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R. Tano, J.S. Yoo, D.S. Tyler, C. Augustine, F. Ali–Osman, H.S. Friedman, S.S. Stinnett, G.J. Jaffe, P. Mruthyunjaya; The in vitro Efficacy of Combining Temozolomide With O6–Benzylguanine in Human Choroidal Melanoma Cell Lines . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2248.
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© ARVO (1962-2015); The Authors (2016-present)
Temozolomide (TMZ) has been previously combined with O6–Benzylguanine (O6–BG), a potent inhibitor of O6–alkylguanine–DNA alkyltransferase (AGT) to overcome TMZ resistance in cutaneous melanoma. However, the efficacy of this chemotherapy combination is unknown in primary human choroidal melanoma (CM). The aim of this study was to determine whether the in vitro efficacy of TMZ could be enhanced when combined with O6–BG in human CM cell lines (92–1, OCM–1, MKT–BR) and a choroidal melanocytic line (UW–1).
Cell lines were treated with 10 µM (92–1) and 5 µM of O6–BG (OCM–1, MKT–BR, UW–1) in combination with increasing concentrations of TMZ (0.5–3.0 mM). Cell survival was measured 24 hours post treatment by the WST–1 colorimetric assay and IC50 was calculated for comparative analysis. Quantitative PCR was performed to measure AGT gene transcripts and compared to a reference cell line with known high AGT activity (HT29).
TMZ significantly reduced the viability of all 4 cell lines. The IC50 of TMZ alone and TMZ combined with O6–BG was, respectively,2.26 mM and 2.16 mM for 92–1, 1.98 mM and 1.88 mM for OCM–1, 2.22 mM and 2.00 mM for MKT–BR, and 2.19 mM and 2.05 mM for UW–1. The addition of O6–BG enhanced cytotoxicity slightly, but not significantly compared to TMZ alone. The relative AGT gene expression of each cell line was low; the transcript levels were only 6, 7, 7 and 10%, respectively, compared to the reference cell line.
TMZ is cytotoxic to cultured human melanoma derived cell lines. Cell toxicity was not significantly enhanced by the addition of O6–BG. The low relative AGT expression in these lines might help explain these observations. Although TMZ–based methylator therapy may provide a novel treatment platform for local control in CM, alternative agents that may help overcome TMZ resistance in CM warrant further consideration.
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