Fischer F344 Rats Research Articles

Abstract 3468: Endoscopic diagnosis and treatment of esophageal tumor using a novel DDS-type photoactivatable agent

Abstract Introduction: Photodynamic diagnosis (PDD) and therapy (PDT) have been clinically used for the early phase cancer of hollow organs. We are advancing the photomedicine to the next stage, in which treatment is carried out immediately after the detection of lesions. For achieving the simultaneous diagnosis and treatment, we established a novel DDS-type photoactivatable agent, micelle-based photosensitizer loading dendrimer porphyrin (DP/M). A micelle-based shell structure is expected to be selectively accumulated in tumors. An intensely emitted fluorescence from porphyrin can be used for lesion detection and a highly quantum yield of porphyrin promises an effective PDT. Apart from the agent, we recently developed a fluorometric system equipped with an ultrathin endoscope that has a spatial resolution of 15,000 pixels even with a diameter of 0.8 mm. The aim of this study was to verify practical effectiveness of the novel photosensitizer combined with the endoscopic fluorometric system for simultaneous diagnosis and treatment. In this study, we targeted esophageal cancer because satisfactory results are not always obtained with conventional therapies and an endoscopic approach is applicable. Materials and Methods: A rat orthotopic esophageal tumor model was established in female Fischer F344 rats by orally administered with NMBuA (N-Nitroso Methylbutylamine) solved in drinking water. After intravenous administration of DP/M, PDD was carried out using the endoscopic fluorometric system (ex. 405 nm, em. > 630 nm). After the detection of lesions, tumors were irradiated using a diffuser tip fiber connected to a 635nm LD laser. Results: About 6 weeks after the administration of NMBuA, polypoid tumors were observed in the esophagus. Pathological analysis revealed that proliferated polymorphonuclear cells were seen: a part of which reached the muscular layer of mucosa. A selective accumulation of DP/M in the tumors was confirmed with the endoscopic fluorometric system. This modality enabled repeated observations of tumors without sacrifice. The following PDT provided a promising result. Conclusions: DP/M combined with the endoscopic fluorometric system brought the simultaneous diagnosis and treatment for the orthotopic rat esophagus tumor models. Acknowledgment: This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3468. doi:1538-7445.AM2012-3468

Abstract 3469: Photodynamic diagnosis (PDD) and therapy (PDT) with dendrimer porphyrin micelle

Abstract [Background and purpose] We previously reported that the efficacy of photodynamic therapy (PDT) with dendrimer phthalocyanine-loaded micelles (DPc/m). However, monitoring of this photosensitizer by the clue of fluorescence is quite difficult because of the low intrinsic fluorescence originating from phthalocyanine, thus other fluorescence dye such as Alexa is required for the monitoring. For resolving this problem, we developed a novel micelle-based photosensitizer loading dendrimer porphyrin (DP). The DP-loaded micelles (DP/m) intensely emit fluorescence from porphyrin without an additional fluorescence labeling. The aim of this study was to verify practical effectiveness of DP/m by comparing that of DP on PDD and PDT. [Materials and Methods] For in vitro cytotoxicity tests, rat bladder carcinoma cells (AY-27) were incubated with DP/m for 4 h and were exposed to laser light (635 nm, 100 J/cm2). One day after PDT, cell viability was measured by WST-8 assay. Distribution of DP/m in cellular organelles was investigated using fluorescence markers of cellular organelles by a confocal laser scanning microscope. In addition, we examined in vivo fluorescence imaging using an orthotopic bladder tumor model established by inoculating AY27 cells in female Fischer F344 rats via transurethral catheterization. Four hours after intravenous administration of DP/m, the bladders were extracted and fluorescence image was obtained of bladder. [Results] DP and DP/m without light irradiation were basically non-toxic (low dark toxicity), showing cell viability being > 85% at the concentration up to 17 μM. However, photoirradiated DP and DP/m induced the high level of cytotoxicity: survival rate was less than 5%. PDT using DP/m showed an approximately 15 times of cytotoxicity when compared with PDT using DP. The results suggested that DP/m highly accumulate in cancer cells and can provide a significant photodynamic effect leading to cell damage. Confocal laser scanning microscopic studies showed that DP/m exhibited preferential accumulation in lysosomes through endocytosis and retention in the cells. When DP/m were injected (i.v.) into the bladder tumor animal models, fluorescence from the DP/m were clearly visualized in accordance with tumor distribution. We detected fluorescence even when a size of tumor was less than 1 mm, thus enabling optical imaging with excellent contrast between tumor and surrounding normal tissue. A study of PDT in vivo is now carried out. [Conclusion] We developed dendrimer porphyrin-loaded micelles (DP/m), which were effective for both PDD and PDT. An excellent detection of tumor was achieved owing to the intense fluorescence of DP/m and highly accumulation in tumors. The preferable DP/m uptake in tumors resulted in effective phototoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3469. doi:1538-7445.AM2012-3469

