878 BRANCHED PEPTIDES AS A NOVEL TUMOR-TARGETING AGENTS FOR BLADDER CANCER

878 BRANCHED PEPTIDES AS A NOVEL TUMOR-TARGETING AGENTS FOR BLADDER CANCER

Vol. 185, No. 4S, Supplement, Monday, May 16, 2011 cer. Although combinations of these agents have shown clinical benefit in advanced urothelial canc...

194KB Sizes 0 Downloads 45 Views

Vol. 185, No. 4S, Supplement, Monday, May 16, 2011

cer. Although combinations of these agents have shown clinical benefit in advanced urothelial cancer, most of patients finally develop recurrent disease. The alternative regimens including cisplatin/paclitaxel, gemcitabine/paclitaxel, and gemcitabine/docetaxel have also shown modest activity. Therefore, development of new combination chemotherapy is needed. This study was undertaken to evaluate the efficacy of combination of gemcitabine and methotrexate in cisplatin-resistant urothelial cancer cells. METHODS: Three human bladder cancer cell lines (KU-1, KU-7, KU19-19) were used. Cisplatin-resistant sub-lines were established by repeated subcultures in the presence of increasing concentrations of cisplatin. The cells were exposed to gemcitabine (50 nM, 500 nM) and methotrexate (1␮M, 10␮M) as single agents and to various combinations (simultaneous exposure, preincubation with methotrexate followed by gemcitabine exposure, preincubation with gemcitabine followed by methotrexate exposure). The concentrations in the combination exposure had the same range as the separate agents. The cytotoxic effects were examined by fluorometric assay. Furthermore, flow cytometric DNA/bromodeoxyuridine measurement was used to evaluate the effect on cell cycle. RESULTS: The fluorometric assay showed that growth inhibitory effect of cisplatin was decreased by 2.5-fold in cisplatin-resistant sublines compared to parental cell lines. Cell cycle analysis demonstrated that exposure of methotrexate synchronized both parental and cisplatin-resistant cells to S-phase. After a 48h exposure, maximal growth inhibition was observed in cells exposed to 1␮M of methotrexate and 50nM of gemcitabine simultaneously, or in cells pre-incubated with 1␮M of methotrexate followed by 50nM of gemcitabine exposure. This effect was higher than that of simultaneous exposure of 10 ␮M of methotrexate and 500 nM of gemcitabine in all three parental cell lines and corresponding cisplatin-resistant sublines. CONCLUSIONS: The combination of methotrexate and gemcitabine demonstrated enhanced growth inhibition on urothelial cancer cells: this inhibition was obtained without increasing the concentration of each agent. This in vitro results support the possible combination of low-dose gemcitabine and methotrexate in the treatment of urothelial cancer patients. Source of Funding: None

877 ROLE OF STEROID HORMONE RECEPTORS ON FORMATION AND PROGRESSION OF BLADDER CARCINOMA Gholamreza Pourmand, Sepehr Salem*, Abdolrasoul Mehrsai, Farid Kosari, Tehran, Iran INTRODUCTION AND OBJECTIVES: Steroid hormone receptors expression, including estrogen (ER), progesterone (PR) and androgen (AR) play essential roles in development, growth, tumorigenesis, and progression of several malignancies; however, there is limited experience with urothelial carcinoma of the bladder (UCB), especially regarding prognostic and therapeutic impact. Furthermore, males have a substantially higher risk of developing UCB than females, whereas its etiology still remains largely obscure. This study sought to further clarify the role of ER/PR/AR expression in UCB, and also to evaluate the possible associations with progression and survival of cancer. METHODS: 120 patients with clinicopathologically confirmed primary UCB, and 132 controls without any malignant disease were evaluated prospectively. Their pathologic specimens were stained immunohistochemically using avidin-biotin-peroxidase technique and monoclonal ER/PR/AR antibodies were used to determine the ER/ PR/AR expression (Dako). Staining characteristics were compared with the clinic-pathological results. Cox regression was used to estimate the adjusted hazard ratios (HR) with 95% confidence intervals (CI), and impact on disease-free survival was analyzed using Kaplan-Meier method. RESULTS: ER/PR expressions were observed in 4.2%/2.5% of cases and 2.3%/1.5% of controls. AR expression was detected in 22% of the patients and all controls were AR-negative. No significant asso-

