Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively.

Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.

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285 Background: Hsp90 is a molecular chaperone responsible for folding many of the proteins directly associated with cancer progression and consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Hsp90 family consist of four isoforms; Hsp90α, Hsp90β, Grp94 and Trap-1. The development of Hsp90 isoform-selective inhibitors represent an alternative approach towards the treatment of cancer that may limit some of the detriments. We demonstrate novel Hsp90 inhibitors, on prostate and bladder cancer cells, which shows both potent antiproliferative effects and specific selectivity for Hsp90β. Methods: PC3MM2, LNCap-LN3, C4-2b, LAPC4 (prostate cancer) and T24, UC3 (bladder cancer) cancer cells were utilized. Cell Titer-Glo luminescent anti-proliferative assay was used to determine the IC50 numbers after 72h treatment. Trypan Blue Cytotoxicity assay was performed for 24h treatment with increasing concentrations of KUNB inhibitors. Effects of KUNB inhibitors on Hsp90’s client protein degradation were investigated by Western Blot. Results: KUNB31 manifested an IC50 of 3.00 µM against UC3 bladder cancer cells, UC3 cells were then evaluated via western blot analyses of known Hsp90α- and Hsp90β-dependent client proteins following treatment with KUNB31 for 24 hours. The data showed that, KUNB31 would not induce the heat shock response like 17AAG, and did cause Hsp90β related protein degradation (CXCR4). Moreover, Hsp27, PKM2, Her2, Hsf-1and Akt all showed degradation to different extent. KUNB105 exhibited potent anti-proliferative in both prostate and bladder cancer cells. IC50 number was determined as 1.24 µM for PC3MM2, 1.18 µM for LNCap-LN3, 1.03 µM for C4-2b, 2.56 µM for LAPC4, 0.20 µM for T24, and 0.30 µM for UC3 cancer cells. Conclusions: KUNB novel Hsp90β selective inhibitors, exhibit potent anti-proliferative and cytotoxic activity along with client protein degradation, without induction of HSR in prostate and bladder cancer cell lines. KUNB compound’s selective inhibition on Hsp90β isomers supports the development of Hsp90-selective inhibitors as a method to overcome the detriments associated with pan-inhibition in cancer treatment.

BACKGROUND AND OBJECTIVE: While radical cystectomy (RC) is the gold-standard treatment for patients with muscle-invasive bladder cancer, it is associated with a significant rate of complications. We aim to develop a prediction model to assess the risk of complications in the postoperative period using routinely collected data in the course of preoperative evaluation in patients undergoing RC for bladder cancer. METHODS: We retrospectively reviewed 508 patients who underwent RC for bladder cancer from January 2008 to October 2016. Potential preoperative risk predictors were collected. Postoperative complications were graded using the Clavien-Dindo scale. Prediction models were developed using variables with the highest predictive value for postoperative complications. The accuracy of themodels was assessed using the area under the receiver operating characteristic curve (AUROC) and cross-validation. RESULTS: Variables achieved the highest prediction accuracy for major postoperative complications in the 31 to 90-day postoperative period. In this period, 14 variables were predictive of major postoperative complications. The three most predictive individual variables were BMI, neoadjuvant chemotherapy, and creatinine with AUROC/odds ratios of 0.643/1.09, 0.609/2.43, and 0.597/1.22, respectively. This postoperative period also had the best performing prediction model for major complications, which utilized four variables to achieve an AUROC of 0.727. CONCLUSION: Routinely collected preoperative variables may be useful for determining patient risk for short-term postoperative complications after RC. Prediction models can help identify patients who may benefit from patient education, counseling and development of risk reduction strategies. Interactions between individual variables should be evaluated to further improve accuracy of the prediction models.