Initial Characterization of the Cellular Basis for Bladder Regeneration in a Rodent Model

PurposeA better understanding of intrinsic mammalian organ regeneration would benefit the field regenerative medicine. Our prior work shows functional bladder recovery in rats after removal of ~70% of the bladder (subtotal cystectomy/STC). Our objective is to begin identifying the spatiotemporal characteristics of cellular response to STC.MethodsTrigone‐sparing STC was performed on 12‐week‐old female Fischer F344 rats. At 1, 3, 5, and 7 days post‐STC, bladders were harvested 2 hours after IP 50mg/kg 5‐Bromo‐2’‐deoxyuridine (BrdU). Fluorescent BrdU labeling was used to quantify and localize S‐phase cells within the bladder wall. Immunohistochemistry for Shh, Gli‐1 and BMP‐4 was also performed. Normal rat bladders served as controls.ResultsSparse BrdU labeling was observed in native bladders (Fig. 1). The total number of BrdU‐positive cells/400x HPF was significantly higher post‐STC at all time points. A time‐depended location change in the percentage of BrdU‐positive cells was seen. Increased positive staining for Shh, Gli‐1 and BMP‐4 was also noted post‐STC.ConclusionsSTC induced a robust proliferative response with quantifiable spatiotemporal characteristics and up‐regulation of key targets in the Shh developmental pathway. Future studies will identify the cell source(s) and details of the molecular pathways that modulate functional bladder regeneration.

260 INITIAL CHARACTERIZATION OF THE CELLULAR BASIS FOR BLADDER REGENERATION IN A RODENT MODEL

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2012260 INITIAL CHARACTERIZATION OF THE CELLULAR BASIS FOR BLADDER REGENERATION IN A RODENT MODEL Charles Peyton, David Burmeister, Karl-Erik Andersson, and George Christ Charles PeytonCharles Peyton Winston Salem, NC More articles by this author , David BurmeisterDavid Burmeister Winston Salem, NC More articles by this author , Karl-Erik AnderssonKarl-Erik Andersson Winston Salem, NC More articles by this author , and George ChristGeorge Christ Winston Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.317AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The significant regenerative capacity of the bladder has facilitated clinical application of tissue engineering technologies for bladder augmentation/reconstruction. Recent studies in a rodent model documented de novo functional and structural bladder regeneration following removal of 70-80% of the bladder (subtotal cystectomy/STC). The aim of this study was to characterize the early cellular response that mediates bladder regeneration following STC in this physiologically relevant mammalian organ system. METHODS Trigone-sparing STC was performed on 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7 days post-STC, bladders were harvested 2 hours after IP injection of 50 mg/kg Bromodeoxyridine (BrdU, a common index of DNA-replication). Fluorescent BrdU labeling was used to quantify BrdU-positive cells within the urothelium, lamina propria and muscularis propria. Immunohistochemistry was performed to evaluate the sonic hedgehog (Shh) pathway and its related transcription factors Gli-1 and bone morphogenic factor-4 (BMP-4). Normal rat bladders served as controls. RESULTS Sparse BrdU labeling was observed in control bladders (Fig. 1A). The percentage of BrdU-positive cells was significantly higher post-STC compared to controls at all time points (Fig. 1C). Interestingly, a time-dependent shift in the location of BrdU-positive cells was observed such that BrdU-positivity was significantly elevated in the urothelium at 1-day post-STC and lower in the muscularis at 3 and 5-days, with similar expression across all layers by 7-days (Fig. 1D). Increased positive staining for Shh, Gli-1 and BMP-4 was noted post-STC compared to normal bladder. CONCLUSIONS STC induced a robust and global proliferative response with quantifiable spatiotemporal characteristics. Up-regulation of key targets in the Shh pathway was also observed. Presumably, increased cellular proliferation and expression of key targets in the Shh developmental signaling pathway provide, at least in part, the cellular basis for functional bladder recovery after STC. Describing the origin, migration and identity of these cells will improve our mechanistic understanding of the intrinsic regenerative capacity of the bladder and could potentially lead to more efficacious therapies. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e105-e106 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Charles Peyton Winston Salem, NC More articles by this author David Burmeister Winston Salem, NC More articles by this author Karl-Erik Andersson Winston Salem, NC More articles by this author George Christ Winston Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

