Rat Bladder Cancer Model Research Articles

Tissue responses to hexyl 5-aminolevulinate-induced photodynamic treatment in syngeneic orthotopic rat bladder cancer model: possible pathways of action

Orthotopic bladder cancer model in rats mimics human bladder cancer with respect to urothelial tumorigenesis and progression. Utilizing this model at pT1 (superficial stage), we analyze the tissue responses to hexyl 5-aminolevulinate-induced photodynamic therapy (HAL-PDT). In comparison to untreated rats, HAL-PDT causes little change in tumor-free rat bladder but induces inflammatory changes with increased lymphocytes and mononuclear cell infiltration in rat bladders with tumor. Immunohistochemistry reveals that HAL-PDT is without effect on proliferating cell nuclear antigen expression within the tumor and increases caspase-3 expression in both normal urothelium and the tumor. Transmission electron microscopy reveals severe mitochondrial damage, formations of apoptotic bodies, vacuoles, and lipofuscin bodies, but no microvillus-formed niches in HAL-PDT-treated bladder cancer rats. Bioinformatics analysis of the gene expression profile indicates an activation of T-cell receptor signaling pathway in bladder cancer rats without PDT. HAL-PDT increases the expression of CD3 and CD45RA in the tumor (determined by immunohistochemistry). We suggest that pathways of action of HAL-PDT may include, at least, activations of mitochondrial apoptosis and autophagy, breakdown of cancer stem cell niches, and importantly, enhancement of T-cell activation.

Allyl isothiocyanate-rich mustard seed powder inhibits bladder cancer growth and muscle invasion

Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G(2)/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 μmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.

Cancer Immunoediting from Immunosurveillance to Tumor Escape in Microvillus-Formed Niche: A Study of Syngeneic Orthotopic Rat Bladder Cancer Model in Comparison with Human Bladder Cancer

Cancer cells can develop an attenuated immunogenicity and/or create an immunosuppressive microenvironment to prevent tumor eradication by host immune system, the so-called “cancer immunoediting” hypothesis. The aim of the present study was to find evidence for this hypothesis by using a rat orthotopic bladder cancer model. Fisher rats were inoculated with AY-27 cells (a Fisher rat bladder cancer cell line). Cultured cancer cells, rat and human bladder cancer tissues, and publicly available microarray data from human bladder cancer were analyzed by means of bioinformatics and morphology. Results showed that 12 of 24 differentially expressed pathways were concordant in connection to cell cycle and proliferation between rats and humans (both non-muscle-invasive and muscle-invasive tumors) and that 11 of the 24 pathways, including major histocompatibility complex, were related to host immunosurveillance with activations of T cells and natural killer cells in rats. The altered pathways and morphogenesis of this rat model corresponded more closely with those of human muscle-invasive rather than non-muscle-invasive tumors. A unique ultrastructure displaying microvillus-formed niches was found in small areas within the tumor of both rats and humans. These niches were interconnected with desmosomes between cancer cells and without infiltration of lymphocytes. The expression of E-cadherin, selectins, PGP9.5, vascular endothelial growth factor, caspase-3, CD133, Oct-4, nestin, CD3, and CD45RA was lower in the tumor than in the adjacent normal epithelium. We suggest that the microvillus-formed niche that harbors a few implanted cancer cells might be the compartment that prevents the tumor eradication by the host immune system.

Abstract 5235: Fluorometric imaging system for visualizing submillimeter level tumors using an ultrathin endoscope

