Rat Bladder Tumor Research Articles

Studies of the photosensitizer disulfonated meso-tetraphenyl chlorin in an orthotopic rat bladder tumor model

Photochemical internalization (PCI) is a novel technology for the release of a therapeutic molecule from endocytic vesicles into the cytosol of a cell. The release of molecules occurs after activation of an endocytic membrane-embedded photosensitizer by light. In this study uptake and localization of the photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) were explored to optimize a PCI protocol in an orthotopic rat bladder tumor model. Female Fischer F344 rats were intravesically instilled with 0.4×10(6) AY-27 transitional carcinoma cells before allowing tumor growth for 14 days. The photosensitizer TPCS2a was intravesically instilled at different concentrations, and bladders were excised after different time intervals. The retention, penetration, and localization of intratumoral TPCS2a were explored ex vivo using fluorescence spectroscopy and fluorescence microscopy to determine an optimal PCI protocol. These results were compared to histological analysis of necrotic areas after activation of intratumoral TPCS2a by red light (652nm, 0.5J/cm(2)). A superficial distribution pattern of the photosensitizer TPCS2a was seen in bladder tumor tissue, and TPCS2a was almost cleared from the tumors after 72h. The highest retention of TPCS2a was found at 24h after instillation when using a concentration of 3mg/ml. An optimal PCI protocol was defined for the tumor model, including a 24-h TPCS2a-to-light interval and a dose of 3mg/ml TPCS2a. This protocol will be utilized for the study of PCI-enhanced therapeutic effects on non-muscle invasive bladder cancer, using a potent chemotherapeutic under an optimal light dose.

DMA(V) in Drinking Water Activated NF-κB Signal Pathway and Increased TGF-β and IL-1β Expressions in Bladder Epithelial Cells of Rats.

Dimethylarsinic acid (DMAV) is the main product of arsenic methylation metabolism in vivo and is rat bladder carcinogen and tumor promoting agent. In this study, we measured the expressions of mRNA and proteins of NF-κB pathway members, IKKα, IKKβ, p65, and p50 in rat bladder epithelium by qRT-PCR and immunohistochemical analysis after rats received drinking water containing 100 and 200 ppm DMAV for 10 weeks. Transforming growth factor-β (TGF-β) immunoexpression in rat bladder epithelium and urine level of IL-1β also were determined. We found that DMAV dramatically increased the mRNA levels of NF-κB p50 and IKKα in the bladder epithelium of rats compared to the control group. Immunohistochemical examinations showed that DMAV increased immunoreactivities of IKKα, IKKβ, and phospho-NF-κB p50 in the cytoplasm and phospho-NF-κB p50 and p65 in nucleus of rat urothelial cells. In addition, DMAV treated rats exhibited significantly increased inflammatory factor TGF-β immunoreactivity in bladder epithelium and IL-1β secretion in urine. These data suggest that DMAV could activate NF-κB signal pathway and increase TGF-β and IL-1β expressions in bladder epithelial cells of rats.

Epigallocatechin-3-gallate prevents tumor cell implantation/growth in an experimental rat bladder tumor model

The aim of this study was to determine the efficacy of epigallocatechin-3-gallate (EGCG) (Polyphenon E®) in comparison with mitomycin C (MMC) to prevent tumor cell implantation/growth in an animal model of superficial bladder cancer and search for possible mechanism(s) of action. Female Fisher 344 rats were used to study the effects of EGCG and mitomycin C for the prevention of transitional cell tumor implantation (AY-27). Twenty rats served as a control, tumor implantation and saline wash only. Sixty rats were treated with EGCG (100, 200 and 400 µM) intravesically for 60 or 120 min after tumor implantation. Thirty other rats were divided equally and pretreated with 400 µM EGCG or saline for 120 min before tumor initiation. In a separate series of experiments, 30 rats were treated 2 weeks after tumor initiation with saline or EGCG (400 µM). In a different experiment 39 rats were treated with: saline (n=10) EGCG (n=9) 400 µM, MMC (n=10) 0.5 µM, MMC (n=10) 400 µM. Rats were sacrificed 3 weeks following treatment. Gross and histological analyses were performed on the bladders. EGCG and mitomycin C prevented intravesical tumor growth in a concentration- and time-dependent manner. EGCG pretreatment or treatment 2 weeks post tumor implantation did not have therapeutic effects. Molecular modeling suggests that EGCG inhibits urokinase and matrix metalloproteinase-9. EGCG prevents intravesical tumor implantation/growth with a slightly better efficacy than mitomycin C in this experimental model. The data suggest that EGCG lowers proteolytic activity and lowers probability of cancer cell implantation rather than direct cancer cell killing.