ABSTRACTThe effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day −1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day −1. Control group III animals (n = 4) were treated by sham electroporation on days −1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao2) (P = .0028; group I vs. groups II and III, P < .01 each). Pao2 was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao2 of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Abstract 4302: Targeting the sphingolipid pathway in NK large granular lymphocyte leukemia

Abstract Large granular lymphocytes (LGL) are a lymphoid subset that consists of 10-15% of peripheral blood mononuclear cells. LGL can be divided into (2) major lineages of CD3- NK cells and CD3+ T Cells. Symptoms may include rheumatoid arthritis and neutropenia which leads to recurrent bacterial infections. The sphingolipid metabolism pathway has been found to be deregulated in NK-LGL leukemia. Glucosylceramide synthase (GCS), a key component of this pathway is an enzyme that catalyzes the conversion of proapoptotic ceramide to inactive glucosylceramide by transferring a glucose residue from UDP-glucose to ceramide. GCS has been found to be upregulated in cancer cells and its overexpression has been associated with multidrug resistance in leukemia cells. Both rat and human leukemic LGL cells have been treated with a combination treatment targeting two key components of the sphingolipid pathway. One of the agents in use is the C6-ceramide (C6) nanoliposome which has led to caspase-dependent apoptosis in a time- and dose-dependent manner. Systemic i.v. delivery of nanoliposomal ceramide led to complete remission in 5/14 syngeneic Fischer F344 rat model of NK-LGL leukemia. However a greater response will be achieved by combination treatment of C6 and through targeting a second key component of ceramide metabolism with DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). PPMP is a GCS inhibitor that prevents the endogenous ceramide already present in the cell from being glycosylated into its inactive form where it can no longer induce cell death. Overall, this combination treatment shows a synergistic effect with increase of ceramide levels in order to induce apoptosis and therefore death of these persistent LGL cells. Cell flow cytometry examining 7AAD/Annexin V staining has shown ∼80% apoptosis when 6.25 uM C6 ceramide and 15 uM PPMP are used in combination on aggressive rat LGL cells. Whereas, only ∼19% is observed with C6 and ∼17% of PPMP are observed individually in treatment. Overall, apoptosis appeared increased in a dose response manner after treatment with PPMP and C6. This is a significant finding of P&amp;lt;0.005, when compared to DMSO and ghost nanoliposome controls which only induce ∼15% apoptosis. In addition, this combination treatment has been utilized on normal human peripheral blood mononuclear cells where only 11.2% apoptosis was induced and 9% in the normal spleen mononuclear cells in rats. As a result, this combination treatment appears promising in specifically targeting LGL cells while sparing normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2011-4302

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Efeito da fotoperiodicidade na taxa de prenhez em ratos isogênicos (F344)

O uso de animais isogênicos apresenta grandes vantagens experimentais, como uniformidade fenotípica e genotípica (reduzindo o número de animais em experimentos) e histocompatibilidade, permitindo, assim, o acúmulo de informações e a repetibilidade dos experimentos. A linhagem isogênica de Rattus norvegicus Fischer 344 existe há 90 anos, entretanto pouco se sabe sobre as razões de seu baixo índice reprodutivo. O presente estudo demonstrou que ratos Fischer F344 são fotorresponsivos quanto à reprodução, tendo seus índices de prenhezes acrescidos com o aumento do fotoperíodo. Os melhores índices são obtidos quando os machos são submetidos a 14 horas de luz e fêmeas a 16 horas de luz, indicando dimorfismo sexual na fotorresponsividade.

