Inducible nitric oxide synthase and PPARγ are involved in bladder cancer progression.

PURPOSE: We evaluated the role of inducible nitric oxide synthase and PPARγ as prognostic factors for bladder cancer. MATERIALS AND METHODS: Inducible nitric oxide synthase and PPARγ were evaluated by Western blot and immunohistochemistry in a mouse bladder cancer model of nonmuscle invasive and invasive MB49-I tumor cells, and in human bladder cancer samples. RESULTS: Inducible nitric oxide synthase expression was negative in mouse normal urothelium and higher in invasive than in noninvasive MB49 tumors. In vitro inducible nitric oxide synthase activity, determined as nitrite, was higher in MB49-I than in MB49 cells (p <0.001). In human samples expression was also associated with tumor invasion. Nuclear PPARγ expression was negative in normal mouse urothelium but higher in nonmuscle invasive MB49 than in MB49-I tumors. Similarly in human tumors low PPARγ was associated with poor prognosis factors, such as high histological grade (p = 0.0160) and invasion status (p = 0.0352). A positive correlation was noted between inducible nitric oxide synthase and PPARγ in low histological grade and nonmuscle invasive tumors (Pearson correlation index 0.6368, p = 0.0351, 0.4438 and 0.0168, respectively). As determined by gene reporter assay, PPARγ activity was induced by nitric oxide only in nonmuscle invasive MB49 cells (p <0.001). CONCLUSIONS: Results suggest that increased PPARγ controls inducible nitric oxide synthase expression at early tumor stages. However, regulation is lost at advanced tumor stages, when the increase in inducible nitric oxide synthase and the decrease in PPARγ seem to be associated with bladder cancer progression.

Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.

Novel invasive orthotopic bladder cancer model with high cathepsin B activity resembling human bladder cancer.

PURPOSE: We developed and characterized an orthotopic invasive bladder tumor model. MATERIAL AND METHODS: The MB49-I invasive bladder tumor cell line was obtained after 13 consecutive in vivo passages of primary tumor obtained by subcutaneous inoculation of MB49 bladder tumor cells in C57Bl/6J male mice. RESULTS: MB49-I tumor local invasiveness, tumor weight and spontaneous metastatic capacity were higher than in MB49 tumors. In MB49-I bladder tumors increased vimentin was observed, suggesting epithelial mesenchymal transition. In vitro the MB49-I cell line showed higher invasive properties associated with an increase in cathepsin B, metalloproteinase 9 and urokinase-type plasminogen activator proteolytic activities. Orthotopic bladder tumors induced by electrocautery of the bladder wall and subsequent instillation of MB49 and MB49-I bladder cancer cells generated superficial and invasive bladder tumors, respectively. CONCLUSIONS: The new murine bladder model described resembles human bladder disease, making it a useful tool for studying the molecular mechanisms of tumor progression and metastasis, and assaying antimetastatic and anti-invasive agents.

Cathepsin B is involved in the apoptosis intrinsic pathway induced by Bacillus Calmette-Guérin in transitional cancer cell lines.

Bacillus Calmette-Guérin (BCG) is the most effective treatment for superficial and in situ transitional bladder cancer. Although the complete mechanisms for its effect are not fully understood yet, both immunological and direct effects on tumor cells have been proposed. It has been proposed that apoptotic tumor cells could be better inducers of immunity than necrotic ones. Thus, apoptosis of bladder cancer cells could contribute to a global response to BCG. Lysosomal hydrolase cathepsin B (CB) is involved in the apoptotic process and has a key role in breast cancer cell programmed death through the activation of a pro-apoptotic protein BID. Truncated BID participates in the mitochondrial apoptotic pathway that involves the activation of pro-caspase 9. The possibility that CB can be involved in apoptosis of TCC line has not been explored yet. Therefore, we analyzed the participation of CB in BCG-induced apoptosis of human and murine TCC lines. Apoptosis was evaluated by a morphologic assay and CB activity by a substrate-specific colorimetric method. Expression of CB, BID and pro-caspase 9 was determined by Western blotting. BCG induced apoptosis of murine (MBT2, MB49) and human (T24) TCC lines. An increase in both CB activity and protein was also observed. The apoptosis of T24 and MB49 cell lines was mediated by activation of pro-caspase 9 and BID, both proteins are involved in mitochondrial apoptosis. Apoptosis and activation of pro-caspase 9 and BID were inhibited by CA-074Me (CA), a cell permeable CB inhibitor. Thus, CB is involved in BCG-induced apoptosis of TCC lines, using at least in part the mitochondrial pathway.