THE JOURNAL OF UROLOGY姞

e351

ciation was found between ER/PR immunoreactive scores and age, tumor size, stage and grade, while statistically significant correlation was revealed between AR expression and tumor stage and grade. AR/PR-positive patients had higher rate of metastasis in comparison with AR/PR-negative patients (p⬍0.05). In multivariate regression analysis, ER/PR was not found to be independent prognostic factors and survival was not affected by their expressions. However, AR-positive patients showed a significantly poorer prognosis than AR-negative cases (log-rank test, p⫽0.02) and it could also be used as a prognostic factor (HR: 2.12; 95%CI: 1.36 – 4.65). CONCLUSIONS: AR expression was found in almost 22% of the tumors and it was significantly associated with high stage, poorly differentiated tumors and unfavorable outcome. Hence, AR could be considered as a potential target for additional hormonal therapy. AR evaluation test could also be regarded as a diagnostic procedure for determining the malignant bladder issues. Moreover, ER and PR expression were not found to have any direct roles on formation and progression of UCBs. Source of Funding: None

878 BRANCHED PEPTIDES AS A NOVEL TUMOR-TARGETING AGENTS FOR BLADDER CANCER Andrea Minervini, Giampaolo Siena*, Florence, Italy; Niccolo` Ravenni, Barbara Lelli, Siena, Italy; Agostino Tuccio, Gianni Vittori, Sergio Serni, Alberto Lapini, Florence, Italy; Jlenia Brunetti, Lisa Bracci, Siena, Italy; Marco Carini, Florence, Italy INTRODUCTION AND OBJECTIVES: Membrane receptors of endogenous peptides like neurotensin (NT) are overexpressed in different human cancers and might be targeted as tumor-specific antigens. Branched peptides (BP), conjugated to different functional units, are effective target-selective carriers, either for in vivo or in vitro cell tracing. The aim of the present study is to verify the efficacy of the NT-tetra-branched form (NT-4) as a targeting agent for tumor imaging in 6 consecutive case-control bladder cancer patients. METHODS: The sequence of NT (NT 1–13) was synthesized through a solid phase chemistry in a tetrabranched form (NT4), where a 3-lysine core enables the covalent bound to the 4 peptide copies, while the C-terminus additional lysine offers a reactive group for functional units coupling. Peptides synthesized in such a oligo-branched form offer the advantage of a multivalent binding and become extremely stable to proteolytic degradation. Samples from 6 patients having bladder cancer surgical resection were stained with NT-4. Healthy tissue was collected from the normal bladder mucosa. Presence of tumor and healthy tissue was checked by the standard staining procedure. The NT-4 binding analysis consists of a previous biotinNT-4 conjugation, followed by Avidin- Fluorescein isothiocyanate, therefore the specimen is observed with confocal microscopy. Nuclei were stained with diamidino-phenylindole. RESULTS: A significant difference between tumor and healthy tissue samples is observed in all the specimens treated with NT-4 (fig A). Indeed, the median of each sample in the green scale of the RGB system results significantly higher in the cancer group (fig B). CONCLUSIONS: NT-4, a protease-resistant tetrabranched form of NT, results as an effective tumor-targeting agent. When NT-4 is conjugated to a fluorophore can efficiently discriminate between tumor and healthy tissue in surgical bladder samples, with a potential in vivo application.

e352

THE JOURNAL OF UROLOGY姞

Vol. 185, No. 4S, Supplement, Monday, May 16, 2011

measured. Statistics was performed by one-way analysis of variance (ANOVA) of natural log transformed bladder weights followed by a post hoc Tukey’s test. RESULTS: I.b. BCG treatment led to an increase in bladder mRNAs of IFN-␥,TNF-␣, IP-10 and perforin and a decrease in bladder mRNAs of TGF-␤. There were no changes in bladder mRNAs of granzyme B, FasL and TRAIL. In MB49-Luc orthotopic tumor model, i.b. BCG treatment reduced bladder weight by 53% (p ⫽ 0.0503 vs. PBS-treated group). In addition, i.b. BCG treatment also led to a delay in tumor growth by bioluminescence compared to PBS-treated group. CONCLUSIONS: Intravesical administration of BCG induced bladder expression of cytotoxic effector molecules IFN-␥, TNF-␣ and perforin, which was correlated with effective BCG treatment of bladder cancer. Increased expression of IP-10 and decreased expression of TGF-␤, together with increased IFN-␥ and TNF-␣, support the role of BCG in the induction of Th1 immune responses in the bladders. Source of Funding: None