Therapeutic Efficacy of FTY720 In a Rat Model of LGL Leukemia

AbstractAbstract 776Natural kill cell large granular lymphocytic (NK-LGL) leukemia is a fatal disorder with death occurring in days to weeks following diagnosis. There is no known curative therapy for this disease. Therefore, there is an urgent unmet need for development of new therapeutics for this deadly leukemia. Fischer F344 rat LGL leukemia model has been established as an important experimental model for the study of NK-LGL leukemia progression and closely resembles human aggressive NK-LGL leukemia exhibiting clonal expansion of CD3-CD8a+ lymphocytes. FTY720, a new immunosuppressant, has been studied for its putative anti-cancer properties in the recent years. At four weeks after transplantation of leukemic LGL cell line, the rats displayed early signs of leukemia, including weight loss, rough hair coat and increased level of neutrophils. By week 5, circulating blasts, anemia, thrombocytopenia, and splenomegaly were observed. These leukemic rats were then injected intraperitoneally with 4.5mg/kg of FTY720 or PBS every day over a 4-week treatment period. Animals died within the next 1 to 2 weeks if treated with PBS. The median survival in PBS treated group was 41 days compared to 51 days in FTY720 treated group (Mantel-Cox test, p<0.0001). Importantly, 5 of 16 leukemic rats treated with FTY720 had maintenance of normal blood counts without circulating blasts suggesting achievement of complete clinical remission. The remaining eleven leukemic rats treated with FTY720 had a transient improvement as evidenced by reduction of white blood cell counts and elevated platelet counts after two weeks treatment. Subsequently, however, blast counts rose and animals died within the following one week. To further determine remission status, the five responsive animals were euthanized after cessation of the treatment. At necropsy, we found these rats had normal levels of CD3-CD8a+ LGL cells in the blood, marrow, and spleen. Of note, the eleven leukemic rats not achieving remission also displayed significant reduction of LGL cells in these tissues, to lesser extent. Examination of spleen sections from rats responsive to FTY720 showed normal splenic histology. In contrast, leukemic rats not achieving remission showed leukemic LGL infiltration of the red pulp and depletion of the white pulp. Interestingly, pro-apoptotic proteins Bax and Bak were dramatically increased while anti-apoptotic protein Mcl-1 was decreased in the spleens of the rats achieving remission; however, they remained unchanged in leukemic rats not achieving remission. These data indicate that in vivo therapeutic efficacy of FTY720 may be a consequence of modulation of anti-apoptosis signaling which led to resolution of leukemic cell infiltration. We then extended these studies to NK LGL from patients. Initial experiments demonstrated that FTY720 displayed dose- and time-dependent apoptotic cell death in PBMC from NK-LGL leukemia patients (CD3−CD56+>80%). In contrast, treatment with 10uM FTY720 did not induce significant cytotoxic effects in PBMC from normal donors, or normal NK cells. In addition, treatment of human and rat NK-LGL leukemia cells with FTY720 led to caspase-dependent apoptosis, generation of reactive oxygen species (ROS), and Mcl-1 degradation which did not occur at the transcriptional level. Of interest, inhibition of ROS rescued FTY720 induced apoptosis in leukemic NK cells. Moreover, efficient knockdown of Mcl-1 resulted in more than two fold increase in apoptotic cell death of NKL, a human NK-LGL leukemia cell line. Collectively, these results indicate efficacy of FTY720 in a rat model of NK LGL leukemia via production of ROS and decreased mcl-1 expression or signaling. Disclosures:No relevant conflicts of interest to declare.

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia

AbstractThe natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca(2+)-independent phospholipase A(2) (iPLA(2)), Ca(2+)-independent plasmalogen PLA(2) or secretory PLA(2 (sPLA2)), but not by Ca(2+)-dependent cytosolic PLA(2) (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca(2+) into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca(2+)-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca(2+) and of cPLA(2). DHA signaling may be mediated by iPLA(2), plasmalogen PLA(2), or other enzymes insensitive to low concentrations of Ca(2+). Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.

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.

Rictor-dependent AKT activation and inhibition of urothelial carcinoma by rapamycin

Objective We previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma. Materials and methods The rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycin's attenuation of urothelial carcinoma was also evaluated by T24 cells. Results Rapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma ( P < 0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA. Conclusions This study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.

