Ubiquitin B在HDAC抑制剂选择性诱导肿瘤细胞凋亡中的机制研究
摘要
研究背景
组蛋白去乙酰化酶(Histone Deacetylases, HDACs)抑制剂是一类新型的非细胞毒类抗肿瘤药物,对多种肿瘤细胞中具有抑制增殖、诱导凋亡和分化的作用。目前,全球有超过100个关于HDAC抑制剂的临床试验正在进行,并已取得令人振奋的结果,显示了该药良好的临床应用前景。2006年,美国食品与药品管理局(FDA)正式批准第一个HDAC抑制剂——Zolinza胶囊(Vorinostat)上市,用于治疗皮肤T细胞淋巴瘤。然而,关于HDAC抑制剂作用机制的研究还相对落后,目前国内外对HDAC抑制剂广谱、特异性抗肿瘤效应的作用靶点的认识仍不十分明确,近年来,虽然有一些研究报道了HDAC抑制剂相关的效应分子,但并不能在HDAC抑制剂与其选择性杀灭肿瘤细胞效应间建立必然的因果逻辑联系。因此,系统性的筛选HDAC抑制剂的作用靶点,不仅有助于阐明HDAC抑制剂选择性抗肿瘤效应的机制,还对进一步发展肿瘤靶向性药物的研究具有重要意义。
目的
通过构建SMART (Suppression of Mortality by Antisense Rescue Technique)文库,以功能效应为基础筛选HDAC抑制剂的作用靶点,阐明HDAC抑制剂选择性诱导肿瘤细胞凋亡的新机制。
方法
建立Trichostatin A选择性诱导肿瘤细胞凋亡模型,构建Trichostatin A诱导MCF-7细胞反义cDNA文库,筛选凋亡效应基因,结合生物信息学分析,确定相应的靶点进行深入机制研究。
结果
成功构建了Trichostatin A选择性诱导肿瘤细胞反义cDNA文库,经过两轮严格筛选,共获得了24个候选靶点。通过对Ubiquitin B基因进行深入机制研究发现：UbiquitinB在肿瘤细胞中可以被Trichostatin A选择性诱导表达升高,Trichostatin A可以引起线粒体膜上BCL-2家族抗凋亡蛋白BCL-2和MCL-1的泛素化降解,改变线粒体膜电位,启动线粒体凋亡途径,最终导致肿瘤细胞凋亡,反义封闭Ubiquitin B可以抵抗Trichostatin A的凋亡诱导作用。而另一类HDAC抑制剂·——Apicidine,也与TrichostatinA具有相同的作用。此外,Trichostatin A还可以引起白血病细胞K562中融合蛋白BCR/ABL发生泛素化降解。
结论
SMART技术作为一种基于功能效应的高通量筛选方法,可以成为研究凋亡相关机制的有效手段。通过筛选获得的Ubiquitin B基因是HDAC抑制剂选择性诱导肿瘤细胞凋亡的重要靶点,HDAC抑制剂通过特异性诱导肿瘤细胞中Ubiquitin B的表达,激活泛素-蛋白酶体系统,引起线粒体膜上BCL-2家族抗凋亡蛋白BCL-2和MCL-1的泛素化降解,改变线粒体膜电位,启动线粒体凋亡途径,最终导致肿瘤细胞凋亡。
Background Histone deacetylase inhibitors (HDACis) are now emerging as a new class of anticancer agents with potent activity in the inhibition of proliferation and induction of apoptosis and differentiation in a wide spectrum of tumors. At least 12 HDACis are under evaluation in over 100 clinical trials and have produced encouraging therapeutic responses with surprisingly good safety profiles. The clinical potential of HDACis has been well exemplified by the successful development of Vorinostat which was recently approved by the US Food and Drug Administration for treatment of cutaneous T-cell lymphoma. Despite the rapid clinical progress achieved, the mechanism of action of HDACis is not yet well understood. One of the central questions is how these agents selectively kill tumor cells while sparing normal cells. Identification of the critical intracellular targets responsible for the tumor selectivity of HDACis will further improve the design of optimized clinical protocols. More attractively, unraveling the potential "death programs" selectively activated in tumor but not in normal cells will have broader implications for the understanding of tumorigenesis and the design of targeted therapies.
Objective To screen some novel functional proapoptotic genes associated with HDAC inhibitors by suppression of mortality by antisense rescue technique and to explore the possible mechanisms of tumor-selective killing by HDAC inhibitors.
Methods MCF-7 cells treated with 250 nmol/L Trichostatin A for different time points were harvested to construct an antisense cDNA library. HeLa cells were transfected with the library, and were selscted with Trichostatin A and Hygromycin B for 2 weeks. Then surviving colonies were amplified and Hirt DNA were extracted for sequencing.
Results Twenty-four genes were identified, the most significant of which was ubiquitin B (UbB). The expression of UbB was selectively up-regulated by TSA in tumor cells, but not non-malignant cells. Further observation indicated that TSA induced a substantial dissipation of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and proteolytic cleavage of caspases 3/9 in HeLa cells which was apparently mediated by ubiquitylation and the subsequent degradation of mitochondrial membrane proteins including BCL-2 and MCL-1. In contrast, knockdown of UbB expression inhibited the TSA-induced apoptotic cascade by abolishing TSA-induced ubiquitylation and the subsequent degradation of mitochondrial membrane proteins. Furthermore, apicidine, another HDACi, exhibited activity similar to that of TSA. Interestingly, TSA induced UbB-dependent proteasomal degradation of BCR-ABL fusion protein in K562 leukemic cells.
Conclusion Ubiquitin B and Ubb-dependent proteasomal protein degradation play an essential role in HDACi-induced tumor selectivity. The mechanism provides a novel starting point for dissecting the molecular mechanism underlying the tumor selectivity of HDACi.
