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Background: Prostate cancer (PCa) bone metastases have been shown to be more resistant to docetaxel than soft tissue metastases. The proinflammatory chemokine receptor CXCR4 has been shown to confer resistance to docetaxel (DOC) in PCa cells. Balixafortide (BLX) is a protein epitope mimetic inhibitor of CXCR4. Accordingly, we hypothesized that BLX would enhance DOC-mediated antitumor activity in PCa bone metastases., Methods: PC-3 luciferase-labeled cells were injected into the tibia of mice to model bone metastases. Four treatment groups were created: vehicle, DOC (5 mg/kg), BLX (20 mg/kg), and combo (receiving both DOC and BLX). Mice were injected twice daily subcutaneously with either vehicle or BLX starting on Day 1 and weekly intraperitoneally with DOC starting on Day 1. Tumor burden was measured weekly via bioluminescent imaging. At end of study (29 days), radiographs were taken of the tibiae and blood was collected. Serum levels of TRAcP, IL-2, and IFNγ levels were measured using ELISA. Harvested tibiae were decalcified and stained for Ki67, cleaved caspase-3, and CD34 positive cells or microvessels were quantified., Results: Tumor burden was lower in the combo group compared to the DOC alone group. Treatment with the combination had no impact on the number of mice with osteolytic lesions, however the area of osteolytic lesions was lower in the combo group compared to the vehicle and BLX groups, but not the DOC group. Serum TRAcP levels were lower in the combo compared to vehicle group, but not the other groups. No significant difference in Ki67 staining was found among the groups; whereas, cleaved caspase-3 staining was lowest in the Combo group and highest in the BLX group. The DOC and combo groups had more CD34+ microvessels than the control and BLX groups. There was no difference between the treatment groups for IL-2, but the combo group had increased levels of IFNγ compared to the DOC group., Conclusions: Our data demonstrate that a combination of BAL and DOC has greater antitumor activity in a model of PCa bone metastases than either drug alone. These data support further evaluation of this combination in metastatic PCa., (© 2023 The Authors. The Prostate published by Wiley Periodicals LLC.)

Background: Raf kinase inhibitor protein (RKIP) has been shown to act as a metastasis suppressor gene in multiple models of cancer. Loss of RKIP expression promotes invasion and metastasis in cell transplantation animal models. However, it is unknown if RKIP expression can impact the progression of cancer in an autochthonous model of cancer. The goal of this study was to determine if loss of RKIP expression in a genetic mouse model of prostate cancer (PCa) impacts metastasis., Methods: Endogenous RKIP expression was measured in the primary tumors and metastases of transgenic adenocarcinoma of the mouse prostate (TRAMP(+) ) mice. RKIP knockout mice (RKIP(-/-) ) were crossbred with (TRAMP(+) ) mice to create RKIP(-/-) TRAMP(+) mice. Mice were euthanized at 10, 20, and 30 weeks for evaluation of primary and metastatic tumor development. To determine if loss of RKIP alone promotes metastasis, RKIP was knocked down in the low metastatic LNCaP prostate cancer cell line., Results: Endogenous RKIP expression decreased in TRAMP(+) mice as tumors progressed. Primary tumors developed earlier in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice. At 30 weeks of age, distant metastases were identified only the RKIP(-/-) TRAMP(+) mice. While prostate epithelial cell proliferation rates were higher at 10 and 20 weeks in RKIP(-/-) TRAMP(+) compared to TRAMP(+) mice, by 30 weeks there was no difference. Apoptosis rates in both groups were similar at all timepoints. Decreased RKIP expression did not impact the metastatic rate of LNCaP in an orthotopic PCa model., Conclusions: These results demonstrate that loss of RKIP decreases latency of tumor development and promotes distant metastasis in the TRAMP mouse model in the context of a pro-metastatic background; but loss of RKIP alone is insufficient to promote metastasis. These findings suggest that in addition to its known metastasis suppressor activity, RKIP may promote tumor progression through enhancing tumor initiation. Prostate 75:292-302, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

