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Accelerated in Vivo Growth of Prostate Tumors that Up-Regulate Interleukin-6 Is Associated with Reduced Retinoblastoma Protein Expression and Activation of the Mitogen-Activated Protein Kinase Pathway

2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63859-x

1525-2191

Hannes Steiner, Sonia Godoy‐Tundidor, Hermann Rogatsch, Andreas P. Berger, Dietmar Fuchs, Barbara Comuzzi, Georg Bartsch, Alfred Hobisch, Zoran Čulig,

Retinoids in leukemia and cellular processes

Interleukin-6 (IL-6) is a multifunctional cytokine that activates the signaling pathways of Janus kinases-signal transducers and activators of transcription (STAT) and/or mitogen-activated protein kinases (MAPK) in various tumors. Thus, it modulates cell growth and apoptosis. IL-6 levels are elevated in tissues and sera from prostate cancer patients and IL-6 receptor expression has been detected in prostate cancer cell lines and clinical specimens. Continuous exposure of prostate cancer cells to IL-6 might alter their responsiveness to this cytokine. To gain more insight into the function of IL-6 in prostate carcinoma, we have inoculated LNCaP-IL-6+ cells, generated after prolonged treatment with IL-6, into nude mice (total n = 16, two independent experiments). Controls included animals bearing LNCaP-IL-6− cells, passaged at the same time as LNCaP-IL-6+ cells without supplementation of IL-6. LNCaP-IL-6+ tumor volumes were larger than those of their counterparts at all time points. There were no signs of cachexia in any of the experimental animals and all mice were free of metastases. To better understand the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we have investigated the expression of cell-cycle regulatory molecules by Western blot analysis. The levels of cyclin-dependent kinase 2 were elevated in LNCaP-IL-6+ cells. There was a strong down-regulation of cyclins D1 and E in the LNCaP-IL-6+ subline. The cell-cycle inhibitor p27 was expressed at a low level in LNCaP-IL-6+ cells and could not be up-regulated by addition of IL-6. Most notably, LNCaP-IL-6+ cells exhibited a reduced expression of the hypophosphorylated form of the retinoblastoma protein (pRb). Accelerated tumor growth in our model system was also associated with alterations in IL-6-signaling pathways. The ability of IL-6 to induce tyrosine phosphorylation of STAT3 was abolished in the LNCaP-IL-6+ subline. In contrast, the levels of the MAPK extracellular signal-regulated kinases 1/2 increased in cells generated after long-term IL-6 treatment. The inhibitor of MAPK kinase PD 98059 retarded the proliferation of LNCaP-IL-6+ but not that of control cells. In summary, we show in the present study that chronic exposure of prostate cancer cells to IL-6 facilitates tumor growth in vivo by abolishment of the growth control by pRb and activation of the MAPK signaling pathway. These findings could be relevant to understand the role of IL-6 in prostate cancer progression. Interleukin-6 (IL-6) is a multifunctional cytokine that activates the signaling pathways of Janus kinases-signal transducers and activators of transcription (STAT) and/or mitogen-activated protein kinases (MAPK) in various tumors. Thus, it modulates cell growth and apoptosis. IL-6 levels are elevated in tissues and sera from prostate cancer patients and IL-6 receptor expression has been detected in prostate cancer cell lines and clinical specimens. Continuous exposure of prostate cancer cells to IL-6 might alter their responsiveness to this cytokine. To gain more insight into the function of IL-6 in prostate carcinoma, we have inoculated LNCaP-IL-6+ cells, generated after prolonged treatment with IL-6, into nude mice (total n = 16, two independent experiments). Controls included animals bearing LNCaP-IL-6− cells, passaged at the same time as LNCaP-IL-6+ cells without supplementation of IL-6. LNCaP-IL-6+ tumor volumes were larger than those of their counterparts at all time points. There were no signs of cachexia in any of the experimental animals and all mice were free of metastases. To better understand the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we