组蛋白去乙酰化酶(Histone Deacetylases, HDACs)抑制剂是一类新型的非细胞毒类抗肿瘤药物,对多种肿瘤细胞中具有抑制增殖、诱导凋亡和分化的作用。目前,全球有超过100个关于HDAC抑制剂的临床试验正在进行,并已取得令人振奋的结果,显示了该药良好的临床应用前景。2006年,美国食品与药品管理局(FDA)正式批准第一个HDAC抑制剂——Zolinza胶囊(Vorinostat)上市,用于治疗皮肤T细胞淋巴瘤。然而,关于HDAC抑制剂作用机制的研究还相对落后,目前国内外对HDAC抑制剂广谱、特异性抗肿瘤效应的作用靶点的认识仍不十分明确,近年来,虽然有一些研究报道了HDAC抑制剂相关的效应分子,但并不能在HDAC抑制剂与其选择性杀灭肿瘤细胞效应间建立必然的因果逻辑联系。因此,系统性的筛选HDAC抑制剂的作用靶点,不仅有助于阐明HDAC抑制剂选择性抗肿瘤效应的机制,还对进一步发展肿瘤靶向性药物的研究具有重要意义。
目的
通过构建SMART (Suppression of Mortality by Antisense Rescue Technique)文库,以功能效应为基础筛选HDAC抑制剂的作用靶点,阐明HDAC抑制剂选择性诱导肿瘤细胞凋亡的新机制。
方法
建立Trichostatin A选择性诱导肿瘤细胞凋亡模型,构建Trichostatin A诱导MCF-7细胞反义cDNA文库,筛选凋亡效应基因,结合生物信息学分析,确定相应的靶点进行深入机制研究。
结果
成功构建了Trichostatin A选择性诱导肿瘤细胞反义cDNA文库,经过两轮严格筛选,共获得了24个候选靶点。通过对Ubiquitin B基因进行深入机制研究发现：UbiquitinB在肿瘤细胞中可以被Trichostatin A选择性诱导表达升高,Trichostatin A可以引起线粒体膜上BCL-2家族抗凋亡蛋白BCL-2和MCL-1的泛素化降解,改变线粒体膜电位,启动线粒体凋亡途径,最终导致肿瘤细胞凋亡,反义封闭Ubiquitin B可以抵抗Trichostatin A的凋亡诱导作用。而另一类HDAC抑制剂·——Apicidine,也与TrichostatinA具有相同的作用。此外,Trichostatin A还可以引起白血病细胞K562中融合蛋白BCR/ABL发生泛素化降解。
结论
SMART技术作为一种基于功能效应的高通量筛选方法,可以成为研究凋亡相关机制的有效手段。通过筛选获得的Ubiquitin B基因是HDAC抑制剂选择性诱导肿瘤细胞凋亡的重要靶点,HDAC抑制剂通过特异性诱导肿瘤细胞中Ubiquitin B的表达,激活泛素-蛋白酶体系统,引起线粒体膜上BCL-2家族抗凋亡蛋白BCL-2和MCL-1的泛素化降解,改变线粒体膜电位,启动线粒体凋亡途径,最终导致肿瘤细胞凋亡。
Background Histone deacetylase inhibitors (HDACis) are now emerging as a new class of anticancer agents with potent activity in the inhibition of proliferation and induction of apoptosis and differentiation in a wide spectrum of tumors. At least 12 HDACis are under evaluation in over 100 clinical trials and have produced encouraging therapeutic responses with surprisingly good safety profiles. The clinical potential of HDACis has been well exemplified by the successful development of Vorinostat which was recently approved by the US Food and Drug Administration for treatment of cutaneous T-cell lymphoma. Despite the rapid clinical progress achieved, the mechanism of action of HDACis is not yet well understood. One of the central questions is how these agents selectively kill tumor cells while sparing normal cells. Identification of the critical intracellular targets responsible for the tumor selectivity of HDACis will further improve the design of optimized clinical protocols. More attractively, unraveling the potential "death programs" selectively activated in tumor but not in normal cells will have broader implications for the understanding of tumorigenesis and the design of targeted therapies.
Objective To screen some novel functional proapoptotic genes associated with HDAC inhibitors by suppression of mortality by antisense rescue technique and to explore the possible mechanisms of tumor-selective killing by HDAC inhibitors.
Methods MCF-7 cells treated with 250 nmol/L Trichostatin A for different time points were harvested to construct an antisense cDNA library. HeLa cells were transfected with the library, and were selscted with Trichostatin A and Hygromycin B for 2 weeks. Then surviving colonies were amplified and Hirt DNA were extracted for sequencing.
Results Twenty-four genes were identified, the most significant of which was ubiquitin B (UbB). The expression of UbB was selectively up-regulated by TSA in tumor cells, but not non-malignant cells. Further observation indicated that TSA induced a substantial dissipation of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and proteolytic cleavage of caspases 3/9 in HeLa cells which was apparently mediated by ubiquitylation and the subsequent degradation of mitochondrial membrane proteins including BCL-2 and MCL-1. In contrast, knockdown of UbB expression inhibited the TSA-induced apoptotic cascade by abolishing TSA-induced ubiquitylation and the subsequent degradation of mitochondrial membrane proteins. Furthermore, apicidine, another HDACi, exhibited activity similar to that of TSA. Interestingly, TSA induced UbB-dependent proteasomal degradation of BCR-ABL fusion protein in K562 leukemic cells.
Conclusion Ubiquitin B and Ubb-dependent proteasomal protein degradation play an essential role in HDACi-induced tumor selectivity. The mechanism provides a novel starting point for dissecting the molecular mechanism underlying the tumor selectivity of HDACi.
引文
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