove any confounding influence of steroids or growth factors otherwise present in the media. The 22Rv1 cell line is a human prostate carcinoma epithelial cell line derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft (21). As described previously for AIPC cell lines (16), flow cytometry confirmed the expression of each of the IL-8 receptors, CXCR1 and CXCR2 in these cells while enzyme-linked immunosorbent assay analysis confirmed a low but detectable level of endogenous IL-8 expression in these cells (data not shown).

Treatment of LNCaP cells with rh-IL-8 for 48 h resulted in a dose-dependent increase in the growth of the cells with a 17, 26 and 45% increase at 1, 3 and 10 nM, respectively (Figure 1a, left panel). In the 22Rv1 cells, the maximal induction of proliferation was observed following treatment with 3 nM IL-8 (25% increase) (Figure 1a, right panel). Addition of higher concentrations of this chemokine did not result in further augmentation of cell proliferation rates. Based on the observations in the two cell lines, a concentration of 3 nM IL-8 was used in all further studies. Flow cytometry experiments were also conducted to determine whether the increase in proliferation rate of the cells was accompanied by an increase in the percentage of cells entering the S phase of the cell cycle. Stimulation of LNCaP cells with 3 nM rh-IL-8 for 24 h resulted in a 2.3-fold upregulation in the percentage of cells in S phase (Figure 1b, left panel). A similar response was observed in the 22Rv1 cell line (Figure 1b, right panel).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. IL-8-induces androgen-independent growth of LNCaP and 22Rv1 cells. (a) Bar graphs presenting cell count analysis of LNCaP cells (left panel) and 22Rv1 cells (right panel) maintained in serum-free, phenol red-free RPMI 1640 medium following treatment with rh-IL-8 (1–10 nM) for 48 h. Values are expressed as the mean ± SEM of four experiments; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Bar graphs presenting the DNA profile analysis (S phase) of LNCaP cells (left panel) and 22Rv1 cells (right panel) maintained in serum-free, phenol red-free RPMI 1640 medium following treatment with 3 nM rh-IL-8 for 24 h. Values are expressed as the mean ± SEM of three experiments; *P < 0.05. (c) Bar graphs presenting cell count analysis of LNCaP cells (left panel) and 22Rv1 cells (right panel) maintained in serum-free, phenol red-free RPMI 1640 medium following treatment with 3 nM rh-IL-8 in the absence or presence of the CXCR2 antagonist AZ10397767 (20 nM) or bicalutamide (1 µM for 22Rv1 cells and 10 µM for LNCaP cells) for 48 h. Values are expressed as the mean ± SEM of four experiments; *P < 0.05, **P < 0.01.

 
Further experiments explored the potential mechanism underlying the IL-8-induced proliferation of the LNCaP and 22Rv1 cells. As before, stimulation of LNCaP cells with 3 nM rh-IL-8 induced a 19% increase in the cell proliferation rate, 48 h postexposure to IL-8 (Figure 1c, left panel). Coadministration of a selective CXCR2 receptor antagonist AZ10397767 with IL-8 abrogated the IL-8-induced increase in proliferation, reducing cell number to below basal levels. The IL-8-promoted increase in LNCaP cell proliferation was also downregulated in the presence of bicalutamide (10 µM). Control experiments confirmed that addition of AZ10397767 or bicalutamide alone was non-toxic to the LNCaP cells at the indicated concentration (data not shown for AZ10397767). Similarly, the IL-8-promoted proliferation of 22Rv1 cells was attenuated in the presence of 20 nM AZ10397767 or 1 µM bicalutamide (Figure 1c, right panel). Therefore, these results confirm that CXCR2-promoted signaling and the activation of AR-dependent signaling contribute in part to the IL-8-promoted increase in the proliferation of both AR-expressing cell lines.

IL-8 signaling increases AR expression
qPCR was used to study the impact of IL-8 signaling upon AR messenger RNA (mRNA) transcript levels. Stimulation of LNCaP cells with 3 nM rh-IL-8 had no effect at early time points but increases ranging from 4.1- to 5.8-fold in AR mRNA expression levels were detected at time points >16 h post-IL-8 stimulation (Figure 2a, left panel). AR mRNA transcript levels were also increased in 22Rv1 cells following stimulation with rh-IL-8 (Figure 2a, right panel). Evidence of an early and statistically significant increase in AR mRNA transcript levels was clearly detectable within 1 h of the IL-8-signaling stimulus in 22Rv1 cells. Further statistically significant increases in AR mRNA transcript levels were detected at time points >6 h postaddition of rh-IL-8 to 22Rv1 cells. IL-8-induced upregulation of AR expression was confirmed at the protein level by immunoblotting experiments. A sustained increase in AR expression was observed at all time points 4 h postaddition of rh-IL-8 in the LNCaP cells (Figure 2b, left panel). Increases in AR expression were also detected within 1 h of adding the rh-IL-8 stimulus to 22Rv1 cells, with a further increase in expression detected 16 h postexposure to rh-IL-8 in these cells (Figure 2b, right panel).