Abstract [Background and purpose] Direct observation of pathological lesions by a relatively noninvasive approach is important for understanding pathophysiological mechanism and for assessing the objective response to therapeutic interventions. On the other hand, it is very difficult to perform direct and relatively noninvasive observation of minimal lesions inside a diseased model using a small live animal. However, recent technological developments in imaging, especially significant progress in fluorescent methods and in endoscopic examinations, have enabled detection and tracing of micro-lesions inside small animal models. We have developed a fluorescence imaging technique using high-resolution ultrathin endoscope, which is accessible to hollow organs of small animals. In addition, we have established an orthotopic rat bladder cancer model in which the cancer cells express a fluorescent protein. The aim of this study was to establish a high-resolution imaging method for visualizing the vascular distribution and mucosal morphology of a bladder tumor. [Materials and Methods] GFP-expressing bladder tumor in a rat orthotopic model: Firstly, we established a cell line with stable integration of GFP constructs that were produced by transfection of pTurboGFP into rat bladder cells (AY27). The stable AY27+tGFP populations were purified by repeated selections using Geneticin (G418). AY27-tGFP cells were implanted in the bladders of female Fischer F344 rats, resulting in development of submillimeter-sized bladder tumors. Development of fluorescence ultrathin endoscopy: We developed a fluorescence ultrathin endoscopic system installed with a 180 W Xenon lamp with excitation filters and 3CCD camera with emission filters. The outer diameter of the ultrathin endoscopic fiber is only 0.8 mm, but its number of pixels reaches 15,000, resulting in a spatial resolution of 50μm. [Results] Fluorescence from AY27-tGFP cells implanted in the bladder was clearly visualized by the ultrathin endoscope placed in the bladder by the transurethral approach. Furthermore, when fluorescence-probed tumor-directive micelles were injected (i.v.) into the animals as a model of drug delivery, fluorescence from the probe agents was visualized in accordance with tumor distribution. In both cases, we detected fluorescence from tumors of less than 1 mm in size. [Conclusion] We established an orthotopic GFP-tumor rat model using a GFP-expressing tumor cell line (AY27-tGFP). Submillimeter tumors in this model were visualized by the newly developed ultrathin endoscopic system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5235.

Abstract A73: Bladder cancer inhibition by allyl isothiocyante

Abstract Nearly all cancers of the urinary bladder originate from its inner surface (epithelium). While most bladder cancers are diagnosed at an early stage, recurrence rate is extremely high. There is an urgent need for agents capable of inhibiting bladder cancer development and recurrence. Allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, was previously shown to kill bladder cancer cells in vitro. Our objective was to evaluate the chemopreventive potential of AITC against bladder cancer. The anticancer activities of AITC were assessed using multiple bladder cancer cell lines in vitro and two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). Its toxicity toward normal human bladder epithelial (NHBE) cells was also examined. Pharmacokinetic study of AITC and histological examination of normal and malignant bladder tissues of AITC-treated rats were undertaken as well. Proliferation of bladder cancer cells was potently inhibited by AITC (IC50 of approximately 3 µM), which was associated with G2/M arrest and apoptosis. AITC was markedly less toxic to NHBE cells (IC50 of 69 µM). Low oral dose of AITC (1 mg/kg), starting one day after cancer cell inoculation significantly inhibited the development of orthotopic bladder cancer, but was ineffective against subcutaneous xenografts of the same cancer cells. This differential effect was explained by our finding that urinary concentrations of AITC equivalent were 2–3 orders of magnitude higher than that in the plasma, and that its levels in the orthotopic bladder cancer tissues were also 3 orders of magnitude higher than that in the subcutaneous cancer tissues. Thus, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development in vivo. Besides its potential use for prevention of primary bladder cancer, AITC appears to represent a novel and very attractive approach in the management of recurrence of superficial bladder cancer, since all currently available intravesicle therapies against bladder cancer recurrence mandate urethral catherization. In addition, this new knowledge opens the possibility for adjuvant or multimodality combination approaches for the prevention and treatment of bladder cancer. Supported by NCI grant CA124627. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A73.

PcDNA3.1tdTomato Is Superior to pDsRed2-N1 for Optical Fluorescence Imaging in the F344/AY-27 Rat Model of Bladder Cancer

Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment. AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems. AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells. In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.

Inhibition of bladder cancer development by allyl isothiocyanate

Bladder cancer is one of the common human cancers and also has a very high recurrence rate. There is a great need for agents capable of inhibiting bladder cancer development and recurrence. Here, we report that allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, potently inhibited the proliferation of bladder carcinoma cell lines in vitro [half maximal inhibitory concentration (IC(50)) of 2.7-3.3 microM], which was associated with profound G(2)/M arrest and apoptosis. In contrast, AITC was markedly less toxic to normal human bladder epithelial cells (IC(50) of 69.4 microM). AITC was then evaluated in two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). The orthotopic model closely mimics human bladder cancer development and recurrence. We show that a low oral dose of AITC (1 mg/kg) significantly inhibited the development and muscle invasion of the orthotopic bladder cancers but was ineffective against the subcutaneous xenografts of the same cancer cells in the same animals. This differential effect was explained by our finding that urinary levels of AITC equivalent were two to three orders of magnitude higher than that in the plasma and that its levels in the orthotopic cancer tissues were also three orders of magnitude higher than that in the subcutaneous cancer tissues. Moreover, we show that AITC is a multi-targeted agent against bladder cancer. In conclusion, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development and invasion.