Effects of a soluble dietary fibre NUTRIOSE® on colonic fermentation and excretion rates in rats

The resistant dextrin NUTRIOSE®, developed from starch, is expected to act as a prebiotic. The aim of this study was to determine the effects of NUTRIOSE® on cecal parameters, short-chain fatty acid (SCFA) concentrations, and fecal excretion in rats. In an initial experiment, twenty-four male Fischer F344 rats were randomly assigned to one of the following four treatments for 14 days: G0 (control diet), G2.5 (control diet + 2.5% of dextrin), G5 (control diet + 5% of dextrin), and G10 (control diet + 10% of dextrin). After 14 days, total cecal weight, cecal content, and cecal wall weight were significantly increased in G5 and G10 compared to G0. At the same time, cecal pH was significantly lower in G10 compared to G0. Total SCFA concentration was significantly higher in G10 than in G5, G2.5, and G0, and significantly higher in G5 than in G0. Acetate, butyrate, and propionate concentrations were significantly increased in G5 and G10 compared to the controls. In a second trial based on a similar design, eighteen male Fischer F344 rats were treated with a control diet supplemented with 5% of dextrin or 5% of fructo-oligosaccharide. The results obtained with NUTRIOSE® were similar to those obtained with the fructo-oligosaccharide. In a third experiment, two groups of 5 Fischer F344 rats were orally treated with 100 and 1,000 mg/kg NUTRIOSE®, respectively, and from 18% to 25% of the dextrin was excreted in the feces. The results of these three studies show that the consumption of NUTRIOSE®, by its effects on total cecal weight, cecal content, cecal wall weight, pH, and SCFA production, could induce healthy benefits since these effects are reported to be prebiotic effects.

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.

Endogenous and Transplanted Small Hepatocytes in Retrorsine-treated/Partially Hepatectomized Rat Liver Show Differences in Growth, Phenotype, and Proximity to Clusters of γ-Glutamyl Transpeptidase-positive Host Hepatocytes

In the present report, we have compared the phenotype and growth of small hepatocyte progenitors (SHPs) induced by retrorsine/partial hepatectomy (R/PH) and small hepatocytes (SHs) isolated from normal adult liver. SHs were isolated by a combination of differential centrifugation and Percoll isodensity fractionation from a liver cell suspension prepared by collagenase perfusion of a dipeptidyl peptidase IV (DPPIV)-positive Fischer F344 rat liver. Following further purification by flow cytometry, the SH-R3 fraction was transplanted via the portal vein into R/PH-treated, DPPIV-negative Fischer F344 rats. Frozen sections from tissue harvested at 5, 7, and 21 days after transplantation were analyzed by indirect immunofluorescence to compare the phenotypic characteristics of colonies formed by exogenous SH-R3s and endogenous SHPs. Colonies of transplanted SHs and endogenous SHPs displayed similar histologies and phenotypes but were distinguished from surrounding hepatocytes by their elevated expression of transferrin receptor. SH-R3 colonies were frequently located within clusters of gamma-glutamyl transpeptidase-positive host hepatocytes. Although significantly smaller at 5 and 7 days after PH, by day 21, SH-R3 colonies were similar in size to those formed by SHPs. The present results suggest that endogenous SHPs are derived, at least in part, from SHPs.

Intravesical Administration of Plasminogen Activator Inhibitor Type-1 Inhibits In Vivo Bladder Tumor Invasion and Progression

The potent effects of PAI-1 on tumorigenesis and angiogenesis in various experimental models are complex, complicated and at times contradictory. We determined the therapeutic potential of PAI-1 for inhibiting bladder tumor invasion under conditions that closely mimic the clinical setting. An orthotopic rat bladder tumor model was established by implanting AY-27 rat transitional carcinoma cells into the bladder lumen of syngeneic Fischer F344 rats. Three weeks after implantation 1 microM PAI-1 was administrated directly into the bladder lumen twice weekly for 2 weeks. Two days after the final treatment tumor size, total bladder weight, tumor stage and angiogenesis were assessed. To assess the uPA axis the levels of active and total uPA, and active and total PAI-1 in tumor extracts were determined 0, 2, 24 and 48 hours after intravesical PAI-1 administration. Intravesical PAI-1 bound and inactivated its molecular target, tumor uPA. There was significant inhibition of bladder tumor progression, as manifested by 53%, 37% and 57% reductions in tumor size, total bladder weight and angiogenesis, respectively. Only 22% of PAI-1 treated tumors invaded muscle vs 79% in controls. No PAI-1 toxicity was detected. To our knowledge this study is the first to demonstrate that intravesical treatment with PAI-1 significantly inhibits tumor progression in an in vivo model of bladder cancer. Further clinical development is warranted for using PAI-1 directly or in combination with current standards, such as bacillus Calmette-Guerin or interferon.

