Publications by authors named "Stephanie Filleur"

25 Publications

  • Page 1 of 1

The Wnt non-canonical signaling modulates cabazitaxel sensitivity in prostate cancer cells.

PLoS One 2020 2;15(6):e0234078. Epub 2020 Jun 2.

Department of Urology, Texas Tech University-Health Sciences Center, Lubbock, Texas, United States of America.

Background: Despite new drugs, metastatic prostate cancer remains fatal. Growing interest in the latest approved cabazitaxel taxane drug has markedly increased due to the survival benefits conferred when used at an earlier stage of the disease, its promising new therapeutic combination and formulation, and its differential toxicity. Still cabazitaxel's mechanisms of resistance are poorly characterized. The goal of this study was thus to generate a new model of acquired resistance against cabazitaxel in order to unravel cabazitaxel's resistance mechanisms.

Methods: Du145 cells were cultured with increasing concentrations of cabazitaxel, docetaxel/ taxane control or placebo/age-matched control. Once resistance was reached, Epithelial-to-Mesenchymal Translation (EMT) was tested by cell morphology, cell migration, and E/M markers expression profile. Cell transcriptomics were determined by RNA sequencing; related pathways were identified using IPA, PANTHER or KEGG software. The Wnt pathway was analyzed by western blotting, pharmacological and knock-down studies.

Results: While age-matched Du145 cells were sensitive to both taxane drugs, docetaxel-resistant cells were only resistant to docetaxel and cabazitaxel-resistant cells showed a partial cross-resistance to both drugs concomitant to EMT. Using RNA-sequencing, the Wnt non-canonical pathway was identified as exclusively activated in cabazitaxel resistant cells while the Wnt canonical pathway was restricted to docetaxel-resistant cells. Cabazitaxel-resistant cells showed a minimal crossover in the Wnt-pathway-related genes linked to docetaxel resistance validating our unique model of acquired resistance to cabazitaxel. Pharmacological and western blot studies confirmed these findings and suggest the implication of the Tyrosine kinase Ror2 receptor in cabazitaxel resistant cells. Variation in Ror2 expression level altered the sensitivity of prostate cancer cells to both drugs identifying a possible new target for taxane resistance.

Conclusion: Our study represents the first demonstration that while Wnt pathway seems to play an important role in taxanes resistance, Wnt effectors responsible for taxane specificity remain un-identified prompting the need for more studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234078PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266300PMC
August 2020

Effect of Serum Deprivation Stress on Signal Induction Regulatory Protein-Alpha (SIRP-Alpha)-Mediated Erythrophagocytosis by Macrophages.

Med Sci Monit Basic Res 2019 03 21;25:100-106. Epub 2019 Mar 21.

Department of Urology, Texas Tech University Health Sciences Center, Lubbock, TX, USA.

BACKGROUND Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome that involves loss of macrophages' self-cells recognition resulting in auto-phagocytosis of erythrocytes, leukocytes, and platelets and leading to multi-system effects. The pathogenesis of HLH is unclear but can be explained by malfunction of the physiologic inhibitory pathway through interaction between macrophage SIRP-alpha and erythrocyte CD 47. The goal of the present study was to evaluate if erythrocytes phagocytosis occurs as a result of altered macrophage SIRP-alpha expression during inflammatory/stressful conditions as seen in HLH. MATERIAL AND METHODS RAW264.7 macrophages were cultured in serum-free media (SFM) and complete media (CM) to simulate stressful and physiologic conditions, respectively. CD47+ mouse erythrocytes were used to test interactions with macrophages at different stages. SIRP-alpha expressions and phagocytosis assays were measured and analyzed at different steps. The study was in vitro and used murine cells to simulate in vivo human interactions. RESULTS SIRP-alpha expressions and phagocytosis rates were higher in SFM compared to CM. Interestingly, after adding SIRP-alpha blocking antibodies (Ab), phagocytosis rates significantly decreased. CONCLUSIONS Serum deprivation and LPS/INF-Gamma induction resulted in increased SIRP-alpha expression and erythrophagocytosis. Using SIRP-alpha Ab during this condition decreased the rate of erythrophagocytosis, which indicates that SIRP-alpha receptor can have pro-phagocytic activity.
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http://dx.doi.org/10.12659/MSMBR.912946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441304PMC
March 2019

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with various efficacy and toxicity.

