IC101 induces apoptosis by Akt dephosphorylation via an inhibition of heat shock protein 90-ATP binding activity accompanied by preventing the interaction with Akt in L1210 cells
IC101 induces apoptosis by Akt dephosphorylation via an inhibition of heat shock protein 90-ATP binding activity accompanied by preventing the interaction with Akt in L1210 cells
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DOI：
10.1124/jpet.104.065979
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发表时间：
2004-09-01
影响因子：
3.5
通讯作者：
Ohizumi, Y
中科院分区：
文献类型：
--
作者：
Fujiwara, H;Yamakuni, T;Ohizumi, Y
为了寻找新的药理学工具，用于分析自然资源中细胞凋亡的分子机制，在本研究中，我们研究了IC 101，从链霉菌属MJ 202 - 72 F3分离的环状缩肽，诱导L1210细胞系凋亡的活性。3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四氮唑溴化物测定显示，IC 101引起浓度依赖性细胞死亡，50%有效浓度值为20 nM。IC 101可诱导细胞皱缩、染色质浓缩、典型的DNA梯状条带和caspase-3表达上调等凋亡生化特征。还观察到IC 101引起Akt和Bad的浓度依赖性去磷酸化，而不影响Akt的上游分子磷脂酰肌醇-3激酶。IC 101使90-kDa蛋白质去磷酸化，如通过使用抗磷酸酪氨酸抗体对细胞提取物进行免疫印迹所测定的。为了鉴定90-kDa蛋白，进行免疫沉淀和直接纳米流液相色谱-串联质谱（LC-MS），以证明该蛋白是热休克蛋白90（HSP 90）。因此，通过使用抗磷酸酪氨酸抗体对与抗HSP 90抗体的免疫沉淀物进行免疫印迹分析，观察到IC 101诱导HSP 90酪氨酸去磷酸化。IC 101引起Raf-1的降解，Raf-1与HSP 90形成复合物。IC 101也以非竞争性方式抑制HSP 90-ATP结合。IC 101以浓度依赖性方式抑制HSP 90与Akt的相互作用。这些结果表明，IC 101通过抑制HSP 90功能使Akt去磷酸化，导致与Akt相互作用以诱导L1210细胞的凋亡细胞死亡。
To find novel pharmacological tools useful for analyzing the molecular mechanism of apoptosis from natural resources, in the present study, we examined the activity of IC101, a cyclic depsipeptide isolated from Streptomyces sp. MJ202-72F3, to induce apoptosis in the L1210 cell line. 3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide assay showed that IC101 caused a concentration-dependent cell death with a 50% effective concentration value of 20 nM. Cell shrinkage, chromatin condensation, a typical DNA ladder pattern, and up-regulation of cleaved caspase-3 expression, which were biochemical characteristics of apoptosis, were induced by IC101. It also was observed that IC101 caused a concentration-dependent dephosphorylation of Akt and Bad without affecting phosphatidylinositol-3 kinase, an upstream molecule of Akt. IC101 dephosphorylated the 90-kDa protein, as assayed by immunblotting of the cell extract by using anti-phosphotyrosine antibody. To identify the 90-kDa protein, immunoprecipitation and direct nano-flow liquid chromatography-tandem mass spectrometry (LC-MS) were performed to demonstrate that this protein was heat shock protein 90 (HSP90). Consistently, it was observed that IC101 induced the HSP90 tyrosine dephosphorylation by immunoblot analysis of immunoprecipitates with anti-HSP90 antibody using anti-phosphotyrosine antibody. IC101 caused the degradation of Raf-1, which formed a complex with HSP90. The HSP90-ATP binding also was inhibited by IC101 in a noncompetitive manner. An interaction of HSP90 with Akt was shown to be inhibited by IC101 in a concentration-dependent manner. These results suggest that IC101 dephosphorylates Akt through an inhibition of HSP90 functions, resulting in the interaction with Akt to induce apoptotic cell death of L1210 cells.