mTOR inhibitors in urinary bladder cancer.

Despite the great scientific advances that have been made in cancer treatment, there is still much to do, particularly with regard to urinary bladder cancer. Some of the drugs used in urinary bladder cancer treatment have been in use for more than 30 years and show reduced effectiveness and high recurrence rates. There have been several attempts to find new and more effective drugs, to be used alone or in combination with the drugs already in use, in order to overcome this situation.The biologically important mammalian target of rapamycin (mTOR) pathway is altered in cancer and mTOR inhibitors have raised many expectations as potentially important anticancer drugs. In this article, the authors will review the mTOR pathway and present their experiences of the use of some mTOR inhibitors, sirolimus, everolimus and temsirolimus, in isolation and in conjunction with non-mTOR inhibitors cisplatin and gemcitabine, on urinary bladder tumour cell lines. The non-muscle-invasive cell line, 5637, is the only one that exhibits a small alteration in the mTOR and AKT phosphorylation after rapalogs exposure. Also, there was a small inhibition of cell proliferation. With gemcitabine plus everolimus or temsirolimus, the results were encouraging as a more effective response was noticed with both combinations, especially in the 5637 and T24 cell lines. Cisplatin associated with everolimus or temsirolimus also gave promising results, as an antiproliferative effect was observed when the drugs were associated, in particular on the 5637 and HT1376 cell lines. Everolimus or temsirolimus in conjunction with gemcitabine or cisplatin could have an important role to play in urinary bladder cancer treatment, depending on the tumour grading.

Trihalomethanes in liver pathology: Mitochondrial dysfunction and oxidative stress in the mouse.

Trihalomethanes (THMs) are disinfection byproducts found in chlorinated water, and are associated with several different kinds of cancer in human populations and experimental animal models. Metabolism of THMs proceeds through enzymes such as GSTT1 and CYP2E1 and gives rise to reactive intermediates, which form the basis for their toxic activities. The aim of this study was to assess the mitochondrial dysfunction caused by THMs at low levels, and the resulting hepatic histological and biochemical changes in the mouse. Male ICR mice were administered with two THMs: dibromochloromethane (DBCM) and bromodichloromethane (BDCM); once daily, by gavage, to a total of four administrations. Animals were sacrificed four weeks after DBCM and BDCM administrations. Blood biochemistry was performed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), albumin (Alb), total protein (TP), creatinine, and urea. Animals exposed to DBCM and BDCM showed elevated ALT and TB levels (p < 0.05) as compared with controls. Histological analysis confirmed the presence of vacuolar degenerescence and a multifocal necrotizing hepatitis in 33% of animals (n = 2). Mitochondrial analysis showed that THMs reduced mitochondrial bioenergetic activity (succinate dehydrogenase (SQR), cytochrome c oxidase (COX), and ATP synthase) and increased oxidative stress (glutathione S-transferase (GST)) in hepatic tissues (p < 0.05). These results add detail to the current understanding of the mechanisms underlying THM-induced toxicity, supporting the role of mitochondrial dysfunction and oxidative stress in liver toxicity caused by DBCM and BDCM. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1009-1016, 2016.

E-cadherin and ß-catenin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in mice.

BACKGROUND: E-cadherin and ß-catenin are adhesion molecules that promote integrity and stability of the urothelium. A decrease in their expression is associated with more aggressive tumour phenotypes with the ability to invade and metastasize. MATERIAL AND METHODS: 45 ICR male mice were used, of which 25 received N-butyl-N-(4-hydroxybutyl)nitrosamine (0.05%) in drinking water for a period of 12 weeks. Immunohistochemical expression was evaluated in all urinary bladder preparations for E-cadherin and for ß-catenin. RESULTS: Preneoplastic lesions showed staining patterns similar to normal urothelium. In simple and nodular hyperplasia, membrane staining was dominant (66.7-78.6 and 50-100%, respectively). In dysplasia a cytoplasmic pattern was prevalent (86.7-100%). Neoplastic lesions exhibit an abnormal staining pattern (100%) with heterogeneous staining (cytoplasmic, nuclear and membrane staining). A strong correlation was observed between both adhesion molecule staining patterns (r = 0.83; p = 0.039). CONCLUSIONS: In mice, as in humans, E-cadherin and ß-catenin are valuable tools to investigate cellular adhesion status of urothelium and can be considered as indicators of tumour aggressiveness and evolution.