have investigated the expression of cell-cycle regulatory molecules by Western blot analysis. The levels of cyclin-dependent kinase 2 were elevated in LNCaP-IL-6+ cells. There was a strong down-regulation of cyclins D1 and E in the LNCaP-IL-6+ subline. The cell-cycle inhibitor p27 was expressed at a low level in LNCaP-IL-6+ cells and could not be up-regulated by addition of IL-6. Most notably, LNCaP-IL-6+ cells exhibited a reduced expression of the hypophosphorylated form of the retinoblastoma protein (pRb). Accelerated tumor growth in our model system was also associated with alterations in IL-6-signaling pathways. The ability of IL-6 to induce tyrosine phosphorylation of STAT3 was abolished in the LNCaP-IL-6+ subline. In contrast, the levels of the MAPK extracellular signal-regulated kinases 1/2 increased in cells generated after long-term IL-6 treatment. The inhibitor of MAPK kinase PD 98059 retarded the proliferation of LNCaP-IL-6+ but not that of control cells. In summary, we show in the present study that chronic exposure of prostate cancer cells to IL-6 facilitates tumor growth in vivo by abolishment of the growth control by pRb and activation of the MAPK signaling pathway. These findings could be relevant to understand the role of IL-6 in prostate cancer progression. Apart from regulation of immune responses, interleukin-6 (IL-6) influences growth of normal and tumor cells. The IL-6 receptor is composed of the ligand-binding subunit gp 80 and the signal-transducing subunit gp 130, which is shared by related cytokines. After binding of IL-6 to its receptor, Janus kinases (JAK) and/or ras-mediated signaling become activated. JAK phosphorylates signal transducers and activators of transcription (STAT) factors, which translocate to the nucleus and activate the transcription of genes containing the STAT response element. Among STAT factors, STAT3 is implicated in IL-6 action in most tissues. The ras-mediated cellular events ultimately lead to activation of the mitogen-activated protein kinase (MAPK)-signaling pathway. Because of different interactions with elements of these pathways in target tissues, IL-6 causes a variety of biological responses ranging from induction of growth arrest to suppression of apoptosis. A consistent growth-promoting effect of IL-6 has been demonstrated in renal cell carcinoma and myeloma.1Angelo LS Talpaz M Kurzrock R Autocrine interleukin-6 production in renal cell carcinoma: evidence for the involvement of p53.Cancer Res. 2002; 62: 932-940PubMed Google Scholar, 2Frassanito MA Cusmai A Iodice G Dammacco F Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis.Blood. 2001; 97: 483-489Crossref PubMed Scopus (137) Google Scholar In several other malignancies, both positive and negative regulation of proliferation of cancer cells were reported. Most interestingly, melanoma cell lines derived from early-stage lesions are inhibited by IL-6, whereas these cells lose sensitivity to IL-6 during tumor progression.3Lu C Vickers MF Kerbel RS Interleukin-6: a fibroblast-derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression.Proc Natl Acad Sci USA. 1992; 89: 9215-9219Crossref PubMed Scopus (168) Google Scholar The initial observation that IL-6 is elevated in sera from patients with advanced carcinoma of the prostate has stimulated research on its expression and function in this most frequently diagnosed tumor in industrialized countries.4Twillie DA Eisenberger MA Carducci MA Hseih W-S Kim WY Simons JW Interleukin-6: a candidate mediator of human prostate cancer morbidity.Urology. 1995; 45: 542-549Abstract Full Text PDF PubMed Scopus (268) Google Scholar More recently, it was reported that the levels of IL-6 and its receptor increase in tissue extracts obtained from patients with clinically localized prostate cancer.5Giri D Ozen M Ittmann M Interleukin-6 is an autocrine growth factor in human prostate cancer.Am J Pathol. 2001; 159: 159-165Google Scholar IL-6 elicits variable responses in established prostate cancer cell lines; in PC-3 cells derived from a bone metastasis, IL-6 acts as an autocrine growth factor.6Chung TD Yu JJ Kong TA Spiotto MT Lin JM Interleukin-6 activates phosphatidylinositol-3 kinase, which inhibits apoptosis in human prostate cancer cell lines.Prostate. 