Eduardo Sandes

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.

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.

Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

The natural flavonoid silybin improves the response to Photodynamic Therapy of bladder cancer cells.

Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments. In the present work, we evaluated the cytotoxicity of the combination of ALA-PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA. Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou-Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells. Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment. Overall, our results suggest that the combination of silybin and ALA-PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.

Role of Peroxisome Proliferator Activated Receptor-Gamma in Bacillus Calmette-Guérin Bladder Cancer Therapy

PURPOSE We evaluated the effects of combined PPARg agonist with bacillus Calmette-Guérin in bladder cancer growth in vitro and in vivo, focusing on the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. MATERIALS AND METHODS PPARs are a superfamily of nuclear receptors that are transcription factors activated by ligands. Activation of PPARg, the γ subtype, causes proliferation inhibition or differentiation of tumor cells. Previously, we reported that the inhibition of murine bladder tumor growth induced by bacillus Calmette-Guérin, which is the standard treatment for patients with nonmuscle invasive, high grade bladder cancer, increased PPARg expression in vitro and in vivo. In vitro the cell growth inhibition induced by bacillus Calmette-Guérin was enhanced by the PPARg agonist 15-d-PGJ2, raising the possibility that PPARg activation may be a therapeutic modality for this disease. RESULTS In MB49 cells bacillus Calmette-Guérin and 15-d-PGJ2 induced PPARg expression, nuclear translocation and transcriptional activity. In vivo bacillus Calmette-Guérin reduced tumor size, an effect that was partially reversed when bacillus Calmette-Guérin was combined with the PPARg agonist rosiglitazone. The same result was found when we analyzed the effect of the PPARg antagonist BADGE (Fluka Chemical, Buchs, Switzerland) combined with bacillus Calmette-Guérin. Analysis of the activation of macrophages and fibroblasts demonstrated that rosiglitazone inhibited the tissue remodeling mechanisms induced by bacillus Calmette-Guérin. CONCLUSIONS Results suggest that PPARg is involved in the antitumor action of bacillus Calmette-Guérin. However, exogenous PPARg agonists would not be a favorable therapeutic modality because they can inhibit the tissue remodeling needed for an overall satisfactory bacillus Calmette-Guérin response.

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.

Animal Models for Basic and Preclinical Research in Bladder Cancer

Bladder cancer is one of the most common cancers in the world. In 2006, there were about 61,240 diagnosed cases of bladder cancer and approximately 13,060 deaths attributable to this disease, being the prevalence estimated worldwide more than 1,000,000 patients (Jemal et al., 2006; Lerner, 2005). Taking into account that its incidence seems to be increasing, bladder cancer is clearly a significant public health issue around the world. Thus, it is necessary to intensify research on this topic. Urinary bladder cancer originates mainly from epithelial cells of the urothelium (LopezBeltran et al., 2004; Montironi et al., 2005). When initially diagnosed, most bladder cancers (about 70%) do not present muscle invasion, and are thus known as non-muscle invasive bladder cancer (pTa and pT1). In these cases, a simple transurethral resection is sufficient to remove the tumor. However, some patients experience recurrence or even tumor progression. The progression of the tumor involves invasion of tumor cells, which penetrate deeper layers of the bladder such as the detrusor muscle (pT2), perivesical tissue (pT3) and extravesical organs (pT4) (Figure 1). Since this progression threatens the patients life, more aggressive therapies are necessary (Sobin et al., 1997). Intensive research in bladder cancer, as well as that in most tumors, is being carried out to elucidate the reason for the appearance of tumors, and to find out which factors are involved in their development and which are related to the tumor progression process. These investigations, which provide insights into the biology of the tumor, are essential for the implementation of new therapeutic and/or preventive modalities (Bhattacharya et al., 2010; Zhang et al., 2011). Research on basic science is focused on the mechanisms that lead cells towards transformation and development of cancer, using simple experimental models where it is easier to interpret the results. Cell culture techniques are widely used to study different oncological processes. The cell culture is the growth of any cell type, usually tumor cells, in with nutrient-containing solutions. The cells grow attached to the plastic surface, forming a monolayer, usually in a two-dimensional way. This technique allows studying processes such as mutagenesis, invasion, migration, and production of proteolytic enzymes. Although cell culture is a very important tool, it has certain limitations. Many biological processes depend on the three-dimensional architecture. In addition, monolayer culture is usually

Flavonoid silybin improves the response to radiotherapy in invasive bladder cancer

Conservative treatment for invasive bladder cancer (BC) involves a complete transurethral tumor resection combined with chemotherapy (CT) and radiotherapy (RT). The major obstacles of chemo‐radiotherapy are the addition of the toxicities of RT and CT, and the recurrence due to RT and CT resistances. The flavonoid Silybin (Sb) inhibits pathways involved in cell survival and resistance mechanisms, therefore the purpose of this paper was to study in vitro and in vivo, the ability of Sb to improve the response to RT, in two murine BC cell lines, with different levels of invasiveness, placing emphasis on radio‐sensitivity, and pathways involved in radio‐resistance and survival. In vitro, Sb radio‐sensitized murine invasive cells through the inhibition of RT‐induced NF‐κB and PI3K pathways, and the increase of oxidative stress, while non‐invasive cells did not show to be sensitized. In vivo, Sb improved RT‐response and overall survival in invasive murine tumors. As Sb is already being tested in clinical trials for other urological cancers and it improves RT‐response in invasive BC, these results could have translational relevance, supporting further research.