SKELETAL MUSCLE SENSITIVITY TO WASTING INDUCED BY UROTHELIAL CARCINOMA.

BACKGROUND: Skeletal muscle wasting is a common phenotypic feature of several types of cancer, and it is associated with functional impairment, respiratory complications, and fatigue. However, equivocal evidence remains regarding the impact of cancer-induced muscle wasting on the different fiber types. AIM: The aim of this study was to investigate the impact of urothelial carcinoma induced in mice on the histomorphometric features and collagen deposition in different skeletal muscles. MATERIALS AND METHODS: Thirteen ICR (CD1) male mice were randomly assigned into two groups: exposed to 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water for 12 weeks, plus 8 weeks of tap water (BBN, n = 8) or with access to tap water for 20 weeks (CONT, n = 5). Tibialis anterior, soleus, and diaphragm muscles were collected from all animals. For cross-sectional area and myonuclear domain analysis, muscle sections were stained with hematoxylin and eosin, and for collagen deposition assessment, muscle sections were stained with picrosirius red. RESULTS: All animals from the BBN group developed urothelial preneoplastic and neoplastic lesions, and the tibialis anterior from these animals presented a reduced cross-sectional area (p < 0.001), with a decreased proportion of fibers with a higher cross-sectional area, increased collagen deposition (p = 0.017), and higher myonuclear domain (p = 0.031). BBN mice also showed a higher myonuclear domain in the diaphragm (p = 0.015). CONCLUSION: Urothelial carcinoma induced muscle wasting of the tibialis anterior, expressed by a decreased cross-sectional area, higher infiltration of fibrotic tissue, and increased myonuclear domain, which also increased in the diaphragm, suggesting that fast glycolytic muscle fibers are more susceptible to be affected by cancer development.

Effects of induced diabetes and the administration of aminoguanidine in the biomechanical retention of implants: a study in rats.

BACKGROUND AND OBJECTIVE: The present study aimed to assess the effects of induced diabetes and the administration of aminoguanidine in the biomechanical retention of implants in rats. MATERIAL AND METHODS: Thirty-six rats were randomly divided into six groups: group 1, healthy rats (no aminoguanidine); group 2 and group 3, healthy rats receiving 10 and 20 mg/kg of aminoguanidine daily, respectively; group 4, diabetic rats (no aminoguanidine); and group 5 and group 6, diabetic rats receiving 10 and 20 mg/kg of aminoguanidine daily, respectively. In each rat an implant was inserted in the femur. After 28 d of healing, the rats were killed. The implants were removed by applying a counter-torque, and the maximum force required for the rupture of the bone-implant interface was recorded using an analog torque meter. The data were evaluated using analysis of variance and the Student's t-test. RESULTS: In the healthy groups, no statistically significant difference could be observed in the average counter-torque values for implant removal, whereas in the diabetic groups, a daily dose of 20 mg/kg of aminoguanidine raised the counter-torque values to the values found in healthy rats. CONCLUSION: The administration of 20 mg/kg of aminoguanidine daily in diabetic rats raised the biomechanical retention of the implants to the level observed in the healthy rat group.

The influence of Castanea sativa Mill. flower extract on hormonally and chemically induced prostate cancer in a rat model.

Prostate cancer (PCa) is one of the most common cancers in men, with a huge impact on their health. The use of Castanea sativa Mill. flowers (CFs) in beverages has been reported, through ancestral claims, as having health benefits. In vitro research has evidenced the properties of CFs, such as antitumor and antioxidant activities. This study aimed to evaluate the effects of CF extract in an animal model of PCa. Forty male Wistar Unilever rats were randomly assigned to four groups: control, induced, control + CF, and induced + CF groups. Animals from the induced groups were exposed to a multistep protocol for PCa induction. The CF extract, rich in trigalloyl-HHDP-glucoside and obtained via decoction, was administered to the CF groups in drinking water (3 mg per animal per day) for 49 weeks. Animals were sacrificed at 61 weeks of age. Regarding the effects of CFs on dorsolateral prostate tumorigenesis, no significant differences were observed between the induced and induced + CF groups. However, animals exposed to the CF extract showed fewer inflammation areas on the dorsolateral prostate lobe than those not exposed to CF. Moreover, the CF extract alleviated the hepatic oxidative stress associated with the multistep protocol, resulting in lower levels of lipid peroxidation. These results suggest that CF extract has antioxidant and anti-inflammatory properties.

