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D. A. Abourbih, S. Di Cesare, S. Bakalian, E. Antecka, B. F. Fernandes, M. N. Burnier, Jr.; The Expression and Inhibition of the Lysyl Oxidase-Like 2 Protein in Human Uveal Melanoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2008;49(13):71. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The lysyl oxidase-like 2 (LOXL2) protein and its homologous family members (LOX, LOXL1, LOXL3, LOXL4) have been implicated in several tumorigenic processes including cell proliferation, invasion, metastasis, and evasion of apoptosis. We sought to characterize the expression of LOXL2 in 5 uveal melanoma (UM) cell lines. The effect of inhibiting LOXL2 enzymatic activity was also assessed using D-Penicillamine (DPA), an FDA approved drug.
Initial characterization was performed using immunocytochemistry against the LOXL2 protein in 4 human UM cell lines (92.1, SP6.5, MKT-BR, OCM-1) and one transformed melanocyte cell line (UW-1). To further localize the expression of LOXL2, we stained with FITC conjugated secondary antibodies. Furthermore, whole cell protein extracts were obtained and western blots were performed. Changes in cellular proliferation were measured using a Sulforhodamine-B assay kit (TOX-6, Sigma-Aldrich Co., St. Louis, Missouri, USA) following treatment with 5 mM of DPA. The invasiveness of treated cells was assessed using a Matrigel invasion assay (BD Biocoat Matrigel Invasion Chambers, BD Biosciences, Mississauga, Ontario, Canada). This assay uses a modified Boyden chamber system consisting of a membrane with 8 µm pores pre-coated with Matrigel, an artificial basement membrane. A solution of 3.0 x 105 cells in serum free RPMI was seeded onto the top portion of the chamber. 10% FBS RPMI was used as a chemo-attractant at the bottom of the wells.
Immunocytochemistry confirmed the expression of LOXL2 in all 5 cell lines. Western Blot analysis showed a prominent band present at 37 kDa corresponding to the mature protein. Treating cells with 5 mM of DPA resulted in a significant decrease (p<0.0001) in cellular proliferation and invasion in all 5 cell lines.
LOXL2 is expressed in all 5 cell lines and appears to be localized to the cell membrane. To the best of our knowledge, this is the first study to use DPA to treat UM cells in an in vitro system. Preliminary results showed on average a 50% decreased cellular proliferation following treatment. More significantly, DPA abolished invasion in the SP6.5 cell line and significantly reduced invasion in the other 4 cell lines tested. Further studies are needed to elucidate the exact role of LOXL2 in UM tumorigenesis and the value of DPA as a therapeutic agent.
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