A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Novel intravesical therapies are needed for superficial bladder cancer that reduce the risk of infection associated with Bacillus Calmette-Guérin (BCG) and further destabilization of the urothelium associated with cytotoxic chemotherapy. Experimental therapies to date have included photodynamic therapy, oncolytic viruses, gene therapy (antisense oligonucleotides and silencing RNA), cytokine therapy, death receptor agonists (tumour-necrosis-factor-related apoptosis-inducing ligand and anti-DR5 monoclonal antibody), naturally occurring substances (curcumin and deguelin) and human α-lactalbumin made lethal to tumour cells (HAMLET). HAMLET, a natural occurring product in milk, induces apoptosis in urothelial cancer cells but has limitations in clinical application because of its human source. A previous study in patients with bladder cancer has demonstrated that intravesical HAMLET (daily for 5 days before tumour resection) caused selective apoptotic tumour cell death. BAMLET, the bovine equivalent of HAMLET, is a complex of bovine α-lactalbumin and oleic acid (bLAC) that has been shown in vitro to accumulate in the endolysosomal compartment of tumour cells and induce leakage of lysosomal cathepsins into the cytosol followed by activation of the pro-apoptotic protein Bax. This is the first in vivo study to show that BAMLET (bLAC) induces apoptosis in urothelial cancer cells and controls the growth of high risk urothelial cancer in a syngeneic rat orthotopic model. This same bladder cancer model system has been used to test other novel therapies, including BCG, and therefore provides a relative comparison of its effectiveness with other intravesical therapies. To investigate the efficacy of a complex of bovine α-lactalbumin and oleic acid (bLAC) to kill urothelial cancer cells in vitro and inhibit tumour growth and progression in a high risk bladder tumour model. The cytotoxicity of bLAC to a large panel of urothelial cell cancer (UCC) cells was tested by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay, using bLA, the folded α-lactalbumin without oleic acid, as a control. The mechanism of bLAC-inducing cell death was evaluated by annexin V staining, TUNEL (terminal deoxynucleotidyl transferase mediated nick end labelling) assay and sub-G1 DNA analysis. The selective bLAC cytotoxicity was examined using multicellular spheroids consisting of UCC and non-transformed fibroblasts. Rats bearing orthotopic tumour received intravesical instillations (twice weekly, for 3 weeks) of bLAC, bLA, BCG or saline, starting 6 days after UCC (AY-27) cell inoculation. Animals were monitored for survival, toxicity and tumour growth control. A dose-dependent bLAC-inducing apoptotic-like cell death was shown in UCC cells tested, including cells refractory to classic apoptosis-inducing agents, whereas bLA showed little cytotoxicity. bLAC selectively destroyed cancer cells in spheroids. Intravesical bLAC therapy demonstrated marked reduction in tumour growth/progression and significantly prolonged animal survival vs saline instillations (P = 0.004, log-rank test) and showed comparable efficacy with BCG (standard) therapy. The findings identify bLAC as a new candidate for UCC therapy and suggests that topical administration of bLAC alone or with BCG to prevent progression of bladder cancer warrants further exploration.

Gleevec, an Abl Family Inhibitor, Produces a Profound Change in Cell Shape and Migration

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

102 Usage of low-intensive laser radiation in the treatment of patients with urological and proctologic diseases

The fluorescence kinetics of photoporphyrin IX (PPIX) following intravesical instillation of 5-aminolaevulinic acid (5-ALA) have been studied in vivo in a rat bladder tumour model. 5-ALA dissolved in NaHCO3 was intravesically instilled for 60 min in tumour-bearing and normal bladders of Wistar rats. The fluorescence was excited with the violet lines of a Kr+-laser and recorded in vivo by means of a fibre coupled optical multichannel analyser. The fluorescence emission bands of PPIX at λ = 636 nm and λ = 708 nm were detected in normal and tumorous orothelium after only 30 min. The maximum fluorescence intensity was obtained in tumorous and normal urothelium 3–4 h after instillation. The ratio of the fluorescence intensity in tumorous to normal urothelium decreased continuously from four to about two during the time range of 6 h. PPIX fluorescence following 5-ALA installation could also be observed in kidney and liver. Fluorescence from further porphyrin species with emission bands at λ = 617 nm and λ = 682 nm was detected in the bladder, indicating an efflux of hydrophilic porphyrins from the hepatic pathway.