2000; 42: 1-7Crossref PubMed Scopus (94) Google Scholar For LNCaP prostate cancer cells, which respond to androgen and express the androgen receptor, contrasting effects of IL-6 on growth have been reported. Several researchers demonstrated that treatment of LNCaP with IL-6 retards the proliferation and increases the percentage of cells in the G1 phase of the cell cycle.7Degeorges A Tatoud R Fauvel Lafeve F Podgorniak MP Millot G de Cremoux P Calvo F Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells, identified as interleukin-6.Int J Cancer. 1996; 68: 207-214Crossref PubMed Scopus (89) Google Scholar, 8Ritchie CK Andrews LR Thomas KG Tindall DJ Fitzpatrick LA The effects of growth factors associated with osteoblasts on prostate carcinoma proliferation and chemotaxis: implications for the development of metastatic disease.Endocrinology. 1997; 138: 1145-1150Crossref PubMed Scopus (78) Google Scholar, 9Levesque 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: 2375-2381Crossref PubMed Scopus (46) Google Scholar, 10Spiotto MT Chung TD STAT3 mediates IL-6-induced growth inhibition in the human prostate cancer cell line LNCaP.Prostate. 2000; 42: 88-98Crossref PubMed Scopus (118) Google Scholar, 11Mori S Murakami-Mori K Bonavida B Interleukin-6 induces G1 arrest through induction of p27 (Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells.Biochem Biophys Res Commun. 1999; 257: 609-614Crossref PubMed Scopus (101) Google Scholar, 12Deeble PD Murphy DJ Parsons SJ Cox ME Interleukin-6 and cyclic-AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells.Mol Cell Biol. 2001; 21: 8471-8482Crossref PubMed Scopus (156) Google Scholar In contrast, other groups showed that the growth of LNCaP cells is stimulated after addition of IL-6.5Giri D Ozen M Ittmann M Interleukin-6 is an autocrine growth factor in human prostate cancer.Am J Pathol. 2001; 159: 159-165Google Scholar, 13Qiu Y Ravi L Kung H-J Requirement of ErbB2 for signalling by interleukin-6 in prostate carcinoma cells.Nature. 1998; 393: 83-85Crossref PubMed Scopus (266) Google Scholar, 14Okamoto M Lee C Oyasu R Interleukin-6 as a paracrine and autocrine growth factor in human prostatic carcinoma cells in vitro.Cancer Res. 1997; 57: 141-146PubMed Google Scholar, 15Lou W Ni Z Dyer K Tweardy DJ Gao AC Interleukin-6 induces prostate cancer cell growth accompanied by activation of stat3 signaling pathway.Prostate. 2000; 42: 239-242Crossref PubMed Scopus (243) Google Scholar In the authors' laboratory, LNCaP cells have constantly been inhibited by IL-6.16Hobisch A Eder IE Putz T Horninger W Bartsch G Klocker H Culig Z Interleukin-6 regulates prostate-specific protein expression in prostate carcinoma cells by activation of the androgen receptor.Cancer Res. 1998; 58: 4640-4645PubMed Google Scholar LNCaP cells up-regulate the expression of prostate-specific antigen after treatment with IL-6 and this effect is antagonized by the nonsteroidal anti-androgen bicalutamide.16Hobisch A Eder IE Putz T Horninger W Bartsch G Klocker H Culig Z Interleukin-6 regulates prostate-specific protein expression in prostate carcinoma cells by activation of the androgen receptor.Cancer Res. 1998; 58: 4640-4645PubMed Google Scholar, 17Lin DL Whitney MC Yao Z Keller ET Interleukin-6 induces androgen responsiveness in prostate cancer cells through up-regulation of androgen receptor expression.Clin Cancer Res. 2001; 7: 1773-1781PubMed Google Scholar Thus, ligand-independent activation of the androgen receptor is associated with cellular differentiation in LNCaP cells. We hypothesized that prolonged stimulation of prostate cancer cells by IL-6 may modify an initial response to this cytokine. For this reason, we have generated a new subline, LNCaP-IL-6+, which does not show a growth-inhibitory response to IL-6 and, in contrast to parental cells, expresses endogenous IL-6.18Hobisch A Ramoner R Fuchs D Godoy-Tundidor S Bartsch G Klocker H Culig Z Prostate cancer cells (LNCaP) generated after long-term interleukin-6 treatment express interleukin-6 