RESUMEN

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.

RESUMEN

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.

Asunto(s)

Ciclina A/genética , Ciclina B/genética , Ciclina D1/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Endotelio Vascular/fisiología , Antígeno Nuclear de Célula en Proliferación/genética , beta 2 Glicoproteína I/metabolismo , Anexina A2/farmacología , Ciclo Celular , División Celular/fisiología , Regulación hacia Abajo , Fibrinolisina/fisiología , Humanos , Neovascularización Fisiológica/fisiología , Venas Umbilicales/citología , Venas Umbilicales/fisiología , Regulación hacia Arriba , beta 2 Glicoproteína I/aislamiento & purificación , beta 2 Glicoproteína I/fisiología

RESUMEN

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.

Asunto(s)

Adhesión Celular/fisiología , Células Endoteliales/citología , Serpinas/genética , Serpinas/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Línea Celular Tumoral , Células Endoteliales/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica/patología , Venas Umbilicales/citología , Neoplasias de la Vejiga Urinaria/fisiopatología

RESUMEN

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.

Asunto(s)

Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/secundario , Neoplasias Pélvicas/patología , Neoplasias Pélvicas/secundario , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/terapia , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Neoplasias Pélvicas/terapia , Estudios Retrospectivos , Neoplasias de la Vejiga Urinaria/terapia

RESUMEN

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.

Asunto(s)

Inhibidores de la Angiogénesis/metabolismo , Neovascularización Patológica/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Angiostatinas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Cistectomía , Endostatinas/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones SCID , Metástasis de la Neoplasia , Neovascularización Patológica/patología , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugía

RESUMEN

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.

Asunto(s)

Inhibidores de la Angiogénesis/uso terapéutico , Carcinoma de Células Transicionales/terapia , Glicoproteínas/metabolismo , Neovascularización Patológica/prevención & control , Neoplasias de la Vejiga Urinaria/terapia , Animales , Anexina A2/metabolismo , Carcinoma de Células Transicionales/irrigación sanguínea , Carcinoma de Células Transicionales/metabolismo , Proliferación Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Fibrinolisina/metabolismo , Glicoproteínas/aislamiento & purificación , Humanos , Immunoblotting , Espectrometría de Masas , Ratones , Ratones SCID , Neovascularización Patológica/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Neoplasias de la Vejiga Urinaria/irrigación sanguínea , Neoplasias de la Vejiga Urinaria/metabolismo , beta 2 Glicoproteína I

RESUMEN

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.

Asunto(s)

Carcinoma/patología , Carcinoma/cirugía , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Estadificación de Neoplasias/métodos , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Anciano , Humanos , Masculino , Persona de Mediana Edad , Prostatectomía , Sensibilidad y Especificidad

RESUMEN

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.

Asunto(s)

Integrina alfa5/biosíntesis , Integrina beta3/biosíntesis , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/biosíntesis , Línea Celular Tumoral , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Endotelio Vascular/citología , Matriz Extracelular/metabolismo , Humanos , Masculino , Fosforilación , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

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.

Asunto(s)

Adhesión Celular , Quimiocinas CXC/metabolismo , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Neoplasias de la Próstata/metabolismo , Quimiocinas CXC/genética , Quimiotaxis de Leucocito/fisiología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neovascularización Patológica , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Hepatocitos/citología , Adulto , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Sustancias de Crecimiento/farmacología , Hepatocitos/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-met/metabolismo

RESUMEN

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.

Asunto(s)

Quimiocinas CXC/genética , Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Receptores de Quimiocina/genética , Adhesión Celular/efectos de los fármacos , Quimiocinas CXC/biosíntesis , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Perfilación de la Expresión Génica , Neoplasias/tratamiento farmacológico , ARN Mensajero/metabolismo , Receptores de Quimiocina/biosíntesis , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Antineoplásicos/farmacología , Línea Celular Tumoral/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferasas/efectos de los fármacos , Ácido Valproico/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/fisiología , Humanos , ARN Mensajero/efectos de los fármacos , Sialiltransferasas/fisiología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Adhesión Celular/efectos de los fármacos , Inmunosupresores/farmacología , Integrina beta1/metabolismo , Ácido Micofenólico/análogos & derivados , Proteínas de Neoplasias/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Ensayos de Selección de Medicamentos Antitumorales , Células HT29 , Humanos , Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfa6beta1/metabolismo , Glicoproteínas de Membrana , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ácido Micofenólico/farmacología , Recurrencia Local de Neoplasia , Complejo GPIb-IX de Glicoproteína Plaquetaria , ARN Mensajero/metabolismo

RESUMEN

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.