Androgen Receptor Signaling Regulates Cell Growth and Vulnerability to Doxorubicin in Bladder Cancer

There are several reports of androgen receptor in bladder cancer cases but androgen receptor expression and the function of androgen/androgen receptor signaling in bladder cancer remain unclear. We investigated androgen receptor expression and the role of androgen/androgen receptor signaling in bladder cancer. We evaluated AR mRNA expression in bladder cancer tissue by quantitative real-time polymerase chain reaction. The role of androgen receptor in cell growth and drug sensitivity was also evaluated in vitro and in vivo in several bladder cancer cell lines. AR mRNA expression inversely correlated with bladder cancer grade, stage and spread. Of several bladder cancer cell lines UMUC3 and MBT-2 markedly expressed androgen receptor transcript and protein. In each cell line androgen/androgen receptor signaling blockade using androgen deprivation, blockade knockdown and antiandrogen agents decreased cell growth, colony formation and cell viability. Androgen receptor expression was implicated in doxorubicin resistance. Inversely androgen receptor deprivation and knockdown made UMUC3 cells sensitive to doxorubicin. Finally, castration slightly suppressed UMUC3 tumor growth in vivo, although this did not attain statistical significance. AR transcript expression inversely correlates with bladder cancer clinicopathological characteristics. Androgen/androgen receptor signaling has an important role in the growth and vulnerability to doxorubicin of bladder cancer cells that express androgen receptor. Androgen/androgen receptor signaling might be a possible prophylactic and therapeutic target for bladder cancer that shows androgen receptor expression.

Letter: The Risk of Bladder Cancer in Korean Diabetic Subjects Treated with Pioglitazone (Diabetes Metab J2012;36:371-8)

Type 2 diabetes mellitus is characterized by insulin resistance as well as progressive pancreatic β-cell dysfunction. Thiazoli dinediones (TZDs) are a class of hypoglycemic medications that influence insulin sensitivity in peripheral tissue [1]. Piogl itazone is a TZD, peroxisome proliferator-activated receptor (PPAR)-γ ligand used in the treatment of type 2 diabetic patients [2]. Many preclinical trials have demonstrated that PPAR-γ is a potential molecular target for the development of bladder cancer. Yoshimura et al. [3] found PPAR-γ mRNA in bladder cancer samples. Furthermore, immunohistochemistry revealed that the protein expression of PPAR-γ was significantly higher with increasing grade and increasing stage of bladder cancer [3]. In male rats exposed to muraglitazar, a dual human PAPR-α/γ agonist, there was a dose-related increased incidence of transitional cell papilloma and carcinoma of the urinary bladder [4]. Lubet et al. [5] also reported that another PPAR-γ agonist, rosiglitazone, enhanced bladder tumors in rats pretreated with N-(4-hydroxybutyl)-N-(butyl)-nitrosamine (BBN), a known DNA-reactive bladder carcinogen in several species. However, the mechanisms underlying this pro-tumor potential of pioglitazone for bladder cancer are not fully understood. In the large prospective pioglitazone clinical trial in macrovascular events (PROactive) study, 14 bladder cancers occurred in the pioglitazone arm (0.5%) versus 6 in the placebo arm (0.2%) [6], and in September 2010, the U.S. Food and Drug

삭카린의 안전성과 각국의 관리 현황

Department of Science and Technology Education for Life, Seoul National University of EducationAbstract Saccharin was reported to cause urinary bladder cancer in male rats when fed at high doses in a two-generationstudy, which led to a ban on the use of saccharin in Canada. However, no carcinogenic effect has been observed in otheranimal experiments conducted with mice, hamsters, or monkeys. Furthermore, numerous epidemiological studies haveindicated that there was no relationship between saccharin consumption and the risk of bladder cancer in the humanpopulation. Sodium saccharin produces urothelial bladder tumors in rats by the formation of a urinary calcium phosphate-containing precipitate, which is not relevant to humans because of critical interspecies difference in urine composition.Consequently, in 1999 IARC (International Agency for Research on Cancer) concluded that saccharin and its salts cannotbe classified as to their carcinogenicity in humans. In 2010, the EPA (Environmental Protection Agency) of the UnitedStates removed saccharin from its list of hazardous substances. It is expected that the use of saccharin in foods might beexpanded because saccharin is currently considered safe.Keywords: saccharin, sodium saccharin, artificial sweetener, safety

