Cytostatic in vitro Effects of DTCM-Glutarimide on Bladder Carcinoma Cells

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 mu g/ml DTCM-glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas.

CD147 overexpression allows an accurate discrimination of bladder cancer patients' prognosis

Background: Urothelial bladder carcinoma (UBC) is a chemo-sensitive tumour, but the response to treatment is heterogeneous. CD 147 has been associated with chemotherapy resistance. We aimed to define tumours with an aggressive phenotype by the combined analysis of clinicopathological and biological parameters.Methods: 77 patients with T1G3 or muscle-invasive UBC treated by radical cystectomy were studied. Immunohistochemistry was performed to detect CD147, heparanase, CD31 (blood vessels identification) and D2-40 (lymphatic vessels identification) expressions. The immunohistochemical reactions were correlated with the clinicopathological and the outcome parameters. 5-year disease-free survival (DFS) and overall survival (OS) rates were estimated using the Kaplan-Meier method. Multivariate analysis was performed by Cox proportional hazards analysis.Results: The 5-year DFS and OS rates were significantly influenced by the classical clinicopathological parameters, and by the occurrence of lymphovascular invasion. CD 147 and heparanase immunoexpression did not affect patients' outcome. However, patients with pT3/pT4 tumours had a median OS time of 14.7 months (95% CI 7.1-22.3, p = 0.003), which was reduced to 9.2 months (95% CI 1.5-17.0, p = 0.008) if the tumours were CD147 positive. We developed a model of tumour aggressiveness using parameters as stage, grade, lymphovascular invasion and CD147 immunoexpression, which separated a low aggressiveness from a high aggressiveness group, remaining as an independent prognostic factor of DFS (HR 3.746; 95% CI 1.244-11.285; p = 0.019) and OS (HR 3.247; 95% CI 1.015-10.388, p = 0.047).Conclusion: CD 147 overexpression, included in a model of UBC aggressiveness, may help surgeons to identify patients who could benefit from a personalized therapeutic regimen. Additional validation is needed. (C) 2011 Elsevier Ltd. All rights reserved.

Local and remote lesions in horses subjected to small colon distension and decompression

The purpose of this study was to observe and characterize colonic and lung lesions in horses subjected to experimental distension and decompression of the small colon. Sixteen healthy adult horses were divided into 2 groups: 9 horses that were subjected to distension of the small colon by means of a latex balloon surgically implanted in the lumen and inflated to a pressure of 40 mm Hg for 4 h, and 7 horses in which the balloon was implanted but not inflated. Colonic biopsy specimens were collected before balloon implantation, at the end of the period of obstruction, and 1.5 and 12 h after decompression and were examined for hemorrhage, edema, and neutrophil infiltration; myeloperoxidase (MPO) activity and hemoglobin concentration were measured as well. At the end of the experiment, lung samples were also collected and examined for neutrophil accumulation and MPO activity. The mucosa was not affected by luminal distension; lesions were restricted to the seromuscular layer. Neutrophil accumulation and edema were observed in the samples from both groups of horses but were greater in those from the distension group, in which there was also hemorrhage, fibrin deposition, and increased MPO activity in the seromuscular layer. Similarly, there was greater accumulation of neutrophils in the lung samples from the distension group than in those from the sham-operated group, as determined by histologic evaluation and MPO assay. These findings provide new evidence of reperfusion injury and a systemic inflammatory response, followed by remote lesions, in horses with intestinal obstruction.

Anti-Angiogenic Effects of the Superantigen Staphylococcal Enterotoxin B and Bacillus Calmette-Guerin Immunotherapy for Nonmuscle Invasive Bladder Cancer