Background: The matricellular protein secreted protein acidic and rich in cysteine (SPARC) plays an important role on tumor metastasis and progression in several cancers. However, the roles of SPARC in prostate cancer (PCa) remain unclear., Methods: To identify SPARC protein in prostate tissue, immunohistochemical analysis of SPARC was conducted using human prostate tissue microarray. To detect SPARC expression in prostate cancer (LNCaP, DU145, and PC-3) and stromal cells, RT-PCR, western blot analysis, and ELISA was conducted. To reveal the function of exogenous SPARC in PCa cells, AKT phosphorylation was confirmed by western blot analysis after coculture with stromal cells. Proliferation and migration of PCa cells were examined by addition of SPARC. The interaction between SPARC and integrin β1 was confirmed by western blot analysis after immunoprecipitation., Results: SPARC protein was expressed well in normal tissue compared with PCa tissue. ELISA showed high secreted SPARC protein in normal prostate-derived stromal cell (PrSC) compared with PCa-derived stromal cell (PCaSC) and PCa. PCa cells cocultured with PrSC showed reduced AKT phosphorylation more than with PCaSC. PCa cells cocultured with PrSC whose SPARC was knocked-down restored AKT phosphorylation. Moreover, PCa cells treated with SPARC led to reduced AKT phosphorylation. Immunoprecipitation with SPARC revealed interaction of SPARC and integrin β1 in PCa cells. Inhibited proliferation and migration of PCa cells by SPARC was restored by integrin β1 neutralizing antibody., Conclusions: Reduced SPARC secretion from stromal cells might affect PCa progression mediating through limiting AKT phosphorylation after interaction with integrin β1., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: No existing animal model fully recapitulates all features of human prostate cancer. The dog is the only large mammal, besides humans, that commonly develops spontaneous prostate cancer. Canine prostate cancer features many similarities with its human counterpart. We sought to develop a canine model of prostate cancer that would more fully represent the features of human prostate cancer than existing models., Methods: The Ace-1 canine prostate cancer cell line was injected transabdominally under transrectal ultrasound (TRUS) guidance into the prostates of immunosuppressed, intact, adult male dogs. Tumor progression was monitored by TRUS imaging. Some dogs were subjected to positron emission tomography (PET) for tumor detection. Time of euthanasia was determined based on tumor size, impingement on urethra, and general well-being. Euthanasia was followed by necropsy and histopathology., Results: Ace-1 tumor cells grew robustly in every dog injected. Tumors grew in subcapsular and parenchymal regions of the prostate. Tumor tissue could be identified using PET. Histological findings were similar to those observed in human prostate cancer. Metastases to lungs and lymph nodes were detected, predominantly in dogs with intraprostatic tumors., Conclusions: We have established a minimally invasive dog model of prostate cancer. This model may be valuable for studying prostate cancer progression and distant metastasis., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Conversion into androgen-hypersensitive state and adaptation to the low concentration of androgen during ADT cause relapse of prostate cancer (PCa). It is important to identify differentially expressed genes between PCa and normal prostate tissues and to reveal the function of these genes that are involved in progression of PCa., Methods: We performed cDNA microarray analysis to identify differentially expressed genes, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2). Immunohistochemical analysis was conducted to investigate the relationship between the CAMKK2 expression level and prognosis. The function of CAMKK2 was assessed by generating CAMKK2 overexpressed LNCaP cells and by knockdown of CAMKK2., Results: We identified CAMKK2 overexpressed six times in PCa more than normal prostate by cDNA microarray analysis. Immunohistochemical analysis of CAMKK2 protein showed that CAMKK2 protein was expressed more in PCa than normal tissue. However, the expression in the high-grade PCa diminished. Moreover, the narrowness of CAMKK2-positive area before ADT was a poor prognostic factor. Androgen-deprivation treatment from the medium in which LNCaP cells were cultured in the presence of 10 nM DHT repressed CAMKK2 expression. CAMKK2 overexpressed LNCaP cells (LNCaP/GFP-CAMKK2) attenuated androgen-sensitivity. Tumorigenesis of LNCaP/GFP-CAMKK2 cells in male SCID mice was decreased compared with control cells irrespective of castration. Finally, knockdown of CAMKK2 mRNA in LNCaP cells induced androgen-hypersensitivity and stimulated LNCaP cell proliferation., Conclusions: Induction of androgen-hypersensitivity after ADT may be involved in down-regulation of CAMKK2. This result may provide new therapeutic approach to keep androgen-sensitivity of PCa after ADT., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer., Methods: A new cell line (Leo) was derived from a dog with spontaneous prostate cancer. Immunohistochemistry and PCR were used to characterize the primary prostate cancer and xenografts in nude mice. Subcutaneous tumor growth and metastases in nude mice were evaluated by bioluminescent imaging, radiography and histopathology. In vitro chemosensitivity of Leo cells to therapeutic agents was measured., Results: Leo cells expressed the secretory epithelial cytokeratins (CK)8, 18, and ductal cell marker, CK7. The cell line grew in vitro (over 75 passages) and was tumorigenic in the subcutis of nude mice. Following intracardiac injection, Leo cells metastasized to the brain, spinal cord, bone, and adrenal gland. The incidence of metastases was greatest to the central nervous system (80%) with a lower incidence to bone (20%) and the adrenal glands (16%). In vitro chemosensitivity assays demonstrated that Leo cells were sensitive to Velcade and an HDAC-42 inhibitor with IC(50) concentrations of 1.9 nm and 0.95 µm, respectively., Conclusion: The new prostate cancer cell line (Leo) will be a valuable model to investigate the mechanisms of the brain and bone metastases., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Osteoblastic bone metastasis is the predominant phenotype observed in prostate cancer patients and is associated with high patient mortality and morbidity. However, the mechanisms determining the development of this phenotype are not well understood. Prostate cancer cells secrete several osteogenic factors including Wnt proteins, which are not only osteoinductive but also oncogenic. Therefore, the purpose of the study was to investigate the contribution of the Wnt signaling pathway in prostate cancer growth, incidence of bone metastases, and osteoblastic phenotype of bone metastases. The strategy involved overexpressing the Wnt antagonist, DKK-1, in the mixed osteoblastic and osteolytic Ace-1 prostate cancer cells., Methods: Ace-1 prostate cancer cells stably expressing human DKK-1 or empty vector were established and transduced with lentiviral yellow fluorescent protein (YFP)-luciferase (Luc). The Ace-1/vector(YFP-LUC) and Ace-1/DKK-1(YFP-LUC) cells were injected subcutaneously, intratibially, or in the left cardiac ventricle in athymic mice., Results: Unexpectedly, DKK-1 significantly increased Ace-1 subcutaneous tumor mass and the incidence of bone metastases after intracardiac injection of Ace-1 cells. DKK-1 increased Ace-1 tumor growth associated with increased phospho46 c-Jun amino-terminal kinase by the Wnt noncanonical pathway. As expected, DKK-1 decreased the Ace-1 osteoblastic phenotype of bone metastases, as confirmed by radiographic, histopathologic, and microcomputed tomographic analysis. DKK-1 decreased osteoblastic activity via the Wnt canonical pathway evidenced by an inhibition of T-cell factor activity in murine osteoblast precursor ST2 cells., Conclusion: The present study showed that DKK-1 is a potent inhibitor of bone growth in prostate cancer-induced osteoblastic metastases., (Copyright © 2010 Wiley-Liss, Inc.)