Prostate 2018 09 10;78(12):905-914. Epub 2018 May 10.

Department of Urology, Texas Tech University-Health Sciences Center, Lubbock, Texas.

Background: Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re-evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti-tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti-angiogenic and anti-neoplastic agent.

Methods: CRPC cells undergoing Taxane treatment were evaluated for cell proliferation, migration and death, and apoptosis using crystal violet staining, chemotaxis, cell cycle, and TUNEL assays. In vitro data were corroborated in CL1 CRPC xenografts where mice received intermittent or metronomic low-doses cabazitaxel ± PEDF.

Results: We found that cabazitaxel inhibits the proliferation of CRPC cells with a higher efficacy than docetaxel in vitro. As expected, high-doses of Taxanes blocked the cells in mitosis. Surprisingly, low-doses of cabazitaxel induced more cell death than docetaxel mainly through apoptosis. In vivo, intermittent cabazitaxel lead to disease stabilization when combined with PEDF. Unexpectedly, low-doses of cabazitaxel delayed tumor growth with severe toxicity for some of the doses tested. Other results showed that PEDF and low-doses of cabazitaxel combination inhibited the migration of tumor cell and increased the tumoricidal activity of macrophages toward prostate tumor cells.

Conclusions: Our findings highlight the great promise of cabazitaxel drug and predict a possible move of cabazitaxel forward within the therapeutic sequence of prostate cancer.
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http://dx.doi.org/10.1002/pros.23647DOI Listing
September 2018

Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche.

Nat Commun 2017 11 6;8(1):1319. Epub 2017 Nov 6.

Department of Cancer Biology, University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA.

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These "non-metastatic" exosomes stimulate an innate immune response through the expansion of Ly6C patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.
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http://dx.doi.org/10.1038/s41467-017-01433-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673063PMC
November 2017

Novel porcine model for calcium oxalate stone formation.

Int Urol Nephrol 2017 Oct 13;49(10):1751-1761. Epub 2017 Jul 13.

Department of Urology, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 7260, Lubbock, TX, 79430-7260, USA.

Background: Mechanisms for calcium-based stone formation are not clearly delineated. Porcine are the most anatomically and physiologically congruent mammal to humans. Our objectives were to develop a cost-effective and easily reproducible porcine model for the study of calcium-based nephrolithiasis.

Methods: Crossbred male pigs (n = 16) were assigned randomly to one of the following treatments: (1) control; (2) ethylene glycol (EG) + vitamin D (VD); (3) EG + ammonium chloride (AC); (4) EG + gentamicin (G); (5) EG + Lasix; (6) EG + VD + AC; (7) EG + VD + G. Treatments were administered for 28 days; blood and urine were collected on day 0, 14, and 28. At the endpoint of the study, renal tissue was collected for gross and microscopic analysis of crystal stone formation and inflammation.

Results: Stone-forming parameters were observed in serum and urine. For control versus all other treatments, by day 28, serum BUN and creatinine were less (P < 0.01), urinary creatinine, citrate and pH were greater (P < 0.01), and urinary oxalate was less (P < 0.01). Histopathological analysis of H&E staining and stone analysis revealed formation of calcium oxalate stones and crystal formation within the renal cortex and medulla for all animals except control. Nephrotoxicity was observed in one animal from treatment EG + G.

Conclusions: The treatments explored in this experiment provided novel examples of cost-effective porcine models for the study of nephrolithiasis. EG + VD had the strongest indicators of nephrolithiasis without nephrotoxicity.
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http://dx.doi.org/10.1007/s11255-017-1657-0DOI Listing
October 2017

PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells in vitro.

PLoS One 2017 12;12(4):e0174968. Epub 2017 Apr 12.

Department of Urology, Texas Tech University-Health Sciences Center, Lubbock, Texas, United States of America.

Background: Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis.

Methods: RAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry.

Results: We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide.

Conclusions: These data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174968PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389654PMC
April 2017

Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy.

Biol Methods Protoc 2017 Jan 9;2(1):bpx002. Epub 2017 Mar 9.

Department of Urology, Texas Tech University-Health Sciences Center, Lubbock, Texas 79430, USA.

Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for methodologies to study the role of cell-cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.
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http://dx.doi.org/10.1093/biomethods/bpx002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994062PMC
January 2017

The clinical usefulness of nuclear matrix protein-22 in patients with end-stage renal disease and microscopic hematuria.

