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Joseph C. Manarang, Alan R. Burns, Alison M. McDermott; Apoptotic Pathways of Antimicrobial Peptide-Induced Death of Uveal Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):511.
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Antimicrobial peptides (AMPs) exhibit cytotoxicity to mammalian cells possibly through an apoptotic process, which appears to be AMP specific and cell dependent. AMPs are also known to exhibit preferential cytotoxicity to tumor cells primarily due to a strong electrostatic interaction, which imparts an exciting potential for anti-tumor therapy. The purpose of this study is to characterize and visualize the apoptotic process and specific pathways involved in AMP-induced cell death of uveal melanoma cells.
OCM3, OCM8, OMM2.5, SP6.5 and MKT-BR uveal melanoma cells were treated for upto 24 hours with 25-500 µg/ml of Human Neutrophil Peptide-1 (HNP-1), cathelicidin LL-37, magainin II, cecropin B or 2 µM staurosporine as a positive control. Active caspase 3 production was quantified by flow cytometry, phosphatidylserine translocation to the outer membrane was determined by Annexin V binding, and loss of mitochondrial transmembrane potential was visualized by JC-1 staining and live cell imaging for upto 14 hours using a Deltavision deconvolution microscope. For all experiments n=2-3 depending on cell type. Statistical analysis was performed using Student’s t-test where applicable with P<0.05 considered significant.
Caspase 3 activation increased proportionally with length of exposure to 2µM staurosporine, from 1.93 + 2.02% active caspase 3-positive cell population in the untreated group, to 75.4 + 13.1% at 24 hours exposure (P=0.0007). AMP treatment did not result in significantly increased caspase 3 activation compared to untreated cells. AMPs did not cause detectable phosphatidylserine translocation as determined by flow cytometry or time-lapse imaging of Annexin V:FITC binding. Live imaging of AMP treated cells showed a shift from punctate red fluorescence of JC-1 dimers in healthy mitochondria, to diffuse green fluorescence of JC monomers in about 3-5 hours indicating loss of mitochondrial transmembrane potential.
Antimicrobial peptide-induced cytotoxicity to uveal melanoma cells is exerted through an intrinsic pathway of apoptosis involving loss of mitochondrial transmembrane potential, and is caspase independent.
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