NSC606985诱导U937细胞凋亡的差异蛋白质组学研究
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摘要
急性髓细胞性白血病(acute myeloid leukemia,AML)是一种异质性恶性血液肿瘤。过去二十年间,人们对于AML在生物学、分子和细胞遗传学等方面的认识取得了巨大进展。与此同时,对于AML病人的治疗也取得了长足进步。然而,大部分AML病人依然不可治愈。因此,开发新的药物对于AML病人来说非常重要。作为一种新型的抗癌药物,喜树碱具有广阔的发展前景。近两年,宋满根博士等在国际上首先研究了一种称为NSC606985的新型喜树碱衍生物对白血病细胞的体内外效应。已有的实验结果显示,在体外,NSC606985能够以极低浓度(纳摩尔浓度水平),通过水解激活PKCδ诱导急性早幼粒细胞性白血病NB4细胞和急性单核细胞性白血病U937细胞凋亡。
为了深入研究NSC606985诱导AML细胞凋亡的分子机制,本文用差异蛋白质组学技术分析50nM NSC606985处理U937细胞36小时后差异表达的蛋白质,结果确定有28种蛋白的表达发生改变。这些差异表达蛋白质的功能涉及细胞骨架、能量代谢、氧化应激、信号转导、RNA代谢、DNA代谢、蛋白质代谢、分化、分子伴侣等。
骨架蛋白在这些差异表达的蛋白中占相当的比例。本文用western blot和二维凝胶电泳+western blot的方法验证骨架蛋白在二维凝胶电泳胶图上的变化。这部分结果显示:β-tubulin、β-actin和transgelin-2的总表达量没有改变;对照组中,这些骨架蛋白有多个等电点,但经NSC606985处理后(实验组),碱性端的等电点都往酸性端偏移,实验组中这些骨架蛋白只有一个等电点(相当于对照组中最偏酸的等电点);鉴于磷酸化修饰是最常见的翻译后修饰,且能使等电点左移,推测这些骨架蛋白在NSC606985诱导U937细胞凋亡过程中发生磷酸化修饰;用碱性磷酸酶处理实验组蛋白,后者有绝大部分往碱性端偏移,符合磷酸化蛋白的特点,证实经NSC606985处理后,这三种蛋白发生磷酸化修饰;鉴于前期工作表明PKCδ水解激活在NSC606985诱导U937细胞凋亡过程中的重要地位和这三种蛋白都具有PKCδ的识别模序(经scansite分析软件预测),观察PKCδ特异抑制剂rottlerin预处理是否能抑制NSC606985诱导的骨架蛋白磷酸化修饰,结果发现rottlerin能抑制β-tubulin和β-actin磷酸化,不能抑制transgelin-2磷酸化;提示β-tubulin和β-actin的磷酸化在PKCδ剪切活化的下游,而transgelin-2磷酸化不是PKCδ剪切活化的下游事件。
抗氧化蛋白peroxiredoxin 1和thioredoxin都明显上调,提示活性氧生成增加。为明确活性氧在NSC606985诱导U937细胞凋亡过程中的变化、机制和意义,本文用DCF法检测活性氧的含量;结果证实NSC606985处理24h,U937细胞活性氧增多;并且这种变化处于PKCδ剪切活化的下游,用rottlerin可抑制这种改变;NAC虽然能完全清除NSC606985作用下的U937细胞中增多的活性氧,却对U937细胞的凋亡无影响。
本研究结果显示了蛋白质组学方法在高通量地筛选凋亡相关蛋白质方面的巨大潜力,以及在探测蛋白翻译后修饰方面的独特优势。上述结果可为进一步探讨NSC606985诱导AML细胞凋亡的机制提供新的线索。
Acute myeloid leukemia(AML)is a heterogenous group of hematopoietic malignancies.Significant advances in understanding the biologic,molecular,and cytogenetic aspects of this malignancy have been achieved in the past 20 years.Meanwhile,substantial progress is also made in the treatment of patients with AML.However,most patients remain incurable.So development of new drugs for treatment of AML is very important.As a promising new class of anticancer drugs, camptothecins have advanced to the forefront of several areas of therapetitic and developmental chemotherapy.The last two years,our task group has studied the potential anti-leukemic effectiveness of NSC606985,a new class of camptothecin analogs,in vitro and in vivo.