Figure 2
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. IL-8 increases AR expression. (a) Bar graphs presenting real-time PCR analysis of AR mRNA transcript levels in LNCaP cells (left panel) and 22Rv1 cells (right panel) cultured in serum-free, phenol red-free RPMI 1640 medium following stimulation with 3 nM rh-IL-8 for the indicated times. Values are expressed as the mean ± SEM of five separate experiments; *P < 0.05, **P < 0.01, ***P < 0.001. (b) Immunoblots determining the level of AR expression in protein lysates extracted from LNCaP cells (left panel) and 22Rv1 cells (right panel) following stimulation with 3 nM rh-IL-8 for the indicated times. Equal protein loading was confirmed by reprobing of the membrane for glyceraldehyde 3-phosphate dehydrogenase expression. (c) Bar graph presenting real-time PCR analysis of AR mRNA transcript levels in LNCaP cells determined under IL-8-stimulated and non-stimulated conditions, in the absence or presence of the CXCR2 antagonist, AZ10397767. Cells were pretreated with AZ10397767 (20 nM) for 4 h prior to stimulation with 3 nM rh-IL-8 for 24 h. Values are expressed as the mean ± SEM of three separate experiments; *P < 0.05, ***P < 0.001. (d) Bar graph presenting real-time PCR analysis of AR mRNA transcript levels in LNCaP cells determined under IL-8-stimulated and non-stimulated conditions, in the absence or presence of signal transduction inhibitors. Cells were pretreated with 5 µM BAY11-7082 or 1 µM JNK inhibitor I for 6 h prior to stimulation with rh-IL-8 for 24 h. Values are expressed as the mean ± SEM of three separate experiments; *P < 0.05.

 
Pretreatment of the LNCaP cells with 20 nM AZ10397767 attenuated the IL-8-induced increase in AR mRNA transcript level measured 24 h poststimulation with IL-8 (Figure 2c). Furthermore, blockade of the transcription factors NF-{kappa}B and AP-1 using the pharmacological inhibitors BAY11-7082 (5 µM) and JNK inhibitor I (1 µM), respectively, also resulted in downregulation of the IL-8-promoted increase in AR mRNA transcript levels at this time point (Figure 2d). Our data suggest that IL-8-promoted regulation of AR expression is mediated in part through a CXCR2-dependent signaling pathway and subsequently via downstream activation of AP-1 and NF-{kappa}B transcription factors.

IL-8 signaling potentiates AR activity
The AR belongs to the superfamily of steroid/nuclear receptors that translocate to the nucleus upon their activation in the cytoplasm and stimulate the transcription of genes having androgen response elements in their promoter regions. Nuclear protein extracts were prepared from rh-IL-8-stimulated LNCaP cells and were assayed for AR expression by immunoblotting. Activation of IL-8 signaling in the LNCaP cells induced a rapid increase in nuclear AR expression, evident within 20 min of adding the stimulus (Figure 3a, left panel). Densitometry analysis confirmed that AR nuclear distribution increased by a peak 3.3-fold over basal levels after 1 and 2 h post-IL-8 stimulation, but remained elevated by a factor of >2-fold for out to 6 h poststimulation (Figure 3a, right panel).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. IL-8 increases transcriptional activity of the AR. (a) Immunoblot illustrating the expression of the AR in nuclear protein lysates extracted from LNCaP cells cultured in serum-free, phenol red-free RPMI 1640 medium and stimulated with 3 nM rh-IL-8 for the indicated times. Equal protein loading was confirmed by reprobing the membranes for the TATA box-binding protein (TBP). Densitometry analysis was performed to quantitate changes in nuclear expression of the AR in stimulated cells (right panel). (b) Bar graphs illustrating the effect of IL-8 signaling upon androgen response element (ARE)-driven luciferase activity in transiently transfected LNCaP cells (left panel) and 22Rv1 cells (right panel). Cells were cotransfected with either ARE luciferase reporter (4 µg) or pGL3 basic vector (4 µg) and Renilla luciferase reporter (0.08 µg). Cells were stimulated 24 h posttransfection with 3 nM rh-IL-8 for the indicated times. Transfection efficiencies were adjusted relative to the Renilla readouts and luciferase activities normalized to pGL3 values. Values are expressed as the mean ± SEM of six separate experiments; *P < 0.05, **P < 0.01, ***P < 0.001. (c) As in section (b) but cells were treated with 20 nM AZ10397767, 5 µM BAY11-7082 or 1 µM JNK inhibitor I for 6 h prior to the addition of 3 nM rh-IL-8 for 24 h. Values are expressed as the mean ± SEM of three separate experiments; *P < 0.05, **P < 0.01.

