KRIBB11 Inhibits HSP70 Synthesis through Inhibition of Heat Shock Factor 1 Function by Impairing the Recruitment of Positive Transcription Elongation Factor b to the hsp70 Promoter
KRIBB11 Inhibits HSP70 Synthesis through Inhibition of Heat Shock Factor 1 Function by Impairing the Recruitment of Positive Transcription Elongation Factor b to the hsp70 Promoter
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DOI：
10.1074/jbc.m110.179440
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发表时间：
2011-01-21
影响因子：
4.8
通讯作者：
Han, Dong Cho
中科院分区：
文献类型：
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作者：
Yoon, Young Ju;Kim, Joo Ae;Han, Dong Cho
热休克因子1 （HSF1）是真核生物热休克蛋白（HSP）表达的主要开关。在热休克元件的控制下，利用荧光素酶报告基因筛选合成化学文库来鉴定HSF1的抑制剂。化合物KRIBB11 （N-2-（1h -吲哚-5-基）- n -6-甲基-3-硝基吡啶-2,6-二胺）具有消除热休克诱导的荧光素酶活性的活性，IC50为1.2 μ mol/l。当细胞在KRIBB11存在下暴露于热休克时，HSF1下游靶蛋白如HSP27和HSP70的诱导被阻断。此外，用KRIBB11处理HCT-116细胞可诱导生长停滞和凋亡。KRIBB11处理后检测细胞凋亡标志物，如cleaved poly（adp -核糖）聚合酶。合成生物素基-KRIBB11作为鉴定KRIBB11靶蛋白的亲和探针。通过亲和层析和竞争分析，发现KRIBB11在体外与HSF1存在关联。染色质免疫沉淀分析显示，KRIBB11抑制了hsf1依赖性的p-TEFb（正转录延伸因子b）向hsp70启动子的募集。最后，以50 mg/kg剂量给裸鼠腹腔注射KRIBB11，对肿瘤生长的抑制率为47.4% (p < 0.05)，且未造成体重减轻。免疫印迹分析显示，HSP70在kribb11处理的肿瘤组织中的表达低于对照组织。由于热休克蛋白在多种肿瘤中都有高水平表达，这些结果加强了靶向HSF1用于癌症治疗的理论依据。
Heat shock factor 1 (HSF1) is the master switch for heat shock protein (HSP) expression in eukaryotes. A synthetic chemical library was screened to identify inhibitors of HSF1 using a luciferase reporter under the control of a heat shock element. A compound named KRIBB11 (N-2-(1H-indazole-5-yl)-N-6-methyl-3-nitropyridine-2,6-diamine) was identified for its activity in abolishing the heat shock-induced luciferase activity with an IC50 of 1.2 mu mol/liter. When the cells were exposed to heat shock in the presence of KRIBB11, the induction of HSF1 downstream target proteins such as HSP27 and HSP70 was blocked. In addition, treatment of HCT-116 cells with KRIBB11 induced growth arrest and apoptosis. Markers of apoptosis, such as cleaved poly(ADP-ribose) polymerase, were detected after KRIBB11 treatment. Biotinyl-KRIBB11 was synthesized as an affinity probe for the identification of KRIBB11 target proteins. Using affinity chromatography and competition assays, KRIBB11 was shown to associate with HSF1 in vitro. Chromatin immuno-precipitation analysis showed that KRIBB11 inhibited HSF1-dependent recruitment of p-TEFb (positive transcription elongation factor b) to the hsp70 promoter. Finally, intraperitoneal treatment of nude mice with KRIBB11 at 50 mg/kg resulted in a 47.4% (p < 0.05) inhibition of tumor growth without body weight loss. Immunoblotting assays showed that the expression of HSP70 was lower in KRIBB11-treated tumor tissue than in control tissues. Because HSPs are expressed at high levels in a wide range of tumors, these results strengthen the rationale for targeting HSF1 in cancer therapy.