Saccharin Deemed “Not Hazardous” in United States and Abroad

Banned in 1981, saccharin has long been considered carcinogenic because it produced bladder tumours in rats. Saccharin is now unbanned, with more than 100 countries worldwide legitimately allowing saccharin as a food sweetening additive[...]

A Comparative Study of the Inhibiting Effects of Mitomycin C and Polyphenolic Catechins on Tumor Cell Implantation/Growth in a Rat Bladder Tumor Model

Mitomycin C (Novaplus®) is often instilled intravesically in the postoperative period to prevent tumor cell implantation/regrowth after transurethral tumor resection. In an earlier study EGCG prevented tumor cell implantation/growth in an experimental bladder tumor model simulating clinical transurethral bladder resection. We compared the efficacy of EGCG (Polyphenon E®) to that of mitomycin C to prevent tumor cell implantation/growth in this model. Mitomycin C and EGCG were studied for their in vitro and in vivo effects. The AY-27 rat urothelial tumor cell line was used for in vitro and in vivo studies. In vitro cell viability studies included trypan blue exclusion, MTT proliferation assay and clonal growth assay. Fischer 344 female rats were used for intravesical tumor implantation/growth assay using an electrocautery injury model. Tumor growth in vivo was assessed in controls treated with phosphate buffered saline and in bladders treated with mitomycin C or EGCG by standard histological techniques using hematoxylin and eosin 4 weeks after injury. Mitomycin C and EGCG showed cytotoxicity on all in vitro assays. They were equivalent for preventing intravesical tumor growth. EGCG prevents intravesical tumor growth with efficacy equivalent to that of mitomycin C in this experimental model.

Thiothymidine combined with UVA as a potential novel therapy for bladder cancer

Background:Thiothymidine (S4TdR) can be incorporated into DNA and sensitise cells to DNA damage and cell death following exposure to UVA light. Studies were performed to determine if the combination of S4TdR and UVA could be an effective treatment for bladder cancer.Methods:Uptake and incorporation of S4TdR was determined in rat and human bladder tumour cell lines. Measures of DNA crosslinking and apoptosis were also performed. In vivo activity of the combination of S4TdR and UVA was investigated in an orthotopic model of bladder cancer in rats.Results:Thiothymidine (200 μM) replaced up to 0.63% of thymidine in rat and tumour bladder cancer cells. The combination of S4TdR (10–200 μM) and UVA (1–5 kJ m−2) caused apoptosis and cell death at doses that were not toxic alone. Addition of raltitrexed (Astra Zeneca, Alderley Edge, Cheshire, UK) increased the incorporation of S4TdR into DNA (up to 20-fold at IC5) and further sensitised cells to UVA. Cytotoxic effect was associated with crosslinking of DNA, at least partially to protein. Intravenous administration of S4TdR, in combination with UVA delivered directly to the bladder, resulted in an antitumour effect in three of five animals treated.Conclusion:These data indicate that the combination of S4TdR and UVA has potential as a treatment for bladder cancer, and give some insight into the mechanism of action. Further work is necessary to optimise the delivery of the two components.

The effects of oral treatment with transfluthrin on the urothelium of rats and its metabolite, tetrafluorobenzoic acid on urothelial cells in vitro

