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Joseph Manarang, Deborah Otteson, Adrian Glasser, Alan Burns, Alison McDermott; Antimicrobial Peptide Expression and Potential Roles in Uveal Melanoma Pathogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4213.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this project was to elucidate the potential roles of AMPs in UM pathogenesis by determining expression of human neutrophil peptide-1 (HNP-1), cathelicidin LL-37, and human beta defensins (hBD) -1, -2, -3 and -4 in UM cells, and their effects on apoptosis induction, cell migration and vasculogenic mimicry (VM).
AMP mRNA expression by UM cell lines OCM8, OMM2.5, SP6.5 and MUM2b and primary normal and UM melanocytes was determined by RT-PCR. The presence of DNA laddering was sought in UM cells treated with HNP-1 (75μg/ml), LL-37 (100μg/ml), Magainin II (250μg/ml) or Cecropin B (800μg/ml) for 24 hrs. To test migration, UM cell monolayers were scratched then HNP-1 (5 and 17.5μg/ml) or LL-37 (5 and 20μg/ml) added. Digital images were collected at 0, 6, 12, 24 and 48 hrs and analyzed by a Matlab program. AMP effects on VM formation were investigated by exposing MUM2b cells growing in a 3D gel matrix to HNP-1 (12.5 and 17.5μg/ml) and LL-37 (5 and 10μg/ml) for upto 21 days. Gels were stained with DAPI and Phalloidin or Calcein AM, then imaged and reconstructed in 3D using a Deltavision microscope running SoftWorx.
LL-37 was expressed by all UM cell lines (n=3). None expressed hBD-1, hBD-2, nor HNP-1 while some expressed hBD-3 and hBD-4. Primary cultured normal and UM melanocytes expressed hBD-4, but variably expressed the other AMPs (n=2-3). DNA laddering characteristic of apoptosis was found in control staurosporine treated cells, but not in AMP treated cells (n=3). In the presence of 10% FBS, UM cells migrated significantly faster than cells in serum free media. However AMPs did not significantly modulate cell migration rates (n=3). Light and fluorescence microscopy revealed MUM2b cells on gels grew in multiple levels forming 3D looping networks with characteristic acellular channels (n=3). These VM patterns were not affected by treatment with AMPs.
AMP mRNA was variably expressed by UM, and there was no marked difference in the expression profiles between UM melanocytes and normal melanocytes. Likewise, our findings suggest that AMPs do not modulate UM pathogenesis with respect to apoptosis induction, cell migration or VM formation. While this indicates that AMPs play no role in UM tumor pathogenesis, their ability to exert a cytotoxic effect on UM cells (Manarang et al. ARVO 2009) may be capitalized upon for development of novel treatments.
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