Técnica del ganglio centinela dinámico en pacientes con cáncer de pene. Evaluación de la experiencia inicial del Instituto de Oncología Angel H. Roffo. U.B.A / Dinamic sentinel limph node technique in patients with penis cancer. Evaluation of initial experience. Instituto de Oncología Angel H. Roffo. U.B.A

Introducción: El manejo de los ganglios linfáticos ha sido durante mucho tiempo, motivo de controversia en el tratamiento del cáncer de pene. Su influencia en el factor pronóstico ha marcado la importancia de realizar un estudio detallado del mismo y tratar de encontrar un indicador que permita determinar un mayor riesgo de diseminación linfática o compromiso ganglionar con baja morbilidad. Objetivo: El propósito de este trabajo, es efectuar una descripción pormenorizada de la técnica dinámica del ganglio centinela (TDGC) y describir nuestra experiencia inicial con la misma, demostrando la franca disminución de la morbilidad que frecuentemente se asocia con la linfadenectomía clásica frente a la TDGC y estableciendo las pautas para que sea una técnica reproducible. Materiales y métodos: Durante el periodo 2000-2004, 20 pacientes con diagnóstico de cáncer de pene han sido sometidos a la aplicación de TDGC. Fueron incluidos pacientes en estadios T1-T3. Resultados: Un grupo de 10 pacientes presentaba adenopatías no palpables y otro grupo, también de 10 pacientes, adenopatías palpables, pequeñas, móviles y no adherentes. En total de 20 pacientes se identificaron 8 casos de metástasis con ganglio centinela positivo (40 porciento) y 12 casos con ganglio centinela negativo (60 porciento). A 6 de los 8 pacientes con ganglio centinela positivo, se les efectuó linfadenectomía iliofemoral, evidenciándose 2 pacientes con metástasis y 4 sin metástasis. En los otros dos pacientes no se realizo linfadenectomía: uno por presencia de metástasis a distancia y el otro por abandono voluntario del tratamiento. El seguimiento mínimo fue de un año y el máximo de 5 años, con una media de 3 años. Ninguno de los pacientes desarrollo recidivas en ganglios linfáticos. Los 4 casos de óbito fueron por recidiva local del tumor primario, sepsis e hipercalcemia. Conclusión: La utilización de TDGC puede ser de gran utilidad en la detección de metástasis ganglionares. Es un buen indicador prec...

Técnica del ganglio centinela dinámico en pacientes con cáncer de pene. Evaluación de la experiencia inicial del Instituto de Oncología Angel H. Roffo. U.B.A / Dinamic sentinel limph node technique in patients with penis cancer. Evaluation of initial experience. Instituto de Oncología Angel H. Roffo. U.B.A