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.

5-Fluorouracil incorporated into the tissue RNA of human and rat bladder carcinoma after administration of 1-(2-tetrahydrofuryl)-5-fluorouracil combined with uracil

UFT is an anticancer agent consisting of 1-(2-tetrahydrofuryl)-5-fluorouracil (5-FU) combined with uracil in a molar ratio of 1: 4. Its mechanisms of action are, presumably, inhibition of deoxyribonucleic acid (DNA) synthesis by thymidylate synthetase (TS) and impairment of ribonucleic acid (RNA) function by the incorporation of 5-fluorouridine-5'-triphosphate into RNA. This study was conducted to examine the TS inhibition rate (TS-IR) and the concentration of 5-FU incorporated in RNA per milligram of tissue treated with UFT (F-RNA). We administered UFT to 12 patients with bladder cancer. We also administered UFT to 20 rats bearing bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), and to 10 BBN-untreated control rats. We then determined the total TS concentration, TS-IR, and F-RNA in the human bladder tumor and normal tissues and in the BBN-treated and BBN-untreated rat organs, including the urinary bladder. In the bladder cancer patients, the mean F-RNAs in the bladder tumor and normal tissue were 0.133 +/- 0.137 and 0.056 +/- 0.062 ng/mg, respectively, without a significant difference (P < 0.1). The mean TS-IR was 35.3 +/- 19.6% in the tumor tissue and 38.0 +/- 16.6% in the normal bladder tissue, and this difference was also not significant. In the rat bladder cancer model, the total TS concentration and F-RNA in the tumor after the administration of UFT were 15.32 pmol/g and 0.780 ng/mg, respectively, being markedly higher than the corresponding values (1.22 pmol/g and 0.129 ng/mg) in the control normal bladder tissue. The impairment of RNA metabolism by F-RNA incorporated in RNA did not seem to be the critical mechanism of the antitumor effect of UFT at the usual clinical dose, as neither TS-IR nor F-RNA (as antitumor parameters) seemed to increase significantly after a clinical dose of UFT. However, the inhibition of DNA synthesis and the impairment of RNA were independent mechanisms of the action of high-dose UFT in the experimental rat bladder tumors, as both total is concentration and F-RNA were increased significantly.

High-resolution imaging diagnosis and staging of bladder cancer: comparison between optical coherence tomography and high-frequency ultrasound

A comparative study between 1.3-microm optical coherence tomography (OCT) and 40-MHz high-frequency ultrasound (HFUS) is presented to enhance imaging of bladder cancers ex vivo. A standard rat bladder cancer model in which transitional cell carcinoma (TCC) was induced by intravesical instillation of AY-27 cells was followed independently with both OCT and HFUS, and the image identifications were compared to histological confirmations. Results indicate that both OCT and HFUS were able to delineate the morphology of rat bladder [e.g., the urothelium (low backscattering/echo) and the underlying lamina propria and muscularis (high backscattering/echo]. OCT differentiated inflammatory lesions (e.g., edema, infiltrates and vasodilatation in lamina propria, hyperplasia) and TCC based on characterization of urothelial thickening and enhanced backscattering or heterogeneity (e.g., papillary features), which HFUS failed due to insufficient image resolution and contrast. On the other hand, HFUS was able to stage large T2 tumors that OCT failed due to limited imaging depth. The results suggest that multimodality cystoscopy combining OCT and HFUS may have the potential to enhance the diagnosis and staging of bladder cancers and to guide tumor resection, in which both high resolution (approximately 10 microm) and enhanced penetration (> 3mm) are desirable.