Background: Recently, we established paclitaxel-resistant prostate cancer cell lines (PC-3-TxR and DU145-TxR). To determine the mechanisms of paclitaxel resistance in PC-3-TxR cells, we compared the gene expression profiles between PC-3 and PC-3-TxR cells. Our results indicated that expression of the C-terminal tensin like protein (CTEN, tensin 4) gene was down-regulated by 10-fold in PC-3-TxR cells. We investigated the possibility that CTEN overexpression restores paclitaxel sensitivity., Methods: We investigated how knockdown and overexpression of CTEN in androgen-independent cell lines affect paclitaxel sensitivity by colony formation assay and growth inhibition assay. To determine the mechanisms by which CTEN affects paclitaxel sensitivity, we investigated the relationships between CTEN and F-actin or epidermal growth factor receptor (EGFR) in PC-3 cells. We also examined the association between expression of CTEN and grade of prostate cancer by immunohistochemistry using tissue microarray analysis., Results: Down-regulation of CTEN, which is located in the cytoskeleton, played an important role in paclitaxel resistance in PC-3-TxR cells. Knockdown of CTEN expression in PC-3 cells induced paclitaxel resistance. Overexpression of CTEN in PC-3-TxR and DU145-TxR cells restored paclitaxel sensitivity. CTEN expression was inversely correlated with F-actin and EGFR expression. Then knockdown of actin and EGFR in PC-3-TxR cells recovered paclitaxel sensitivity, indicating that CTEN down-regulation mediates paclitaxel resistance through elevation of EGFR and actin expression. Moreover, CTEN expression was inversely correlated with Gleason score., Conclusions: These results strongly suggested that CTEN plays an important role in paclitaxel sensitivity and that CTEN expression level may be a prognostic predictive factor for PCa patients., ((c) 2009 Wiley-Liss, Inc.)