Genotyping in the Brazilian Criollo Horse Stud Book: resources and perspectives.

The goal of this research was to evaluate the ability of the genotyping information available in the Brazilian Criollo Horse Stud Book to describe the genetic variability of the breed and the exclusion probability determined in comparative tests. Altogether, two softwares were used in the analyses of the available genotypes: Cervus 3.0.3 and Genepop 4.0. Eight microsatellite markers totaled 109 alleles, with an average of 13.6 +/- 0.6 alleles per locus. Large differences between expected and observed heterozygosity were ubiquitous (0.821 +/- 0.07 and 0.470 +/- 0.17, respectively). Although the estimated null allele frequency caused initial concern (0.284 +/- 0.199), it is likely that it was a reflection of the inbreeding coefficients found (0.432 +/- 0.184). All loci showed significant deviation from Hardy-Weinberg equilibrium, with heterozygote deficit (P < 0.0001) and genotypic linkage disequilibrium with at least one marker. The high polymorphic information content (0.798 +/- 0.088) could not warrant exclusion power for three loci (HMS7, HMS6 and HTG4) above 50% (0.491 +/- 0.158). However, combined exclusion probability reached 99.61%, a level close to ideal. The results demonstrate the excellent performance of the markers assessed in describing the genetic status of the breed and suggest the considerable ability to establish parentage.

P53 in skin cancer: From a master player to a privileged target for prevention and therapy.

The increasing incidence of skin cancer (SC) is a global health concern. The commonly reported side effects and resistance mechanisms have imposed the pursuit for new therapeutic alternatives. Moreover, additional preventive strategies should be adopted to strengthen prevention and reduce the rising number of newly SC cases. This review provides relevant insights on the role of p53 tumour suppressor protein in melanoma and non-melanoma skin carcinogenesis, also highlighting the therapeutic potential of p53-targeting drugs against SC. In fact, several evidences are provided demonstrating the encouraging outcomes achieved with p53-activating drugs, alone and in combination with currently available therapies in SC. Another pertinent perspective falls on targeting p53 mutations, as molecular signatures in premature phases of photocarcinogenesis, in future SC preventive approaches. Overall, this review affords a critical and timely discussion of relevant issues related to SC prevention and therapy. Importantly, it paves the way to future studies that may boost the clinical translation of p53-activating agents, making them new effective alternatives in precision medicine of SC therapy and prevention.

HPV infection as a risk factor for atherosclerosis: A connecting hypothesis.

Atheromatous plaques occurring in large arteries are common and life-threatening lesions. Multiple factors are involved in the pathogenesis of atheromatous plaques, such as hyperlipidaemia and hypercholesterolaemia, high blood pressure and chronic systemic inflammation. Recent findings have suggested that infection with high-risk human papillomavirus (HPV) may increase the risk of developing atheromatous plaques. However, HPV is considered a tissue-specific virus with a strong tropism towards squamous epithelial cells, and the mechanisms whereby it may promote the development of atheromas remain unclear. Here, we propose a connecting hypothesis to explain the possible causative role of HPV on atheroma development. We hypothesize that HPV infection may promote atheroma formation in infected patients by enhancing systemic inflammation or by directly targeting blood vessels via nucleic acids carried by extracellular vesicles such as exosomes. The pro-inflammatory effects of HPV and the release of extracellular vesicles by HPV-transformed cells are well documented in scientific literature. Possible experimental approaches to test this hypothesis are also discussed, especially experiments employing transgenic mice bearing HPV16 transgenes. If correct, this hypothesis would have major implications for the prevention of cardiovascular diseases, especially due to the preventable nature of HPV infection through vaccination.

