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A. M. Alegret, Y. Pina, T. G. Murray, F. Suarez, B. Hayden, C. Cebulla, W. Feuer, H. Boutrid, M.-E. Jockovich; Differential Induction of Apoptosis in the Treatment of LHBETATAG Retinoblastoma Following Vessel Targeting. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1600.
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To evaluate the mechanisms and timing of tumor cell death in the LHBETATAG mouse model of retinoblastoma following treatment with two vascular targeting therapies: anecortave acetate (AA) and combretastatin A4P (CA4P).
The study protocol was approved by the University of Miami, Miller School of Medicine Animal Care and Use Review Board. All experiments in this study were conducted in accordance with the Association for Research in Vision and Ophthalmology guidelines for the use of animals in ophthalmologic and vision research. Eyes from LHBETATAG mice of 4, 8, 10 and 16 weeks of age (n=5 per group) were analyzed for cell death. A. Mechanisms of tumor cell death were evaluated using TUNEL assays, or immunofluorescence analysis of activated caspase 3, to detect apoptosis, and by histopathologic analysis to identify areas of necrosis. B. To assess cell death following vascular targeting, 16 week-old LHBETATAG mice (n=20) were treated with a single subconjunctival injection of either AA (300µg /ml) or CA4P (1.5 mg/ml) and eyes were analyzed at 1-day and 7-days post-treatment (n=5 per group).
A. Analysis of tumor cells during tumor progression suggests that death by apoptosis increases with tumor progression in this animal model. Results show that apoptosis increases exponentially with age, reaching a peak at 12 weeks. There is a gradual decrease of apoptotic cells in advanced disease (16 weeks). No significant necrosis was detected by analysis of H&E-stained sections. B. Treatment with vessel targeting agents resulted in an acute increase of apoptosis; CA4P showed a 13-fold increase and AA a 5.5-fold increase of apoptotic cell density at 1-day post treatment (No treatment control = 2 cells per high-power field (HPF); 1-day post-treatment of AA = 11 cells per HPF; 1-day post-treatment of CA4P = 26 cells per HPF; p<0.001). However, there was a significant decline in apoptosis at 1-week post-treatment with both treatment modalities (CA4P & AA) (7-days post-treatment of AA = 3.5 cells per HPF; 7-days post-treatment of CA4P = 3 cells per HPF; p<0.05). No significant necrosis was detected following treatment with either agent.
Vascular targeting agents significantly increase cell death via apoptosis, while not having a significant effect on necrosis in this murine model of transgenic retinoblastoma.
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