In order to explore further the molecular mechanisms of NSC606985 inducing AML cell apoptosis,the technology of differential proteomics has been used to analyze the differential proteins expression in U937 cells treated with(36 hours)or without 50nM NSC606985.As a result,we identified 28 proteins that were significantly deregulated by NSC606985. These proteins were mainly involved in cytoskeleton,RNA metabolism, DNA metabolism,protein metabolism,carbohydrate metabolism,energy metabolism,signal transduction,stress response,oxidation and reduction, differentiation and others.
Cytoskeleton proteins occupied a great proportion of these differential proteins.We used western blot and two-dimensional gel electrophoresis plus western blot to validate the changes of cytoskeleton proteins in 2D-GE.The results revealed that the protein expression ofβ-tubulin,β-actin and transgelin-2 did not be changed.In tthe control group,these cytoskeleton proteins had several isoelectric points.After treated by NSC606985(the experimental group),the isoelectric points in the basic end transferred to the acid end and each of these cytoskeleton proteins had only one isoelectric point in the experimental group(it is equal to the acidest point in the control group).Because the phosphorylation is the commonest post-translational modification and it can induce isoelectric points to move to left,we presumed that these cytoskeleton proteins was been phosphorylated.Treated the total proteins of the experimental group by alkaline phosphotase,the great mass ofβ-tubulin,β-actin and transgelin-2 transferred to the basic end.It was according with the characteristic of phosphorylated proteins and it was proved that these three cytoskeleton proteins were been phosphorylated in the apoptotic process which was induced by NSC606985.Because the preliminary work proved that active cleaved PKCδplayed a key role in the apoptotic process and all of these three cytoskeleton proteins had recognizing motif (forcasting by scansite software),we observed that whether rottlerin(a specific inhibitor of PKC)pretreatment could inhibit the phosphorylationof these three cytoskeleton proteins.The results showed that rottlerin could totally inhibited the phosphorylation ofβ-tubulin andβ-actin,but it was unable to inhibited the phosphorylation of transgelin-2.It was suggested that the phosphorylation ofβ-tubulin andβ-actin were downstream to PKCδactivation,the phosphorylation of transgelin-2 was not one of the events which were,downstream to PKCδactivation.
Peroxiredoxin 1 and thioredoxin,two antioxidant proteins,were increased significantly.It was suggested that ROS was produced.To confirm the change,mechanism and significance of ROS in NSC606985 inducing apoptotsis,we mensurated the content of ROS in the cells with DCF.The results showed that ROS of U937 cells was increased after NSC606985 treating 24 hours.This event was downstream to PKCδactivation because it was totally inhibited by rottlerin.Though NAC could eliminate ROS of U937 cells,it had no influence on NSC606985 inducing apoptotsis.
In summary,this study showed that the proteomic technique was a powerful method to identify apoptotic related proteins,and it has unique superiority to explore post-translation modifications of proteins.The results of the present study are worthing further investigation to understand the mechanisms ofNSC606985 inducing apoptotsis.
为了深入研究NSC606985诱导AML细胞凋亡的分子机制,本文用差异蛋白质组学技术分析50nM NSC606985处理U937细胞36小时后差异表达的蛋白质,结果确定有28种蛋白的表达发生改变。这些差异表达蛋白质的功能涉及细胞骨架、能量代谢、氧化应激、信号转导、RNA代谢、DNA代谢、蛋白质代谢、分化、分子伴侣等。
骨架蛋白在这些差异表达的蛋白中占相当的比例。本文用western blot和二维凝胶电泳+western blot的方法验证骨架蛋白在二维凝胶电泳胶图上的变化。这部分结果显示:β-tubulin、β-actin和transgelin-2的总表达量没有改变;对照组中,这些骨架蛋白有多个等电点,但经NSC606985处理后(实验组),碱性端的等电点都往酸性端偏移,实验组中这些骨架蛋白只有一个等电点(相当于对照组中最偏酸的等电点);鉴于磷酸化修饰是最常见的翻译后修饰,且能使等电点左移,推测这些骨架蛋白在NSC606985诱导U937细胞凋亡过程中发生磷酸化修饰;用碱性磷酸酶处理实验组蛋白,后者有绝大部分往碱性端偏移,符合磷酸化蛋白的特点,证实经NSC606985处理后,这三种蛋白发生磷酸化修饰;鉴于前期工作表明PKCδ水解激活在NSC606985诱导U937细胞凋亡过程中的重要地位和这三种蛋白都具有PKCδ的识别模序(经scansite分析软件预测),观察PKCδ特异抑制剂rottlerin预处理是否能抑制NSC606985诱导的骨架蛋白磷酸化修饰,结果发现rottlerin能抑制β-tubulin和β-actin磷酸化,不能抑制transgelin-2磷酸化;提示β-tubulin和β-actin的磷酸化在PKCδ剪切活化的下游,而transgelin-2磷酸化不是PKCδ剪切活化的下游事件。
抗氧化蛋白peroxiredoxin 1和thioredoxin都明显上调,提示活性氧生成增加。为明确活性氧在NSC606985诱导U937细胞凋亡过程中的变化、机制和意义,本文用DCF法检测活性氧的含量;结果证实NSC606985处理24h,U937细胞活性氧增多;并且这种变化处于PKCδ剪切活化的下游,用rottlerin可抑制这种改变;NAC虽然能完全清除NSC606985作用下的U937细胞中增多的活性氧,却对U937细胞的凋亡无影响。