Background: Tranilast is a therapeutic agent used in treatment of allergic diseases, although it has been reported to show anti-tumor effects on some cancer cells. To elucidate the effects of tranilast on prostate cancer, we investigated the mechanisms of its anti-tumor effect on prostate cancer., Methods: The anti-tumor effects and related mechanisms of tranilast were investigated both in vitro on prostate cancer cell lines and bone-derived stromal cells, and in vivo on severe combined immunodeficient (SCID) mice. We verified its clinical effect in patients with advanced hormone refractory prostate cancer (HRPC)., Results: Tranilast inhibited the proliferation of LNCaP, LNCaP-SF, and PC-3 cells in a dose-dependent manner and growth of the tumor formed by inoculation of LNCaP-SF in the dorsal subcutis and in the tibia of castrated SCID mice. Flow cytometry and TUNEL assay revealed induction of cell cycle arrest and apoptosis by tranilast. Tranilast increased expression of proteins involved in induction of cell cycle arrest and apoptosis. Coculture with bone-derived stromal cells induced proliferation of LNCaP-SF cells. Tranilast also suppressed secretion of transforming growth factor beta1 (TGF-beta1) from bone-derived stromal cells, which induced their differentiation. Moreover, tranilast inhibited TGF-beta1-mediated differentiation of bone-derived stromal cells and LNCaP-SF cell migration induced by osteopontin. In the clinical investigation, PSA progression was inhibited in 4 of 16 patients with advanced HRPC., Conclusions: These observations suggest that tranilast may be a useful therapeutic agent for treatment of HRPC via the direct inhibitory effect on cancer cells and suppression of TGF-beta1-associated osteoblastic changes in bone metastasis.

Background: Bone metastasis occurs frequently in advanced prostate cancer (PCa) patients; however, it is not known why this happens. The epidermal growth factor receptor (EGFR) ligand EGF is available to early stage PCa; whereas, TGF-alpha is available when PCa metastasizes. Since the microenvironment of metastases has been shown to play a role in the survival of the tumor, we examined whether the ligands had effects on cell survival and proliferation in early and late PCa., Methods: We used LNCaP cells as a model of early stage, non-metastatic PCa and the isogenic C4-2B cells as a model of late stage, metastatic PCa., Results: We found that the proliferation factor MAPK and the survival factor AKT were differentially activated in the presence of different ligands. TGF-alpha induced growth of C4-2B cells and not of the parental LNCaP cells; however, LNCaP cells expressing a constitutively active AKT did proliferate with TGF-alpha. Therefore, AKT appeared to be the TGF-alpha-responsive factor for survival of the late stage PCa cells. LNCaP cells exposed to EGF produced more osteoprotegerin (OPG), an inhibitor of bone remodeling, than C4-2B cells with TGF-alpha, which had increased expression of RANKL, an activator of bone remodeling. In concordance, TGF-alpha-treated C4-2B conditioned medium was able to differentiate an osteoclast precursor line to a greater extent than EGF-treated C4-2B or TGF-alpha-treated LNCaP conditioned media., Conclusion: The switch in EGFR ligand availability as PCa progresses affects cell survival and contributes to bone remodeling., ((c) 2009 Wiley-Liss, Inc.)