Introducción: El manejo de los ganglios linfáticos ha sido durante mucho tiempo, motivo de controversia en el tratamiento del cáncer de pene. Su influencia en el factor pronóstico ha marcado la importancia de realizar un estudio detallado del mismo y tratar de encontrar un indicador que permita determinar un mayor riesgo de diseminación linfática o compromiso ganglionar con baja morbilidad. Objetivo: El propósito de este trabajo, es efectuar una descripción pormenorizada de la técnica dinámica del ganglio centinela (TDGC) y describir nuestra experiencia inicial con la misma, demostrando la franca disminución de la morbilidad que frecuentemente se asocia con la linfadenectomía clásica frente a la TDGC y estableciendo las pautas para que sea una técnica reproducible. Materiales y métodos: Durante el periodo 2000-2004, 20 pacientes con diagnóstico de cáncer de pene han sido sometidos a la aplicación de TDGC. Fueron incluidos pacientes en estadios T1-T3. Resultados: Un grupo de 10 pacientes presentaba adenopatías no palpables y otro grupo, también de 10 pacientes, adenopatías palpables, pequeñas, móviles y no adherentes. En total de 20 pacientes se identificaron 8 casos de metástasis con ganglio centinela positivo (40 porciento) y 12 casos con ganglio centinela negativo (60 porciento). A 6 de los 8 pacientes con ganglio centinela positivo, se les efectuó linfadenectomía iliofemoral, evidenciándose 2 pacientes con metástasis y 4 sin metástasis. En los otros dos pacientes no se realizo linfadenectomía: uno por presencia de metástasis a distancia y el otro por abandono voluntario del tratamiento. El seguimiento mínimo fue de un año y el máximo de 5 años, con una media de 3 años. Ninguno de los pacientes desarrollo recidivas en ganglios linfáticos. Los 4 casos de óbito fueron por recidiva local del tumor primario, sepsis e hipercalcemia. Conclusión: La utilización de TDGC puede ser de gran utilidad en la detección de metástasis ganglionares. Es un buen indicador prec...(AU)

Bacillus Calmette-Guérin induces the expression of peroxisome proliferator-activated receptor gamma in bladder cancer cells.

Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial and in situ bladder cancer. The exact mechanism of the antitumor activity of BCG is not completely understood. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that is involved in cell growth and differentiation as well as inflammatory processes. PPARgamma is expressed in normal urothelium and a lack of expression was associated with bladder cancer progression. We analyzed whether PPARgamma is involved in the inhibition of bladder cancer cell survival by BCG. PPARgamma expression in murine MB49 and human T24 bladder cancer cells was evaluated employing immunofluorescence and immunohistochemistry techniques. In vitro cell viability and nitric oxide (NO) production was evaluated by using MTS and Griess reagent respectively. Our results show that BCG induced the cytoplasmatic expression of PPARgamma in bladder tumor cells in vitro and in vivo. BADGE, antagonist of this receptor, abrogated in vitro BCG-mediated cell cytotoxicity. Natural agonist 15-deoxy-Delta12,14 prostaglandin J2 (15-d-PGJ2) but not rosiglitazone (RO), a synthetic agonist, induced in vitro inhibition of cell viability of both cancer cell lines and the effect was partially reversed by BADGE. We also determined whether the activation of PPARgamma could inhibit NO production, which is considered a survival factor for bladder tumor cells. Both 15-d-PGJ2 and RO significantly inhibited the NO production in T24 and MB49 cells by PPARgamma-independent pathway since it was not antagonized by BADGE. Thus, our results show that BCG induces functional PPARgamma in bladder tumor cells in vivo and in vitro, being these receptors intrinsically involved in the antitumor activity of BCG.

Expression of inducible nitric oxide synthase in tumoral and non-tumoral epithelia from bladder cancer patients.

We have previously demonstrated that nitric oxide (NO) is elevated in the urine from bladder cancer patients. As the inducible nitric oxide synthase (iNOS) produces high NO output, the aim of this study was to examine iNOS expression and activity in tumoral (BT) and non-tumoral bladder tissue (NT). iNOS expression was determined by Western blot in 42 BT, 22 NT, and 4 normal bladders (normal B). iNOS activity was evaluated by conversion of [(14)C]l-arginine to [(14)C]l-citrulline plus NO, in additional 15 BT, 8 NT, and 1 normal B. iNOS tissue localization was studied by immunohistochemistry. iNOS expression and activity were found in almost 50% of bladder cancer patients, in both BT and in NT. A similar positive or negative iNOS expression in each pair of NT and BT tissue compared was observed, suggesting that high urine NO levels could be generated by an active iNOS present not only in the tumor but also in the non-tumoral bladder tissue. By immunohistochemistry, heterogeneous iNOS staining was detected in tumor cells from superficial and invasive tumors, while it was not evident in the normal bladder epithelium. A follow-up of 21 patients during 2 years showed recurrences in 80% with positive iNOS. On the contrary, no recurrences were observed in 73% of iNOS negative patients. Our results suggest that iNOS expression in bladder tissue may predispose to cancer recurrences.

