17-AAG and 17-DMAG-induced inhibition of cell proliferation through B-Raf downregulation in WTB-Raf -: Expressing uveal melanoma cell lines
17-AAG and 17-DMAG-induced inhibition of cell proliferation through B-Raf downregulation in WTB-Raf -: Expressing uveal melanoma cell lines
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DOI：
10.1167/iovs.07-1305
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发表时间：
2008-06-01
影响因子：
4.4
通讯作者：
Mascarelli, Frederic
中科院分区：
文献类型：
--
作者：
Babchia, Narjes;Calipel, Armelle;Mascarelli, Frederic
目的。HSP90抑制剂17-烯丙氨基-17-去甲氧基格尔达那霉素(17-AAG)通过降解B-Raf的V600E突变体(B-V600E-Raf)在皮肤黑色素瘤细胞系中具有良好的抗肿瘤活性。然而，根据肿瘤细胞中B-WT-Raf的激活水平，它对野生型B-Raf(B-WT-Raf)有不同的影响。葡萄膜黑色素瘤细胞表达B-WT-Raf，仅极少数表达B-V600E-Raf。本研究旨在探讨热休克蛋白90对葡萄膜黑色素瘤细胞株的抑制作用。方法：人葡萄膜黑色素瘤细胞株分别用热休克蛋白90抑制剂17-AAG和17-dimethylaminoethylamino-17-demethoxy-geldanamycin(17-DMAG)处理。用四甲基偶氮唑盐(四甲基偶氮唑蓝)染色检测细胞增殖，流式细胞仪检测细胞凋亡率。Western印迹分析B-Raf热休克蛋白90的表达及MEK/ERK下游信号通路的激活情况。结果：在表达B-WT-Raf的葡萄膜黑色素瘤细胞系中，HSP90的抑制作用使B-Raf表达下调，细胞增殖减少，MEK/ERK活性降低。HSP90抑制也降低了Akt的表达，但Akt的抑制对细胞增殖没有影响，排除了Akt在17-AAG诱导的细胞增殖抑制中的作用。Cdc37的下调不影响MEK/ERK信号转导和细胞增殖，表明辅伴侣不是HSP90控制的B-Raf稳定所必需的。HSP90抑制后，c-Kit的表达也下调。17-DMAG联合c-Kit抑制剂伊马替尼对B-WT-Raf葡萄膜黑色素瘤细胞株的增殖有协同抑制作用。结论靶向HSP90联合c-Kit抑制可能是一种有前景的葡萄膜黑色素瘤治疗方法。
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 (B-V600E-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (B-WT-Raf), according to the B-WT-Raf activation levels in the tumor cells. Uveal melanoma cells express B-WT-Raf and only rarely express B-V600E-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 B-WT-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 B-WT-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.