Evidence for benefits and risks of tadalafil as a non-prescription medicine: review and evaluation using the Group Delphi technique to achieve consensus amongst clinical experts.

An evidence-based consensus meeting was held with urologists, a pharmacist and a cardiologist to perform a structured benefit-risk analysis of reclassifying tadalafil, a phosphodiesterase type 5 (PDE5) inhibitor for treatment of erectile dysfunction (ED), to be available without prescription in Germany. As per the Brass process endorsed by regulatory authorities, an evidence-based Brass value tree was developed, which identified the incremental benefits and risks that should be considered above the safety and efficacy evidence required for prescription medicines. During the Group Delphi consensus meeting, the expert panel rated the likelihood and clinical impact of each benefit and risk on a scale of 0 (none) to 3 (high). Overall attribute scores were calculated from the product of the mean likelihood and mean clinical impact scores giving a possible score of 0-9. The overall benefit attribute scores ranged from 2.8 to 5.4. The overall risk attribute scores ranged from 0.2 to 2.2 though most were 1.0 or less (3 or more is generally considered to be of concern). On balance, the independent meeting scored the benefits of reclassification of tadalafil higher than the risks and considered the risk mitigation strategies of the packaging label and patient information leaflet (PIL) sufficient.

Maspin modulates adhesion of bladder carcinoma cells to vascular endothelium.

PURPOSE: Maspin belongs to the serpin family and has been shown to suppress tumor growth and metastasis in several tumor types. The role of maspin in bladder carcinoma has not been fully elucidated, and the object of this study was to investigate whether maspin contributes to bladder tumor adhesion to vascular endothelial cells (HUVEC). METHODS: Expression of maspin-coding mRNA was evaluated in a panel of bladder carcinoma cell lines. Maspin distribution in maspin mRNA(high) versus maspin mRNA(low) cells was further analyzed by flow cytometry and confocal microscopy. Adhesion to HUVEC was measured in a coculture model and correlated with the surface-bound maspin. RESULTS: Maspin(high) (RT-4, RT-112) cell lines strongly attached to HUVEC, whereas maspin(low) (UMUC-3, MGH-U1) cell lines poorly adhered to HUVEC. Distinct cytoplasmic maspin accumulation and moderate surface-bound maspin was found in RT-4 cells. Blocking maspin surface receptors prevented tumor cell attachment to HUVEC, indicating that surface-bound maspin is responsible for triggering cell adhesion. PMA-triggered elevation of surface-bound maspin was accompanied by an enhanced adhesion capacity of RT-4 cells, compared to controls. Finally, exposing the bladder carcinoma cells to the differentiation-inducing agent valproic acid led to a surface-bound (but not cytoplasmic) maspin decrease, paralleled by a significant reduction in tumor cell binding to HUVEC. CONCLUSION: Surface-bound maspin directly controls bladder carcinoma cell adhesion to the vascular wall. Blocking this process may prevent transendothelial migration and tumor cell dissemination. Therefore, therapeutic down-regulation of surface-bound maspin might become an option to prevent tumor spread into distant organs.

Expression of angiogenesis inhibitors in human bladder cancer may explain rapid metastatic progression after radical cystectomy.

Angiogenesis is essential for tumor growth and progression. It has been demonstrated that the expression of angiogenesis stimulators (e.g. basic fibroblast growth factor and vascular endothelial growth factor) correlates to tumor progression in various human tumor types. Furthermore, endogenous angiogenesis inhibitors (e.g. angiostatin and endostatin) have been isolated from human tumor models and have been successfully used to treat tumors in mice and humans. In the present study, the expression of angiostatin, endostatin and thrombospondin-1 in four different human bladder cancer cell lines with different tumorigenic potential (MGH-U4, RT-4, RT-112 and UMUC-3) were investigated. A subset of bladder carcinoma patients demonstrates rapid metastatic progression after removal of the primary tumor, although no evidence of metastasis is diagnosed before the surgical procedure. A potential mechanism to explain this phenomenon is suggested. Angiostatin, endostatin and thrombospondin-1 was detected in the conditioned media of four human bladder cancer cell lines using Western blotting. Angiostatin was purified and amino acid sequenced via mass spectrometry. The biological activity of angiostatin was determined by proliferation assays using endothelial cells, smooth muscle cells and fibroblasts. Tumor characteristics of the four human bladder carcinoma models were investigated in vitro and in vivo. All the bladder carcinoma cell lines employed in this study produced two biologically active variants of the angiostatin molecule (38 and 49 kDa). Endostatin and thrombospondin-1 were only produced by the low malignancy MGH-U4 and RT-4 bladder carcinoma models. This study identified the expression of different antiangiogenic molecules in human bladder carcinoma. The expression of antiangiogenic molecules seems to be a characteristic of low malignancy bladder carcinomas. The sudden lack of expression of antiangiogenic molecules as a consequence of surgical removal of highly malignant bladder carcinomas may explain the rapid metastatic progression of a subset of bladder carcinomas.