本研究结果显示了蛋白质组学方法在高通量地筛选凋亡相关蛋白质方面的巨大潜力,以及在探测蛋白翻译后修饰方面的独特优势。上述结果可为进一步探讨NSC606985诱导AML细胞凋亡的机制提供新的线索。
Acute myeloid leukemia(AML)is a heterogenous group of hematopoietic malignancies.Significant advances in understanding the biologic,molecular,and cytogenetic aspects of this malignancy have been achieved in the past 20 years.Meanwhile,substantial progress is also made in the treatment of patients with AML.However,most patients remain incurable.So development of new drugs for treatment of AML is very important.As a promising new class of anticancer drugs, camptothecins have advanced to the forefront of several areas of therapetitic and developmental chemotherapy.The last two years,our task group has studied the potential anti-leukemic effectiveness of NSC606985,a new class of camptothecin analogs,in vitro and in vivo.
In order to explore further the molecular mechanisms of NSC606985 inducing AML cell apoptosis,the technology of differential proteomics has been used to analyze the differential proteins expression in U937 cells treated with(36 hours)or without 50nM NSC606985.As a result,we identified 28 proteins that were significantly deregulated by NSC606985. These proteins were mainly involved in cytoskeleton,RNA metabolism, DNA metabolism,protein metabolism,carbohydrate metabolism,energy metabolism,signal transduction,stress response,oxidation and reduction, differentiation and others.
Cytoskeleton proteins occupied a great proportion of these differential proteins.We used western blot and two-dimensional gel electrophoresis plus western blot to validate the changes of cytoskeleton proteins in 2D-GE.The results revealed that the protein expression ofβ-tubulin,β-actin and transgelin-2 did not be changed.In tthe control group,these cytoskeleton proteins had several isoelectric points.After treated by NSC606985(the experimental group),the isoelectric points in the basic end transferred to the acid end and each of these cytoskeleton proteins had only one isoelectric point in the experimental group(it is equal to the acidest point in the control group).Because the phosphorylation is the commonest post-translational modification and it can induce isoelectric points to move to left,we presumed that these cytoskeleton proteins was been phosphorylated.Treated the total proteins of the experimental group by alkaline phosphotase,the great mass ofβ-tubulin,β-actin and transgelin-2 transferred to the basic end.It was according with the characteristic of phosphorylated proteins and it was proved that these three cytoskeleton proteins were been phosphorylated in the apoptotic process which was induced by NSC606985.Because the preliminary work proved that active cleaved PKCδplayed a key role in the apoptotic process and all of these three cytoskeleton proteins had recognizing motif (forcasting by scansite software),we observed that whether rottlerin(a specific inhibitor of PKC)pretreatment could inhibit the phosphorylationof these three cytoskeleton proteins.The results showed that rottlerin could totally inhibited the phosphorylation ofβ-tubulin andβ-actin,but it was unable to inhibited the phosphorylation of transgelin-2.It was suggested that the phosphorylation ofβ-tubulin andβ-actin were downstream to PKCδactivation,the phosphorylation of transgelin-2 was not one of the events which were,downstream to PKCδactivation.
Peroxiredoxin 1 and thioredoxin,two antioxidant proteins,were increased significantly.It was suggested that ROS was produced.To confirm the change,mechanism and significance of ROS in NSC606985 inducing apoptotsis,we mensurated the content of ROS in the cells with DCF.The results showed that ROS of U937 cells was increased after NSC606985 treating 24 hours.This event was downstream to PKCδactivation because it was totally inhibited by rottlerin.Though NAC could eliminate ROS of U937 cells,it had no influence on NSC606985 inducing apoptotsis.
In summary,this study showed that the proteomic technique was a powerful method to identify apoptotic related proteins,and it has unique superiority to explore post-translation modifications of proteins.The results of the present study are worthing further investigation to understand the mechanisms ofNSC606985 inducing apoptotsis.
引文
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