880 SPLICE VARIANT VASCULAR ENDOTHELIAL GROWTH FACTOR-A LEVEL AS PROGNOSTIC INDICATOR OF PROGRESSION IN BLADDER CANCER Hugues Bittard*, Isabelle Lascombe, Ste´phane Bernardini, Eric Chabannes, Sylvie Fauconnet, Besancon, France

Source of Funding: None

879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG) Matthew Knudson*, Jonathan Henning, Michael O’Donnell, Yi Luo, Iowa City, IA INTRODUCTION AND OBJECTIVES: Bladder expression of cytokines and chemokines in response to intravesical (i.b.) BCG has been well documented. However, studies on BCG induction of bladder expression of cytotoxic effector molecules are underdeveloped. We investigated a panel of cytotoxic effector molecules, including IFN-␥, TNF-␣, perforin, granzyme B, Fas ligand (FasL), and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), in the BCG-treated bladders to determine whether expression of these molecules was correlated with the effect of BCG in treating bladder cancer. METHODS: C57BL/6 female mice were treated i.b. with Pasteur strain BCG (0.1 OD/dose) twice weekly for a total of 7 treatments. Mice treated with PBS served as a control. Mice were sacrificed for bladder RNA extraction after treatments 2, 4, 6 and 7. RT-PCR was used to analyze mRNA levels of IFN-␥ TNF-␣, perforin, granzyme B, FasL and TRAIL. IP-10 (a Th1 chemokine) and TGF-␤ (an inhibitory cytokine) were included. GAPDH was used as an internal control. ImageJ software was used to quantify PCR products on agarose gels. In a parallel experiment C57BL/6 female mice were implanted i.b. with 5 X 105 syngeneic bladder cancer MB49 cells that expressed luciferase (MB49-Luc) at day 0 and treated i.b. with BCG (0.1 OD/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with PBS. Bioluminescence was measured once weekly with Xenogen IVIS. At day 23 mice were euthanized and bladder weights

INTRODUCTION AND OBJECTIVES: angiogenesis is required for growth as well as expansion of solid tumor and is mediated by soluble angiogenic factors such as vascular endothelial growth factor (VEGF). The present investigation was conducted to determine whether correlation exists between VEGF-A expression within the bladder tumor and clinicopathologic parameters and whether the tumor aggressiveness is depending on a specific VEGF-A isoform expression. METHODS: total RNA was isolated from 37 specimens of bladder cancer and VEGF-A transcripts were detected by Northernblotting analysis. Progression-free survival curve was plotted after mRNA quantification. VEGF-A splice variants were determined by southern-blotting and the expression intensity of each isoform was evaluated by quantitative real time RT-PCR in 20 new fresh frozen recent tumors. RESULTS: northern-blotting analysis detected three VEGF-A transcripts of 5.2, 4.5 and 1.7 kb in length respectively. The VEGF-A transcripts levels were greater in cancer tissues than in normal urothelium, significantly higher in pT2-T4 than in pTa and pT1 urothelial tumors and thus, were correlated to the pathologic stage. Patients with higher VEGF-A mRNA levels had a significantly shorter survival without progression compared to those with lower levels. Three VEGF-A splice variants were detected namely, VEGF121, 165 and 189. VEGF121 and VEGF165 were expressed at the similar level. On the contrary, they were significantly more expressed than VEGF189 (p⬍0.05). The three isoforms were higher expressed in pT2 bladder cancers than in pTa tumors (p⬍0.05). There was only a significant correlation between the increased expression level of VEGF121 and 165 and the histological grade of the lesion (p⬍0.05). CONCLUSIONS: VEGF-A mRNA level could serve as a prognostic indicator of progression in bladder cancer as well as the expression level of the different VEGF-A splice variants. Source of Funding: PHRC(MNER France), Ligue contre le cancer