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Aizhan Tapenbayeva, Julia Lüke, Matthias Lüke, Salvatore Grisanti, Aysegul Tura; Outcomes Of Rho-kinase Inhibition On The Metastatic Profile Of A Uveal Melanoma Cell Line. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6873.
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© ARVO (1962-2015); The Authors (2016-present)
Uveal melanoma is the most common primary intraocular tumor in adults with a high rate of mortality due to metastasis. Rho-kinase is an intracellular signalling cascade effector, which is activated in a wide variety of tumor types. In this study, we evaluated the outcomes of Rho-kinase inhibition on the viability, proliferation and migration of the uveal melanoma cell line 92.1 as well as the expression of several metastatic proteins.
The 92.1 cells were cultured in DMEM/F-12 supplemented with 10% serum and treated with the specific pharmacological Rho-kinase inhibitor H1152 at a concentration range of 0.1-10 µM. Viability and proliferation were assessed by Live-Dead staining, MTT assay, and Ki-67 immunostaining. The organization of the actin cytoskeleton was evaluated by phalloidin staining. The extent of Rho-kinase activity was determined by an in vitro kinase assay. Migration was analyzed by the scratch assay on the confluent monolayers of cells grown in 6-well plates. The levels of beta-catenin, N-cadherin, and vimentin were determined by immunostainings on cells grown on fibronectin coated 8-well slides and by Western blot.
H1152 resulted in a dose-dependent reduction in the Rho-kinase activity at the concentrations of 0.1 and 1 µM, whereas the administration of the inhibitor at 10 µM weakened this effect possibly due to the interference with other signalling pathways and resulted in a more aberrant, oncotic morphology together with a slight decrease in viability. H1152 at 1 µM significantly suppressed the proliferation of the 92.1 cells by 50% (p<0.05) without exerting any toxicity and interfered with the migration of the cells into the wound area after 2 days. The levels of vimentin and beta-catenin underwent a significant reduction of 20-30%, respectively, in response to 1 µM H1152 already after 1 day of incubation. Moreover, the weak immunoreactivity to N-cadherin was further reduced by 20% in the cells treated with 1 µM H1152 for 1 day.
These findings provide evidence to the involvement of Rho-kinase in the proliferation and migration of the 92.1 cells and demonstrate the efficacy of Rho-kinase inhibition in suppressing the metastatic potential of these cells as a novel alternative for the treatment of uveal melanoma.
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