HSP90抑制剂AUY922逆转氟维司群在乳腺癌细胞中的反馈性激活效应

发布时间：2018-03-07 05:34

本文选题：氟维司群　切入点：反馈性激活效应　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及目的:内分泌治疗作为乳腺癌辅助治疗重要的治疗策略,能够明显改善激素受体阳性乳腺癌患者的预后[1],氟维司群(Fulvestrant)是常用的内分泌药物[2]。蛋白组学分析发现,长期雌激素剥夺细胞(long-term estrogen deprivation cells,LTED cells)的Erb B家族膜受体介导的PI3K/m TOR信号通路活性增强。证据表明,在雌激素治疗耐药的乳腺癌患者中存在PI3K/AKT信号通路的激活[3-7],从而在细胞的生长增殖中发挥重要作用。然而,从目前的研究结果来看,在雌激素耐药的细胞中,单独应用EGFR抑制剂吉非替尼并不能抑制AKT的反馈性激活[8]。目前的信号调控网络相关研究表明肿瘤细胞生长增殖及耐药等过程十分复杂,单纯的干扰或者抑制某一特定的靶点对于抑制细胞增殖杀灭癌细胞的作用可能十分有限。因此,如果能够同时抑制肿瘤细胞中生长相关的多个关键蛋白对于抑制杀灭肿瘤似乎更为有效。HSP90是细胞内广谱表达的伴侣蛋白,许多肿瘤细胞恶性表型的相关蛋白都依赖于HSP90的折叠功能从而发挥其生物学效应。临床相关资料表明,肿瘤组织中HSP90的表达相比较于癌旁正常组织要高2-3倍,而且与临床不良预后明显相关[9,10]。研究发现,HSP90伴侣蛋白的高表达与乳腺癌的不良预后具有密切相关,且是影响乳腺癌预后的独立因素。HSP90的高表达与HER2阳性、ER阳性、肿瘤较大、组织学分级较高以及淋巴结转移具有明显相关性[11]。研究表明,HSP90能够增强乳腺癌细胞对于激素治疗的抵抗性,与激素治疗的耐药性相关[12]。AUY922是第二代HSP90抑制剂,它可以竞争性结合于HSP90的ATP结合位点,从而抑制HSP90的生物学活性,导致细胞内很多关键蛋白不能被正常折叠从而失去生物学功能。由于氟维司群的反馈性激活效应是乳腺癌细胞发生耐药的重要原因,而且多个通路及多种通路相关蛋白参与了这一过程,然而,目前仍然没有关于AUY922是否能够逆转这一反馈性激活效应的相关研究。我们推测,AUY922可能能够通过抑制HSP90的活性,从而抑制Erb B家族及PI3K/AKT及ERK信号通路,逆转氟维司群带来的反馈性激活效应,为联合用药相关临床试验提供新的基础理论支持。研究方法:1.利用western blotting技术检测了氟维司群单药作用对于MCF-7和T47D细胞的ER水平的影响,以及短时间内对于Erb B家族蛋白的反馈性激活,进而进一步活化下游的PI3K/AKT和ERK信号通路。并且利用RT-PCR检测了Erb B家族的m RNA水平在氟维司群处理后的变化。2.利用western blotting技术检测了AUY922单药对于PI3K/AKT和ERK信号通路的抑制作用。3.Western blotting技术检测在氟维司群处理的基础上联合AUY922能够成功抑制并逆转氟维司群带来的Erb B家族蛋白反馈性激活效应,进而抑制下游增殖相关PI3K/AKT和ERK信号通路。4.利用MTT实验、平板克隆实验检测双药联合对于细胞生长的抑制作用,利用流式细胞技术检测双药联合作用对于细胞周期的阻滞。5.人源肿瘤组织的离体验证实验中用western blotting技术进一步证实了AUY922对于氟维司群的反馈激活效应的逆转作用。研究结果:1.氟维司群能够在48小时内降低MCF-7和T47D细胞中的ER的水平。2.氟维司群在降低MCF-7和T47D细胞ER水平的同时能反馈性激活Erb B家族蛋白,同时p-EGFR、p-HER2、p-HER3及p-HER4的表达量增加,表明其功能活化。这一反馈性激活作用在氟维司群作用的短时间(48小时)内引起的。3.AUY922单药能够有效抑制MCF-7和T47D中的HSP90活性,进一步检测发现氟维司群对于HSP90活性并无影响。AUY922能够有效抑制PI3K/AKT和ERK信号通路的活性。4.在氟维司群作用24小时之后的基础上,进一步联合AUY922能够成功抑制并逆转氟维司群引起的Erb B家族蛋白的反馈性激活效应,并且有效抑制了下游增殖相关PI3K/AKT和ERK信号通路的活性。5.在细胞功能学实验中,双药联合能够有效抑制细胞生长,在平板克隆形成实验中发现,双药联合能够更加有效抑制细胞克隆形成。细胞周期检测中发现,在ER+乳腺癌细胞中双药联合能够更有效的阻滞细胞周期。6.人源肿瘤组织的体外验证实验证明,氟维司群的治疗确实能够反馈性激活Erb B家族蛋白,引起下游PI3K/AKT和ERK信号通路的活化,联合应用AUY922能够成功逆转这种反馈性激活效应。进一步证实了细胞系中的实验结论。研究结论:本研究发现并证实了Erb B受体家族及其下游增殖相关通路PI3K/AKT和ERK信号通路在氟维司群治疗后能够发生反馈性激活,而HSP90抑制剂AUY922能够多靶点抑制这些反馈性活化的蛋白活性,同时,这一实验结果在乳腺癌细胞系及新鲜乳腺癌离体标本中均得到了证实。
[Abstract]:Background and objective: endocrine therapy as adjuvant therapy for breast cancer treatment strategies that can significantly improve the prognosis of [1] patients with hormone receptor positive breast cancer, fulvestrant (Fulvestrant) is a group of common endocrine drugs [2]. protein analysis found that long-term estrogen deprived cells (long-term estrogen deprivation cells, LTED cells) the enhanced Erb B family of receptor mediated PI3K/m TOR signaling pathway activity. There is evidence that the activation of [3-7] PI3K/AKT signaling pathway in estrogen treatment of drug-resistant breast cancer patients, and thus play an important role in cell growth and proliferation. However, the results from the current study, the estrogen resistant cells, alone the application of EGFR inhibitor gefitinib and activation of signal regulatory networks [8]. current related research shows that AKT feedback cannot inhibit tumor cell growth and proliferation resistance The process is very complicated, simple interference or inhibition of a specific target for the inhibition of cell proliferation to kill cancer cells may be very limited. Therefore, if can also inhibit tumor cell growth in a number of key proteins for inhibiting the growth of tumor.HSP90 expression seems to be more effective broad-spectrum intracellular chaperone protein folding. Proteins related to many malignant phenotype of tumor cells are dependent on HSP90 to exert its biological effects. The related clinical data showed that the expression of HSP90 in tumor tissue compared with adjacent normal tissue was 2-3 times higher, but with a poor clinical outcome was significantly related to the [9,10]. study found that high expression and poor prognosis of breast cancer HSP90 chaperone the effect of.HSP90 is closely related to, and independent factors of prognosis of breast cancer with high expression of HER2 positive, ER positive, larger tumor size, histology High grade and lymph node metastasis of [11]. has significant correlation showed that