 
To confirm this change in AR distribution coincided with an increase in AR activity, we used an androgen response element-luciferase reporter assay to examine the effects of IL-8 treatment on AR-dependent transcription. In LNCaP cells, we observed an initial increase in AR activity 2 h postexposure to rh-IL-8 (Figure 3b, left panel). Further increases in AR activity were detected 4, 6 and 24 h postexposure to IL-8. In 22Rv1 cells, significant increases in luciferase activity in the order of 1.3- to 1.8-fold over basal activity were detected at 2, 6, 16 and 24 h posttreatment with IL-8 in these cells (Figure 3b, right panel).

In a further experiment conducted on LNCaP cells, the IL-8-promoted increase in AR activation observed after 24 h stimulation was shown to be sensitive to pharmacological inhibition of CXCR2, NF-{kappa}B and AP-1 signaling using AZ10397767, BAY11-7082 and JNK inhibitor I, respectively (Figure 3c). In these experiments, blockade of CXCR2 signaling was shown to inhibit the ability of IL-8 signaling to activate the AR. In contrast, each of the signal transduction inhibitors was shown to reduce the IL-8-promoted transcriptional activity of the AR to below basal levels. Our observations are consistent with the previously characterized effects of AZ10397767 and these signal transduction inhibitors in attenuating the IL-8-promoted transcription of the AR gene. Accordingly, this suggests that the IL-8-mediated increase in AR transcriptional activity observed 24 h poststimulation is, at least in part, associated with the IL-8-promoted increase in AR expression.

IL-8 signaling increases the expression of AR-regulated genes
PSA is a serine protease synthesized by the prostate gland. The levels of PSA in the serum of patients have been shown to correlate positively with clinical stage of the disease (22). Since PSA is one of the most widely characterized AR-regulated genes, the effect of IL-8 signaling upon PSA expression in LNCaP cells was determined. Treatment of LNCaP cells with 3 nM rh-IL-8 had no immediate effect on the transcription of the PSA gene. However, we observed a marked increase in PSA mRNA expression at later time points, with a 3-fold increase in transcript levels of this gene observed >16 h postexposure to rh-IL-8 (Figure 4a). The regulation of the PSA gene by IL-8 signaling was also confirmed at the protein level. Using chemiluminescent microparticle immunoassay technology, IL-8 signaling was shown to increase the secretion of PSA by LNCaP cells at all time points >16 h poststimulation (Figure 4b).


Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. IL-8 signaling increases PSA and Cdk2 gene transcription. (a) Bar graph presenting real-time PCR analysis of PSA and Cdk2 mRNA transcript levels in LNCaP cells cultured in serum-free, phenol red-free RPMI 1640 medium following stimulation with 3 nM rh-IL-8 for the indicated times. Values are expressed as the mean ± SEM of four separate experiments; *P < 0.05, ***P < 0.001. (b) Bar graph presenting PSA secretion levels as determined by chemiluminescent microparticle immunoassay technology from LNCaP cells cultured in serum-free, phenol red-free RPMI 1640 medium following stimulation with 3 nM rh-IL-8. PSA levels were normalized to cell number. Values are expressed as mean ± SEM of four separate experiments; *P < 0.05, **P < 0.01. (c) Bar graph presenting real-time PCR analysis of IL-8-regulated PSA and Cdk2 mRNA transcript levels in LNCaP cells following transfection with AR gene-targeted siRNA oligonucleotides (final concentration of 100 nM) or a scrambled control oligonucleotide (100 nM). Cells were transfected with siRNA oligonucleotides 48 h prior to stimulation with 3 nM rh-IL-8 for 24 h. Values are expressed as the mean ± SEM of three separate experiments; *P < 0.05. (d) Bar graph demonstrating the effect of treating LNCaP cells with 20 nM AZ10397767 on IL-8-induced transcription of the PSA and Cdk2 genes. Cells were treated with the CXCR2 antagonist for 4 h prior to stimulation with 3 nM rh-IL-8. Values are expressed as the mean ± SEM of four separate experiments; *P < 0.05.