Purpose: We compared and characterized the effects of intravesical bacillus Calmette-Guerin and/or staphylococcal enterotoxin B for nonmuscle invasive bladder cancer.Materials and Methods: A total of 75 female Fisher 344 rats were anesthetized. of the rats 15 received 0.3 ml saline (control) and 60 received 1.5 mg/kg MNU (N-methyl-n-nitrosourea) intravesically every other week for 6 weeks. The rats were divided into 5 groups. The MNU and control groups received 0.3 ml saline. The bacillus Calmette-Guerin group received 10(6) cfu bacillus Calmette-Guerin. The staphylococcal enterotoxin B group received 10 mu g/ml staphylococcal enterotoxin B. The bacillus Calmette-Guerin plus staphylococcal enterotoxin B group received the 2 treatments simultaneously. Each group was treated intravesically for 6 weeks. At 15 weeks all bladders were collected for histopathological and immunological evaluation, and Western blot.Results: Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (carcinoma in situ) were more common in the MNU group. Papillary hyperplasia was more common in the bacillus Calmette-Guerin and enterotoxin groups. Flat hyperplasia was more common in the bacillus Calmette-Guerin plus enterotoxin group. No significant toxicity was observed. The apoptosis and cellular proliferation indexes decreased in the bacillus Calmette-Guerin, enterotoxin and bacillus Calmette-Guerin plus enterotoxin groups compared to the MNU group. Intensified vascular endothelial growth factor, matrix metalloproteinase-9, Ki-67 and insulin-like growth factor receptor-1 immunoreactivity was verified in the MNU group, moderate in the bacillus Calmette-Guerin and enterotoxin groups, and weak in the bacillus Calmette-Guerin plus enterotoxin and control groups. In contrast, intense endostatin immunoreactivity was verified in the control and bacillus Calmette-Guerin plus enterotoxin groups.Conclusions: Bacillus Calmette-Guerin and staphylococcal enterotoxin B showed similar anti-angiogenic effects. Bacillus Calmette-Guerin plus enterotoxin treatment had additional activity compared to that of monotherapy. It was more effective in restoring apoptosis and balancing cellular proliferation, and it correlated with increased endostatin, and decreased vascular endothelial growth factor, matrix metalloproteinase-9, Ki-67 and insulin-like growth factor receptor-1 reactivity.

Urinary bladder washings in autopsies. A cytopathologic study of 63 cases

The sediment from urinary bladder washings from 63 consecutive autopsies was cytologically studied in order to achieve a better understanding of the changes in urothelial cells collected from hospital populations. The observed alterations were correlated with alterations in the urinary system and with therapy preceding death. The specimens obtained were of good quality. In 39.7% of the cases, the sediment contained giant superficial multinucleated cells. Three of nine cases previously subjected to radiation or chemotherapy showed atypical urothelial cells. In three cases with immunosuppression, there was cytologic evidence of subclinical infection by polyomavirus, and virus particles were identified by electron microscopy of the vesical mucosa. The study of the smear background offered additional information: the sediment contained hyaline or hematic or hyaline-cellular casts in 17.4% of the cases, in all of which there were renal tubulopathies when the kidney sections were studied. The method is useful for a good evaluation of the autopsy as well as for training in urinary cytopathology.

Expression of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: possible relationship with the prognosis.

Transitional cell carcinoma (TCC) of the bladder is a neoplasm with variability in its clinical behavior. Although there are several studies correlating stage and ABO isoantigen expression with invasiveness, there is no single predictor factor to assess the potential invasiveness, especially in the low grade, non-invasive TCC. In the present study we evaluated the correlation of histological grade plus stage and the expression of beta human chorionic gonadotropin (beta-hCG), in 100 cases of TCC, with the clinical behavior. These features were correlated with tumor progression in patients with at least two years of follow up. We observed more aggressiveness in G4 group (high grade and invasive) (93% had tumor progression) when compared to G1 group (low grade and superficial) (11% had tumor progression). However in 25.5% of the TCC cases (groups G2: low grade and invasive and G3: high grade and superficial) the clinical behavior was intermediate, showing some limitation in using grading and staging only, as a predictive factor. There was an expression of beta-hCG in 21.4% of the cases in up to 25% of the tumor cells without any trophoblastic morphology. These beta-hCG producing TCC had a strong correlation with aggressiveness: 39.1% and 12.8% of the TCC expressed beta-hCG with and without tumor progression, respectively.

Frequent p53 mutations and occasional loss of chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-hydroxybutil)nitrosamine in B6D2F1 mice

B6D2F1 mice (45/group) were treated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) or uracil as follows: Group 1 received 0.05% BBN in drinking water for the entire experiment, Group 2 received 5 mg of BBN by gastric gavage in 0.1 mL of 20% ethanol twice per week for 10 wk, Group 3 received a 2.5% uracil-containing diet for the entire experiment, and Group 4 was controls (received 0.1 mL of 20% ethanol by gavage twice per week for 10 wk). The surviving mice in Group 1 were killed after week 26 and those in the other groups after week 30. By week 15, three of 11 Group 1 and one of 15 Group 2 mice had bladder carcinoma. By 26 and 30 wk, respectively, invasive carcinomas were observed in 33 of 34 and six of 21 mice in Groups 1 and 2 and renal pelvic carcinomas in 11 of 34 and three of 21 mice in Groups 1 and 2. Four of 19 uracil-treated mice had bladder nodular hyperplasia. By polymerase chain reaction-single-strand conformation polymorphism and sequence analyses, 16 of 20 and two of five bladder carcinomas from Groups 1 and 2, respectively, showed mutations in the p53 gene. Ha-ras mutation was present in one case. Loss of heterozygosity analysis with simple-sequence length polymorphism markers for chromosome 4 showed that 10 of 21, two of 15, and nine of 13 mice in Groups 1-3, respectively, had heterozygous or homozygous deletions. B6D2F1 mice are therefore susceptible to the urothelial carcinogenic effects of BBN and develop frequent p53 mutations and chromosome 4 deletions. Chromosome 4 deletions were also seen with uracil.