Ren Fail 2013 14;35(1):72-6. Epub 2012 Nov 14.

Department of Urology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-726, USA.

Objectives: To evaluate the sensitivity, specificity, and false-positive rate of the nuclear matrix protein-22 (NMP22) test in patients with end-stage renal disease (ESRD) and microscopic hematuria in order to avoid unnecessary follow-up tests for patients with false-positive NMP22 test results.

Patients And Methods: Patients with ESRD were screened for microscopic hematuria as part of the pre-transplant workup. Patients with documented microscopic hematuria underwent workup as recommended by the American Urological Association.

Results: Between January 2006 and April 2012, 277 patients with ESRD were referred to the Department of Urology for pre-transplant evaluation. Fifty-seven (22.6%) patients were found to have microscopic hematuria and underwent further testing. Nineteen (33.3%) patients demonstrated a positive NMP22 test result and 38 (66.7%) had a negative NMP22 test result. The false-positive rate was 32.7%. The sensitivity and specificity of the NMP22 test in this patient population were 50% and 67%, respectively. The positive predictive value of the test was 52.6% and the negative predictive value 97.3%. Especially noteworthy, the two detected transitional cell cancers of the urinary bladder were both demonstrated during cystoscopy, independent of their NMP22 or urine cytology test result.

Conclusions: Our study revealed a significantly increased NMP22 test false-positive rate, low sensitivity, and specificity in the setting of high prevalence of microscopic hematuria, proteinuria, and low glomerular filtration rate in patients with ESRD. Therefore, cystoscopy remains the gold standard for patients with ESRD and microscopic hematuria for pre-transplant evaluation.
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http://dx.doi.org/10.3109/0886022X.2012.741648DOI Listing
July 2013

Positive correlation between PEDF expression levels and macrophage density in the human prostate.

Prostate 2013 Apr 4;73(5):549-61. Epub 2012 Oct 4.

Department of Urology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6591, USA.

Background: In this study, we investigated the capacity of pigment epithelium-derived factor (PEDF) to modulate the recruitment and the differentiation of monocytes/macrophages both in vitro and in human prostate.

Methods: Using Boyden chambers, we assessed PEDF effect on the migration of monocytes and chemically activated RAW 264.7 macrophages. Normal, prostatitis, and prostate cancer specimens were retrospectively selected and examined by immunohistochemistry for PEDF expression and infiltration of immune CD68 + macrophagic cells. PEDF expression and macrophage density were then correlated with each other and clinicopathological parameters. M1 and M2 differentiation markers were quantified by qRT-PCR, Western blotting, and ELISA.

Results: In chemotaxis, PEDF induced the migration of monocytes/macrophages. In immunohistochemistry, macrophages were markedly increased in prostatitis and malignant compared to normal tissues. PEDF was expressed at variable levels in the stroma and epithelium. PEDF mRNA was down-regulated in both prostate cancer and prostatitis compared to normal tissues. In correlation studies, macrophage density and PEDF expression were respectively positively and negatively associated with prostate size. Most importantly, PEDF expression positively correlated with macrophage density. Finally, PEDF stimulated the expression of iNOS, IL12, and TNFα; and inhibited IL10 and arginase 1 in mouse and human macrophages confirming a M1-type differentiation.

Conclusions: Our data demonstrate that PEDF acts directly on monocytes/macrophages by inducing their migration and differentiation into M1-type cells. These findings suggest a possible role of macrophages in PEDF anti-tumor properties and may support further development of PEDF-based anti-cancer therapy.
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http://dx.doi.org/10.1002/pros.22595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600115PMC
April 2013

AFM nano-mechanics and calcium dynamics of prostate cancer cells with distinct metastatic potential.

Biochim Biophys Acta 2012 Jul 16;1820(7):1111-20. Epub 2012 Feb 16.

Department of Physics, Texas Tech University, Box 41051, Lubbock, TX 79409, United States.

Background: Despite recent advances, it is not clear to correlate the mechanical compliances and the metastatic potential of cancer cells. In this study, we investigated combined signatures of mechanical compliances, adhesions, and calcium dynamics correlated with the metastatic potential of cancer cells.