 
Cdk2 regulates cell proliferation and progression through the cell cycle and has been shown to be positively regulated by AR signaling (23,24). Analysis of Cdk2 mRNA transcript levels following IL-8 treatment yielded a similar trend of induction as that observed for PSA. Initially, no effect was seen on Cdk2 mRNA transcript levels following IL-8 treatment. However, the Cdk2 mRNA expression level was increased 2.7- and 3.5-fold in the LNCaP cells 24 and 48 h poststimulation with rh-IL-8, respectively (Figure 4a).

To demonstrate that the IL-8-induced increase in PSA and Cdk2 transcript levels was mediated by the AR, qPCR analysis was repeated in LNCaP cells depleted of AR expression by transient transfection with an AR-targeted siRNA oligonucleotide pool at a concentration of 100 nM. Inhibition of AR expression had no effect on the basal mRNA transcript levels detected for either of these two genes in comparison with the effect of a scrambled, non-targeting oligonucleotide sequence. However, inhibition of AR expression abrogated the IL-8-induced upregulation of both PSA and Cdk2 transcript levels, reducing them to levels observed in unstimulated controls in the case of Cdk2 and below basal levels in the case of PSA (Figure 4c). In further experiments, coadministration of the CXCR2 antagonist, AZ10397767 (20 nM) abrogated the rh-IL-8-promoted induction of PSA and the Cdk2 genes, though statistical significance was only accomplished with the former gene in these replicates (Figure 4d).

Blockade of IL-8 signaling increases the efficacy of bicalutamide
At present, treatment options for CaP following the failure of endocrine therapy are extremely limited and at best offer palliation. Approaches that may reverse an acquired resistance to antiandrogens are therefore of great potential. We examined the consequence of blocking IL-8 signaling using the CXCR2 receptor antagonist, AZ10397767, on the effect of bicalutamide on LNCaP and 22RV1 cell viability, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Bicalutamide alone was largely ineffective in reducing the viability of LNCaP cells with an IC10 value in excess of 10 µM (Figure 5a). Higher concentrations of the drug were not used due to the observed toxicity of the equivalent DMSO concentration in the vehicle control. However, when coadministered with AZ10397767 (20 nM), bicalutamide was shown to reduce the viability of the LNCaP cells with a calculated IC30 value of 0.25 µM. The 22Rv1 cells were slightly more sensitive to bicalutamide as a single agent than the LNCaP cell line. Although IC20 values were not reached at the concentrations used, an IC10 value of 4.84 µM was achieved for bicalutamide alone (Figure 5b). Coadministration with AZ10397767 (20 nM) increased the sensitivity of the cells to bicalutamide by 120-fold, with a new IC10 value of 40.6 nM being achieved, and an IC20 value of 0.33 µM now being reached. Control experiments confirmed that addition of AZ10397767 alone was non-toxic to both cell lines at the indicated concentration (data not shown).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Inhibition of IL-8 signaling sensitizes LNCaP and 22Rv1 cells to bicalutamide. Bar graphs representing cell viability in (a) LNCaP cells and (b) 22Rv1 cells as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, conducted after a 72 h exposure to increasing concentrations of bicalutamide. The effect of inhibiting IL-8 signaling upon the effect of bicalutamide on cell viability was determined by coadministration of the CXCR2 antagonist AZ10397767 at a final concentration of 20 nM. Values are expressed as the mean ± SEM of five separate experiments; *P < 0.05, **P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Although CaP tumor growth can initially be controlled by depletion of androgen levels and antagonism of androgen signaling, in many patients, the tumor is able to re-establish growth in an ablation-resistant manner. AR expression and AR-regulated genes such as PSA are detected in AIPC, suggesting that the AR-signaling pathway is still functional in these cancers despite their progression to a castration-resistant state (25–27). Multiple strategies linked with the AR-signaling pathway have been described that enable CaP cells to survive and grow under androgen-depleted conditions. These include the amplification of the AR gene to increase the efficiency of signaling in androgen-depleted conditions, mutations of the AR gene that permit it to be activated by other steroidal hormones, alterations in the interactions between the AR and its coactivators, epigenetic changes in gene structure and finally, through ligand-independent activation of the AR (1–8). An increased understanding of the events promoting these responses and their potential association with one another will theoretically lead to advances in our ability to control the progression of CaP to an ablation-resistant state.

