Hsp90抑制剂SNX-2112诱导AML细胞分化与凋亡的研究
发布时间：2019-02-09 14:17
【摘要】：急性髓系白血病(Acute myeloid leukemia,AML)是造血干/祖细胞起源的一组异质性克隆性肿瘤,目前临床上对AML病人的标准联合化疗方案存在诸多问题。热休克蛋白(Hsp)90在AML中高表达,在细胞内起到维护癌蛋白稳定的作用,使细胞最终恶性转化为白血病细胞。研究表明Hsp90可作为靶向治疗AML的良好靶点。本项目研究新型Hsp90抑制剂SNX-2112抗人急性骨髓系白血病(AML)细胞的活性。首先,采用CCK8检测SNX-2112对两株AML细胞株增殖的影响;然后通过流式细胞术和western blot检测SNX-2112对AML细胞的细胞周期和凋亡影响;并且通过流式细胞术检测分化相关表面标记物CD11b的表达,western blot和qRT-PCR,软琼脂克隆形成以及瑞氏-吉姆萨染色等方法用于探究SNX-2112对两株细胞分化的影响;最后采用western blot和激光共聚焦来初步的分析和观察SNX-2112作用于AML细胞的作用机制。结果表明,SNX-2112能够显著的抑制AML细胞的增殖,且效果要较经典的Hsp90抑制剂-17-AAG显著,对人正常细胞的细胞毒性较小;SNX-2112能诱导AML细胞的G2/M期阻滞,且下调周期蛋白A的表达,同时SNX-2112能诱导AML细胞发生凋亡,引起凋亡相关蛋白caspase3等的激活;此外SNX-2112可诱导AML细胞的分化,抑制原癌基因C-Myc的表达,并上调分化相关转录因子PU.1、C/EBPα的表达;机制研究表明,SNX-2112抑制了Hsp90的客户蛋白IKK、Akt的表达,并抑制相关下游信号通路的激活。结论:本研究表明了SNX-2112体外诱导AML周期阻滞、凋亡以及分化,具有抗急性髓性白血病的潜在效果,其作用机制可能是通过抑制PI3K/NF-κB信号通路的激活,下调c-Myc的表达,激活分化相关转录因子的表达。本研究为SNX-2112治疗急性髓性白血病奠定了研究基础,为SNX-2112的临床前应用提供理论研究。
[Abstract]:Acute myeloid leukemia (Acute myeloid leukemia,AML) is a heterogeneous clonal tumor of hematopoietic stem / progenitor cell origin. At present, there are many problems in the standard combination chemotherapy regimen for AML patients. Heat shock protein (Hsp) 90 (HSP90) is highly expressed in AML and plays a role in maintaining the stability of oncoprotein in the cells, which leads to the malignant transformation of the cells into leukemia cells. Studies have shown that Hsp90 can be used as a good target for targeted treatment of AML. The aim of this study was to investigate the activity of SNX-2112, a novel Hsp90 inhibitor, against human acute myeloid leukemia (AML) cells. Firstly, the effects of SNX-2112 on the proliferation of two AML cell lines were detected by CCK8, then the effects of SNX-2112 on the cell cycle and apoptosis of AML cells were detected by flow cytometry and western blot. The expression of CD11b, western blot and qRT-PCR, soft Agar clone formation were detected by flow cytometry, and the effects of SNX-2112 on the differentiation of two cell lines were investigated by Ricker-Gimsa staining. Finally, western blot and laser confocal focus were used to analyze and observe the mechanism of SNX-2112 acting on AML cells. The results showed that SNX-2112 could significantly inhibit the proliferation of AML cells, and the effect was more significant than that of classical Hsp90 inhibitor-17-AAG, and the cytotoxicity to human normal cells was less. SNX-2112 could induce G _ 2 / M arrest and down-regulate the expression of cyclin A in AML cells, and SNX-2112 could induce apoptosis of AML cells and induce the activation of apoptosis-related protein caspase3. In addition, SNX-2112 could induce the differentiation of AML cells, inhibit the expression of proto-oncogene C-Myc and up-regulate the expression of differentiation related transcription factor PU.1,C/EBP 伪. SNX-2112 inhibits the expression of Hsp90 client protein IKK,Akt and inhibits the activation of related downstream signaling pathways. Conclusion: this study suggests that SNX-2112 induces AML cycle arrest, apoptosis and differentiation in vitro, which has the potential effect of anti-acute myeloid leukemia, and its mechanism may be by inhibiting the activation of PI3K/NF- 魏 B signaling pathway. The expression of c-Myc was down-regulated and the expression of differentiation related transcription factors was activated. This study lays a foundation for the treatment of acute myeloid leukemia with SNX-2112 and provides a theoretical study for the preclinical application of SNX-2112.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
[Abstract]:Acute myeloid leukemia (Acute myeloid leukemia,AML) is a heterogeneous clonal tumor of hematopoietic stem / progenitor cell origin. At present, there are many problems in the standard combination chemotherapy regimen for AML patients. Heat shock protein (Hsp) 90 (HSP90) is highly expressed in AML and plays a role in maintaining the stability of oncoprotein in the cells, which leads to the malignant transformation of the cells into leukemia cells. Studies have shown that Hsp90 can be used as a good target for targeted treatment of AML. The aim of this study was to investigate the activity of SNX-2112, a novel Hsp90 inhibitor, against human acute myeloid leukemia (AML) cells. Firstly, the effects of SNX-2112 on the proliferation of two AML cell lines were detected by CCK8, then the effects of SNX-2112 on the cell cycle and apoptosis of AML cells were detected by flow cytometry and western blot. The expression of CD11b, western blot and qRT-PCR, soft Agar clone formation were detected by flow cytometry, and the effects of SNX-2112 on the differentiation of two cell lines were investigated by Ricker-Gimsa staining. Finally, western blot and laser confocal focus were used to analyze and observe the mechanism of SNX-2112 acting on AML cells. The results showed that SNX-2112 could significantly inhibit the proliferation of AML cells, and the effect was more significant than that of classical Hsp90 inhibitor-17-AAG, and the cytotoxicity to human normal cells was less. SNX-2112 could induce G _ 2 / M arrest and down-regulate the expression of cyclin A in AML cells, and SNX-2112 could induce apoptosis of AML cells and induce the activation of apoptosis-related protein caspase3. In addition, SNX-2112 could induce the differentiation of AML cells, inhibit the expression of proto-oncogene C-Myc and up-regulate the expression of differentiation related transcription factor PU.1,C/EBP 伪. SNX-2112 inhibits the expression of Hsp90 client protein IKK,Akt and inhibits the activation of related downstream signaling pathways. Conclusion: this study suggests that SNX-2112 induces AML cycle arrest, apoptosis and differentiation in vitro, which has the potential effect of anti-acute myeloid leukemia, and its mechanism may be by inhibiting the activation of PI3K/NF- 魏 B signaling pathway. The expression of c-Myc was down-regulated and the expression of differentiation related transcription factors was activated. This study lays a foundation for the treatment of acute myeloid leukemia with SNX-2112 and provides a theoretical study for the preclinical application of SNX-2112.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
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