Background: Prostate cancer (PCa) frequently metastasizes to the bone and induces osteoblastic lesions. We previously demonstrated through over-expression of the Wnt inhibitor dickkopf-1 (DKK-1) that Wnts contribute to the osteoblastic component of PCa osseous lesions in vivo., Methods: To test the clinical significance of DKK-1 expression during PCa progression, tissue microarrays were stained for DKK-1 protein by immunohistochemistry., Results: DKK-1 expression index (EI) was found to increase in PIN and primary lesions compared to non-neoplastic tissue (106 +/- 10 vs. 19 +/- 6, respectively, where the EI is the product of the percent expression and staining intensity). DKK-1 expression was also found to be higher in all PCa metastatic lesions (56 +/- 21 EI) compared to non-neoplastic tissues but was significantly decreased versus primary PCa lesions (P < 0.008). The decline in DKK-1 correlated with a shift of beta-catenin staining from the nucleus to the cytoplasm suggesting possible mechanism for the observed decrease in DKK-1 levels during PCa progression. Within metastatic lesions, DKK-1 expression was least abundant in PCa bone metastases relative to all soft tissue PCa metastatic lesions except lymph node metastases. High DKK-1 expression within PCa metastases was further associated with shorter over-all patient survival., Conclusions: Taken together, these data demonstrate that elevated DKK-1 expression is an early event in PCa and that as PCa progresses DKK-1 expression declines, particularly in advanced bone metastases. The decline of DKK-1 in bone metastases can unmask Wnts' osteoblastic activity. These data support a model in which DKK-1 is a molecular switch that transitions the phenotype of PCa osseous lesions from osteolytic to osteoblastic., ((c) 2008 Wiley-Liss, Inc.)

Background: Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models., Methods: The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model., Result: The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone., Conclusion: The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa., ((c) 2008 Wiley-Liss, Inc.)

Background: Metastases to bone are a frequent complication of human prostate cancer and result in the development of osteoblastic lesions that include an underlying osteoclastic component. Previous studies in rodent models of breast and prostate cancer have established that receptor activator of NF-kappaB ligand (RANKL) inhibition decreases bone lesion development and tumor growth in bone. RANK is essential for osteoclast differentiation, activation, and survival via its expression on osteoclasts and their precursors. RANK expression has also been observed in some tumor cell types such as breast and colon, suggesting that RANKL may play a direct role on tumor cells., Methods: Male CB17 severe combined immunodeficient (SCID) mice were injected with PC3 cells intratibially and treated with either PBS or human osteprotegerin (OPG)-Fc, a RANKL antagonist. The formation of osteolytic lesions was analyzed by X-ray, and local and systemic levels of RANKL and OPG were analyzed. RANK mRNA and protein expression were assessed on multiple prostate cancer cell lines, and events downstream of RANK activation were studied in PC3 cells in vitro., Results: OPG-Fc treatment of PC3 tumor-bearing mice decreased lesion formation and tumor burden. Systemic and local levels of RANKL expression were increased in PC3 tumor bearing mice. PC3 cells responded to RANKL by activating multiple signaling pathways which resulted in significant changes in expression of genes involved in osteolysis and migration. RANK activation via RANKL resulted in increased invasion of PC3 cells through a collagen matrix., Conclusion: These data demonstrate that host stromal RANKL is induced systemically and locally as a result of PC3 prostate tumor growth within the skeleton. RANK is expressed on prostate cancer cells and promotes invasion in a RANKL-dependent manner., ((c) 2007 Wiley-Liss, Inc.)