Intravesical Ultrasonography for Tumor Staging in an Orthotopically Implanted Rat Model of Bladder Cancer

Noninvasive and serial evaluation of tumor development and growth is important in an orthotopic animal bladder tumor model. However, to our knowledge a reliable method has not been established. We determined the usefulness of intravesical ultrasonography for tumor staging and volumetric assessment in an animal model. Tumors with various stages were formed in 20 female Fischer F344 rats by implanting AY-27 rat bladder carcinoma cells. Cells were implanted by instilling a suspension (4x10 cells) into the bladder cavity after urothelial denudation using hydrochloric acid or by directly injecting the cell suspension into the bladder wall. Tumor volume and invasion depth were measured by intravesical ultrasonography using an ultrathin 2.5Fr intraluminal ultrasound catheter via the urethra 7 to 10 days after cancer cell implantation. The rats were then sacrificed for histopathological examination. All rats showed bladder tumors 7 to 10 days after cancer cell implantation, of which stages varied from superficial to advanced disease. Intravesical ultrasonography showed a clear cross-sectional view of all layers of the bladder wall and enabled visualization of bladder tumors (minimal 0.5 mm in diameter). This approach also provided an accurate diagnosis of tumor invasion into muscle layers and perivesical tissues with precise invasion depth and tumor size. The positive predictive ratio regarding tumor staging reached 85%. Repeat examinations were feasible without noticeable adverse effects. Intravesical ultrasonography is a reliable and appropriate noninvasive method for evaluating tumor stage and size in an orthotopic rat bladder tumor model.

Preclinical evaluation of superantigen (staphylococcal enterotoxin B) in the intravesical immunotherapy of superficial bladder cancer

Superantigens are potent activators of T lymphocytes; therefore, their characteristics can be exploited in diseases where immunomodulation is known to be effective. In this study, we evaluated a new approach for the intravesical therapy of superficial bladder cancer. We investigated in coculture experiments if staphylococcal enterotoxin B (SEB)-activated PBMCs are able to induce apoptosis in human transitional cell carcinoma (TCC) cells. Additionally, we tested the toxicity and efficacy of SEB dissolved in NaCl 0.9% administered intravesically once weekly for 6 weeks in a rat bladder cancer model. To validate the coculture in vitro findings, we evaluated tumor stage, grade, apoptotic cells in the urothelium and stroma of the bladder and infiltration of the bladder wall by lymphocytes, macrophages and mononuclear cells. Coculture experiments revealed that SEB-activated PBMCs are able to kill TCC cells by inducing apoptosis. The intravesical toxicity study with a maximum dose of 100 microg/ml SEB demonstrated no side effects. In the intravesically SEB-treated animals (10 microg/ml), only 3 tumors remained vs. 15 persisting tumors in the control group. The remaining tumors of the therapy group showed a significant amount of apoptosis and granulocytes, mainly in the urothelium, whereas no relevant apoptosis or infiltration of the bladder with lymphocytes or macrophages was found in the control group. These preclinical findings suggest that SEB might be an interesting candidate for further clinical evaluation.

Diagnosis of biological tissue morphology and function with endoscopic optical coherence tomography.

We present some of the recent technological advances in our MEMS-based endoscopic optical coherence tomography (OCT) system for the enhancement of image fidelity and diagnosis. The endoscopic OCT system permits simultaneous cross-sectional OCT imaging and en face white-light visual guidance as well as fluorescence imaging guidance. The transverse and axial resolutions of the OCT scope are roughly 12μm and 10μm, respectively, and the axial resolution can be enhanced to 3um if connecting it to our recent custom sub-8fs Ti:Al2O3laser. To test the endoscopic OCT system for imaging diagnosis of early epithelial cancers, rat bladder cancer models were used and the results show over 90% sensitivity and specificity. Applications in imaging of bladder functions and engineering tissue growth are demonstrated.

