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Narjes Babchia, Armelle Calipel, Frédéric Mouriaux, Anne-Marie Faussat, Frédéric Mascarelli; 17-AAG and 17-DMAG–Induced Inhibition of Cell Proliferation through B-Raf Downregulation in WTB-Raf–Expressing Uveal Melanoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2008;49(6):2348-2356. doi: 10.1167/iovs.07-1305.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (V600EB-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WTB-Raf), according to the WTB-Raf activation levels in the tumor cells. Uveal melanoma cells express WTB-Raf and only rarely express V600EB-Raf. This study was conducted to investigate the effects of HSP90 inhibition on uveal melanoma cell lines.
methods. Human uveal melanoma cell lines were treated with the HSP90 inhibitors 17-AAG and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG). Cell proliferation was assessed by MTT staining, and apoptosis was quantified by flow cytometry. Analysis of the expression of HSP90 and activation of the MEK/ERK downstream signaling of B-Raf was performed by Western blot. Effects of the downregulation of the HSP90 cochaperone, cdc37, on cell proliferation and activation of MEK/ERK was investigated by siRNA strategy.
results. The inhibition of HSP90 downregulated B-Raf, decreased cell proliferation, and reduced activation of MEK/ERK in uveal melanoma cell lines expressing WTB-Raf. HSP90 inhibition also reduced the expression of Akt, but the inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG–induced inhibition of cell proliferation. The downregulation of cdc37 did not affect MEK/ERK signaling and cell proliferation, demonstrating that the cochaperone was not required for HSP90-controlled stability of B-Raf. c-Kit was also downregulated after HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in WTB-Raf uveal melanoma cell lines.
conclusions. These results suggest that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.
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