ANTICANCER RESEARCH 25: 3809-3816 (2005)

Osteoprotegerin is Expressed in Colon Carcinoma Cells

INGVILD PETTERSEN, WENCHE BAKKELUND, BÅRD SMEDSRØD and BALDUR SVEINBJØRNSSON

Department of Experimental Pathology, Medical Faculty, University of Tromsø, N-9037, Tromsø, Norway

Abstract. Background: Osteoprotegerin (OPG), a soluble preventing it from activating its cognate receptor RANK, member of the tumor necrosis factor family, is produced by which is the receptor essential for osteoclast various cell types and tissues and plays a key role in the maturation, activation and survival (1-4). physiological regulation of osteoclast differentiation and OPG is produced and secreted by and activity. Also, OPG is a soluble decoy receptor for tumor marrow stromal cells, but is also expressed in other types of necrosis factor-related -inducing factor (TRAIL). In human tissues including liver, lung, heart, bone, kidney, the present study we investigated whether the human colon placenta, stomach,thyroid gland, dendritic cells, B-cells, cancer cell lines HT-29 and SW-480 produce and secrete OPG chondrocytes and blood vessels (3-5). in vitro. Materials and Methods: Expression of OPG mRNA OPG and its cognate ligands have been shown to play a was examined by RT-PCR. OPG protein was analysed by significant role within the immune system (6). The binding ELISA assay and immunostaining methods. The effect of of RANKL to RANK augments survival and promotes the OPG secretion on TRAIL-mediated apoptosis was also immunostimulatory capacity of dendritic cells (2, 7), and investigated. Results: By RT-PCR, it was demonstrated that modulates activated T cells (8), whereas OPG has been mRNA transcripts for OPG were produced by both cell lines. shown to antagonize these functions (9). OPG also has been By ELISA analysis, OPG was detected in the culture medium; shown to be a decoy receptor for tumor necrosis factor- and treatment of cells with proinflammatory cytokines TNF-· related apoptosis-inducing ligand (TRAIL) (10). TRAIL is and IL-1‚ increased OPG secretion significantly. Tumor expressed by immune cells and effectively induces apoptosis xenografts in nude mice also were shown to express OPG by in tumor cells, whereas most normal cells appear to be immunohistochemistry. When RANKL, which selectively binds resistant towards its cytotoxicity (11). Thus, OPG may act OPG, was added to cell cultures along with recombinant as a survival factor by blocking TRAIL-mediated apoptosis TRAIL, apoptosis was shown to increase significantly. in malignant cells (10, 12, 13). Conclusion: These data indicate that OPG may be involved in The expression of OPG has previously been reported in tumorigenesis and the progression of colon cancer. several tumor cell lines such as melanoma (14), prostate (12, 15 ,16), osteosarcoma (17), malignant gliomas (18) and Osteoprotegerin (OPG) is a new member of the tumour neuroblastoma (19). Recently, expression of OPG has been necrosis factor (TNF) family which plays a key role in the shown to correlate with aggressiveness and poor prognosis physiological regulation of osteoclastic bone resorption. of gastric carcinoma (20). OPG is also a soluble decoy receptor for RANKL (receptor In the present study, the expression of OPG in human activator of NFÎ B ligand), and neutralizes its function by colon carcinoma cell lines was investigated.

Materials and Methods Abbreviations: OPG, osteoprotegerin; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; RANKL, receptor- Reagents. Recombinant human TRAIL (375-TEC), recombinant activator of NFÎ ‚ ligand. human OPG, recombinant human TNF-· (210-TA), recombinant human IL-1‚ (201-LB) and recombinant human RANKL were Correspondence to: Baldur Sveinbjørnsson, Department of purchased from R&D Systems (Abingdon, UK). Monoclonal anti- Experimental Pathology, Medical Faculty, University of Tromsø, human OPG (IMG-103) for immunocytochemistry was purchased N-9037, Tromsø, Norway. Fax: + 47-77-64-54-00, e-mail: baldur@ from Imgenex (San Diego, CA, USA). Mouse isotype controls, fagmed.uit.no secondary biotinylated antibody, streptavidin-Fitc and streptavidin- HRP were from Zymed (USA). MTT (3-(4,5-dimethylthiazol-2-yl)- Key Words: Colon cancer, osteoprotegerin, RANKL, TNF, IL-1‚, 2,5-diphenyl-tetrazolium bromide) was purchased from Sigma immunohistochemistry. Chemical Company (St. Louis, MO, USA).