Comparative Study of Apoptotic Antitumor Effect between Angiogenesis Inhibitor AGM-1470 and MVAC Chemotherapy on Rat Urinary Bladder Cancer

Objectives: To investigate the antitumor apoptotic effect of AGM-1470 by comparing it to that induced by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy, currently the standard chemotherapy for bladder cancer, in a rat bladder cancer model. Materials and Methods: A total of 45 six-week-old female rats were divided into 3 equal groups: those receiving AGM-1470 treatment; those receiving MVAC treatment, and controls. All rats were cystectomized to evaluate the therapeutic effect with regard to macroscopic tumor findings, hematoxylin and eosin pathology, apoptosis detection, and immunohistochemistry (IHC) for bcl-2. Results: Our experimental protocol succeeded in producing invasive bladder tumors in the majority of rats. Tumor volume was significantly reduced in the AGM-1470 and MVAC treatment groups compared with that in the control group. The apoptotic indices of tumor cells was significantly higher in the AGM-1470 and MVAC treatment groups than in the control group. IHC for bcl-2 demonstrated no statistical difference in expression among the groups. However, the apoptotic indices of high-level bcl-2 expression were significantly lower than the indices of low-level expression in the AGM-1470 group. Conclusions: AGM-1470 and MVAC appear to exert a prominent mass reduction effect via tumor cell apoptosis in cases of invasive bladder tumor, although these therapies did not demonstrate any obvious modulation of bcl-2 protein expression status. Bcl-2 overexpression might be an obstacle to AGM-1470 therapy because of its significant inhibitory effect on apoptosis.

Mitochondrial DNA mutations in chemical carcinogen-induced rat bladder and human bladder cancer

Mitochondrial (mt) DNA mutations have been described recently in different tumors, whereas similar studies focusing on bladder cancer are scarce. In an effort to understand the significance of mtDNA mutations in bladder cancer, we investigated the mtDNA alterations in both clinical human bladder cancer and in a carcinogen-induced rat bladder cancer model. Human bladder cancer tissues were obtained by radical cystectomy and transurethral resection of bladder tumors. Rat bladder tumors were induced in SD rats by treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for 24-28 weeks. Genomic DNA was extracted from tumor specimens and microdissected normal bladder mucosae. Mitochondrial genes and D-loop region were amplified by PCR. The amplified PCR fragments were either cloned into plasmid vector or used for direct DNA sequencing. The results of DNA sequence revealed numerous point mutations in the non-coding D-loop region and different mtDNA genes in both human and rat bladder cancers. In addition, we also detected deletions of variable lengths in mononucleotide repeats in the D-loop region, ND2, ATPase 8 and COIII genes in human bladder cancer samples. Our results show that mtDNA exhibits a high rate of mutations in both human and rat bladder cancers. We also demonstrate that the repetitive sequences of mononucleotides within the mt genome are unstable and subjected to deletions. The high incidence of mtDNA mutations in bladder cancer suggests that mtDNA and mitochondria could play an important role in the process of carcinogenesis and also mtDNA could be valuable as a marker for early bladder cancer diagnosis.

A review of animal model studies of tomato carotenoids, lycopene, and cancer chemoprevention.

There are relatively few reports on the cancer chemopreventive effects of lycopene or tomato carotenoids in animal models. The majority, but not all, of these studies indicate a protective effect. Inhibitory effects were reported in two studies using aberrant crypt foci, an intermediate lesion leading to colon cancer, as an end point and in two mammary tumor studies, one using the dimethylbenz(a)anthracene model, and the other the spontaneous mouse model. Inhibitory effects were also reported in mouse lung and rat hepatocarcinoma and bladder cancer models. However, a report from the author's laboratory found no effect in the N-nitrosomethylurea-induced mammary tumor model when crystalline lycopene or a lycopene-rich tomato carotenoid oleoresin was administered in the diet. Unfortunately, because of differences in routes of administration (gavage, intraperitoneal injection, intra-rectal instillation, drinking water, and diet supplementation), species and strain differences, form of lycopene (pure crystalline, beadlet, mixed carotenoid suspension), varying diets (grain-based, casein based) and dose ranges (0.5-500 ppm), no two studies are comparable. It is clear that the majority of ingested lycopene is excreted in the feces and that 1000-fold more lycopene is absorbed and stored in the liver than accumulates in other target organs. Nonetheless, physiologically significant (nanogram) levels of lycopene are assimilated by key organs such as breast, prostate, lung, and colon, and there is a rough dose-response relationship between lycopene intake and blood levels. Pure lycopene was absorbed less efficiently than the lycopene-rich tomato carotenoid oleoresin and blood levels of lycopene in rats fed a grain-based diet were consistently lower than those in rats fed lycopene in a casein-based diet. The latter suggests that the matrix in which lycopene is incorporated is an important determinant of lycopene uptake. A number of issues remain to be resolved before any definitive conclusions can be drawn concerning the anticancer effects of lycopene. These include the following: the optimal dose and form of lycopene, interactions among lycopene and other carotenoids and fat soluble vitamins such as vitamin E and D, the role of dietary fat in regulating lycopene uptake and disposition, organ and tissue specificity, and the problem of extrapolation from rodent models to human populations.