Scope Of Review: We used the lowly (LNCaP) and highly (CL-1, CL-2) metastatic human prostate cancer cells. The AFM-based nanomechanics was performed to determine the elastic moduli and the cell-to-substrate adhesion. The intracellular calcium dynamics was evaluated by fluorescence spectroscopy. Cell migration and the distribution of cytoskeleton were evaluated using the wounded monolayer model and immunofluorescence, respectively. The elastic moduli, the calcium dynamics, and the migratory ability are greater in CL-1 and CL-2 than LNCaP. CL-1 and CL-2 also display a significantly larger area of cell-to-substrate adhesions while the LNCaP displays a limited adhesion. These properties were slightly reduced in CL-2 compared with CL-1 cells. The enhanced elastic moduli and calcium dynamics found in CL-1 and CL-2 can be consistently explained by the intensified tensile stress generated by actin cytoskeletons anchored at more focal adhesion sites.

Major Conclusions: Although the suppressed mechanical compliance of highly metastatic cells may not support the enhanced cancer metastasis, the enhanced adhesion and calcium dynamics are favorable for invasion and extra-vasation required for malignant progression.

General Significance: Our results suggest that the mechanical compliance alone may fail to indicate the metastatic progression, but the combined biomechanical signatures of mechanical compliance, adhesion, and calcium dynamics can provide critical clues to determine the metastatic potential of cells.
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http://dx.doi.org/10.1016/j.bbagen.2012.02.006DOI Listing
July 2012

PEDF inhibits IL8 production in prostate cancer cells through PEDF receptor/phospholipase A2 and regulation of NFκB and PPARγ.

Cytokine 2011 Aug 13;55(2):202-10. Epub 2011 May 13.

Department of Urology, Texas Tech University-Health Sciences Center, Lubbock, TX 79430, USA.

Interleukin-8 (IL8/CXCL8) has been described as a key effector in prostate cancer progression and resistance to standard chemotherapeutic drugs. In the present study, we investigated the effect of the natural, angio-inhibitory and anti-tumoral Pigment Epithelium-Derived Factor (PEDF) on the expression of IL8 cytokine by prostate cancer cells. Using a cytokine antibody array and ELISA, in addition to IL8 quantitative RT PCR, we showed that PEDF inhibits the production of IL8 in human hormone-refractory prostate cancer cells, and delays the growth of these cells in vitro. IL8 reduction was mimicked in cancer cells treated with PPARγ agonist and NFκB-specific inhibitors. Accordingly, PPARγ expression increased in response to PEDF, whereas RelA/p65 expression and nuclear localization, and NFκB transcriptional activity decreased. NFκB deactivation was reversed by the PPARγ antagonist GW9662 and PPARγ (Leu(468)/Glu(471)) dominant negative suggesting a PPARγ-dependent process. We also investigated PEDF Receptor/PLA2 as key player in this pathway by small interference RNA. PEDFR knock down in prostate cancer cells reversed PEDF-induced PPARγ up-regulation, and NFκB and IL8 inhibition compared to non-targeting control siRNA. We conclude that by binding to PEDFR, PEDF up-regulates PPARγ, leading subsequently to suppressed NFκB-mediated transcriptional activation, reduced production of IL8 and limited proliferation of prostate cancer cells. These results reinforce PEDF's therapeutic potential and imply that blocking IL8 could represent a novel alternative for prostate cancer treatment.
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http://dx.doi.org/10.1016/j.cyto.2011.04.010DOI Listing
August 2011

INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling.

Cancer Cell Int 2009 Nov 11;9:28. Epub 2009 Nov 11.

Texas Tech University Health Sciences Center, Department of Urology, Lubbock, TX, USA.

Background: The gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells.

Results: Markedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1.

Conclusion: These results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.
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http://dx.doi.org/10.1186/1475-2867-9-28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779787PMC
November 2009

Clinical outcome of patients with docetaxel-resistant hormone-refractory prostate cancer treated with second-line cyclophosphamide-based metronomic chemotherapy.

Med Oncol 2010 Jun 14;27(2):363-7. Epub 2009 Apr 14.

Department of Urology, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 7260, Lubbock, TX 79430-7260, USA.