DNA cytometry and kinetics of rat urothelial lesions during chemical carcinogenesis.

The aims of this study were to evaluate the DNA content of chemically-induced rat urothelial lesions and their relationship to the proliferation index and histological patterns. Sixty female Fisher 344 rats were divided randomly into six groups, four groups were exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine for a period of 10 and 20 weeks, and two groups of ten rats were used as control animals. Paraffin sections were Feulgen stained and analyzed using DNA image cytometry analysis; histograms were classified as either diploid or aneuploid. Ki-67 immunoreactivity was determined by means of the streptavidin-biotin-complex immunoperoxidase method. All normal urothelium from the control groups were found to have diploid DNA content. The same histogram pattern was found in the simple hyperplasia group. As regards the other histological lesions, the frequency of the aneuploidy varied depending on the lesion type: 20% of aneuploidy were nodular hyperplasia, 32% of aneuploidy were dysplasias, 25% of aneuploidy were papilloma, 44% of aneuploidy were papillary neoplasm of low malignant potential, 22% of aneuploidy were low-grade papillary carcinoma, 100% of aneuploidy were high-grade papillary carcinoma and 100% of the aneuploidy were invasive carcinoma. Our results revealed the existence of a statistically significant relationship between DNA ploidy and histological pattern lesions (r=0.3, p<0.023). The Ki-67 proliferation index was significantly higher in aneuploid lesions than in diploid (r=0.56, p=0.01). There was also a statistically significant difference in the Ki-67 proliferation index in relation to the histopathological pattern (r=0.751, p<0.01). DNA content was associated with the Ki-67 proliferation index and histopathological grade. DNA content and prolife-ration index have critical roles to play during urothelial carcinogenesis.

Ozone therapy prevents the onset of dysplasia in HPV16-transgenic mice-A pre-clinical efficacy and safety analysis.

Infection with high-risk human papillomavirus (HPV), most often HPV16, is associated with the development of anogenital and oropharyngeal cancers. Recently, ozone therapy was reported to have considerable efficacy against rabbit VX2 tumors, induced by the cottontail rabbit papillomavirus. The present study aims to determine whether similar results can be obtained in HPV16-transgenic mice, possibly paving the way for new therapeutic options against HPV-induced cancers. HPV16-transgenic and wild-type, female, 20 weeks-old mice were injected intraperitoneally with medical O3/O2 (80░mL/kg, at O3 50░µg/mL), once a day, for 5 consecutive days. The animals were sacrificed at 25 weeks-old, and skin samples were analyzed histologically to study tumour progression. Blood and internal organ samples were used to study toxicological parameters. 85.7% of untreated transgenic mice showed dysplastic skin lesions, compared with 28.6% of O3-treated mice. This was associated with a marked reduction of dermal inflammation associated with those lesions. No significant changes were observed in any toxicological parameters. These preliminary results support the hypothesis that O3 therapy is effective against papillomavirus-induced lesions, particularly against those induced by the most common high-risk virus, HPV16. Further studies are needed to confirm the mechanisms underlying these effects.

Laurus nobilis (laurel) aqueous leaf extract's toxicological and anti-tumor activities in HPV16-transgenic mice.

Cancers induced by human papillomavirus (HPV) infection remain a significant public health threat, fueling the study of new therapies. Laurel (Laurus nobilis) compounds and extracts recently showed in vitro activity against HPV-transformed cell lines. This work aims to evaluate the in vivo efficacy and hepatic toxicity of a laurel extract in a transgenic mouse model of HPV16-induced cancer. The extract was administered in drinking water (20 mg per animal per day) for three consecutive weeks, using four experimental groups (n = 10) (group I: HPV16-/- without treatment, group II: treated HPV16-/-, group III: HPV16+/- without treatment and group IV: treated HPV16+/-). Following the treatment period, animals were sacrificed and skin samples were used to classify skin lesions histologically. Toxicological parameters included hematological and biochemical blood markers, splenic and hepatic histology and hepatic oxidative stress. The extract did not prevent the progression of HPV16-induced cutaneous lesions in this model. The treated wild-type animals showed mild hepatitis, while transgenic animals suffered weight loss. However, there were no changes concerning hematological, biochemical and hepatic oxidative stress markers.