Transfluthrin, a pyrethroid insecticide, induced urinary bladder tumors in rats but not in mice in 2-year bioassays. We investigated the urothelial effects of transfluthrin in vivo in rats and the effects of its major metabolite tetrafluorobenzoic acid (TFBA) in vitro on rat (MYP3) and human (1T1) urothelial cell lines. Rats were fed diet containing 0, 2000 or 5000 (with and without 1.25% NH4Cl) ppm transfluthrin for 4weeks or 0 or 2000ppm transfluthrin for 13weeks. After 4weeks, there was no evidence of hyperplasia or increased proliferation in any treatment group. After 13weeks treatment with 2000ppm, cytotoxicity and necrosis of the rat urothelial superficial layer were detected by scanning electron microscopy. The urinary concentration of TFBA in rats fed 2000ppm transfluthrin was 2.94±0.67mM. The LC50 of TFBA was 2.25mM for MYP3 cells and 2.43mM for 1T1 cells. These studies support cytotoxicity and regenerative proliferation as the mechanism for induction of bladder tumors with high oral doses of transfluthrin due to metabolism of transfluthrin to the weakly cytotoxic TFBA which is excreted at high concentrations in the urine of rats administered high doses of transfluthrin (⩾2000ppm) for an extended period.

Abstract A106: Efficacy of EGFr inhibitors gefitinib on development of OH-BBN-induced rat bladder tumors: Dose dependency and modulation of biomarkers

Abstract The preventive effects of the EGFr inhibitor Iressa on the induction of urinary bladder cancers induced by OH-BBN was determined. Treatment of rats with Iressa (10 mg/kg BW/day) beginning two weeks after the last dose of OH-BBN decreased the incidence of large palpable bladder cancer lesions by 95% at an 8-month observation period. Treatment with lower doses of Iressa (4 and 1.33 mg/kg BW/day) decreased the incidence of large tumors by 80 and 65%, respectively. This preventive efficacy was similarly observed when one examined an incidence curve; i.e., a large increase in tumor latency. At 3 1/2 months after the last OH-BBN dose, 75% of the rats had already developed microscopic cancers. Iressa treatment beginning at this time period also lowered the incidence of large tumors; implying that most of the effects of Iressa were during the latter periods of tumor progression. We also examined potential biomarkers modulated by short-term exposure to Iressa (10 mg/kg BW/day) in urinary bladder cancers. Three approaches were taken to identify biomarkers: (1) Alterations in EGFr related proteins directly associated with the efficacy of Iressa by immunohistochemistry. Levels of various biomarkers (phosphorylated EGFr, phosphorylated AKT, but not phosphorylated ERK levels were significantly decreased in the bladder tumors of treated rats. (2) Alterations in protein expression when comparing control and Iressa treated tumors. Employing two-dimensional gel electrophoresis and tandem mass spectrometry, it was found that proteins associated with the angiogenic effects of VEGF were decreased in tumors from treated animals. These included cytoskletal proteins nucleolin, annexin A1 and the MAP kinase (MAPKK). (3) Alterations in gene expression employing array gene technology. Numerous metabolic and cellular pathways were markedly altered by Iressa treatment, most particularly cell cycle related genes; i.e., there were strong inhibition of expression of CDC20 and both of the aurora kinases. Thus, we have identified biomarkers that might be relevant as pharmacodynamic or efficacy biomarkers in clinical prevention trials of urinary bladder cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A106.

An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells

Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI). We found that the presence of DiI did not alter the in vitro AY-27 cell proliferation and that the DiI label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 days. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1 to 3 days after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1mM MM-C survived the final endpoint of 30 days after intravesical MM-C. Moreover, 10 and 30 days after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of DiI-loaded AY-27 cells and cellular debris by macrophages and related immune cells.

A novel Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system exhibits antitumor activity in a rat model of bladder cancer

Bladder cancer is the ninth most common malignancy in the world. Successful clinical management remains a challenge. In order To search for novel targeted and efficacious treatment, we sought to investigate anti-tumor activity of BI-TK suicide gene therapy system in a rat model of bladder tumors. We first constructed and tested an anaerobic Bifidobacterium infantis-mediated thymidine kinase (BI-TK) suicide gene therapy system. To test the in vivo efficacy of this system, we established a rat model of bladder tumors, which was induced by N-methyl-nitrosourea perfusion. Bifidobacterium infantis containing the HSV-TK (i.e., BI-TK) were constructed by transformation of recombinant plasmid pGEX - TK. The engineered BI-TK was injected into tumor-bearing rats via tail vein, followed by intraperitoneal injection of ganciclovir (GCV). Using the rat model of bladder tumors, we found that bladder tumor burdens were significantly lower in the rats treated with BI-TK/GCV group than that treated with normal saline control group (p <0.05). While various degrees of apoptosis of the tumor cells were detected in all groups using in situ TUNEL assay, apoptosis was mostly notable in the BI-TK/GCV treatment group. Immunohistochemical staining further demonstrated that the BI-TK/GCV treatment group had the highest level of caspase3 protein expression than that of the empty plasmid group and normal saline group (p < 0.05). Thus, our results demonstrate that the Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system can effectively inhibit rat bladder tumor growth, possibly through increasing caspase 3 expression and inducing apoptosis.