High expression of cathepsin B in transitional bladder carcinoma correlates with tumor invasion.

BACKGROUND: Cathepsin B (CB) is a lysosomal cysteine proteinase synthesized as a zymogen of 39-47 kilodaltons (kD), which is subsequently converted into an active single- chain form of 33 kD (CB33) and, by additional processing, into the active 2-chain form containing a heavy chain of 27-29 kD (CB(27-29)) and a light chain of 4-6 kD. Increased or altered CB expression has been documented in a variety of tumor cells, but to the authors' knowledge only one study published to date has reported clinicopathologic significance for CB in transitional cell carcinoma (TCC) of the bladder. METHODS: In this work, CB expression was determined by Western blot analysis in TCC bladder tissue from 30 patients. Nontumor bladder tissue was also analyzed for CB expression. RESULTS: The study results demonstrate higher expression of CB in TCC invasive tumors than in superficial bladder carcinoma. Furthermore, whereas normal bladder only expressed the 29-kD CB protein, tumor and peritumoral tissue demonstrated the 27- to 29-kD CB form. Immunohistochemical staining did not evidence changes in CB localization between tumor and nontumor tissue. CONCLUSIONS: According to the results of the current study, bladder tumor progression appears to be associated with quantitative changes in CB protein expression, as well as with qualitative changes related to the type of CB expressed.

Nitric oxide in patients with transitional bladder cancer.

BACKGROUND AND OBJECTIVES: One of the current challenges in clinical oncology is the identification of patients with superficial transitional bladder carcinoma (TBC) at high risk of recurrence or myoinvasive disease. Recently, inducible nitric oxide synthase (iNOS) expression was detected in urinary bladder cancers. Because iNOS produces a high concentration of nitric oxide (NO), we thought it possible that urine from TBC patients produces high levels of NO. The aim of this study was to determine urine NO levels in TBC compared with healthy controls and with patients bearing other nonrelated tumors, as well as to examine iNOS expression in bladder cancer tissue. METHODS: This study evaluated patients with TBC (n = 33), with gynecological tumors (GT) (n = 19), TBC patients with no evidence of tumor (no evidence of disease [NED]) (n = 19), and healthy subjects (n = 39). Urine NO levels were determined by Griess reagent, expressed as microM NO(2) (-)/100 mg creatinine. RESULTS: TBC patients produced significantly higher urine NO median values (4.2 microM; range, 2.1-91.6) than were produced by healthy individuals (2.1 microM; range, 0.4-4.9), by the NED group (1.7 microM; range 1.2-5.4), and by GT patients (2.0 microM; range, 0.8-58.1) (P = 0.000, Kruskal-Wallis test). iNOS was detected by Western blot in 52% (13/25) of bladder tumors examined. CONCLUSIONS: Although a wider study is necessary, our results suggest that the enhanced NO levels could perhaps be considered as a putative marker in TBC patients.

Metástasis torácicas de carcinomas testiculares: cirugía de rescate / Thoracic metastases in patients with advanced germ cell testicular tumors