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Cells. Human colon carcinoma cells, HT-29 (ATTC, HTB-38) media were assessed for 3 days. OPG secretion into the media was and SW-480 (ATCC, CCL-228), were cultured in monolayers in measured by ELISA assay, according to the manufacturer’s RPMI 1640 supplied with 10% fetal calf serum (FCS) and instruction (R&D Systems). antibiotics. The cultures were kept at 37ÆC in an atmosphere of 5% CO2. Animals. Specific pathogen-free Balb/c nude mice, about 10 weeks of age, were purchased from Harlan, The Netherlands. The mice Treatment. Colon carcinoma cells (3x104/well) were treated with were kept on a standard laboratory chow and water diet. These recombinant human TRAIL (20 ng/ml) in RPMI 1640 experiments were conducted in accordance with the Norwegian supplemented with 1% FCS medium alone, or together with "Animal Protection Ordinance" (1977) and "The European sRANKL (100ng/ml) for 24 h at 37ÆC. Convention for the protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes of March 18th, 1986". Cytotoxicity assay. Cytotoxicity against tumor cells was assayed by The protocol was approved by the Norwegian Ethics Committee the MTT assay, according to procedures previously described (21). on Animal Research.

RT-PCR. Total RNA was extracted from the cultured colon Immunofluorescence analysis. Identification of OPG-positive carcinoma cells using the TRIZOL reagent (Life Technologies, cells was performed by using monoclonal anti-human OPG Inc., Grand Island, NY 14072, USA), according to the antibody. HT-29 and SW-480 cells were grown on fibronectin- manufacturer’s protocol. cDNA was synthesized using 2.0 Ìg total coated chamber slides (Nunc, Denmark) for 24 hours in RPMI RNA that was reverse transcribed in a final volume of 50 Ìl using 1640 medium supplied with 10% FCS. The cultures were then the superScript Preamplification (Life Technologies, Inc., washed and fixed for 12 hours in 4% paraformaldehyde in Gaithersburg, MD, USA). PCR was performed in 50 Ìl of reaction phosphate buffer containing 0.2 M sucrose, followed by fixation mixture containing 1 Ìl cDNA (from isolated RNA), 2.5 units of in cold methanol for 30 minutes. After washing with TBS buffer, Taq DNA polymerase (Promega), 10 mM Tris-HCl (pH 8.0), 100 the antibody was diluted 1:100 and incubated with the cultures ® mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 1 mM overnight at 4ÆC. After rinsing in TBS, sections were incubated deoxynucleotide triphosphate mix, and 1 M of each primer. PCR for 45 minutes with secondary goat anti-mouse biotinylated was performed at 94ÆC for 5 minutes (first denaturation/Hot start) antibody and, thereafter, with the streptavidin-fluorescein and then at 94ÆC for 1 minute (denaturation), 52ÆC for 1.5 minutes complex. The cultures were examined using an Axiophot (annealing) and 72ÆC for 1 minute (extension), for 35 cycles with a photomicroscope (Carl Zeiss, Oberkochen, Germany). The 10-minute final extension at 72ÆC. PCR amplifications were photomicrographs were taken with a Nikon Coolpix 4500 digital performed in a PTC-200 Peltier Thermal Cycler (MJ Research camera. Inc., Waltham, MA, USA). The PCR products were analysed by agarose gel (1.5%) electrophoresis and photographed under UV Immunohistochemistry. Tumors were established in nude mice. light. The nucleotide sequences of the PCR primers used were as The tumor cells were harvested and washed in RPMI 1640 follows: osteoprotegerin (OPG) 5’-GCTAACCTCACCTTCGAG- medium before injection. Each animal received a single 3’ (sense) and 5’-TGATTGGACCTGGGTTACC-3’ (antisense); inoculation of 106 tumor cells subcutaneously in the abdomen. 324 bp fragment expected (22). Approximately one week later, the mice were killed with CO2 RT-PCR for ‚-actin was performed as follows, using the same and then perfused intracardially with cold PBS (pH 7.2), PCR mixture as previously described. The following primer pair for followed by 5 minutes of cold 4% paraformaldehyde containing ‚-actin was used; ‚-actin, 5’-TGACGGGGTCACCCACACTGT 0.2 mol/L sucrose in phosphate buffer. The tumor tissue was GCCCATCTA-3’ (sense) and 5’-ACTCGTCATACTCCTGCTTG excised and immersed in the same fixative at 4ÆC for 48 hours, CTGATCCA-3’ (antisense); 625 bp fragment expected. PCR was embedded in paraffin, and sectioned at 5 Ìm thickness onto poly- performed at 94ÆC for 5 minutes (first denaturation/Hot start) and L-lysine-coated slides. Sections were deparaffinized by xylene then at 94ÆC for 1 minute (denaturation), 55ÆC for 1 minute and graded alcohol and immersed in distilled water. After (annealing) and 72ÆC for 1 minute (extension), for 27 cycles with a antigen retrieval in 10 mM citrate buffer (pH 6.5) at 100ÆC for 5 10-minute final extension at 72ÆC. PCR amplifications were minutes, the sections were washed three times with TBS and performed in a PTC-200 Peltier Thermal Cycler (MJ Research subsequently incubated in TBS containing 1% bovine serum Inc.). The PCR products were analyzed by agarose gel (1.5%) albumin (1% TBSA) for 30 minutes. The primary monoclonal electrophoresis and photographed under UV light. antibody was diluted 1:50 and incubated with the sections overnight at 4ÆC. As a control for non-specific background Cell culture and stimulation. Colon carcinoma cells (HT-29, SW- staining, some sections were incubated with mouse IgG isotype 480) were seeded in 96-well culture plates at a density of 3x104 control. After rinsing in TBS, endogenous peroxidase activity was cells/well in RPMI-1640 supplemented with 10% FCS and 2 mM blocked by 5-minute incubation in 3% H2O2 methanol at room L-glutamine. temperature. The sections were incubated for 45 minutes with For stimulation experiments, the cells were made quiescent by secondary biotinylated antibody and, thereafter, with serum starvation (0.5% FCS) for 24 hours. Stimulation with streptavidin-horseradish peroxidase complex. The reaction cytokines (IL-1‚, TNF-·) was performed in RPMI-1640 products were visualized with the DAB substrate chromogen containing 0.5% FCS for 16 hours. For measurment of basal OPG system (Dako, CA, USA). The sections were examined using an secretion, cells were seeded in 75-cm2 culture flasks in medium Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany). containing 10% serum and were left to adhere overnight before The photomicrographs were taken with a Nikon Coolpix 4500 addition of medium containing 0.5% serum. Samples from the digital camera.