Background: Although paclitaxel is used for hormone-resistant prostate cancer, relapse definitely occurs later. Details of the molecular mechanism responsible for paclitaxel- resistance remain unclear., Methods: We established paclitaxel-resistant cells, DU145-TxR and PC-3-TxR from parent DU145 and PC-3. To characterize these cells, we examined cross-resistance to other anticancer drugs. Expression of several potential genes that had been related to drug-resistance was compared with parent cells by RT-PCR and Western blotting. Methylation analysis of multiple drug resistance (MDR1) promoter was carried out using bisulfite-modified DNA from cell lines. Knockdown experiments using small interfering RNA (siRNA) were also performed to confirm responsibility of drug-resistance. Finally, cDNA microarray was performed to quantify gene expression in PC-3 and PC-3-TxR cells., Results: The IC(50) for paclitaxel in DU145-TxR and PC-3-TxR was 34.0- and 43.4-fold higher than that in both parent cells, respectively. Both cells showed cross-resistance to some drugs, but not to VP-16 and cisplatin. Methylation analysis revealed that methylated CpG sites of MDR1 promoter in DU145 and PC-3 cells were demethylated in DU145-TxR cells, but not in PC-3-TxR cells. Knockdown of P-glycoprotein (P-gp), which was up-regulated in resistant cells, by MDR-1 siRNA restored paclitaxel sensitivity in DU145-TxR but not in PC-3-TxR, indicating that up-regulation of P-gp was not always main cause of paclitaxel-resistance. Microarray analysis identified 201 (1.34%) up-regulated genes and 218 (1.45%) out of screened genes in PC-3-TxR., Conclusions: Our data will provide molecular mechanisms of paclitaxel-resistance and be useful for screening target genes to diagnose paclitaxel sensitivity.

Background: Diminished expression of Raf kinase inhibitor protein (RKIP), an inhibitor of the Raf signaling cascade, promotes prostate cancer (PCa) metastasis in a murine model, suggesting that it is a metastasis suppressor gene. However, the prognostic significance of RKIP expression and its association with metastasis in PCa patients is unknown., Methods: To investigate RKIP protein expression is a prognostic marker in PCa we performed immunohistochemical staining for RKIP expression in tissue microarrays consisting of 758 non-neoplastic prostate tissues, primary tumors and metastases from 134 PCa patients. The Cox proportional-hazards model was used to adjust for covariates including Gleason score, tumor volume, tumor weight, clinical stage, digital rectal exam findings, serum PSA level and surgical margins., Results: RKIP expression was low in approximately 5%, 48%, and 89% of non-neoplastic prostate, primary tumors and metastases, respectively. Low RKIP expression in primary tumors was a strong positive predictive factor for PCa recurrence based on PSA levels. In patients whose primary tumors expressed high RKIP levels, the 7-year PSA recurrence rate was <10%; whereas in patients with tumors with low RKIP expression the recurrence rate was 50% (P<0.001). Multivariate analysis revealed RKIP was an independent prognostic factor (P<0.001)., Conclusion: In contrast to increased expression of pro-tumorigenic genes, these results demonstrate decreased protein expression of a gene, for example, RKIP, can serve as a prognostic marker in PCa patients., (Copyright (c) 2005 Wiley-Liss, Inc.)

Background: Current animal models of prostate cancer (CaP) bone metastasis do not allow measurement of either tumor growth in bone over time or activation of gene promoters in intraosseous tumors. To develop these methods, we used bioluminescent imaging (BLI) to determine if expression of receptor activator of NF-kappaB ligand (RANKL), a pro-osteoclastogenic factor that promotes CaP bone metastases, is modulated by the bone matrix protein transforming growth factor-beta (TGF-beta) in vivo., Methods: C4-2B human CaP cells were treated with TGF-beta in vitro and RANKL mRNA and protein production were measured by polymerase chain reaction (PCR) and ELISA, respectively. Then C4-2B cells stably transfected with the RANKL promoter driving luciferase (lux) were injected intra-tibially into severe combined immundeficient (SCID) mice. Tumors were subjected to BLI every 2 weeks for 6 weeks and serum prostate specific antigen (PSA) was measured using ELISA. Vehicle (V), 1,25 dihydroxyvitamin D (VitD), or TGF-beta was administered to mice with established tumors and BLI to measure RANKL promoter activity was performed. Tumors were then subjected to immunohistochemistry for lux and assayed for RANKL mRNA levels., Results: TGF-beta induced RANKL protein and mRNA expression and activated the RANKL promoter activity in a dose-dependent manner in vitro. BLI demonstrated an increase in intraosseous tumor size over time, which correlated with serum PSA levels. Administration of TGF-beta and VitD to mice with established intraosseous tumors increased lux activity compared to V. Intratibial tumor RANKL mRNA expression paralleled the increased promoter activity. Immunohistochemistry confirmed the presence of lux in the intraosseous tumors., Conclusions: These results demonstrate the ability to measure intraosseous tumor growth over time and gene promoter activation in an established intraosseous tumor in vivo and also demonstrate that TGF-beta induces activates the RANKL promoter. These results provide a novel method to explore the biology of CaP bone metastases., (Copyright 2004 Wiley-Liss, Inc.)

