Nickel pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via both Bcr/Abl-dependent and Bcr/Abl-independent mechanisms.
Nickel pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via both Bcr/Abl-dependent and Bcr/Abl-independent mechanisms.
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吡啶硫酮镍通过 Bcr/Abl 依赖性和 Bcr/Abl 独立机制诱导对伊马替尼耐药的慢性粒细胞白血病细胞凋亡
DOI：
10.1186/s13045-016-0359-x
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发表时间：
2016-11-25
影响因子：
28.5
通讯作者：
Liu J
中科院分区：
文献类型：
--
作者：
Lan X;Zhao C;Chen X;Zhang P;Zang D;Wu J;Chen J;Long H;Yang L;Huang H;Carter BZ;Wang X;Shi X;Liu J
获得性伊马替尼（IM）耐药通常以Bcr-Abl突变为特征，该突变影响慢性髓性白血病（CML）患者的IM结合和激酶抑制。Bcr-Abl-T315I突变是获得性耐药的主要机制。因此，迫切需要寻找其他方法和靶向策略来克服IM耐药性。我们最近报道了镍吡硫酮（NiPT）通过靶向19S蛋白酶体相关的去泛素酶（UCHL5和USP14）有效抑制泛素蛋白酶体系统，而不影响20S蛋白酶体。在本研究中，我们研究了一种新型蛋白酶体去泛素酶抑制剂NiPT对携带Bcr-Abl- t315i或野生型Bcr-Abl的CML细胞存活或凋亡的影响。方法采用MTS法和台色蓝法检测KBM5、KBM5R、K562、BaF3-p210-WT、BaF3-p210-T315I细胞及NiPT处理的CML患者骨髓样本的细胞活力。采用Annexin V-FITC/PI和罗丹明-123染色检测CML细胞凋亡，荧光显微镜和流式细胞术检测细胞凋亡相关蛋白。采用western blotting和real-time PCR分析CML细胞中Bcr-Abl的表达水平。用特异性荧光底物测定20S蛋白酶体肽酶活性。利用活性位点定向标记蛋白酶体DUBs以及USP14的磷酸化来评估NiPT对DUBs活性的抑制作用。分析小鼠KBM5和KBM5R细胞异种移植模型，并通过western blots和/或免疫组织学分析检测bcr - abl相关蛋白和肿瘤组织中与增殖、分化和粘附相关的蛋白生物标志物。结果snipt诱导CML细胞凋亡，抑制裸鼠抗im Bcr-Abl-T315I异种移植物的生长。从机制上讲，NiPT诱导Bcr-Abl蛋白的减少，这与Bcr-Abl转录的下调以及活化的半胱天酶对Bcr-Abl蛋白的切割有关。nipt诱导的泛素蛋白酶体系统抑制在im耐药和im敏感的CML细胞中诱导caspase激活，并且caspase激活是nipt诱导的Bcr-Abl下调和凋亡细胞死亡所必需的。结论这些发现支持NiPT可以通过bcr - abl依赖和bcr - abl非依赖机制克服IM耐药，为CML治疗提供了潜在的新选择。
BackgroundAcquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.MethodsCell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients’ bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses.ResultsNiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death.ConclusionsThese findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.
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影响因子：
6.4
作者：
Morinaga, Koji;Yamauchi, Takahiro;Ueda, Takanori
通讯作者：
Ueda, Takanori
影响因子：
11.4
作者：
通讯作者：
--
影响因子：
9
作者：
通讯作者：
--
影响因子：
6
作者：
Hisatomi, Toshio;Nakao, Shintaro;Miller, Joan W.
通讯作者：
Miller, Joan W.
影响因子：
8
作者：
Danial, NN;Rothman, P
通讯作者：
Rothman, P