Long-term exercise training prevents mammary tumorigenesis-induced muscle wasting in rats through the regulation of TWEAK signalling.

AIM: Exercise training has been suggested as a non-pharmacological approach to prevent skeletal muscle wasting and improve muscle function in cancer cachexia. However, little is known about the molecular mechanisms underlying such beneficial effect. In this study, we aimed to, firstly, examine the contribution of TWEAK signalling to cancer-induced skeletal muscle wasting and, secondly, evaluate whether long-term exercise alters TWEAK signalling and prevents muscle wasting. METHODS: Female Sprague-Dawley rats were randomly assigned to control and exercise groups. Fifteen animals from each group were exposed to N-Methyl-N-nitrosourea carcinogen. Animals in exercise groups were submitted to moderate treadmill exercise for 35 weeks. After the experimental period, animals were killed and gastrocnemius muscles were harvested for morphological and biochemical analysis. RESULTS: We verified that exercise training prevented tumour-induced TWEAK/NF-κB signalling in skeletal muscle with a beneficial impact in fibre cross-sectional area and metabolism. Indeed, 35 weeks of exercise training promoted the upregulation of PGC-1α and oxidative phosphorylation complexes. This exercise-induced muscle remodelling in tumour-bearing animals was associated with less malignant mammary lesions. CONCLUSION: Data support the benefits of an active lifestyle for the prevention of muscle wasting secondary to breast cancer, highlighting TWEAK/NF- κB signalling as a potential therapeutic target for the preservation of muscle mass.

E-cadherin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in rats.

An alteration in the expression of E-cadherin has been observed in many epithelial neoplasms. No data exist, however, for the expression of this protein in an animal model for urinary bladder cancer. The present study investigated the expression of E-cadherin in rat urothelial preneoplastic lesions and tumours induced by oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine, during 10, 15 and 20 weeks. Simple hyperplasia and squamous metaplasia showed a similar E-cadherin pattern when compared with normal urothelium, with its expression confined to cell membrane. Thirty eight percent of the nodular hyperplasia, 41.4% of the dysplasia and 100% of the papillomas showed a weak E-cadherin expression. All papillary neoplasm of low malignant potential, low-and high-grade papillary carcinoma, and invasive carcinoma revealed an abnormal staining pattern with an increase in cytoplasm reactivity and discontinuous cell membrane positivity. The loss of expression for low-grade papillary carcinoma versus simple hyperplasia, nodular hyperplasia and dysplasia was statistically significant (p = 0.0001, p = 0.007 and p=0.008, respectively). There was a similar decrease in E-cadherin expression for papillary neoplasm of low malignant potential versus simple hyperplasia, nodular hyperplasia and dysplasia (p = 0.0001; p = 0.001 and p=0.0001, respectively). These results suggest that alteration in the expression of this adhesion molecule in rat may be indicative of tumour progression in N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder cancer.

Challenges with in vitro and in vivo experimental models of urinary bladder cancer for novel drug discovery.

INTRODUCTION: Urinary bladder cancer (UBC) is the second most frequent malignancy of the urinary system and the ninth most common cancer worldwide, affecting individuals over the age of 65. Several investigations have embarked on advancing knowledge of the mechanisms underlying urothelial carcinogenesis, understanding the mechanisms of antineoplastic drugs resistance and discovering new antineoplastic drugs. In vitro and in vivo models are crucial for providing additional insights into the mechanisms of urothelial carcinogenesis. With these models, various molecular pathways involved in urothelial carcinogenesis have been discovered, allowing therapeutic manipulation. AREAS COVERED: This paper provides critical information on existing in vitro and in vivo models to screen the efficacy and toxicity of innovative UBC therapies and point out the challenges for new and improved models. EXPERT OPINION: In our opinion, results obtained with in vitro and in vivo models should be interpreted together, as a set of delicate biological tools that can be used at different stages in the drug discovery process, to address specific questions. With the development of new technologies, new assays and biomarkers are going to play an important role in the study of UBC. The molecular diagnostics and genomic revolution will not only help to develop new drug therapies, but also to achieve tailored therapies.