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix.

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.

Combined MRI and MR spectroscopy of the prostate before radical prostatectomy.

OBJECTIVE: The purpose of this study was to evaluate a routine protocol for combined MR and spectroscopic imaging of the prostate for staging accuracy. SUBJECTS AND METHODS: Fifty patients with biopsy-proven prostate carcinoma were examined with our sequence protocol, which consisted of T2-weighted fast spin-echo sequences and a pelvic T1-weighted spin-echo sequence. For spectroscopy, we used a 3D chemical shift imaging (CSI) spin-echo sequence. Image interpretation was performed by two radiologists. The total number of tumor voxels and tumor voxels per slice were counted to estimate the tumor volume in every patient. The potential of MR spectroscopy to differentiate between T2 and T3 tumors, based on the estimated tumor volumes, was compared with the staging performance of MRI. RESULTS: The MR measurement time was 19.01 minutes, and the total procedure time averaged 35 minutes. Seventy-six percent of the spectroscopic examinations were successful. Statistically significant differences in the number of tumor voxels per slice and tumor volumes were found between T2 and T3 tumors. The descriptive parameters of MRI and MR spectroscopy did not differ significantly; sensitivity and specificity were 75% and 87%, respectively, for MRI and 88% and 70%, respectively, for MR spectroscopy. The combination of both methods resulted in only a slight improvement in staging performance and was not statistically significant. CONCLUSION: Combined MRI and MR spectroscopy of the prostate has no diagnostic advantage in staging performance over MRI alone. The mean tumor volumes, estimated by MR spectroscopy, differ statistically significantly between T2 and T3 tumors.

Radiation abscopal antitumor effect is mediated through p53.

The observation that radiation treatment to a local area of the body results in an antitumor effect for tumors distant to the radiation site has been termed the "abscopal effect." To understand the mechanism of this unusual phenomenon, we examined whether the effect was mediated through p53, a protein complex up-regulated in irradiated cells. Non-tumor-bearing legs of C57BL/6 (wild-type p53) and p53 null B6.129S2-Trp53(tm1Tyj) mice were irradiated to determine whether an abscopal effect could be observed against Lewis lung carcinoma (LLC) and T241 (fibrosarcoma) implanted at a distant site. In mice with wild-type p53, both LLC and T241 tumors implanted into the midline dorsum grew at a significantly slower rate when the leg of the animal was exposed to five 10-Gy fractions of radiation compared with sham-irradiated animals, suggesting that the abscopal effect is not tumor specific. When the radiation dose to the leg was reduced (twelve fractions of 2 Gy each), the inhibition of LLC tumor growth was decreased indicating a radiation-dose dependency for the abscopal effect. In contrast, when the legs of p53 null animals or wild-type p53 mice treated with pifithrin-alpha (a p53 blocker) were irradiated (five 10-Gy fractions), tumor growth was not delayed. These data implicate p53 as a key mediator of the radiation-induced abscopal effect and suggest that pathways downstream of p53 are important in eliciting this response.

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy.

BACKGROUND: Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. METHODS: Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 microM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. RESULTS: Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 muM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. CONCLUSION: We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.

Valproic acid modulates NCAM polysialylation and polysialyltransferase mRNA expression in human tumor cells.