Background: Cyclooxygenase (COX) -2, an inducible isoform of COX, has been observed to be expressed in prostate cancer. Several studies have reported that COX-2 overexpression is associated with carcinogenesis, cell growth, angiogenesis, apoptosis, and invasiveness in a variety of tumor types., Methods: To investigate the function of COX-2 in prostate cancer directly, we stably transfected human full-length COX-2 cDNA into LNCaP cells (LNCaP-COX-2), which express low levels of endogenous COX-2., Results: The level of COX-2 mRNA and protein and the COX activity in COX-2 LNCaP-COX-2 cells was significantly increased compared with parent and control-transfected cells. Overexpression of COX-2 increased both proliferation in vitro and tumor growth rate in vivo. However, the pro-tumor effect was neither associated with changes of androgen receptor (AR) expression level nor AR activity. Furthermore, addition of the major metabolites of COX-2-mediated arachidonic acid metabolism did not alter the proliferation of LNCaP-COX-2 cells in vitro. LNCaP-COX-2 cells had increased secretion of vascular endothelial growth factor (VEGF) protein, suggesting that angiogenesis induced by COX-2 stimulates tumor growth in vivo., Conclusion: These data demonstrate that COX-2 contributes to prostate cancer progression and suggest that it mediates this effect, in part, through increased VEGF., (Copyright 2002 Wiley-Liss, Inc.)

Background: Progressive prostate cancer typically metastasizes to bone where prostate cancer cells gain an osteoblast-like phenotype and induce osteoblastic metastases through unknown mechanisms. To investigate the biology of prostate cancer skeletal metastases, we compared gene expression between the non-metastatic LNCaP cell line and its derivative cell line C4-2B that metastasizes to bone., Methods: Total RNA from LNCaP and C4-2B cell lines was isolated and used to probe membrane-based gene arrays (Comparison 1). Additionally, LNCaP cells were incubated in the absence or presence of conditioned media (CM) from a human osteoblast-like cell line (HOBIT) and total RNA from these cells was used to probe gene arrays (Comparison 2). Differential expression of genes was confirmed by RT-PCR., Results: Of the 1,176 genes screened, 35 were differentially expressed between LNCaP and C4-2B cells (Comparison 1). HOBIT-CM induced differential expression of 30 genes in LNCaP cells (Comparison 2). Interestingly, 19 genes that were differentially expressed in C4-2B vs. LNCaP also displayed a similar expression pattern in LNCaPs grown in HOBIT-CM. These genes are primarily involved in motility, metabolism, signal transduction, tumorigenesis, and apoptosis., Conclusions: These results suggest that osteoblasts produce soluble factors that contribute to the progression of prostate cancer skeletal metastases, including their transition to an osteoblast-like phenotype. Additionally, these data provide targets to explore for further investigations towards defining the biology of skeletal metastases., (Copyright 2002 Wiley-Liss, Inc.)

Background: Despite clinical associations and in vitro data suggesting that autocrine interleukin-6 (IL-6) production contributes to prostate cancer progression or chemotherapy resistance, there have been no reports that explore the role of IL-6 on prostate tumors in vivo. In the present study, we investigated the effect of IL-6 inhibition on the growth of human prostate cancer xenografts in nude mice., Methods: To determine if autocrine IL-6 production contributes to prostate cancer growth and chemotherapy resistance in vivo, xenografts of a human prostate cancer cell line that produces IL-6 (PC-3) were established in nude mice. The mice were randomly divided into four treatment groups: (1) saline (vehicle control) + murine IgG (isotype control); (2) etoposide + murine IgG; (3) saline + anti-IL-6 monoclonal antibody; and (4) etoposide + anti-IL-6 monoclonal antibody. Tumors were measured twice weekly during a 4-week treatment period. At the conclusion of the study, all mice were sacrificed, and in addition to final volume, tumors were evaluated for the degree of apoptosis by TUNEL analysis., Results: Anti-IL-6 Ab (with saline or etoposide) induced tumor apoptosis and regression ( approximately 60% compared to initial tumor size). Etoposide alone did not induce tumor regression or apoptosis in this animal model, and there was no synergy between anti-IL-6 Ab and etoposide., Conclusions: These studies suggest that IL-6 contributes to prostate cancer growth in vivo, and that targeting IL-6 may contribute to prostate cancer therapy., (Copyright 2001 Wiley-Liss, Inc.)