Long-term exercise training as a modulator of mammary cancer vascularization.

BACKGROUND: Breast cancer remains a leading cause of death by cancer worldwide. It is commonly accepted that angiogenesis and the expression of angiogenic factors such as vascular endothelial growth factor-A (VEGF-A) is associated with the increased risk of metastasis and poor patient outcome. OBJECTIVE: This work aimed to evaluate the effects of long-term exercise training on the growth and vascularization of mammary tumors in a rat model. MATERIALS AND METHODS: Fifty female Sprague-Dawley rats were divided into four groups: two N-methyl-N-nitrosourea (MNU)-exposed groups (exercised and sedentary) and two control groups (exercised and sedentary). MNU was administered once, intraperitoneally at 7 weeks-old. Animals were then exercised on a treadmill for 35 weeks. Mammary tumors were evaluated using thermography, ultrasonography [Power Doppler (PDI), B Flow and contrast-enhanced ultrasound (CEUS)], and immunohistochemistry (VEGF-A). RESULTS: Both, MNU sedentary and exercised groups showed 100% of tumor incidence, but exercised animals showed less tumors with an increased latency period. Exercise training also enhanced VEGF-A immunoexpression and vascularization (microvessel density, MVD) (p<0.05), and reduced histological aggressiveness. Ultrasound and thermal imaging analysis confirmed the enhanced vascularization of tumors on exercised animals. CONCLUSION: Long-term exercise training increased VEGF-A expression, leading to enhanced tumor vascularization and reduced tumor burden, multiplicity and histological aggressiveness.

Intake, digestibility, performance, and nitrogen metabolism of feedlot-finished young bulls () fed diets containing peanut cake.

This aim of this study was to evaluate the use of peanut cake as a dietary substitute for soybean meal and to determine the effects on intake, apparent digestibility, performance, and N metabolism in feedlot-finished young bulls. Thirty-two young Nellore bulls with an average initial BW of 390 ± 43.5 kg were distributed in a completely randomized design and individually housed in stalls. The young bulls were fed Tifton 85 hay and 4 concentrate mixes containing 0, 33, 66, or 100% peanut cake replacing soybean meal. The diets were formulated to be isonitrogenous, containing 150 g/kg CP, and isocaloric (65% TDN), to have a 40:60 forage:concentrate ratio, and were provided as a total mixed ration. The experiment lasted 90 d and data were collected every 28 d. Blood and urine samples were taken during the last 6 d. Intakes of DM ( = 0.005), OM ( = 0.006), CP ( = 0.002), NDF ( = 0.022), nonfiber carbohydrates ( = 0.002), and TDN ( = 0.018) linearly decreased as the dietary inclusion of peanut cake in the diet was increased. Conversely, intake and ether extract digestibility linearly increased ( < 0.035). The average daily weight gain decreased ( = 0.015) as the peanut cake levels were increased. Plasma urea N decreased (linearly; = 0.005). Peanut cake may not be used to replace soybean meal in the diet of young feedlot-finished Nellore bulls.

Evaluation of DNA content in preneoplastic changes of mouse urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

Using image cytometry analysis we analysed the deoxyribonucleic acid content of preneoplastic lesions induced in C3H/He female mice. Female mice (n = 30) were given 0.05% BBN in their drinking water for 4, 8 and 12 weeks, and were then euthanized. At the 4th week 90% hyperplasias, 90% dysplasias and 10% papillary tumours developed selectively in the bladder, after 8 weeks mice developed 80% hyperplasias and 80% dysplasias and 12 weeks we identified 70% hyperplasias, 70% dysplasias, 10% papillary tumours and 20% squamous metaplasia. Among the ones who developed simple hyperplasia, 58.3% were diploid and 41.7% were aneuploid. In the dysplasia lesions 29.2% were diploid and 70.8% were aneuploid (p = 0.041). All papillary tumours were aneuploid and all epidermoid metaplasias were diploid. These results suggest that aneuploidization, DNA content alteration of preneoplastic lesions, might be considered as a prognostic factor.