3810 Pettersen et al: Osteoprotegerin is Expressed in Colon Carcinoma Cells

Figure 1. RT-PCR expression analysis of OPG (A) and ‚-actin (B) in HT-29 and SW-480 cells. Negative control (N) without cDNA template in the sample was included.

Results

To identify OPG mRNA transcripts in the colon cancer cell lines HT-29 and SW-480, RT-PCR analysis was performed using a specific primer for human OPG. A strong 324 bp fragment for OPG mRNA was detected in both cell lines (Figure 1). The OPG levels in the cell supernatants were measured by ELISA. Basal secretion of OPG was linear with time within the time-period investigated and the cell lines secreted OPG at very different levels (Figure 2). The effect of pro-inflammatory cytokines (TNF-·, IL-1) on the secretion of OPG from cultured colon carcinoma cells was Figure 2. Time-course of basal level secretion of OPG in colon carcinoma investigated. Cells were stimulated for 16 hours and both cell cells. SW-480 cells (A) and HT-29 cells (B). Data are expressed as the lines showed elevated secretion upon stimulation with cytokines mean±SEM. (Figure 3). Both cell lines demonstrated immunoreactivity for OPG after cultivation in vitro (Figure 4A, 4B). Colon cancer xenograft tumor tissue showed significant cytoplasmic OPG Discussion immunoreactivity in tumor epithelial cells of both tumor types (Figure 5A, 5B). Some stromal cells also stained positive. In all OPG is a member of the tumor necrosis factor receptor cases, the isotype control stained negative. family and inhibits by binding to RANKL, We have previously shown that addition of recombinant thereby inhibiting the activation of RANK, which is the OPG to the culture medium decreased the level of TRAIL- osteoclast receptor vital for osteoclast differentiation, mediated apoptosis of the SW-480 and HT-29 cell lines, activation and survival (3). OPG mRNA can be detected in respectively (23). In the present study, both cell lines were many different tissues and, in contrast to other members of shown to secrete OPG into the culture medium. We the tumor necrosis receptor family members, OPG lacks therefore investigated whether TRAIL-mediated apoptosis transmembrane and cytoplasmic domains and is secreted as could be increased by neutralizing OPG in the medium. a soluble protein (24). Cultures treated with TRAIL in the presence of RANKL OPG may also be involved in certain aspects of neoplastic showed significantly decreased numbers of viable cells disease. Recently, clinical studies have determined that compared with those treated with TRAIL alone (Figure 6). serum OPG levels are dramatically elevated in some