Polysialic acid (PSA) is a dynamically regulated carbohydrate modification of the neural cell adhesion molecule NCAM, which has been linked to cancer development and dissemination. Two enzymes, the polysialyltransferases ST8SiaIV and ST8SiaII, are known to be involved in the polysialylation of NCAM. The antiepileptic drug valproic acid (VPA) is associated with anti-cancer activity. In this study, VPA blocked the adhesion of several neuroectodermal tumor cell lines to human umbilical vein endothelial cells. Furthermore, VPA induced intracellular PSA accumulation and enhanced expression of PSA-NCAM on the cell surface. Using a semiquantitative RT-PCR strategy, VPA was shown to up-regulate ST8SiaIV mRNA, whereas ST8SiaII mRNA was down-regulated by this compound. Our data indicate that increased expression of ST8SiaIV enables accelerated polysialylation of NCAM, which might be coupled to a loss of adhesive functions of tumor cells.

Modulation of the CXC-chemokine expression profile on tumor cells by the immunosuppressive drug mycophenolate mofetil.

The most undesirable complication of an effective immunosuppressive therapy is neoplastic tumor recurrence or the development of de novo cancer. Though the immunosuppressive drug, mycophenolate mofetil (MMF), has been introduced into clinical practice, no data dealing with the influence of MMF on tumor cell malignancy are available. We analyzed the adhesion capacity of colon, pancreas and kidney carcinoma cell lines to endothelium, as well as their chemokine profile before and after MMF treatment. Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1, 1, and 10 microM MMF and compared to unstimulated controls. Chemokine analysis concentrated on the CXC family, including 6 CXC-receptors (CXCR) and 15 CXC-ligands (CXCL), and was carried out by reverse transcriptase-polymerase chain reaction and flow cytometry. MMF strongly diminished the adhesion capacity of HT-29 colon tumor cells but not of DanG pancreas tumor cells to endothelium. MMF also had a strong impact on the chemokine profile of colon, kidney and pancreas carcinomas, whereby individual changes were observed, depending on the tumor type. Down-regulating effects on chemokines did not correlate with down-regulating effects on tumor cell adhesion. Since several of the chemokines investigated are regulatory elements in the process of cell transformation, dissemination and angiogenesis, we speculate that MMF might prevent post-transplant tumor recurrence and transendothelial migration. However, the efficacy of MMF might differ according to the tumor type.

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants.

AIM: To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS: MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC) monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion. RESULTS: Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice variants CD44v4, CD44v5, and CD44v7 were all up-regulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION: CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.

AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.

Human cytomegalovirus infection of tumor cells downregulates NCAM (CD56): a novel mechanism for virus-induced tumor invasiveness.

Pathologic data indicate that human cytomegalovirus (HCMV) infection might be associated with the pathogenesis of several human malignancies. However, no definitive evidence of a causal link between HCMV infection and cancer dissemination has been established to date. This study describes the modulation of the invasive behavior of NCAM-expressing tumor cell lines by HCMV. Neuroblastoma (NB) cells, persistently infected with the HCMV strain AD169 (UKF-NB-4AD169 and MHH-NB-11AD169), were added to endothelial cell monolayers and adhesion and penetration kinetics were measured. The 140- and 180-kDa isoforms of the adhesion receptor NCAM were evaluated by flow cytometry, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides or NCAM transfection. HCMV infection profoundly increased the number of adherent and penetrated NB, compared to controls. Surface expression of NCAM was significantly lower on UKF-NB-4AD169 and MHH-NB-11AD169, compared to mock-infected cells. Western-blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoform. An inverse correlation between NCAM expression and adhesion capacity of NB has been shown by antisense and transfection experiments. We conclude that HCMV infection leads to downregulation of NCAM receptors, which is associated with enhanced tumor cell invasiveness.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

BACKGROUND: The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. METHODS: The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. RESULTS: MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 microM, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. CONCLUSIONS: MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.

ADAMTS-13 activity in patients with brain and prostate tumors is mildly reduced, but not correlated to stage of malignancy and metastasis.