We have reported previously that CaP cells are subjected to an autocrine/paracrine IL-8-signaling stimulus as a consequence of the increased expression of IL-8 and each of its receptors, CXCR1 and CXCR2, in tumor cells of human prostate biopsy tissue (16). This stimulus is detected in low Gleason grade CaP but reaches a maximal level in androgen-independent disease, suggesting that elevated autocrine/paracrine IL-8 signaling may contribute to the acquisition of an androgen-independent state of disease. In our current study, we initially confirmed that administration of exogenous IL-8 to two AR-expressing CaP cell lines (LNCaP and 22Rv1) resulted in the proliferation of these cells under steroid-depleted conditions. Furthermore, the IL-8-promoted proliferation of either cell line was abrogated following blockade of the AR with the AR antagonist, bicalutamide. Our findings in both LNCaP and 22Rv1 cells validate two prior reports illustrating the capacity of IL-8 signaling to induce androgen-independent proliferation of the LNCaP cell line (18,28). However, our study has investigated at greater depth the association of IL-8 signaling with regard to the regulation of AR expression and activity in both of these AR-expressing prostate cell lines.

Real-time PCR analysis initially confirmed that IL-8 signaling upregulated the expression of mRNA transcripts encoding the AR gene in LNCaP and 22Rv1 cells. A preliminary mechanistic study suggests that the IL-8-promoted transcription of the AR gene is mediated in part through NF-{kappa}B and AP-1-dependent mechanisms. Pharmacological inhibition of NF-{kappa}B or AP-1 activation had minimal effect on basal AR gene expression alone but resulted in almost total blockade of AR gene transcription in the presence of exogenous IL-8. The very pronounced effect of IL-8 signaling in abrogating AR gene expression when the NF-{kappa}B and AP-1 transcription factors are inhibited suggests a complex regulation of the gene. Currently, we can only speculate that IL-8 signaling may simultaneously activate additional repressive transcription factors, the effect of which only becomes apparent when NF-{kappa}B or AP-1 transcriptional activity is inhibited. NF-{kappa}B has been shown to positively regulate AR expression (29,30) but no role for AP-1 has yet been demonstrated or reported with regards to the transcriptional regulation of the AR, although c-jun is known to enhance AR-induced transactivation by acting as a cofactor (31,32). Consistent with the role of these factors in underpinning IL-8-promoted increases in AR gene transcription, we have observed that administration of exogenous IL-8-induces NF-{kappa}B and AP-1 transcription in CaP cells (Wilson et al., manuscript submitted; J.Pettigrew and D.Waugh, unpublished data). However, more detailed studies will need to be conducted to confirm the dynamics with which NF-{kappa}B, AP-1 and other as yet unidentified transcription factors co-ordinate the IL-8-promoted transcription of the AR gene.

Immunoblotting experiments conducted on lysates extracted from 22Rv1 cells demonstrate an early induction (1–2 h) and late induction (>16 h) in the expression of the AR that is coincident with increased transcript levels of the gene. In LNCaP cells, although increases in AR mRNA levels were not seen until later time points (>16 h), IL-8-promoted increases in AR protein expression were observed as early as 4 h post-IL-8 treatment, suggesting that IL-8 also controls AR expression through a non-transcriptional mechanism. We have shown previously that IL-8 signaling can positively influence protein translation in AIPC cell lines (33). Thus, IL-8 signaling may increase expression of the AR through regulation of both the transcription of this gene and subsequent translation of the encoding mRNA transcript. Interestingly, we have also shown that IL-8 signaling increases the expression of the molecular chaperone Hsp90 in CaP cell lines (A.Seaton and D.Waugh, unpublished data). The AR is a well-characterized client protein of Hsp90, assisting in maintaining the receptor in a high affinity state (34,35). Therefore, it is also possible that IL-8 signaling may effect an Hsp90-mediated posttranslational stabilization of the AR in these cells.

In addition to regulating expression of the AR, our studies confirmed that IL-8 signaling induced changes in both the distribution of the AR and in its transcriptional activity. Luciferase reporter assays revealed that IL-8 signaling increases AR transcriptional activity at early (2 h), intermediate (6 h) and late (24 h) time points, ultimately inducing the transcription of two well-characterized AR-regulated genes, PSA and Cdk2. The mechanism through which IL-8 signaling promotes AR activation may be multifaceted. Although the requirement of a direct phosphorylation of the AR for its transcriptional activity remains controversial (36,37), signaling kinases including p42/44 mitogen-activated protein kinase and phosphatidylinositol-3 kinase have been shown to regulate activation of the AR (38–40). If not mediated through direct phosphorylation of the AR, these signaling pathways may promote AR activation via phosphorylation of the coactivators of the AR, the majority of which are phosphoproteins (41,42). We have already confirmed that IL-8 signaling induces a sustained activation of both the p42/44 mitogen-activated protein kinase and phosphatidylinositol-3 kinase signal transduction cascades in AIPC cells (32). In addition, IL-8 signaling has been shown to positively regulate the activity of known transcriptional coactivators of the AR, including STAT-3 (43). Further research is underway to characterize the importance of these pathways in regulating IL-8-induced AR transcriptional activity in LNCaP cells and determining whether the activation of AR transcription is mediated through phosphorylation of the AR or many of its known coactivators.