For patients with docetaxel-resistant hormone-refractory prostate cancer (HRPC) no standard chemotherapeutic treatment exists. In this study, we evaluate the efficacy of cyclophosphamide (CP)-based metronomic chemotherapy in this patient population. Patients with metastatic HRPC with disease progression under docetaxel-based chemotherapy were eligible. The primary endpoint was prostate-specific antigen (PSA) response. Secondary endpoints were survival and toxicity. Low-dose CP (50 mg/d) and dexamethasone (1 mg/d) were administered orally in a metronomic manner. Treatment was continued until disease progression or intolerable side effects occurred. Seventeen patients were enrolled in this study. The median follow-up was 12 weeks (range: 4-60). Median age was 68 years (range: 42-85). Median PSA at study entry was 134 ng/ml (range: 46.0-6554). Nine patients had a PSA response (median 44.4%), four patients >or=50% and five patients <50%. Eight patients had a PSA progression. Overall survival was 24 months. Five patients reported a decrease in bone pain after 4 weeks' treatment. No grade 3 and 4 toxicities were noted. In this study, low-dose metronomically administered CP demonstrated efficacy as a second-line treatment in patients with docetaxel-resistant HRPC. The treatment was well tolerated and almost without toxicity. Further advantages of low-dose CP were its convenient oral administration, dosing schedule, low cost, and low-toxicity profile. These attributes in combination with immunoregulatory and antiangiogenic potentials make CP also a prime candidate for combination with other treatment regimens.
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http://dx.doi.org/10.1007/s12032-009-9218-8DOI Listing
June 2010

Pigment epithelium-derived factor and interleukin-6 control prostate neuroendocrine differentiation via feed-forward mechanism.

J Urol 2008 Jun 23;179(6):2427-34. Epub 2008 Apr 23.

Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.

Purpose: PEDF (pigment epithelium-derived factor) promotes the differentiation and survival of neuronal cells, and expands the adult neuronal stem cell niche. In the prostate PEDF is suppressed by androgen with unclear physiological consequences. We report that PEDF induced the neuroendocrine differentiation of prostate cancer cells, which was accompanied by neurite outgrowth and chromogranin A expression.

Materials And Methods: We performed neuroendocrine differentiation assay, Western blot analysis, immunostaining and reverse transcriptase-polymerase chain reaction in the human prostate cancer cell lines LNCaP, PC-3 and DU145, and the prostate epithelial strain RWPE-1 (ATCC).

Results: Ectopic and endogenous PEDF caused neuroendocrine differentiation of prostate cancer cells, as manifested by neurite-like outgrowths and chromogranin A expression. The transdifferentiated cells expressed axonal and dendritic markers, as ascertained by immunoblotting for specific markers. Neuroendocrine cells formed multiple synaptophysin positive protrusions resembling dendritic spines and vesicles containing serotonin, pointing to possible synapse formation. The known transdifferentiating agent interleukin-6 induced PEDF secretion. Moreover, PEDF neutralizing antibodies abolished the transdifferentiation of interleukin-6 treated cells, suggesting an autocrine loop. Neurogenic events were independent of cyclic adenosine monophosphate. Instead, PEDF activated in this order RhoA, nuclear factor kappaB and Stat3. Inhibitors of the Rho, nuclear factor kappaB and STAT pathways abolished differentiation and synapse formation. Additionally, nuclear factor kappaB activation caused interleukin-6 expression.

Conclusions: We discovered that nuclear factor kappaB controls the formation of neuronal communications in the prostate due to PEDF. We defined a feed-forward loop, in which nuclear factor kappaB induction elicits Stat3 activation and pro-differentiating interleukin-6 expression causes the further expansion of neuroendocrine communications. Our findings point to the role of nuclear factor kappaB and PEDF in coordinated prostate development.
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http://dx.doi.org/10.1016/j.juro.2008.01.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849797PMC
June 2008

Impact of PSA flare-up in patients with hormone-refractory prostate cancer undergoing chemotherapy.

Int Urol Nephrol 2008 30;40(1):97-104. Epub 2007 Jun 30.

Department of Urology, Texas Tech University Health Sciences Center, Medical Office Plaza, Suite 260, 3502 9th Street, Lubbock, TX 79415, USA.

Objectives: The intention of this study is to describe the impact and underlying potential basis of the prostate-specific antigen (PSA) flare-up phenomenon in patients with hormone-refractory prostate cancer (HRPC) treated with docetaxel-based chemotherapy.

Methods: We retrospectively identified 74 consecutive patients who received docetaxel/estramustine-based chemotherapy at our institution. Patients were evaluated based on modified criteria from the Prostate-Specific Antigen Working Group regarding survival and toxicity. Additionally, two androgen receptor mutations derived from patients with advanced disease were analyzed for promiscuous transactivation activity.