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apoptosis in colon cancer cell lines in vitro (36, 23) and in vivo xenograft models (36), whereas normal colon epithelium seems to be resistant to TRAIL-induced apoptosis (35). Regarding OPG, only few investigations have been reported concerning the level of expression of this soluble decoy receptor within tumors (37, 15, 20, 18). In the present study, we demonstrated the expression of OPG by human colon cancer cells in vitro. As shown in Figure 1, both the HT-29 and SW-480 cell lines expressed OPG mRNA, as assessed by RT-PCR. The regulation of OPG mRNA levels and protein by cytokines has been studied in several human cell lines and primary cultures. Stimulation with proinflammatory cytokines such as TNF-·/‚ and IL-1 leads to enhanced expression of OPG in the osteosarcoma cell line MG-63 (38-40), human prostate cancer cell line (16), human stromal bone marrow cells (41), human microvascular endothelial cells (42), human thyroid follicular cells (43), human aortic smooth muscle cells (44, 45) and human umbilical vein endothelial cells (46) in vitro. Also, TGF-‚ and IL-18 have been reported to enhance the expression of OPG in murine stromal bone marrow cells (47, 48). Here, it was demonstrated that human colon cancer cells produce significant amounts of OPG in vitro with time and that its secretion is potently up-regulated by inflammatory cytokines such as TNF-· and IL-1 (Figure 3). These data are in accordance with recent studies on OPG secretion of human intestinal epithelial cells (49). Therefore, TNF-· and IL-1, along with other inflammatory cytokines produced by tumor stromal cells such as macrophages and fibroblasts (50), may contribute to the further induction of OPG secretion by the cancer cells. Also, both types of tumor xenografts were positively stained for OPG (Figure 5). We have reported previously that the two cell lines used in this study, HT-29 and SW-480, are sensitive to TRAIL-mediated apoptosis (23). Here, we show that TRAIL-mediated apoptosis may be enhanced by neutralizing the endogenous level of OPG (Figure 6), suggesting that OPG may act as a suppressor of TRAIL Figure 3. Secretion of OPG in colon carcinoma cells after stimulation with different doses of TNF-· (A) and IL-1‚ (B). SW-480 (hatched bars), activity. A similar function for OPG has been suggested HT-29 (open bars). OPG levels were measured with ELISA. Data are in earlier studies on human prostate (12) and myeloma expressed as the mean±SEM. tumor cells (13). The fact that OPG secretion is also enhanced by inflammatory cytokines implies that cancer cells may advantageously exploit macrophage products and simultaneously limit the capacity of the host cells to patients with advanced prostate cancer (25-27) and liver restrict the growth of the tumor. metastases (28). OPG expression also has been detected in Experimental studies have proposed that TRAIL plays a several human cancer cell lines (12, 14-19). substantial role in suppressing tumor growth (51-54). A number of studies has demonstrated death receptor However, it is unclear to what degree OPG secretion by expression in tumors (29-34). TRAIL has been shown to be tumor cells in vivo can result in decrement of TRAIL- expressed highly in the colon, although its function in this mediated apoptosis. The sensitivity of tumors to TRAIL and tissue has not been fully elucidated (35). TRAIL induces other death receptor ligands may be determined by both the

3812 Pettersen et al: Osteoprotegerin is Expressed in Colon Carcinoma Cells

Figure 4. Immunocytochemical localization of OPG in (A) SW-480 and (B) HT-29 cells. Scale bar 10 Ìm.