The specificity of the observed responses to IL-8 was proven using a CXCR2-selective antagonist. Blockade of CXCR2 signaling was shown to (i) abrogate the IL-8-promoted proliferation of the LNCaP and 22Rv1 cells; (ii) attenuate the IL-8-promoted increases in AR expression and (iii) reverse the IL-8-promoted increases in AR transcriptional activity to basal levels in both luciferase and target gene mRNA analysis. The inability of the CXCR2 antagonist to effect a complete abolition of IL-8-induced AR expression suggests that IL-8 signaling through the CXCR1 receptor may also contribute to the IL-8-promoted phenotype. The absence of a selective CXCR1 receptor antagonist prevents a current pharmacological-based investigation of this receptor's role in regulating AR expression.

Finally, we have shown that the inhibition of CXCR2 signaling also enhances the effect of bicalutamide in reducing LNCaP and 22Rv1 cell viability. This observation is consistent with recent studies reporting that bicalutamide-mediated inhibition of DNA synthesis in LAPC-4 and LNCaP cells is decreased in IL-8 overexpressing clones of these cell lines (27). The authors of this study concluded that this may result from a decreased dependence of IL-8 overexpressing clones upon the AR for proliferation since they observed that IL-8 signaling reduced AR expression. Our data in contrast show that IL-8 increases the expression and activity of the AR in these cells and enable us to propose that the reinforcement of ligand-independent AR activity by IL-8 signaling may underpin a functional antagonism of AR-targeted therapeutic strategies. Hence, the blockade of IL-8 signaling within tumors of the prostate gland may have tangible benefits in preventing the progression of CaP and in addition, enhancing the sensitivity of this disease to currently exploited antiandrogen therapies.

Our current studies add to our increasing understanding of the importance of IL-8 signaling in contributing to CaP progression. We have now shown that IL-8 signaling promotes the proliferation of both androgen dependent and AIPC cells (16). Our past demonstration that IL-8 signaling can stimulate the proliferation of AR-deficient PC3 cells suggests that this chemokine can exploit additional mechanisms besides that of the AR to underpin progressive tumor growth, including the activation of multiple proliferation-associated signal transduction pathways (16,33). Irrespective, the capacity for IL-8 signaling to increase the expression and promote a ligand-independent activation of the AR suggests that elevated signaling of this CXC chemokine is a significant contributor to early progression and subsequent transition of the disease to an androgen-independent, castrate-resistant stage of disease.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Ulster Cancer Foundation to D.J.J.W.; Action Cancer to A.S.; Association for International Cancer Research (AICR-04-516).