Results: The 74 patients were stratified into four groups: response, partial response, flare-up-initial PSA elevation, and progression. Median survival in the flare-up group (n=8) was 20 months and did not differ from the response group (p=0.564). The flare-up group showed a maximum PSA elevation from baseline between 3.4 and 28.3% (between three and six weeks) followed by PSA decline >or=50% from the baseline level in seven of the eight patients. The androgen receptor mutations AR(877) and AR(715) displayed a 37.5- and 5.2-fold increase in transactivation activity by progesterone and a 12.6- and 5.4-fold increase by estrogen compared to the AR(WT), respectively.

Conclusions: A considerable portion of HRPC patients experience an initial PSA flare-up under systemic chemotherapy. In this study, occurrence of flare-up phenomenon did not impact survival. Chemotherapy should be continued a minimum of six weeks before removing patients from a docetaxel-based regimen. We showed evidence that co-medication with dexamethasone/prednisolone and/or estramustine itself can induce an initial PSA flare-up via androgen receptor mutations.
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http://dx.doi.org/10.1007/s11255-007-9221-yDOI Listing
September 2008

Androgen receptor targets NFkappaB and TSP1 to suppress prostate tumor growth in vivo.

Int J Cancer 2007 Sep;121(5):999-1008

Department of Urology, Texas Tech University Health Sciences Center, Texas Tech University, Lubbock, TX.

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NFkappaB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NFkappaB suppression, since it was restricted to the cells lacking nuclear (active) NFkappaB. Thus we for the first time identified combined decrease of NFkappaB and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (c) 2007 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/ijc.22802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810747PMC
September 2007

Evaluation of peri-operative peripheral and renal venous levels of pro- and anti-angiogenic factors and their relevance in patients with renal cell carcinoma.

BJU Int 2007 Jul 8;100(1):209-14. Epub 2007 Apr 8.

Department of Urology, Otto-von-Guericke-Universität, Magdeburg, Germany.

Objective: To evaluate peri-operative peripheral and renal venous plasma levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor (TGF)-beta1, endostatin, and thrombospondin-1 (TSP-1) in relation to pathological variables and prognosis, as pro- and anti-angiogenic factors are important for tumour growth and treatment of patients with renal cell carcinoma (RCC).

Patients And Methods: The study included 74 consecutive patients with sporadic RCC who had tumour nephrectomy. Peripheral venous blood was drawn 1 day before, immediately and 1, 3 and 5 days after surgery. Renal venous blood was collected in a subgroup of 33 patients during surgery. The variables were analysed using quantitative enzyme-linked immunoassay kits, and associated with pathological variables and disease-specific survival.

Results: Soon after surgery, peripheral venous VEGF, PDGF-BB and TGF-beta1 levels were decreased, whereas endostatin levels were significantly increased. Renal venous VEGF, PDGF-BB and TGF-beta1 levels were higher than in the general venous blood pool. Renal venous VEGF levels were correlated with tumour diameter and associated with grade and vascular invasion. After a mean follow-up of 30 months, higher peripheral preoperative, early peripheral postoperative and renal venous VEGF levels were associated with a poorer prognosis. However, in a multivariate analysis only Tumour-Node-Metastasis stage and Eastern Cooperative Oncology Group performance status were independent prognosticators of disease-specific survival.

Conclusions: Circulating pro- and anti-angiogenic factors change early after nephrectomy. VEGF, PDGF-BB and TGF-beta1 are higher in the renal vein than in the general venous blood pool. Higher renal venous and peripheral levels of VEGF might be associated with a poorer prognosis.
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http://dx.doi.org/10.1111/j.1464-410X.2007.06871.xDOI Listing
July 2007

A randomized study of docetaxel and dexamethasone with low- or high-dose estramustine for patients with advanced hormone-refractory prostate cancer.

BJU Int 2006 Sep;98(3):580-5

Department of Urology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany.

Objective: To test the combination of docetaxel with two different doses of estramustine in patients with hormone-refractory prostate cancer (HRPC), to improve response rates and to lower side-effects, as docetaxel-based chemotherapy is an increasing option for men with advanced HRPC, and alone or combined with estramustine, docetaxel improves median survival.