Effects of uracil calculi on cell growth and apoptosis in the BBN- initiated Wistar rat urinary bladder mucosa

The different potential of initiated and non-initiated urinary bladder mucosa (UBM) to develop neoplasia was quantitatively evaluated in the male Wistar rat. Initiation of carcinogenesis was accomplished with N-butyl-N-(4- hydroxybutyl)-nitrosamine (BBN). Stimuli for cell proliferation and apoptosis were obtained by exposure followed by withdrawal of 3% Uracil in the diet. The proliferation index (PI) was estimated in UBM immunostained for the proliferating nuclear cell antigen (PCNA). The apoptotic index (AI) and the density of papillary/nodular hyperplasia (PNH) were estimated in hematoxilin- eosin stained sections. PNH was the main proliferative response to the mechanical irritation by uracil, irrespective of previous initiation with BBN. Uracil exposure induced higher PI and PNH density in the initiated rats. After uracil withdrawal, there was a significant increase of the AI in both uracil-treated groups, which correlated well to the respective PNH density. However, at the end of the experiment, PNH incidence and density were significantly higher in the BBN-initiated mucosa, which also presented 18% incidence of papillomas and 27% of carcinomas. Therefore, under prolonged uracil calculi trauma, the UBM of BBN-initiated Wistar rats gives rise to epithelial proliferative lesions that progress to neoplasia through acquired resistance to apoptosis.

Anatomical features of the urethra and urinary bladder catheterization in female mice and rats. An essential translational tool

PURPOSE: To present fundamental anatomical aspects and technical skills necessary to urethra and urinary bladder catheterization in female mice and rats. METHODS: Urethral and bladder catheterization has been widely utilized for carcinogenesis and cancer research and still remains very useful in several applications: from toxicological purposes as well as inflammatory and infectious conditions to functional aspects as bladder dynamics and vesicoureteral reflux, among many others. RESULTS: Animal models are in the center of translational research and those involving rodents are the most important nowadays due to several advantages including human reproducibility, easy handling and low cost. CONCLUSIONS: Although technical and anatomical pearls for rodent urethral and bladder access are presented as tackles to the advancement of lower urinary tract preclinical investigation in a broaden sight, restriction to female animals hampers the male microenvironment, demanding future advances.

Low RKIP expression associates with poor prognosis in bladder cancer patients

Urothelial bladder cancer (UBC) is a heterogeneous type of disease. It is urgent to screen biomarkers of tumour aggressiveness in order to clarify the clinical behaviour and to personalize therapy in UBC patients. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor, and its downregulation is associated with metastatic events in an increasing number of solid tumours. We evaluated the clinical and prognostic significance of RKIP expression in patients with high risk of progression UBC. Using immunohistochemistry, we determined RKIP expression levels in a series of 81 patients with high-grade pT1/pTis or muscle-invasive UBC. Staining of CD31 and D2-40 was used to assess blood and lymphatic vessels, in order to distinguish between blood and lymphatic vessel invasion (LVI). We found that 90 % of pT1/pTis tumours, 94 % of non-muscle invasive papillary tumours and 76 % of the cases without LVI occurrence expressed RKIP in >10 % of cells. In this group, we observed a subgroup of tumours (42 %) in which the tumour centre was significantly more intensely stained than the invasion front. This heterogeneous pattern was observed in 63 % of the cases with LVI. Low RKIP expression was associated with poorer 5-year disease-free and overall survival rates, and remained as an independent prognostic factor for disease-free survival. Loss of RKIP expression may be an important prognostic factor for patients with high risk of progression bladder cancer. © 2013 Springer-Verlag Berlin Heidelberg.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma

Objective: To understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients. Methods: Tumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses-all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region. Results and Conclusions: HR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods free-of-neoplasia. Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis. © 2013 Elsevier Inc.