   Acknowledgments
 
The authors acknowledge Prof. Nancy Weigel of Baylor University for provision of the androgen response element-luciferase construct, Margaret McDonnell of the Clinical Biochemistry Laboratory, Belfast City Hospital for carrying out the analysis of PSA protein secretion and AstraZeneca for provision of bicalutamide and AZ10397767.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Debes JD, et al. Mechanisms of androgen refractory prostate cancer. N. Engl. J. Med. (2004) 351:1488–1490.[Free Full Text]
  2. Pienta KJ, et al. Mechanisms underlying the development of androgen-independent prostate cancer. Clin. Cancer Res. (2006) 12:1665–1671.[Free Full Text]
  3. Culig Z, et al. Expression, structure and function of androgen receptor in advanced prostatic carcinoma. Prostate (1998) 35:63–70.[CrossRef][Web of Science][Medline]
  4. Cronauer MV, et al. The androgen receptor in hormone-refractory prostate cancer: relevance of different mechanisms of androgen receptor signalling. Int. J. Oncol. (2003) 23:1095–1102.[Web of Science][Medline]
  5. Taplin ME, et al. Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J. Cell. Biochem. (2004) 91:483–490.[CrossRef][Web of Science][Medline]
  6. Chen CD, et al. Molecular determinants of resistance to anti-androgen therapy. Nat. Med. (2004) 10:33–39.[CrossRef][Web of Science][Medline]
  7. Jia L, et al. Locus-wide chromatin remodeling and enhanced androgen receptor-mediated transcription in recurrent prostate tumor cells. Mol. Cell. Biol. (2006) 26:7331–7341.[Abstract/Free Full Text]
  8. Scher HI, et al. Targeting the androgen receptor: improving outcomes for castration-resistant prostate cancer. Endocr. Relat. Cancer (2004) 11:459–476.[Abstract/Free Full Text]
  9. Culig Z, et al. Androgen receptor activation in prostatic tumour cell lines by insulin-like growth factor-I, keratinocyte growth factor and epidermal growth factor. Cancer Res. (1994) 54:5474–5478.[Abstract/Free Full Text]
  10. Lee LF, et al. Neuropeptide-induced androgen independence in prostate cancer cells: roles of non-receptor tyrosine kinases Etk/Bmx, Src and focal adhesion kinase. Mol. Cell. Biol. (2001) 21:8385–8397.[Abstract/Free Full Text]
  11. Hobisch A, et al. Interleukin-6 regulates prostate-specific protein expression in prostate carcinoma cells by activation of the androgen receptor. Cancer Res. (1998) 58:4640–4645.[Abstract/Free Full Text]
  12. Inoue K, et al. Interleukin-8 expression regulates tumourigenicity and metastases in androgen-independent prostate cancer. Clin. Cancer Res. (2000) 6:2104–2119.[Abstract/Free Full Text]
  13. Kim SJ, et al. Expression of interleukin-8 correlates with angiogenesis, tumourigenicity and metastasis of human prostate cancer cells implanted orthotopically in nude mice. Neoplasia (2001) 3:33–42.[CrossRef][Web of Science][Medline]
  14. Veltri RW, et al. Interleukin-8 serum levels in patients with benign prostatic hyperplasia and prostate cancer. Cancer (1999) 91:2322–2328.
  15. McCarron SL, et al. Influence of cytokine gene polymorphisms on the development of prostate cancer. Cancer Res. (2002) 62:3369–3372.[Abstract/Free Full Text]
  16. Murphy C, et al. Non-apical and cytoplasmic expression of interleukin-8, CXCR1 and CXCR2 correlates with cell proliferation and microvessel density in prostate cancer. Clin. Cancer Res. (2005) 11:4117–4127.[Abstract/Free Full Text]
  17. Uehara H, et al. Expression of interleukin-8 gene in radical prostatectomy specimens is associated with advanced pathologic stage. Prostate (2005) 64:40–49.[CrossRef][Web of Science][Medline]
  18. Lee LF, et al. Interleukin-8 confers androgen-independent growth and migration of LNCaP: differential effects of tyrosine kinases Src and FAK. Oncogene (2004) 23:2197–2205.[CrossRef][Web of Science][Medline]
  19. Lee SO, et al. Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway. Oncogene (2003) 22:7981–7988.[CrossRef][Web of Science][Medline]
  20. McDonald S, et al. Ligand responsiveness in human prostate cancer: structural analysis of mutant androgen receptors from LNCaP and CWR22 tumours. Cancer Res. (2000) 60:2317–2322.[Abstract/Free Full Text]
  21. Sramkoski RM, et al. A new human prostate carcinoma cell line, 22RV1. In Vitro Cell. Dev. Biol. Anim. (1999) 35:403–409.[Web of Science][Medline]
  22. Stamey TA, et al. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N. Engl. J. Med. (1987) 317:909–916.[Abstract]
  23. Lu J, et al. Regulation of androgen-dependent prostatic cancer cell growth: androgen regulation of CDK2, CDK4 and CKI p16 genes. Cancer Res. (1997) 57:4511–4516.[Abstract/Free Full Text]
  24. Gregory CW, et al. Androgen receptor regulation of G1 cyclin and cyclin-dependent kinase function I the CWR22 human prostate cancer xenograft. J. Androl. (2001) 22:537–548.[Abstract]
  25. Hobisch A, et al. Distant metastases from prostatic carcinoma express androgen receptor protein. Cancer Res. (1995) 55:3068–3072.[Abstract/Free Full Text]
  26. van der Kwast TH, et al. Androgen receptors in endocrine-therapy-resistant human prostate cancer. Int. J. Cancer (1991) 48:189–193.[Web of Science][Medline]
