Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan.
Life Sci. 2010 Jan 30;86(5-6):199-206. doi: 10.1016/j.lfs.2009.12.003. Epub 2009 Dec 22.
Bortezomib is a potent proteasome inhibitor currently used to treat various malignancies with promising results. To explore the role of bortezomib in reducing cancer cell migration and inducing apoptosis, we evaluated the effects of bortezomib on the expression of focal adhesion kinase (FAK).
Various types of cancer cells including lung cancer A549, H1299; a breast cancer MCF7; a hepatocellular carcinoma Huh7, and a tongue squamous cell carcinoma SCC-25 were treated with different concentrations of bortezomib or MG-132 as indicated for 24h. Protein and mRNA levels were determined by Western blotting and real-time PCR. Apoptosis was analyzed by caspase 3 cleavage and activity. FAK promoter and NFkappaB binding activities were measured by luciferase-reporter method. NFkappaB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis.
Both bortezomib and another proteasome inhibitor, MG-132, significantly reduced FAK expression, suppressed cancer cell migration and increased cell apoptosis. Results of real-time PCR and promoter activity assay revealed that bortezomib decreased FAK expression through transcriptional inactivation. Results of FAK promoter activity and ChIP assays in A549 and H1299 cells indicated that bortezomib suppressed FAK activity through a p53-independent pathway. Furthermore, reduction of NFkappaB binding capacity demonstrated by EMSA and ChIP assay suggested that NFkappaB plays an important role in bortezomib suppressing FAK expression.
These results suggested that FAK is downregulated by bortezomib through a proteasome-dependent NFkappaB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy.
硼替佐米是一种有效的蛋白酶体抑制剂，目前用于治疗多种恶性肿瘤，疗效显著。为了探讨硼替佐米在降低癌细胞迁移和诱导细胞凋亡中的作用，我们评估了硼替佐米对黏着斑激酶（FAK）表达的影响。
用不同浓度的硼替佐米或 MG-132 处理各种类型的癌细胞，包括肺癌 A549、H1299；乳腺癌 MCF7；肝癌 Huh7 和舌鳞状细胞癌 SCC-25；24 小时后，通过 Western blot 和实时 PCR 测定蛋白和 mRNA 水平。通过 caspase 3 切割和活性分析来检测细胞凋亡。通过荧光素酶报告基因法测定 FAK 启动子和 NFkappaB 结合活性。通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析测定 NFkappaB 亚基 p65 的结合能力。
硼替佐米和另一种蛋白酶体抑制剂 MG-132 均显著降低了 FAK 表达，抑制了癌细胞迁移并增加了细胞凋亡。实时 PCR 和启动子活性测定结果表明，硼替佐米通过转录失活降低了 FAK 表达。A549 和 H1299 细胞中的 FAK 启动子活性和 ChIP 分析结果表明，硼替佐米通过 p53 非依赖性途径抑制了 FAK 活性。此外，EMSA 和 ChIP 分析显示 NFkappaB 结合能力降低表明 NFkappaB 在硼替佐米抑制 FAK 表达中起重要作用。
这些结果表明，硼替佐米通过依赖蛋白酶体的 NFkappaB 抑制机制下调 FAK 的表达。因此，FAK 可能是硼替佐米治疗策略的潜在分子靶标。