Background: Prostate cancer frequently metastasizes to bone. However, unlike many other tumors that produce osteolytic lesions, prostate cancer produces osteoblastic lesions through unknown mechanisms. In the current study, we explored the ability and mechanism of an osteotropic prostate cancer cell line (C4-2B) to induce mineralization., Methods: C4-2B cells were grown in promineralization media. Mineral deposition was characterized using von Kossa staining, calcium retention, alizarin red staining, Raman spectroscopy, and electron microscopy. Expression of osteoblast-related proteins was determined by RT-PCR. The nuclear level of the bone-specific transcription factor Cbfa1 was determined using western analysis and the effect of inhibiting Cbfa1 function, using a "decoy" Cbfa1 response element oligo, on mineralization was determined., Results: The studies demonstrated that C4-2B cells, but not its nonosteotropic parent cell line LNCaP, has an osteoblastlike phenotype including production of alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteoprotegerin (OPG), and OPG ligand. Most importantly, the C4-2B cells produced hydroxyapatite mineral in vitro. Furthermore, C4-2B cells expressed high nuclear levels of the bone-specific transcription factor Cbfa1, compared to LNCaP cells, which accounts for their ability to produce bone-specific proteins. Inhibition of Cbfa1, using decoy DNA Cbfa1 response elements, abrogated the ability of C4-2B to produce mineral. Finally, we determined that C4-2B cells express bone morphogenic protein-7, a known inducer of Cbfa1 expression., Conclusions: These data demonstrate a novel mechanism through which prostate cancer cells may directly contribute to the osteoblastic component that characterize their skeletal metastatic lesions. Prostate 47:212-221, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Background: One of the mechanisms through which prostate cancers relapse during anti-androgen therapy may involve adaptation to low concentrations of androgen induced by anti-androgen therapies. Recent studies from our laboratory have reported that tumor necrosis factor-alpha (TNFalpha) is secreted from prostate cancer epithelial cells and LNCaP cells. We hypothesized that TNFalpha changes androgen-sensitivity in LNCaP cells., Methods: We cultured LNCaP cells for more than 3 months in the presence of 50 ng/ml TNFalpha and established TNFalpha-resistant LNCaP cells (LN-TR2). Sensitivity to androgen was examined by the cell proliferation assay. We also transfected LNCaP and LN-TR2 cells with a luciferase reporter plasmid driven by prostate-specific antigen (PSA) promoter and compared PSA promoter activity. Nuclear localization of AR protein that binds to target genes was also examined by Western blotting., Results: LN-TR2 cells had increased sensitivity to dihydrotestosterone (DHT) (i.e., proliferation and PSA promoter activation) than LNCaP cells. Total AR mRNA and AR protein levels were decreased in LN-TR2 cells. However, LN-TR2 cells demonstrated increased levels of nuclear AR compared to LNCaP cells. At 1 nM DHT, the anti-androgen bicalutamide stimulated LN-TR2 and inhibited LNCaP proliferation., Conclusions: Long-term exposure of TNFalpha causes hypersensitivity to DHT in LNCaP and this was associated with increased nuclear AR protein. Furthermore, hypersensitivity to androgen caused anti-androgen withdrawal phenomenon in the presence of DHT although bicalutamide itself did not stimulate LNCaP proliferation without androgen. This result may be one possible mechanism for the anti-androgen withdrawal phenomenon., (Copyright 2001 Wiley-Liss, Inc.)