HSP90 can enhance the breast cancer cells for resistance to hormone therapy, and drug resistance related [12].AUY922 hormone therapy is the second generation of HSP90 inhibitors, which can compete with HSP90 in ATP binding sites, thereby inhibiting the biological activity of HSP90, resulting in many of the key protein in cells can not be folded to lose normal biological function. Due to the feedback activation effect of fulvestrant is an important cause of breast cancer cells and drug resistance, multiple pathways and multiple pathway related proteins involved in this process, however, there is still no about whether AUY922 can reverse the feedback activation effect of related research. We speculate that AUY922 may be able to inhibit the activity of HSP90, thereby inhibiting the Erb B family and PI3K/AKT and ERK signaling pathway, the reversal of fluoride The activation effects brought by our group dimension feedback, provide relevant clinical trials support new theory basis for combination therapy. Methods: 1. using Western blotting technique to detect effects of fulvestrant monotherapy for MCF-7 and T47D cells at the ER level, and within a short period of time for the Erb feedback activation of B family proteins further, the activation of PI3K/AKT and downstream of the ERK pathway. And RT-PCR is used to detect the changes of the.2. Erb B family m RNA level in fulvestrant treatment after the detection of AUY922 single drug for the detection of PI3K/AKT and ERK signal pathway of the inhibitory effect of.3.Western Blotting Technology Based on fulvestrant treatment combined with AUY922 can inhibit and reverse fulvestrant brought Erb B protein family feedback activation effect using Western Blotting Technology, and then inhibit the downstream proliferation related PI3K/AKT signal pathways of ERK and.4. Experiments using MTT assay colony, the two drug combination for inhibition of cell growth, tissue block.5. for human tumor cell cycle by flow cytometry double medicine combined effect of in vitro experiments using Western blotting technology further confirmed that AUY922 for fulvestrant feedback activation effect of reversal effect results: 1.. Fulvestrant can reduce MCF-7 and T47D cells in ER within 48 hours of the level of.2. of fulvestrant in MCF-7 and T47D cells and reduce the level of ER can feedback activation of Erb B protein family, and p-EGFR, p-HER2, expression of p-HER3 and p-HER4 increase, show that its functions activation. This feedback activation of fulvestrant in short time (48 hours) the role of.3.AUY922 in the single drug can effectively inhibit MCF-7 and T47D activity in HSP90, further detection of fulvestrant For does not affect the activity of HSP90.AUY922 can effectively inhibit PI3K/AKT and ERK pathway in.4. based fulvestrant 24 hours later, further combined with AUY922 can successfully inhibit and reverse fulvestrant feedback activation effect induced by Erb B protein family, and can inhibit the activation of.5. and downstream proliferation related PI3K/AKT and the ERK signaling pathway in cell function experiments, two drug combination can effectively inhibit the cell growth and colony formation in the two drug combination can effectively inhibit the formation of cell clones. Found that cell cycle detection, in ER+ breast cancer cells by two drug combination can prove the in vitro experiments of cell cycle arrest.6. in human tumor tissue more efficiently, the treatment of fulvestrant can feedback activation of Erb B protein family, caused by the downstream PI3K/AKT and ERK signaling pathway The activation of the combined application of AUY922 can successfully reverse the feedback activation effect. Experimental results further confirmed the cell line. Conclusion: This study found and confirmed the Erb B receptor and its downstream pathways related to PI3K/AKT proliferation and ERK signaling pathways occur feedback activation in the treatment of fulvestrant can, HSP90 inhibitor AUY922 can these feedback target inhibition activated protein activity, at the same time, this results in breast cancer cell lines and fresh breast cancer specimens were confirmed.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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