The orthotopic Fischer/AY-27 rat bladder urothelial cell carcinoma model to test the efficacy of different apaziquone formulations

Objectives Apaziquone used intravesically showed significant activity in phase I and II marker lesion studies in non-muscle-invasive bladder cancer. The objective of this study was to assess antitumor activity and safety of 3 different formulations of intravesical apaziquone in an orthotopic rat bladder cancer model. Materials and methods Female Fischer F344 rats were instilled with 1.5 × 10 6 AY-27 urothelial cell carcinoma cells and divided in 3 treatment groups ( n = 10) and 1 placebo group ( n = 6). Intravesical treatment was administered for 1 hour on days 2 and 5. Rats were treated with apaziquone in the formulation used in phase I/II clinical trials (group 1); apaziquone with an altered buffering capacity being used in phase III clinical trials (group 2), and apaziquone as in group 2, but without propylene glycol in the diluent (group 3). On days 5 and 14, the bladder wall was inspected by cystoscopy and evaluated for macroscopic tumor growth. After sacrificing the rats (day 14), cystectomy was performed and the bladders were investigated. Results There were no signs of any toxicity due to the study drug. On histopathologic examination of the bladders 0, 1, and 2 tumors per group were found in group 1, 2, and 3, respectively. In the placebo-treated group, 60% of animals developed tumor, which is comparable to untreated animals. Conclusions Apaziquone showed an excellent antitumor activity. The effectiveness of apaziquone in this orthotopic rat bladder tumor model corroborates the clinical observations and implies the validity of this model.

Effects of Pioglitazone, a Peroxisome Proliferator–Activated Receptor Gamma Agonist, on the Urine and Urothelium of the Rat

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which belong to the nuclear receptor superfamily. Some PPARgamma agonists, such as pioglitazone, and dual PPARgamma/PPARalpha agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyuridine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPARgamma agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat-specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Experimental rat bladder urothelial cell carcinoma models

Bladder cancer is a major public health problem. Currently available therapeutic options seem to be unable to prevent bladder cancer recurrence and progression. To enable preclinical testing of new intravesical therapeutic agents, a suitable bladder tumor model that resembles human disease is highly desirable. The aim of this topic paper was to discuss the problems associated with current in vivo animal bladder tumor models, focusing on the orthotopic syngeneic rat bladder tumor model. In the second part of the paper the development of a potential new orthotopic rat bladder tumor model is described.

Preventing Bladder Tumor Implantation With Photodynamic Therapy in a Rat Model Mimicking Post-Fluorescence Guided Transurethral Resection

Fluorescence guided transurethral resection has gained acknowledgment from the urological community and it is progressively becoming more applied. It has been shown to decrease the recurrence rate of nonmuscle invasive bladder cancer due to incomplete resection due to lack of visualization. The implantation of viable tumor cells seeded during transurethral resection is another reason for recurrence. We investigated whether applying photodynamic therapy on sensitized tumor cells would decrease the amount of viable intraluminal cells and tumor cell implantation. Two models were designed to mimic the situation after fluorescence guided transurethral resection, including partly or fully de-epithelialized bladders and circulating tumor cells loaded with protoporphyrin IX. Photodynamic therapy was performed. Controls consisted of no drug with no light, light only and drug only. Immediately after photodynamic therapy the intravesical contents were retrieved and clonogenic assays were performed on cells. Bladders were harvested 10 days after cell administration and subjected to pathological analysis. In the photodynamic therapy and control groups tumor volume was proportional to the instilled cell load. Clonogenic assays showed that viable cells were decreased a tenth of the initial administered amount. Tumor implantation decreased to less than a fifth of control values. Photodynamic therapy can effectively decrease the amount of viable tumor cells in the bladder lumen. This results in a significant decrease in tumor implantation. This technique could possibly be used to further decrease the recurrence rate of nonmuscle invasive bladder cancer.