Asunto(s)

Adhesión Celular/fisiología , Ciclo Celular/fisiología , Células Endoteliales/metabolismo , Receptores de Hialuranos/metabolismo , Isoformas de Proteínas/metabolismo , Neoplasias Gástricas , Empalme Alternativo , Línea Celular Tumoral , Células Endoteliales/citología , Humanos , Receptores de Hialuranos/genética , Isoformas de Proteínas/genética

RESUMEN

Angiogenesis, the induction of vessel growth is involved in numerous physiological and pathological processes. While the anti-tumor effect of angiogenesis inhibitors has been extensively investigated in malignant tumors, there is very little information on the effect of angiogenesis inhibitors on inflammation induced angiogenesis. In this report, we utilized a murine model of acute chemically induced cystitis to investigate the ability of three different angiogenesis inhibitors, angiostatin, endostatin and TNP-470, to inhibit the angiogenesis stimulated by this injury. We demonstrate herein, that prophylactic application of the angiogenesis inhibitors led to a significant reduction of each of the inflammatory parameters that were measured. We conclude that anti-angiogenic therapy with angiostatin, endostatin and TNP-470 inhibits inflammation associated angiogenesis induced in this model. We also propose that anti-angiogenic agents may serve as a valuable addition to a standard cyclophosphamid chemotherapy regimen to help reduce the chemotherapy-related side effects while potentially adding an anti-tumor effect.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Ciclofosfamida/efectos adversos , Cistitis/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Angiostatinas/farmacología , Animales , Permeabilidad Capilar , Ciclohexanos , Cistitis/inducido químicamente , Cistitis/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Endostatinas/farmacología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/prevención & control , O-(Cloroacetilcarbamoil) Fumagilol , Sesquiterpenos/farmacología

RESUMEN

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.

Asunto(s)

Antígeno CD56/genética , Infecciones por Citomegalovirus/patología , Citomegalovirus , Endotelio Vascular/fisiología , Neoplasias/genética , Secuencia de Bases , Adhesión Celular , Línea Celular Tumoral , Infecciones por Citomegalovirus/genética , Cartilla de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Neoplasias/patología , Neuroblastoma , Oligonucleótidos Antisentido , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Venas Umbilicales

RESUMEN

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.

Asunto(s)

Colágeno , Receptores ErbB/metabolismo , Receptores ErbB/fisiología , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-met/fisiología , Western Blotting , Comunicación Celular , Técnicas de Cultivo de Célula , Células Cultivadas/fisiología , Citometría de Flujo , Hepatocitos , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Fosforilación , Transducción de Señal

RESUMEN

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.

Asunto(s)

Adhesión Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Secuencia de Bases , Células Cultivadas , Técnicas de Cocultivo , Cartilla de ADN , Endotelio Vascular/efectos de los fármacos , Humanos , Cinética , Moléculas de Adhesión de Célula Nerviosa/genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Células Tumorales Cultivadas , Venas Umbilicales

RESUMEN

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.

Asunto(s)

Neoplasias Encefálicas/enzimología , Metaloendopeptidasas/análisis , Metaloendopeptidasas/deficiencia , Neoplasias de la Próstata/enzimología , Proteínas ADAM , Proteína ADAMTS13 , Adulto , Anciano , Biomarcadores de Tumor , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Femenino , Glioblastoma/sangre , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Masculino , Neoplasias Meníngeas/sangre , Neoplasias Meníngeas/enzimología , Neoplasias Meníngeas/patología , Meningioma/sangre , Meningioma/enzimología , Meningioma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Oligodendroglioma/sangre , Oligodendroglioma/enzimología , Oligodendroglioma/patología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Valores de Referencia , Factor de von Willebrand

RESUMEN

In experienced hands, laparoscopic pyeloplasty is an effective alternative treatment for symptomatic ureteropelvic junction obstruction (UPJO). Although laparoscopic surgery can clearly benefit patients, laparoscopic pyeloplasty using conventional instrumentation is complex. The purpose of this report is to evaluate the feasibility of robot assisted laparoscopic surgery. Eleven pyeloplasties for UPJO were performed via a laparoscopic transperitoneal approach exclusively with the da Vinci Surgical System. The mean procedure time was 197 min (range 110-310 min). All operations were completed laparoscopically with no intraoperative complications and negligible blood loss. All patients recovered rapidly after surgery with excellent functional results at the 1 year follow-up. Our initial experience suggests that robot assisted Anderson-Hynes pyeloplasty is a safe and effective alternative to conventional laparoscopic surgery. In our opinion, robot assisted surgery will allow urologists to perform complex procedures with greater precision, confidence, and better results, as well as enable them to adapt the whole spectrum of laparoscopic procedures to their field.

Asunto(s)

Pelvis Renal/cirugía , Robótica/instrumentación , Obstrucción Ureteral/cirugía , Diseño de Equipo , Estudios de Factibilidad , Estudios de Seguimiento , Humanos , Factores de Tiempo