and acquire an interleukin-6-partially resistant phenotype.Clin Cancer Res. 2001; 7: 2941-2948PubMed Google Scholar However, in LNCaP-IL-6+ cells, IL-6 was capable of stimulating androgen receptor-mediated reporter gene activity.18Hobisch A Ramoner R Fuchs D Godoy-Tundidor S Bartsch G Klocker H Culig Z Prostate cancer cells (LNCaP) generated after long-term interleukin-6 treatment express interleukin-6 and acquire an interleukin-6-partially resistant phenotype.Clin Cancer Res. 2001; 7: 2941-2948PubMed Google Scholar For the development of novel therapeutical approaches based on interference with IL-6 action, it is necessary to obtain more information about IL-6 regulation of prostate cancer in vivo. In the present study, we have investigated the growth of LNCaP-IL-6+ tumors in nude mice and analyzed alterations in the expression of key cell-cycle regulators after continuous treatment with IL-6. LNCaP-IL-6+ cells were maintained in culture as previously described.18Hobisch A Ramoner R Fuchs D Godoy-Tundidor S Bartsch G Klocker H Culig Z Prostate cancer cells (LNCaP) generated after long-term interleukin-6 treatment express interleukin-6 and acquire an interleukin-6-partially resistant phenotype.Clin Cancer Res. 2001; 7: 2941-2948PubMed Google Scholar High subline passages were used for in vivo experiments. LNCaP-IL-6− cells were passaged at the same time as LNCaP-IL-6+ cells in the absence of IL-6.18Hobisch A Ramoner R Fuchs D Godoy-Tundidor S Bartsch G Klocker H Culig Z Prostate cancer cells (LNCaP) generated after long-term interleukin-6 treatment express interleukin-6 and acquire an interleukin-6-partially resistant phenotype.Clin Cancer Res. 2001; 7: 2941-2948PubMed Google Scholar In terms of IL-6 responsiveness, they do not differ from parental LNCaP cells. Male nude mice (nu/nu) were purchased from Charles River Laboratories (Sulzfeld, Germany). To assess the growth of LNCaP-IL-6+ cells in vivo, two independent experiments were performed. Before transplantation, mice were supplemented with slow-release testosterone pellets (12.5 mg, 60-day release; Innovative Research of America, Sarasota, FL). In each experiment, eight animals were inoculated subcutaneously with LNCaP-IL-6+ and eight animals with LNCaP-IL-6− cells. The experimental protocol was approved by the Committee of the Austrian Federal Ministry of Education, Science, and Culture. Cells (1 × 106) were inoculated with Matrigel (Pharmingen Becton Dickinson, San Diego, CA). Animal weight and tumor size were monitored weekly. Tumor size was measured using a caliper and tumor volume was calculated according to the formula: length × width2 of a tumor area/2. After 6 weeks, nude animals were sacrificed by beheading and the tumors and lymph nodes were removed for pathohistological examination. The animals were X-rayed to detect metastatic spread. Blood samples were collected, frozen, and stored for IL-6 determination. For Western blot analyses, the following antibodies were purchased: monoclonal anti-cyclin-dependent kinase (cdk) 2 and cdk 4 and anti-phospho-Tyr1022/1023 JAK (pJAK) antibodies from Biosource International, Camarillo, CA. The monoclonal antibodies anti-retinoblastoma (pRb), which recognizes both hypo- and hyperphosphorylated forms of the protein, and anti-cyclin D1 were obtained from Lab Vision Neomarkers (Freemont, CA). Monoclonal anti-cyclin E, anti-p27, and anti-STAT3 antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). The phospho-STAT3 (Y705) (pSTAT3) antibody was a product of Upstate Biotechnology (Lake Placid, NY) and the monoclonal antibody that reacts with both phosphorylated and nonphosphorylated forms of the extracellular signal-regulated kinases (ERK) 1/2 was from Zymed Laboratories (San Francisco, CA). The MAPK kinase inhibitor PD 98059 was purchased from VWR International (Darmstadt, Germany). Human recombinant IL-6 and IL-6 enzyme-linked immunosorbent assay were from R&D Systems (Minneapolis, MN). The secondary anti-mouse horseradish peroxidase-linked antibody was from Amersham Pharmacia Biotech (Freiburg, Germany) and the anti-rabbit secondary antibody from Santa Cruz Biotechnologies. LNCaP-IL-6+ and