Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI.
Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI.
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DOI：
10.1016/j.bcp.2008.03.017
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发表时间：
2008-06
影响因子：
5.8
通讯作者：
M. Choi;F. Najafi;A. Safa;H. Drexler
中科院分区：
文献类型：
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作者：
M. Choi;F. Najafi;A. Safa;H. Drexler
蛋白酶体抑制剂在体外和体内均表现出有效的抗肿瘤和抗血管生成特性。然而，蛋白酶体抑制剂杀死肿瘤细胞的机制仍然相当难以捉摸，治疗耐药性的分子基础也是如此。为了解决这些问题，我们采用高通量蛋白质印迹程序来分析早幼粒细胞白血病细胞系 HL-60 在用蛋白酶体抑制剂 PSI (Z-Ile-Glu(OtBu)-Ala-Leu-醛) 处理后约 800 个蛋白质的亚蛋白质组的变化，并将所选靶蛋白的变化与两种多重耐药 HL-60 变体的变化相关联。总共有 105 个蛋白质在 PSI 处理后上调超过 1.5 倍，而 79 个蛋白质则下调。在 HL-60 细胞凋亡过程中，caspases-3 和 -8 的激活、Bcl-2 家族成员的调节以及应激信号通路的刺激非常显着。我们还发现了以前未知的受蛋白酶体抑制剂影响的蛋白质丰度的变化。相比之下，两种过度表达MRP1或P-糖蛋白的多重耐药HL-60细胞系对PSI诱导的细胞凋亡有很大抵抗力，并且不能被药物外排泵MK571或PSC833的药理学抑制剂重新敏化。耐药性也与 Bad 的上调无关。因此，多药耐药蛋白、P-糖蛋白和 MRP-1 的过度表达不足以解释 HL-60 细胞对蛋白酶体抑制剂 PSI 治疗的耐药性，这仍然与低水平的 Bax 表达和无法激活 JNK 密切相关。因此，必须考虑获得 PSI 抗性的替代途径。
Proteasome inhibitors display potent anti-neoplastic and anti-angiogenic properties both in vitro and in vivo. The mechanisms, however, by which proteasome inhibitors kill tumor cells are still fairly elusive as is the molecular basis of resistance to treatment. To address these questions, we employed a high-throughput Western blotting procedure to analyze changes in a subproteome of ∼800 proteins in the promyelocytic leukemia cell line HL-60 upon treatment with the proteasome inhibitor PSI (Z-Ile-Glu(OtBu)-Ala-Leu-aldehyde) and correlated the changes of selected target proteins with the changes in two multidrug-resistant HL-60 variants. In total, 105 proteins were upregulated more than 1.5-fold after PSI treatment, while 79 proteins were downregulated. Activation of caspases-3 and -8, modulation of members of the Bcl-2 family as well as stimulation of stress signaling pathways was prominent during HL-60 apoptosis. We also identified changes in the abundance of proteins previously not known to be affected by proteasome inhibitors. In contrast, two multidrug-resistant HL-60 cell lines, overexpressing either MRP1 or P-glycoprotein were largely resistant to PSI-induced apoptosis and could not be resensitized by the pharmacological inhibitors of the drug efflux pumps MK571 or PSC833. Drug resistance was also independent of the upregulation of Bad. Overexpression of multidrug resistance proteins, P-glycoprotein and MRP-1 is thus not sufficient to explain resistance of HL-60 cells to treatment with proteasome inhibitor PSI, which remains more closely related to a low level of Bax expression and to the inability to activate JNK. Alternative routes to the acquisition of resistance to PSI have therefore to be considered.