Decreased Invasion of Urothelial Carcinoma of the Bladder by Inhibition of Matrix-Metalloproteinase 7.

OBJECTIVES: To measure and to modulate the invasive potential of urothelial carcinoma of the bladder (UCB) cells in a standardized preclinical setting using broad-spectrum matrix-metalloproteinase (MMPs) inhibitors and specific targeting of MMP7. MATERIALS AND METHODS: MMP expression levels in UCB cells were determined by quantitative real-time PCR (qRT-PCR) and gel zymographies of cell supernatants (MMP9, MMP2 and MMP1) and cell lysates (MMP7). The invasiveness of human UCB cells (HT1197 and T24/83) and human benign urothelial cells (UROtsa) was modulated by a broad-spectrum MMP inhibitor (4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic acid; AHA) and by MMP7 specific siRNAs. MMP7 knockdown efficiency was assessed by qRT-PCR and western blot. Invasive potential of UCB cells was measured by a standardized trans-epithelial electrical resistance (TEER) assay. RESULTS: Different MMP secretion profiles were measured in UCB cells. The active form of MMP7 was exclusively detected in HT1197 cells. Characteristic TEER breakdown patterns were observed in UCB cells when compared to benign cells. Invasive potentials were significantly higher in HT1197 cells than in T24/83 and in UROtsa cells [14.8±5.75 vs. 1.5±0.56 and 1.2±0.15, respectively; p < 0.01]. AHA treatment reduced the invasive potential of HT1197 cells. Also the specific downregulation of MMP7 by siRNA lowered the HT1197 cell invasiveness [20±1.0 vs. 16±2.8; p < 0.05]. Neither AHA nor MMP-7 siRNA transfection altered the invasive potential of T24/83 cells. CONCLUSIONS: Invasion of UCB is partially dependent on MMPs. Specific targeting of MMP7 by siRNA reduces the invasive potential in a subgroup of UCB cells. Therefore, MMP7 represents a potential therapeutic target which warrants further investigation.

Assessing the invasive potential of bladder cancer: development and validation of a new preclinical assay.

PURPOSE: We developed and validated an electrophysiological method for standardized preclinical assessment of the invasive potential of urothelial carcinoma of the bladder. MATERIALS AND METHODS: Human UMUC-3, RT-112, HT-1197 and T24/83 bladder urothelial carcinoma cells, and UROtsa benign urothelial cells were co-cultivated with high resistance MDCK-C7 cells seeded below a 0.4 µm pore membrane of an insert to avoid physical contact and cellular migration. Transepithelial electrical resistance in Ω cm(2) across the MDCK-C7 monolayer was measured longitudinally. Invasive potential coefficients were calculated based on the secretion of proteolytic factors by invading cells. RESULTS: Consistent transepithelial electrical resistance breakdown patterns were reproduced in 14 or more independent samples of each cell line. Coefficients of invasive potential were significantly higher in bladder urothelial carcinoma than UROtsa cells, including a mean ± SD of 1.5 ± 0.32 vs 9.9 ± 4.97 in UMUC-3, 12.5 ± 6.61 in T24/83, 20.5 ± 4.24 in RT-112 and 21.0 ± 5.15 in HT-1197 cells (p <0.001). No correlation was found between the secretion patterns of matrix metalloproteinase-1, 2 and 9, and invasive potential. Stimulation of UROtsa cells with recombinant human epidermal growth factor up-regulated matrix metalloproteinase-9 secretion and significantly increased invasive potential a mean of 1.3 ± 0.22 vs 14.6 ± 3.28 after stimulation with 10 ng/ml epidermal growth factor (p <0.001). CONCLUSIONS: We developed a highly sensitive translational tool to study the initial process of metastatic spread of urothelial carcinoma of the bladder. The presented electrophysiological invasion assay enables reliable quantification of the invasive potential of bladder urothelial carcinoma cells before physical transmigration. It can be used to identify key molecules for bladder urothelial carcinoma invasion and develop new therapeutic strategies.

Cytokine profile after lung transplantation: correlation with allograft injury.

BACKGROUND: Post-lung transplant reperfusion edema (PLTRE) and its more severe form, primary graft failure (PGF), occur in 10% to 60% of lung transplant recipients. We hypothesized that PLTRE and PGF would be associated with an elevated proinflammatory cascade and that the allograft would be the source of cytokine appearance in the circulation. METHODS: Pulmonary arterial and systemic arterial samples were obtained at baseline and at 4, 8, and 24 hours after reperfusion. Post-lung transplant reperfusion-edema and PGF were defined as PaO2/FiO2 less than 300 with a mild or moderate infiltrate, or less than 200 with a severe infiltrate and ventilator dependence after 72 hours, respectively. Tumor necrosis factor alpha (TNFalpha), interleukin (IL)-6, IL-8, and IL-10 concentrations were determined by immunoassay. RESULTS: Fifteen single and 6 bilateral lung recipients were studied. Six (29%) had PLTRE and 4 (19%) had PGF; these patients had an overall elevation in plasma IL-6, IL-8, and IL-10 concentrations (all p < 0.05). Subgroup analysis revealed a significantly greater elevation in IL-6, IL-8, and IL-10 levels in PGF patients (all p < 0.01) versus PLTRE. In the PGF group, TNFalpha and IL-10 concentrations were significantly greater in the systemic versus the pulmonary arterial samples (p < 0.05). CONCLUSIONS: Patients with PLTRE and PGF exhibited graded increases in IL-6, IL-8, and IL-10 concentrations. The PGF patients had higher TNFalpha and IL-10 systemic arterial concentrations overall, consistent with the allograft being a source of this cytokine production.

Five-year results of a phase II trial of hyperfractionated radiotherapy and concurrent daily cisplatin chemotherapy for stage III non-small-cell lung cancer.

The purpose of this study was to evaluate the 5-year results for a phase II trial of hyperfractionated radiotherapy (RT) and concurrent daily cisplatin chemotherapy. Between August 1994 and December 1999, 63 patients with stage IIIA and stage IIIB non-small-cell lung cancer were treated with RT to a dose of 69.6 Gy at 1.2 Gy twice daily with daily cisplatin at 6 mg/m. Thirty-seven patients elected to receive consolidation carboplatin and paclitaxel chemotherapy. Recurrence and survival outcomes were evaluated by Kaplan-Meier analysis. Acute and late side effects were scored by the Radiation Therapy Oncology Group (RTOG) grading system. Radiographic complete or partial tumor response was achieved in 34 of 63 (54%) of cases. Median absolute survival was 20.1 months. Median time to local recurrence and distant metastases were 10.6 and 8.6 months, respectively. Overall survival rates were 57%, 35%, and 23% at 1, 3, and 5 years, respectively. Survival was significantly greater for patients receiving consolidation chemotherapy (50% versus 20% at 3 years). Only 5 patients (7%) experienced Grade 3 or 4 esophagitis. There were 16 cases of Grade 1 or 2 pneumonitis; steroid therapy resolved symptoms in 9 patients. This regimen of hyperfractionated RT and chemotherapy achieved significant response, and 5-year survival rates with acceptable toxicity.