  27. Agoulnik IU, et al. Androgen receptor action in hormone-dependent and recurrent prostate cancer. J. Cell. Biochem. (2006) 99:362–372.[CrossRef][Web of Science][Medline]
  28. Araki S, et al. Interleukin-8 is a molecular determinant of androgen-independence and progression in prostate cancer. Cancer Res. (2007) 67:6854–6862.[Abstract/Free Full Text]
  29. Zhang L, et al. Nuclear factor-{kappa}B activates transcription of the androgen receptor gene in sertoli cells isolated from testes of adult rats. Endocrinology (2004) 145:781–789.[Abstract/Free Full Text]
  30. Lee SO, et al. Requirement of NF-{kappa}B in interleukin-4-induced androgen receptor activation in prostate cancer cells. Prostate (2005) 64:160–167.[CrossRef][Web of Science][Medline]
  31. Bubulya A, et al. c-Jun can mediate androgen receptor-induced transactivation. J. Biol. Chem. (1996) 271:24583–24589.[Abstract/Free Full Text]
  32. Chen SY, et al. c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation. Oncogene (2006) 25:7212–7223.[CrossRef][Web of Science][Medline]
  33. MacManus CF, et al. Interleukin-8 signaling promotes translational regulation of cyclin D in androgen-independent prostate cancer cells. Mol. Cancer Res. (2007) 5:737–748.[Abstract/Free Full Text]
  34. Fang Y, et al. Hsp90 regulates androgen receptor hormone binding affinity in vivo. J. Biol. Chem. (1996) 271:28697–28702.[Abstract/Free Full Text]
  35. Georget V, et al. Mechanism of anti-androgen action: key role of hsp90 in conformational change and transcriptional activity of the androgen receptor. Biochemistry (2002) 41:11824–11831.[CrossRef][Web of Science][Medline]
  36. Gioeli D, et al. Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J. Biol. Chem. (2002) 277:29304–29314.[Abstract/Free Full Text]
  37. Gioeli D. Signal transduction in prostate cancer progression. Clin. Sci. (2005) 108:293–308.[CrossRef][Web of Science][Medline]
  38. Weber MJ, et al. Ras signalling in prostate cancer progression. J. Cell. Biochem. (2004) 91:13–25.[CrossRef][Web of Science][Medline]
  39. Bakin RE, et al. Constitutive activation of the Ras/mitogen-activated protein kinase signalling pathway promotes androgen hypersensitivity in LNCaP prostate cancer cells. Cancer Res. (2003) 63:1981–1989.[Abstract/Free Full Text]
  40. Lu S, et al. PI3K-Akt signalling is involved in the regulation of p21WAF/CIP expression and androgen-independent growth in prostate cancer cells. Int. J. Oncol. (2006) 28:245–251.[Web of Science][Medline]
  41. Ueda T, et al. Ligand-independent activation of the androgen receptor by interleukin-6 and the role of steroid receptor coactivator-1 in prostate cancer cells. J. Biol. Chem. (2002) 277:38087–38094.[Abstract/Free Full Text]
  42. Gregory CW, et al. Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer. Cancer Res. (2004) 61:2892–2898.
  43. Burger M, et al. KSHV-GPCR and CXCR2-transforming capacity and angiogenic responses are mediated through a JAK2-STAT3 dependent pathway. Oncogene (2005) 24:2067–2075.[CrossRef][Web of Science][Medline]
  44. Maxwell PJ, et al. HIF-1 and NK-{kappa}B-mediated upregulation of CXCR1 and CXCR2 expression promotes cell survival in hypoxic prostate cancer cells. Oncogene (2007) 26:7333–7345.[CrossRef][Web of Science][Medline]
Received December 20, 2007; revised April 11, 2008; accepted April 28, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Endocr Relat CancerHome page
D. Vindrieux, P. Escobar, and G. Lazennec
Emerging roles of chemokines in prostate cancer
Endocr. Relat. Cancer, September 1, 2009; 16(3): 663 - 673.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
H. Neuwirt, M. Puhr, F. R. Santer, M. Susani, W. Doppler, G. Marcias, V. Rauch, M. Brugger, A. Hobisch, L. Kenner, et al.
Suppressor of Cytokine Signaling (SOCS)-1 Is Expressed in Human Prostate Cancer and Exerts Growth-Inhibitory Function through Down-Regulation of Cyclins and Cyclin-Dependent Kinases
Am. J. Pathol., May 1, 2009; 174(5): 1921 - 1930.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
K. Malinowska, H. Neuwirt, I. T Cavarretta, J. Bektic, H. Steiner, H. Dietrich, P. L Moser, D. Fuchs, A. Hobisch, and Z. Culig
Interleukin-6 stimulation of growth of prostate cancer in vitro and in vivo through activation of the androgen receptor
Endocr. Relat. Cancer, March 1, 2009; 16(1): 155 - 169.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
D. J.J. Waugh and C. Wilson
The Interleukin-8 Pathway in Cancer
Clin. Cancer Res., November 1, 2008; 14(21): 6735 - 6741.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
C. Wilson, T. Wilson, P. G. Johnston, D. B. Longley, and D. J.J. Waugh
Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells
Mol. Cancer Ther., September 1, 2008; 7(9): 2649 - 2661.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/6/1148    most recent
bgn109v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Seaton, A.
Right arrow Articles by Waugh, D. J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seaton, A.
Right arrow Articles by Waugh, D. J. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?