LNCaP-IL-6− cells were maintained in culture in the presence or absence of IL-6, respectively. For detection of pJAK and pSTAT3, the cells were stimulated for 30 minutes before harvesting. Western blots for cdk, cyclins, p27, and pRb were performed after 48 hours of treatment. ERK 1/2 Western blots were performed after both 30 minutes and 48 hours of treatment. The experiments were performed in whole cell extracts with the exemption of p27 and STAT3 that were detected in nuclear preparations. Nuclear extracts were prepared from 2 × 106 cells using nuclear and cytoplasmatic extraction reagents (Pierce, Rockford, IL). After the treatment, protein content in whole cell extracts was measured according to Bradford.19Bradford M A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding.Anal Biochem. 1978; 72: 248-254Crossref Scopus (214455) Google Scholar For this purpose, samples were lysed in a buffer containing 20 mmol/L NaH2PO4, pH 7.5, 1 mmol/L ethylenediaminetetraacetic acid, and 10% glycerol. For the electrophoresis, aliquots of the samples were then diluted in NuPAGE LDS sample buffer (Invitrogen, Leek, The Netherlands) according to the manufacturer's instructions. Afterward, they were sonicated and boiled for 10 minutes at 70°C. The lysates were loaded onto 3 to 8% Tris-acetate gels and run for 1 hour at 150 V with 1× NuPAGE sodium dodecyl sulfate (for cdk and pRb Western blots) or 4 to 12% Bis-Tris gels and run with a buffer containing 3-(N-morpholino)-propanesulphonic acid buffer (Invitrogen) for 50 minutes to 1 hour for the other experiments. The proteins were then transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA) with help of the Xcell blot module for 1 hour at 30 V using NuPAGE transfer buffer. After the transfer procedure, the membranes were washed in Tris-buffered saline (TBS) once for 5 minutes and in TBS with 0.05% Tween-20 (TBST) three times for 5 minutes. Then the membranes were blocked with 5% skim milk in TBST (TBSTM) for 1 hour and incubated with the respective primary antibody overnight at 4°C. After two washes in TBS and five in TBST, the membranes were incubated with the respective secondary antibody. The antibodies were diluted as follows: anti-cdk 2 1:100, anti-cdk 4 1:50, both followed by a secondary anti-mouse antibody diluted 1:2000; the anti-cyclin D1 antibody was diluted 1:100 followed by the secondary anti-mouse antibody diluted 1:1000; the dilution of anti-cyclin E antibody was 1:100 and that of the secondary anti-mouse antibody 1:2000; the anti-p27 antibody was diluted 1:100 and the secondary anti-mouse antibody 1:1000; anti-pRb was diluted 1:100 and the secondary antibody 1:1000; anti pJAK was used diluted 1:1000 and the secondary anti-rabbit antibody 1:5000; anti pSTAT3 was applied diluted 1:500 and the secondary anti-rabbit antibody 1:8000; the dilution of anti-STAT3 was 1:2000 and that of the secondary anti-mouse antibody 1.8:4000; the anti-ERK 1/2 antibody was diluted 1:500 followed by the anti-mouse antibody diluted 1.8:4000. The membranes were washed as described above, with an additional final wash step with TBS. Western blots were developed by means of the ECL Plus+ substrate (Amersham). As a control for equal protein loading, Western blots for β-actin were performed. LNCaP-IL-6+ and LNCaP-IL-6− cells were grown on six-well plates (80,000 cells/well) in the absence or presence of the MAPK kinase inhibitor PD 98059 for 48 hours and counted afterward. The Mann-Whitney t-test was used for assessment of statistical significance. The growth of LNCaP-IL-6+ and LNCaP-IL-6− cells in host animals was evaluated in two independent experiments. Each group consisted of eight animals at the beginning of the experiment. In both experiments, all mice bearing LNCaP-IL-6+ tumors survived, whereas two animals inoculated with control cells died during the performance of the first experiment and one during the second experiment. There was no evidence of tumor in those dead mice. Tumor incidence was 100% after 3 weeks in the group bearing LNCaP-IL-6+ cells. In the control group, tumors were formed in five of six animals in the first and in six of seven mice in the