Antecedentes: La aplicación de poliquimioterapia cisplatino produce remisión completa de las matástasis torácicas de muchos de los portadores de carcinomas germinales de testículo en estadio III. Otros también se consideran curados porque las masas residuales que persisten no contienen neoplasia. En un grupo de pacientes sin embargo, se mantiene tumor viable en las metástasis, cuya resección total puede derivar en una prolongada sobrevida libre de enfermedad. Población y métodos: De 172 pacientes con carcinomas germinales avanzados de testícul, asistidos, 23 fueron sometidos a rescate quirúrgico por metástasis torácicas. Se practicaron 28 operaciones, porque a 4 pacientes se le realizaron 2 sucesivas y otro fue sometido a 3 intervenciones. Los procedimientos fueron videotoracoscópicos y a cielo abierto, en su mayoría para efectuar resecciones segmentarias atípicas. Resultados: Se reconoció que en las piezas operatorias de 9 casos no existía tejido tumoral remanente, hecho demostrativo de la efectividad de la poliquimioterapia previamente suministrada. En los otros, hubo diferentes tipos histológicos, 4 de los cuales eran teratomas maduros y 5 teratomas inmaduros, lo cual en conjunto integra un grupo de 18 respuestas oncológicas completas. De los 5 pacientes con tejido tumoral viable, hubo 1 con más de 5 años de supervivencia libre de enfermedad, que puede considerrarse como curación. En dos pacientes con fibrosis/necrosis en retroperitoneo, se hallaron teratomas en las metástasis pulmonares. Conclusiones: La persistencia de nódulos pulmonares y de formaciones en mediatino o pared del tórax, en pacientes ya antes sometidos a orquiectomías y quimioterapia pro carcinomas germinales, es indicación de tratamiento quirúrgico, aun cuando en linfadenectomía retroperitoneal previa no se hubiera hallado tejido tumoral. La exéresis completa de las metástasis torácicas con tumor viable, es capaz de producir supervivencias prolongadas libres de enfermedad

Metástasis torácicas de carcinomas testiculares: cirugía de rescate / Thoracic metastases in patients with advanced germ cell testicular tumors

Antecedentes: La aplicación de poliquimioterapia cisplatino produce remisión completa de las matástasis torácicas de muchos de los portadores de carcinomas germinales de testículo en estadio III. Otros también se consideran curados porque las masas residuales que persisten no contienen neoplasia. En un grupo de pacientes sin embargo, se mantiene tumor viable en las metástasis, cuya resección total puede derivar en una prolongada sobrevida libre de enfermedad. Población y métodos: De 172 pacientes con carcinomas germinales avanzados de testícul, asistidos, 23 fueron sometidos a rescate quirúrgico por metástasis torácicas. Se practicaron 28 operaciones, porque a 4 pacientes se le realizaron 2 sucesivas y otro fue sometido a 3 intervenciones. Los procedimientos fueron videotoracoscópicos y a cielo abierto, en su mayoría para efectuar resecciones segmentarias atípicas. Resultados: Se reconoció que en las piezas operatorias de 9 casos no existía tejido tumoral remanente, hecho demostrativo de la efectividad de la poliquimioterapia previamente suministrada. En los otros, hubo diferentes tipos histológicos, 4 de los cuales eran teratomas maduros y 5 teratomas inmaduros, lo cual en conjunto integra un grupo de 18 respuestas oncológicas completas. De los 5 pacientes con tejido tumoral viable, hubo 1 con más de 5 años de supervivencia libre de enfermedad, que puede considerrarse como curación. En dos pacientes con fibrosis/necrosis en retroperitoneo, se hallaron teratomas en las metástasis pulmonares. Conclusiones: La persistencia de nódulos pulmonares y de formaciones en mediatino o pared del tórax, en pacientes ya antes sometidos a orquiectomías y quimioterapia pro carcinomas germinales, es indicación de tratamiento quirúrgico, aun cuando en linfadenectomía retroperitoneal previa no se hubiera hallado tejido tumoral. La exéresis completa de las metástasis torácicas con tumor viable, es capaz de producir supervivencias prolongadas libres de enfermedad (AU)

Cirugía de rescate en cáncer de testículo / Ranson surgery in testicular neoplasm

Se presentan datos sobre 16 cirugías de rescate en 15 pacientes portadores de tumor de testículo avanzado. Se evalúan los datos de estadificación, evolución y seguimiento. Se efectúan consideraciones sobre la táctica quirúrgica realizada, los hallazgos operatorios y la histología de las piezas resecadas, valorando además la utilidad de los exámenes complementarios

Cirugía de rescate en cáncer de testículo / Ranson surgery in testicular neoplasm

Se presentan datos sobre 16 cirugías de rescate en 15 pacientes portadores de tumor de testículo avanzado. Se evalúan los datos de estadificación, evolución y seguimiento. Se efectúan consideraciones sobre la táctica quirúrgica realizada, los hallazgos operatorios y la histología de las piezas resecadas, valorando además la utilidad de los exámenes complementarios (AU)