Detection of tumorigenesis in rat bladders with optical coherence tomography.

Optical coherence tomography (OCT) is a novel technique that enables noninvasive cross-sectional imaging of biological tissues. Because of its high resolution (approximately 10 microm), superior dynamic range (140 dB in our case) and up to 2-3 mm penetration depth, OCT is potentially useful for noninvasive screening of superficial lesions. Bladder cancer arises within the transitional epithelium. Despite the ability to visualize the epithelium via cystoscopy, it is often difficult to detect early epithelial cancers and to determine their penetration to the underlying layers. To investigate the potential of OCT to enhance imaging of bladder cancers and other epithelial lesions, we applied OCT to normal and diseased bladder epithelium, and correlated the results with histological findings. OCT images of porcine bladder (a close homolog of human bladder) confirm the ability of this method to image human tissues. To determine whether OCT can track the course of bladder cancer, a standard rat model of bladder cancer in which Fisher rats are exposed to methyl-nitroso-urea (MNU), was followed both with OCT and histological studies. Our results show that the micro morphology of porcine bladder such as the urothelium, submucosa and muscles is identified by OCT and well correlated with the histological evaluations. OCT detected edema, inflammatory infiltrates, and submucosal blood congestion as well as the abnormal growth of urothelium (e.g., papillary hyperplasia and carcinomas). By contrast, surface imaging, which resembles cystoscopy, provided far less sensitivity and resolution than OCT. This is the first OCT study of any tumor documented in a systematic fashion, and the results suggest the potential of OCT for the noninvasive diagnosis of both bladder inflammatory lesions and early urothelial abnormalities, which conventional cystoscopy often misses, by imaging characterization of the increases in urothelial thickening and backscattering. However, because of the depth limitation, OCT may have limited applications in staging the invasion of higher-state urothelial cancers, especially for papillary carcinomas.

MMP 2 and MMP 9 Expression and Basement Membrane Degradation in the Rat Model of Bladder Cancer

MMP 2 and MMP 9 Expression and Basement Membrane Degradation in the Rat Model of Bladder Cancer

MVAC chemotherapy-induced apoptosis and p53 alterations in the rat model of bladder cancer

Objectives. To investigate the induction of apoptosis by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy as well as p53 alterations in a rat model of bladder cancer induced by N-butyl- N-(4-hydroxybutyl)nitrosamine (BBN). Methods. At 20 and 28 weeks after starting the administration of BBN, 60 rats (30 at 20 weeks, and 30 at 28 weeks) were divided into an MVAC treatment group (20 rats) and a control group of untreated rats (10 rats). After one intraperitoneal injection of MVAC, the rat bladder was obtained on day 1 or 7 for evaluation of apoptosis by biohistochemical and electron microscopic observations, and of p53 alterations immunohistochemically. Results. All tumors were noninvasive transitional cell carcinoma (TCC) at 20 weeks and invasive TCC at 28 weeks. In comparison with untreated tumors, a threefold increase of apoptotic indexes (AIs) was noted in invasive TCC and a twofold increase in noninvasive TCC on day 1 after MVAC therapy. Both decreased AIs and a frequent occurrence of apoptotic necrosis were observed on day 7. Occurrence of tumor necrosis was not affected by MVAC therapy, and the extent of necrosis was not related to apoptosis. Detection of p53 alterations, 45% and 40% in MVAC treated and untreated tumors, respectively, did not correlate with AIs. Conclusions. MVAC therapy may act through the induction of apoptosis, and invasive TCC cells may be much more sensitive to MVAC therapy in the rat model of bladder cancer. Neither spontaneous nor MVAC-induced apoptosis may be related to p53 alterations in the rat model of bladder cancer.