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J. C. Manarang, R. Rodriguez, A. R. Burns, A. M. McDermott; Induction of Apoptosis in Ocular Tumor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5985.
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Uveal melanoma and retinoblastoma are the most common forms of ocular tumors. Antimicrobial peptides (AMPs) have been previously shown to be cytotoxic to corneal epithelial cells and OCM8 uveal melanoma cells, and their preferential cytotoxicity to cancer cells might be capitulated upon for future anti-cancer treatments. This study aims to extend the cytotoxicity profiles of AMPs to include other uveal melanoma and retinoblastoma cell lines, and to determine if camptothecin or staurosporine, known inducers of apoptosis, could be used as a positive control to investigate the mechanisms of AMP cytotoxicity.
Cytotoxicity profiles of human neutrophil peptide-1 (HNP-1), LL-37, Magainin II and Cecropin B to uveal melanoma cell lines OMM2.5, OCM3, SP6.5, MKT-BR and retinoblastoma cells Y79 and WERI Rb1 were determined by MTT cytotoxicity assays. Cells were treated with camptothecin or staurosporine, cytotoxicity was determined by MTT assay and induction of apoptosis was determined by Annexin V-PE quantification by flow cytometry, and visualization of morphologic changes by live cell imaging using a Deltavision deconvolution microscope.
AMPs displayed concentration-dependent cytotoxicity to all the ocular tumor cell lines tested starting at 10-25 ug/ml for HNP-1 and LL-37, and 100ug/ml for Magainin II and Cecropin B (n=2-3). Staurosporine, but not camptothecin, induced cytotoxicity in ocular tumor cells in a dose dependent manner, starting at 500nM (19-36% cell death) and reaching maximal cytotoxicity (80-87% cell death) at 2uM (n=1-3). Staurosporine increased Annexin-V positive cells in OCM3, OCM8, OMM2.5 uveal melanoma cell lines by 1.92 - 8.5 fold and in Y79 and WERI-Rb1 retinoblastoma cell lines by 1.39 - 1.57 fold (n=1-2). Staurosporine induced classical morphological features of apoptosis, including cell shrinkage, chromatin condensation, contraction of cell processes, violent membrane blebbing, and loss of membrane integrity, in OCM3 and OCM8 cells.
HNP-1, LL-37, Magainin II and Cecropin B exhibit concentration-dependent cytotoxicity to uveal melanoma and retinoblastoma cell lines. Staurosporine initiated cell death through an apoptotic mechanism and can be used as a positive control for understanding the mechanisms through which AMPs are cytotoxic.
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