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L. Asnaghi, M. L. Coonfield, K. C. Schreck, E. E. Bar, J. Handa, S. Merbs, K. Ebrahimi, J. Harbour, C. G. Eberhart; Notch Signaling: A New Potential Target in the Treatment of Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3803.
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To investigate the role of Notch signaling in promoting uveal melanoma cellular proliferation and invasion.
Growth, invasion and clonogenicity of cell lines were examined using MTT, transwell migration and soft agar assays. Expression of Notch pathway member mRNA and protein was measured using qPCR and Western blotting.
We examined five established uveal melanoma cell lines, and found using qPCR that the Notch 1-2-3 receptors, Jag1-2 ligands and the pathway target Hes1 were expressed to varying degrees in all lines. We then blocked Notch signaling using a gamma-secretase inhibitor (GSI), and found that only three of the lines (OCM1, OCM3, OCM8) had their growth inhibited by the drug. Interestingly, these three lines had significantly higher levels of Hes1 mRNA as compared to the two lines resistant to GSI treatment (Mel285, Mel290). GSI treatment induced a dose-dependent reduction of anchorage-independent clonogenic growth, with 50% inhibition in OCM1, 70% in OCM3, and 40% in OCM8, as well as a significant reduction in Hes1 mRNA and protein levels. Apoptosis, as measured by cleavage and activation of caspase-9, was also induced. Finally, we observed inhibition of cellular invasion in GSI treated cultures using transwell assays. Preliminary studies of snap-frozen primary tumors indicate that Notch pathway components are expressed in uveal melanoma in vivo as well. In addition, oligonucleotide microarray analysis showed a significant increase in Jag2 and Notch3 expression levels in primary uveal melanomas that metastasized as compared to those that did not.
Our findings suggest that the Notch pathway plays an important role in inducing cellular proliferation and invasion in uveal melanoma, and that inhibiting this pathway using pharmacological agents may be effective in preventing tumor growth and metastasis.
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