Systemic Delivery of Nanoliposomal Ceramide Displays Anti-Leukemic Activity in a Rat Model of Large Granular Lymphocytic Leukemia

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

EVALUATION OF AN ORTHOTOPIC RAT BLADDER UROTHELIAL CELL CARCINOMA MODEL BY CYSTOSCOPY

OBJECTIVES: To enable preclinical testing of intravesical therapies against non-muscle-invasive bladder cancer (NMIBC) in an orthotopic rat bladder tumour model, augmented by the use of serial cystoscopy for in vivo tumour assessment and follow-up. MATERIALS AND METHODS: Fischer F344 rats had a 16-G transurethral cannula placed. The bladder mucosa was conditioned with an acid rinse, followed by a 1-h instillation of 1.5 x 10(6) AY-27 rat bladder urothelial cell carcinoma (UCC) cells (day 0). Cystoscopy (1 mm) was done on day 0 (control) and at 3, 4, 5, 6, 7, 10, 13 and 17 days. At the scheduled times the rats were killed after cystectomy (four at each time) for histopathological examination of the bladder. RESULTS: Overall, tumour establishment was >80%, with predominantly carcinoma in situ preceding or concomitant with invasive tumour growth. All tumours were formed at 3-5 days, and remained non-muscle-invasive up to 5 days. From 6 days, tumours progressed to muscle-invasive disease in 40% of the rats. Visibility at cystoscopy was excellent and tumours were apparent in >90% of rats from 5 days on, with a specificity and sensitivity of >90%. Cystoscopy could not distinguish NMIBC from muscle-invasive disease. CONCLUSIONS: This is a reliable model of orthotopic rat bladder UCC, with early high-grade NMIBC growth, immediately followed by muscle-invasive growth, i.e. the recommended time to start intravesical therapy would be 5 days after tumour cell inoculation. Tumour growth can easily be monitored by cystoscopy, but cannot be used to distinguish NMIBC from muscle-invasive bladder cancer.

Evaluation of an orthotopic rat bladder urothelial cell carcinoma model by cystoscopy

To enable preclinical testing of intravesical therapies against non-muscle-invasive bladder cancer (NMIBC) in an orthotopic rat bladder tumour model, augmented by the use of serial cystoscopy for in vivo tumour assessment and follow-up. Fischer F344 rats had a 16-G transurethral cannula placed. The bladder mucosa was conditioned with an acid rinse, followed by a 1-h instillation of 1.5 x 10(6) AY-27 rat bladder urothelial cell carcinoma (UCC) cells (day 0). Cystoscopy (1 mm) was done on day 0 (control) and at 3, 4, 5, 6, 7, 10, 13 and 17 days. At the scheduled times the rats were killed after cystectomy (four at each time) for histopathological examination of the bladder. Overall, tumour establishment was >80%, with predominantly carcinoma in situ preceding or concomitant with invasive tumour growth. All tumours were formed at 3-5 days, and remained non-muscle-invasive up to 5 days. From 6 days, tumours progressed to muscle-invasive disease in 40% of the rats. Visibility at cystoscopy was excellent and tumours were apparent in >90% of rats from 5 days on, with a specificity and sensitivity of >90%. Cystoscopy could not distinguish NMIBC from muscle-invasive disease. This is a reliable model of orthotopic rat bladder UCC, with early high-grade NMIBC growth, immediately followed by muscle-invasive growth, i.e. the recommended time to start intravesical therapy would be 5 days after tumour cell inoculation. Tumour growth can easily be monitored by cystoscopy, but cannot be used to distinguish NMIBC from muscle-invasive bladder cancer.