second experiment (83.3% and 85.7%, respectively). However, in the control group two tumors appeared after 3 weeks, two after 4 weeks, and one after 6 weeks in the first experiment. In the second experiment, all control animals developed tumor lesions after 3 weeks. Most notably, differences in tumor volume were appreciable between the IL-6-producing tumors and the controls. The tumor volumes in the LNCaP-IL-6+ group were significantly larger at all time points (Figure 1). At the time when the experiments were terminated, the mean tumor volume in the LNCaP-IL-6+ group was 0.93 cm3 in the first and 0.6 cm3 in the second experiment. In the control group, the volumes were 0.16 and 0.09 cm3, respectively. In one animal bearing LNCaP-IL-6+ tumor, skin ulceration was noted. The tumors were examined pathohistologically. In both groups, there were evident areas of necrosis. Lymph nodes removed from the vicinity of the tumors were tumor-free and X-ray imaging did not provide any evidence of the presence of bone metastases (data not shown). There was no statistically significant difference in weight between the animals inoculated with LNCaP-IL-6+ versus those inoculated with LNCaP-IL-6− cells. At the end of the first experiment, the mean weight of the mice bearing LNCaP-IL-6+ tumors was 31.5 g and that of their counterparts 31.6 g. The respective weights at the end of the second experiment were 26.6 g and 29.7 g. The ability of LNCaP-IL-6+ cells to secrete IL-6 in vivo was confirmed; serum IL-6 levels of 45.6 ± 5.5 pg/ml were measured in the first and 148.7 ± 35.7 pg/ml in the second experiment. IL-6 levels measured in sera from nude mice inoculated with LNCaP-IL-6− cells were below the detection limit. To gain more insight into the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we first investigated the expression of cell-cycle regulatory molecules. Cdk 4-cyclin D complexes and cdk 2-cyclin E complexes govern the transition through the G1 phase of the cell cycle. In parental LNCaP cells, IL-6 down-regulates the expression and activity of cdk 2 and cdk 4 and up-regulates p27 expression.11Mori S Murakami-Mori K Bonavida B Interleukin-6 induces G1 arrest through induction of p27 (Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells.Biochem Biophys Res Commun. 1999; 257: 609-614Crossref PubMed Scopus (101) Google Scholar In control cells, IL-6 treatment diminished the levels of both cdk (Figure 2), consistent with the findings reported by Mori and associates.11Mori S Murakami-Mori K Bonavida B Interleukin-6 induces G1 arrest through induction of p27 (Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells.Biochem Biophys Res Commun. 1999; 257: 609-614Crossref PubMed Scopus (101) Google Scholar As shown in Figure 2, there were differences in the regulation of both kinases in LNCaP-IL-6+ cells. The expression of cdk 2 but not that of cdk 4 increased in untreated LNCaP-IL-6+ compared to LNCaP-IL-6− cells. In the LNCaP-IL-6+ subline, addition of IL-6 did not cause any effect on cdk expression. In parental LNCaP cells, there was a slightly reduced expression of cyclins D1 and E after IL-6 treatment.11Mori S Murakami-Mori K Bonavida B Interleukin-6 induces G1 arrest through induction of p27 (Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells.Biochem Biophys Res Commun. 1999; 257: 609-614Crossref PubMed Scopus (101) Google Scholar In LNCaP-IL-6− control cells, we have observed a minor inhibitory effect of IL-6 on cyclin D1 levels. Interestingly, continuous IL-6 treatment yielded a considerably reduced expression of both cyclins D1 and E (Figure 3). The cell-cycle inhibitor p27 was slightly induced by IL-6 in controls (Figure 4). In contrast, its expression was negligible in the LNCaP-IL-6+ subline and it could not be increased by addition of IL-6 to culture media.Figure 3Western blot of cyclin D1 and cyclin E in controls and IL-6-resistant cells. Cyclins D1 and E were probed with monoclonal antibodies in extracts from LNCaP-IL-6− and LNCaP-IL-6+ cells that were cultured in the absence (lane 1) or presence of IL-6 (A: lane 2, 10 ng/ml; B: lane 2, 1 ng/ml; lane 3, 15 ng/ml).