Impaired Von Willebrand factor cleaving activity of ADAMTS-13 was demonstrated in patients with metastasizing and malignant tumors. To investigate the relevance of ADAMTS-13 for tumor progression, we determined ADAMTS-13 activity and VWF:Ag in 80 patients with various malignancies: 30 patients with benign brain tumors, 30 patients with malignant brain tumors, 10 patients with local prostate tumors and 10 patients with metastatic prostate tumors. We found mild ADAMTS-13 deficiency in 17/80 tumor patients, but there was no significant difference in ADAMTS-13 activity between the age- and sex-matched patients with benign and malignant brain tumors nor between the age matched patients with local and metastatic prostate tumors. Patients with malignant brain tumors could only be distinguished from patients with benign tumors by elevated levels of VWF:Ag (p=0.018). Our data are hence in variance to previous studies, which found a distinct inverse correlation between ADAMTS-13 activity and level of metastasis and/or malignancy in diverse cancer-types.

Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.

Plasmin-clipped beta(2)-glycoprotein-I inhibits endothelial cell growth by down-regulating cyclin A, B and D1 and up-regulating p21 and p27.

beta(2)-Glycoprotein-I (beta(2)gpI), an abundant plasma glycoprotein, functions as a regulator of thrombosis. Previously, we demonstrated that plasmin-clipped beta(2)gpI (cbeta(2)gpI) exerts an anti-angiogenic effect on human umbilical vein endothelial cells (HUVEC). The present study was focused on the molecular background responsible for this phenomenon. cbeta(2)gpI strongly reduced HUVEC growth and proliferation as evidenced by the MTT and BrdU assay and delayed cell cycle progression arresting HUVEC in the S-and G2/M-phase. Western blot analysis indicated that cbeta(2)gpI inhibited cyclin A, B and D1, and enhanced p21 and p27 expression. Activity of p38 was down-regulated independently from the cbeta(2)gpI incubation time. Phosphorylation of ERK1/2 was not changed early (30 and 60 min) but became enhanced later (90 min, 4h). JNK activity was reduced rapidly after cbeta(2)gpI treatment but compared to controls, increased thereafter. Annexin II blockade prevented growth inhibition and cell cycle delay evoked by cbeta(2)gpI. We assume that cbeta(2)gpI's effects on HUVEC growth is mediated via cyclin A, B and D1 suppression, up-regulation of p21 and p27 and coupled to modifications of the mitogen-activated protein (MAP) kinase signalling pathway. cbeta(2)gpI may represent a potential endogenous angiogenesis-targeted compound, opening the possibility of a novel tool to treat cancer.

Association of intravesical tumor location with metastases to the pelvic lymph nodes in transitional cell cancer of the bladder.

INTRODUCTION: In this study, we aimed to determine those clinical and pathologic features that are associated with pelvic lymph node metastasis in patients with transitional cell cancer of the bladder. Unlike previous studies, we particularly focused on intravesical tumor location. METHODS: We included 173 patients who underwent radical cystectomy and bilateral pelvic lymphadenectomy for muscle-invasive or high-risk superficial bladder cancer. Fifty patients (28.9%) presented with lymph node metastases. Tumor-related and personal characteristics were analyzed. RESULTS: Lymph node positive disease occurred in association with an increasing pathologic tumor stage (P < 10(-6)) and with a decreasing differentiation status (P = 0.008). The rate of pelvic lymph node metastasis differed in primary tumors growing on different intravesical locations. Cancers located exclusively on the lateral bladder walls (P < 10(-5)) and tumors involving the lateral walls (P = 0.042) were highly correlated with lymph node positive disease. Posterior wall tumors were least associated with lymph node metastases compared with other tumor locations (P = 0.015). Focal tumor growths located on the lateral bladder wall and an increasing pathologic tumor stage and decreasing differentiation-status were identified as independent risk factors for the pelvic lymph node status. CONCLUSIONS: For the first time we present the association of intravesical tumor location and the rate of lymph node metastasis in transitional cell cancer of the bladder. Our findings may ultimately contribute to a more individualized patient management.

An endogenous inhibitor of angiogenesis derived from a transitional cell carcinoma: clipped beta2-glycoprotein-I.