Patients And Methods: In all, 72 patients with metastatic HRPC were randomly assigned to receive docetaxel (70 mg/m(2) intravenously, on day 2 every 21 days) and estramustine (3 x 280 mg/day oral starting 1 day before docetaxel, for 5 consecutive days) for arm A, or estramustine (3 x 140 mg/day oral starting 1 day before docetaxel, for 3 consecutive days) for arm B. Premedication with oral dexamethasone at a total daily dose of 16 mg, in divided doses twice a day was administered in arm A on day 1-5 and in arm B on day 1-3. Initially, six cycles were administered. Chemotherapy was restarted after a significant increase in prostate-specific antigen (PSA) level. Patients were monitored for any measurable PSA response and toxicity.

Results: Between the arms there was no statistically significant difference in time to progression and overall survival. However, treatment B had less treatment-related toxicity than A. Independent prognostic variables were baseline factors like PSA level, haemoglobin level, Eastern Cooperative Oncology Group performance status, and bone pain at presentation.

Conclusions: In this randomized phase II study the combination of docetaxel and estramustine had substantial activity in HRPC, with a significant incidence of severe toxicity, both haematological and not. Nevertheless, treatment-related toxicity was predictable and manageable. There was no better effect with a higher dose of estramustine with docetaxel than for a lower dose. There was a slight tendency to higher toxicity for high-dose estramustine but this was not statistically significant. The present results support the assertion that estramustine is not necessary in docetaxel-based treatment regimens.
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http://dx.doi.org/10.1111/j.1464-410X.2006.06324.xDOI Listing
September 2006

Characterization of prognostic factors and efficacy in a phase-II study with docetaxel and estramustine for advanced hormone refractory prostate cancer.

Onkologie 2005 Nov;28(11):573-8

Department of Urology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.

Background: Docetaxel based chemotherapy not only reduces pain and improves quality of life in advanced hormone refractory prostate cancer (HRPC), but it also improves survival. We investigated the combination of docetaxel and estramustine in patients with HRPC regarding efficacy and prognostic parameters.

Patients And Methods: We conducted a phase-II trial, administering docetaxel (70 mg/m(2) i.v., day 2, every 3 weeks) and estramustine (280 mg 3 times daily p.o., 1 day prior to docetaxel, on 5 consecutive days) to patients with HRPC. Patients were monitored for PSA (prostate-specific antigen) response and toxicity.

Results: 62 patients were treated. The median age was 67.5 years, the median PSA was 177.9 ng/ml. The median number of cycles was 6. The median time to progression (TTP) and median survival time were 14 (+/-2) and 24 (+/-5) months, respectively. A = 50% decrease in PSA levels from baseline occurred in 38 (61.3%) patients of whom 25 (40.3%) had a = 75% PSA decrease. The main grade 3-4 hematologic toxicities were neutropenia 34% and anemia 18%.

Conclusions: The combination of docetaxel and estramustine exerts substantial activity in HRPC suggesting an overall survival benefit with manageable toxicity. This trial also demonstrated a survival advantage for patients with early chemotherapeutic intervention. We identified PSA relapse, baseline PSA and hemoglobin as valuable prognostic factors in this setting.
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http://dx.doi.org/10.1159/000088297DOI Listing
November 2005

Two functional epitopes of pigment epithelial-derived factor block angiogenesis and induce differentiation in prostate cancer.

Cancer Res 2005 Jun;65(12):5144-52

Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs' three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L19VEEED24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH2-kinase-dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34- and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDF-based tumor therapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-3744DOI Listing
June 2005

Human breast tumors override the antiangiogenic effect of stromal thrombospondin-1 in vivo.

Int J Cancer 2005 Sep;116(5):686-91

INSERM Research Unit 664, Laënnec School of Medicine, Lyon, France.

The antiangiogenic extracellular matrix protein thrombospondin-1 (TSP-1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP-1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP-1 in breast cancer, we first used a breast tumorigenesis model in which tumor-associated stromal fibroblasts were engineered to produce high levels of TSP-1. We demonstrate here that stromal TSP-1 delayed human MDA-MB-231/B02 breast tumor growth. However, this delay in MDA-MB-231/B02 tumor growth upon exposure to TSP-1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP-1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP-1. TSP-1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse-free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP-1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP-1, leading to disease progression.
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http://dx.doi.org/10.1002/ijc.20584DOI Listing
September 2005

Androgens repress the expression of the angiogenesis inhibitor thrombospondin-1 in normal and neoplastic prostate.