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Western blot analysis of the cell-cycle inhibitor p27. Nuclear extracts were prepared from both LNCaP sublines cultured without (lane 1) or with IL-6 (lane 2, 10 ng/ml) and analyzed with a p27 monoclonal antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Rb gene product prevents unrestricted proliferation by binding to the transcription factor E2F. In untreated LNCaP-IL-6− cells, pRb was detected in its hypophosphorylated form, in concordance with the results published by Hofman and colleagues.20Hofman K Swinnen JV Verhoeven G Heyns W E2F activity is biphasically regulated by androgens in LNCaP cells.Biochem Biophys Res Commun. 2001; 283: 97-101Crossref PubMed Scopus (44) Google Scholar In our study, there were striking differences in the expression of the Rb protein; its levels were strongly down-regulated in LNCaP-IL-6+ versus control cells (Figure 5). The two major signaling pathways of IL-6 are the JAK-STAT and the MAPK pathway. Spiotto and Chung,10Spiotto MT Chung TD STAT3 mediates IL-6-induced growth inhibition in the human prostate cancer cell line LNCaP.Prostate. 2000; 42: 88-98Crossref PubMed Scopus (118) Google Scholar, 21Spiotto MT Chung TD STAT3 mediates IL-6-induced neuroendocrine differentiation in prostate cancer cells.Prostate. 2000; 42: 186-195Crossref PubMed Scopus (158) Google Scholar who investigated the regulation of cellular events in LNCaP cells by IL-6, found that the phosphorylation of STAT3 is associated with growth inhibition and neuroendocrine differentiation. We evaluated the effect of IL-6 on the levels of phosphorylated and total STAT3 in LNCaP-IL-6+ cells and in controls. In control cells, there was a strong induction of pSTAT3 detected after treatment with IL-6 (Figure 6). In sharp contrast, there was no STAT3 phosphorylation observed in LNCaP-IL-6+ cells. The expression of total STAT3 was slightly lower in the LNCaP-IL-6+ than in the LNCaP-IL-6− subline. Consistent with these findings, there was no increase in the phosphorylation of the upstream kinase JAK1 at Tyr1022/1023 in LNCaP-IL-6+ cells (data not shown). The expression of the MAPK ERK 1/2 in LNCaP-IL-6+ cells was investigated using an antibody that reacts with both nonphosphorylated and phosphorylated kinases. Interestingly, MAPK expression was up-regulated in LNCaP-IL-6+ cells compared to controls (Figure 7) and the presence of IL-6 in culture either during 30 minutes (Figure 7A) or 48 hours (Figure 7B) did not cause a major effect on MAPK levels. As our antibody recognizes both MAPK forms, we asked whether elevated MAPK expression has a functional significance. For this purpose, we treated LNCaP-IL-6+ cells and controls with the MAPK kinase inhibitor PD 98059. As shown in Figure 8, this compound had no effect on LNCaP-IL-6− cells. In contrast, PD 98059 caused a statistically significant inhibition of proliferation of LNCaP-IL-6+ cells.Figure 8The effect of the MAPK kinase inhibitor PD 98059 on proliferation of LNCaP-IL-6− (open symbols) and LNCaP-IL-6+ (shaded symbols) cells. The cells were cultured in the absence or presence of the inhibitor for 48 hours and counted afterward (three independent experiments; *, P < 0.05, Mann-Whitney t-test); bars, ±SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In a series of recent studies, IL-6 was identified as an important growth regulator in human prostate cancer.5Giri D Ozen M Ittmann M Interleukin-6 is an autocrine growth factor in human prostate cancer.Am J Pathol. 2001; 159: 159-165Google Scholar, 7Degeorges A Tatoud R Fauvel Lafeve F Podgorniak MP Millot G de Cremoux P Calvo F Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells, identified as interleukin-6.Int J Cancer. 1996; 68: 207-214Crossref PubMed Scopus (89) Google Scholar, 8Ritchie CK Andrews LR Thomas KG Tindall DJ Fitzpatrick LA The effects of growth factors associated with osteoblasts on prostate carcinoma proliferation and chemotaxis: implications for the development of metastatic disease.Endocrinology. 1997; 138: 1145-1150Crossref PubMed Scopus (78) Google Scholar, 10Spiotto MT Chung TD STAT3 mediates IL-6-induced growth inhibition in the human pro