Figure 5. Immunohistochemical localization of OPG within tumor xenografts showing significant immunoreactivity of OPG in cell cytoplasm (A) SW- 480 and (B) HT-29. Scale bar 40 Ìm.

net abundance and affinity of functional death receptors dendritic cells, that can mediate survival of dendritic cells, versus decoy receptors and the balance between intracellular may be disrupted by OPG (2,7,8,9). Furthermore, OPG has apoptotic and anti-apoptotic mechanisms (55). been reported to inhibit the proliferation of myeloid A recent study demonstrated that OPG is an important progenitor cells (60). In summary, we have shown that survival factor for endothelial cells (56-58), which suggests a human colon carcinoma cells express OPG mRNA and role for OPG in stabilizing and promoting tumor protein and its expression may be enhanced by angiogenesis. In addition, OPG may inhibit survival signals proinflammatory cytokines. How OPG expression within the propagated by binding of RANKL to RANK. By binding to tumor environment may influence tumorigenesis warrants RANKL, OPG may down-regulate maturation, further investigation into the role of death receptors and differentiation and efficient antibody response (59). Also, their ligands in both pathology and normal physiology of the interactions between RANKL on T cells and RANK on colon epithelium.

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48 Makiishi-Shimobayashi C, Tsujimura T, Iwasaki T, Yamada N, 55 Cappello P, Novelli F, Forni G and Giovarelli M: Death Sugihara A, Okamura, H, Hayashi S and Terada N: Interleukin- receptor ligands in tumors. J Immunotherapy 25: 1-15, 2001. 18 up-regulates osteoprotegerin expression in stromal/osteoblastic 56 Malyankar UM, Scatena M, Suchland KL, Yun TJ, Clark EA cells. Biochem Biophys Res Commun 281: 361-366, 2001. and Giachelli CM: Osteoprotegerin is an ·v‚3-induced, NF-Î B- 49 Vidal K, Serrant P, Schlosser B, van den Broek P, Lorget F and dependent survival factor for endothelial cells. J Biol Chem 275: Donnet-Hughes A: Osteoprotegerin production by human 20959-20962, 2000. intestinal epithelial cells: a potent regulator of mucosal immune 57 Scatena M and Giachelli C: The ·v‚3 , NF-Î B, responses. Am J Physiol Gastrointest Liver Physiol 287: G836- osteoprotegerin endothelial cell survival pathway. Potential role G844, 2004. in angiogenesis. Trends Cardiovasc Med 12: 83-88, 2002. 50 Seljelid R, Jozefowski S and Sveinbjørnsson B: Tumor stroma 58 Pritzker LB, Scatena M and Giachelli CM: The role of review. Anticancer Res 19: 4809-4822, 1999. osteoprotegerin and tumor necrosis factor-related apoptosis- 51 Takeda K, Hayakawa Y, Smyth MJ, Kayagaki N, Yamaguchi N, inducing ligand in human microvascular endothelial cell Kakuta S, Iwakura Y, Yagita H and Okumura K: Involvement survival. Mol Biol Cell 15: 2834-2841, 2004. of tumor necrosis factor-related apoptosis-inducing ligand in 59 Yun TJ, Tallquist MD, Aicher A, Rafferty KL, Marshall AJ, surveillance of tumor metastasis by liver natural killer cells. Moon JJ, Ewings ME, Mohaupt M, Herring SW and Clark EA: Nature Med 7: 94-100, 2001. Osteoprotegerin, a crucial regulator of bone metabolism, also 52 Takeda K, Smyth MJ, Cretney E, Hayakawa Y, Kayagaki N, regulates B cell development and function. J Immunol 166: Yagita H and Okumura K: Critical role for tumor necrosis 1482-1491, 2001. factor-related apoptosis-inducing ligand in immune surveillance 60 Ra JS, Broxmeyer HE, Kim MW, Han I-S, Choi SW and Kwon against tumor development. J Exp Med 195: 161-169, 2002. BS: Osteoprotegerin inhibits proliferation of myeloid 53 Cretney E, Takeda K, Yagita H, Glaccum M, Peschon JJ and progenitor cells. J Hematother Stem Cell Res 12: 33-38, 2003. Smyth MJ: Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice. J Immunol 168: 1356-1361, 2002. 54 Seki N, Hayakawa Y, Brooks AD, Wine J, Wiltrout RH, Yagita H, Tanner JE, Smyth MJ and Sayers TJ: Tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis is an important endogenous mechanism for resistance to liver metastases in murine renal cancer. Cancer Received July 6, 2005 Res 63: 207-213, 2003. Accepted September 1, 2005

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