Cancer Res 2005 Jan;65(1):300-8

Institut National de la Sante et de la Recherche Medicale, Research Unit 403, Laënnec School of Medicine, Lyon, France.

In order to understand why the angiogenesis inhibitor thrombospondin-1 (TSP1) is often, although not always, associated with prostatic tumors, we have investigated its relationship with the testosterone and the vasculature on which both normal and tumorigenic prostatic epithelia depend. In vivo, androgen withdrawal led to increased TSP1 production and decreased vascularization in the normal rat prostate which was reversed by androgen replacement. Androgen repression of TSP1 production occurred at the transcriptional level and was dependent on the presence of the first intron of the TSP1 gene. In an experimental model of prostate tumorigenesis, TSP1, when delivered by admixed stromal fibroblasts, markedly delayed LNCaP tumor growth and limited tumor vascularization. However, prolonged exposure to TSP1 resulted in the growth of tumors secreting high levels of vascular endothelial growth factor in the bloodstream of tumor-bearing animals and tumor growth was no longer sensitive to TSP1 inhibitory effects. Clinical evidence also suggested that prostate carcinomas are able to adapt to escape the antiangiogenic effects of TSP1. In human androgen-dependent localized prostate carcinomas, TSP1 expression was inversely correlated with blood vessel density. Androgen deprivation in patients with hormone-responsive tumors led to increased TSP1 expression and vascular regression. In contrast, despite a sustained expression in the tumor bed, TSP1 was no longer associated with decreased vascularization in hormone-refractory prostate tumors. Overall, these results suggest that the high in situ TSP1 exposure triggered by androgen deprivation in patients with prostate cancer could lead to early tumor resistance. Such patients could benefit from a combination of androgen deprivation and antiangiogenic therapy in order to minimize the induction of such tumor escape.
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January 2005

Nuclear factor of activated T cells balances angiogenesis activation and inhibition.

J Exp Med 2004 Jun;199(11):1513-22

Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial-derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis. We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export. We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain-like interleukin 1beta-converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.
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http://dx.doi.org/10.1084/jem.20040474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211785PMC
June 2004

SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth.

Cancer Res 2003 Jul;63(14):3919-22

CNRS UPR 9079, 94801 Villejuif, France.

In the past few years, several laboratories have developed antiangiogenic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate.
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July 2003

Bisphosphonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats.

Cancer Res 2002 Nov;62(22):6538-44

Institut National de la Santé et de la Recherche Médicale, Research Unit 403, Faculty of Medicine Laënnec, Lyon 69372, France.

Bisphosphonates (BPs) are used currently in the treatment of patients with bone metastases because these compounds inhibit bone resorption. We examined here the effects of BPs on inhibition of endothelial cell functions in vitro and in vivo. Treatment of endothelial cells with BPs (clodronate, risedronate, ibandronate, and zoledronic acid) reduced proliferation, induced apoptosis, and decreased capillary-like tube formation in vitro. Quantification of blood vessels in bone biopsy specimens from patients with Paget's disease before and after clodronate treatment showed a 40% reduction of the vascularization after BP treatment. However, such a decreased vascularity could be secondary to a reduction of bone resorption. Therefore, the tissue distribution of [14C]BPs in male rats was examined to develop an angiogenesis model in a noncalcified tissue where BPs could accumulate. [14C]BPs (zoledronic acid, ibandronate, and clodronate) not only accumulated in bone but also transiently accumulated in the prostate. The effects of BPs on testosterone-induced revascularization of the prostate gland in castrated rats were then studied. Testosterone in combination with ibandronate or zoledronic acid induced a 17-35% reduction of the prostate weight compared with castrated rats treated with testosterone alone. Blood vessel immunostaining on prostate tissue sections revealed that both ibandronate and zoledronic acid induced a 50% reduction of the revascularization of the prostate gland. Moreover, zoledronic acid did not alter testosterone-induced activity of a luciferase gene reporter construct transfected in androgen-dependent prostatic cells, indicating that this BP did not directly interfere with testosterone. In conclusion, BPs have in vivo antiangiogenic properties, which could be of relevance to improve therapy and prevention of bone metastasis. In addition, our results extend the potential clinical use of BPs to patients with early prostate cancer.
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November 2002