BACKGROUND: Invasive cell carcinoma of the bladder often develops after complete transurethral excision of superficial transitional cell carcinoma. It has been postulated that primary tumors release angiogenesis-blocking proteins which suppress distant metastases. We have identified an endogenous protein which might be responsible for tumor dormancy. METHODS: A transitional cell carcinoma cell line was developed (UMUC-3i) which inhibits the growth of a tumor implant at a distant site in SCID mice. Conditioned media of UMUC-3i cultured cells was first pooled and then fractioned, and the capacity of individual components to block endothelial cell growth was tested. The protein fraction responsible for blocking endothelial cell growth was identified by N-terminal amino acid sequencing as well as by mass-spectrometry. The effects of the purified protein in preventing endothelial cell proliferation and tube formation in an in vitro angiogenesis assay was investigated. RESULTS: The plasma protein beta(2)-glycoprotein-I (beta(2)gpI) was isolated and identified from conditioned medium of UMUC-3i cultured cells. Based on the in vitro angiogenesis assay, beta(2)gpI strongly inhibited endothelial cell growth and tube formation, whereby the inhibitory activity corresponded to the clipped version of beta(2)gpI (cbeta(2)gpI). Clipping was induced by adding plasmin at a molar ratio 1:15 (plasmin:substrate). Further analysis indicated that cbeta(2)gpI effects were mediated by annexin II surface receptors expressed on endothelial cells. CONCLUSIONS: cbeta2gpI may be involved in blocking angiogenic processes and bladder cancer progression. In this case, cbeta2gpI may be a promising tool in bladder cancer therapy.

CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins.

The mechanisms leading to prostate cancer metastasis are not understood completely. Although there is evidence that the CXC chemokine receptor (CXCR) 4 and its ligand CXCL12 may regulate tumor dissemination, their role in prostate cancer is controversial. We examined CXCR4 expression and functionality, and explored CXCL12-triggered adhesion of prostate tumor cells to human endothelium or to extracellular matrix proteins laminin, collagen, and fibronectin. Although little CXCR4 was expressed on LNCaP and DU-145 prostate tumor cells, CXCR4 was still active, enabling the cells to migrate toward a CXCL12 gradient. CXCL12 induced elevated adhesion to the endothelial cell monolayer and to immobilized fibronectin, laminin, and collagen. Anti-CXCR4 antibodies or CXCR4 knock out significantly impaired CXCL12-triggered tumor cell binding. The effects observed did not depend on CXCR4 surface expression level. Rather, CXCR4-mediated adhesion was established by alpha5 and beta3 integrin subunits and took place in the presence of reduced p38 and p38 phosphorylation. These data show that chemoattractive mechanisms are involved in adhesion processes of prostate cancer cells, and that binding of CXCL12 to its receptor leads to enhanced expression of alpha5 and beta3 integrins. The findings provide a link between chemokine receptor expression and integrin-triggered tumor dissemination.

Serum levels of basic fibroblast growth factor reflect disseminated disease in patients with testicular germ cell tumors.

The potential role of angiogenesis stimulators in the pathogenesis of different tumor entities has been confirmed in several studies. We measured the serum levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) in 51 patients with testicular germ cell tumors and in 39 healthy volunteers. Serum concentrations of bFGF, VEGF and PDGF-AB were determined by enzyme-linked immunosorbent assay. The median serum bFGF level for tumor patients was 3.46 pg/ml (range 0-61.6) compared to 0.7 pg/ml (0-11) in the control group (P<0.01). In patients with metastatic disease, the median serum bFGF level was 10.3 pg/ml (0-61.6) in contrast to 2.8 pg/ml (0-50) in patients with localized disease (P<0.01). The median serum VEGF and PDGF levels were 270 pg/ml (0-1,903) and 37,837 pg/ml (9,075-108,800), respectively, for tumor patients and 200 pg/ml (44-585) and 23,000 pg/ml (4,250-70,650) in the control group ( P<0.05). Our data suggest that angiogenesis, as reflected by serum concentrations of bFGF, VEGF and PDGF, plays a functional role in the growth and progression of testicular germ cell tumors.