摘要
研究背景
虽然在过去的几十年中,有关非霍奇金淋巴瘤(non-Hodgkin's lymphoma,NHL)的治疗策略有了很多改进,仍旧有40%～70%的进展期和高分级的NHL患者不能达到长期无病生存(long-term disease-free survival,DFS),并且对于低分级的NHL,目前还没有比较公认的合适的治疗策略。来源于人Burkitt淋巴瘤的Raji细胞是具有典型多药耐药特征的NHL细胞系,其p53基因突变率发生率高,且高表达BCL-2蛋白,是研究NHL耐药及凋亡的理想模型。
化学治疗是NHL治疗方案的重要组成部分,阿霉素(Adiramycin,ADR)作为一个经典的蒽环类抗生素被广泛应用于NHL的联合化疗方案中如CHOP(环磷酰胺+阿霉素+长春新碱+强的松)。然而,相当一部分的NHL患者对于阿霉素的化学耐药性和剂量相关的心脏毒性是导致治疗失败的重要原因。阿霉素作用于肿瘤细胞的机制被证实为诱导凋亡及导致DNA损伤修复缺陷,此外,由阿霉素导致的活性氧簇(reactive oxygen species,ROS)的释放和肌原纤维变性也是与阿霉素剂量相关的心脏毒性的主要原因。由于线粒体在凋亡、DNA损伤修复及ROS的释放中的重要调控作用,因此被认为与阿霉素的作用机制密切相关。此外,随着肿瘤化疗药物的广泛应用,肿瘤的耐药性问题也越来越突出,已成为肿瘤化疗失败的主要因素之一。肿瘤细胞的多药耐药(multidrug-resistance,MDR)是细胞耐药的常见方式,MDR产生的机制可能有:药物的外排增加和亚细胞分布改变、药物作用的靶标如拓扑异构酶的改变、细胞的损伤修复能力增强、抗凋亡相关通路的改变、细胞增殖速率变化,以及体内药代动力学因素等一系列的问题,这一系列的原因也与线粒体的功能密切相关。
此外,NHL是一类放射敏感性肿瘤,放射治疗(radiation therapy,RT)可单独用于根治Ⅰ期和Ⅱ期的惰性NHL患者,对于进展期和侵袭性NHLs,RT常和化学治疗联合应用。放射治疗的目的是诱导细胞凋亡以最终达到增殖性细胞死亡(proliferative cell death,PCD,也称为“secondary apoptosis”)及导致不可逆性DNA损伤。对比于其他实体瘤,虽然NHL细胞对放射治疗更敏感,但是由于细胞对于射线的应答机制很复杂,缺乏完全且有力的凋亡启动及维持可能是导致放射抵抗的重要原因,也是制约对NHL达到理想治疗目的的一大障碍。由于线粒体在细胞凋亡中的重要作用,因此也与放射抵抗机制密切相关。
线粒体是普遍存在于真核细胞中的一种重要的细胞器,参与很多重要的细胞过程,除了在生物合成(例如血红素、核酸和氨基酸)和降解(例如尿素循环)中的重要作用外,还参与包括ATP的产生、脂肪酸代谢、细胞内钙离子稳态的调节、以及细胞信号传导和凋亡等过程。近年来,已经证实线粒体可以通过释放一些介质如细胞色素C和凋亡诱导因子(apoptosis-inducing factor,AIF)等而启动凋亡,这些介质激活caspase家族蛋白酶而导致凋亡。线粒体的主要化学成分是蛋白质和脂类,其中蛋白质占干重的65%～70%,脂类占25-30%。线粒体也是细胞内含酶最多的细胞器,约有120余种,其中,外膜中含有单胺氧化酶以及参与糖和脂肪代谢的各种转移酶,内膜中富含执行呼吸链氧化还原反应的酶系,基质中则含有大量的多种酶的复合体。据推测,人类线粒体大约包含2000-2500种蛋白质或多肽,除了其中的13个蛋白复合体由线粒体基因组(mtDNA)编码外,其余都由核基因组(ntDNA)编码,包括大多数的参与线粒体氧化磷酸化(OXPHOS)的蛋白酶、代谢酶、DNA和RNA聚合酶、核糖体蛋白、mtDNA调节因子等,这些蛋白在被运入线粒体之前,是作为蛋白前体而存在的,在内质网合成后被转运至线粒体。
由于蛋白质是细胞生命活动及其功能的主要执行者,因此直接从蛋白质整体水平来研究肿瘤细胞的化学或放射抵抗的产生机制、筛选耐药相关的蛋白质分子对于揭示并逆转肿瘤细胞的MDR无疑具有重要价值。随着人类基因组全序列测定工作的完成,生命科学进入了后基因组时代,作为功能基因组学的重要内容之一的蛋白质组学应运而生。蛋白质组学分为结构蛋白质组学和功能蛋白质组学,差异蛋白质组学是结构蛋白质组学的一个分支,其目的是寻找和筛选任何有意义的因素引起的两个样本间的差异蛋白质谱,揭示细胞生理和病理状态的进程与本质、对外界环境刺激的反应途径,以及细胞调控机制,同时获得对某些关键蛋白的定位和功能分析。蛋白质分离技术、蛋白质鉴定技术和生物信息学是蛋白质组学的三大基石。双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)是目前唯一能同时将数千种蛋白质同时分离与展示的技术,是目前蛋白质组学研究中的主要分离蛋白质的方法,常和质谱(mass spectrometry,MS)技术结合进行蛋白质定量鉴定。2-DE的优点是可根据凝胶上点的图形变化比较蛋白质组的变化,找出差异蛋白,但同时它又有不少缺点,如费时、重复性差,且在分离分子量极大(＞200kDa)或极小(＜10kDa)、极酸或极碱性及疏水性蛋白等方面都有它的局限性。荧光双向差异凝胶电泳(fluorescence two-dimensional differential gelelectrophoresis,DIGE)技术是2-DE的发展,DIGE是将待比较的蛋白质样品经不同的荧光染料(Cy2,Cy3,Cy5)标记后,等量混合进行双向电泳。蛋白量的差异可以通过蛋白点不同荧光信号间的比率来决定。因为荧光染料灵敏度高,故所需样品量非常少,而且一张胶可以同时分析3个样品,省去了胶图之间的匹配问题,减少了工作量,不仅重复性提高,而且提高了分析通量。蛋白质组学的发展离不开完善高效的生物信息学发展,其核心是蛋白质数据库,目前最常应用的是SWISS-PROT和GENEPETP等组成的NRDB数据库及由美国国家生物技术信息中心和欧洲生物信息学研究所(NCBI/EBI)等共同编辑的dbEST数据库。MITOMAP是包含线粒体编码、核编码的线粒体的基因和蛋白质的综合性数据库,其中收录有1150种线粒体相关的基因和对应的蛋白质。总之,由于线粒体在生命活动中的重要作用,线粒体蛋白质组学研究已经成为亚细胞蛋白质组学的研究热点。在本研究中,我们拟运用比较蛋白质组学技术分别分离鉴定阿霉素及射线处理前后的Raji细胞线粒体的差异表达蛋白质,为从亚细胞器蛋白质组水平进一步阐明阿霉素及射线的作用机制及抵抗机制奠定基础,同时对筛选出的化学耐药相关蛋白进行功能研究,验证这些差异蛋白作为阿霉素相关的NHL治疗靶点的可行性。
第Ⅰ部分阿霉素作用下Raji细胞线粒体的差异蛋白质组学研究
研究目的
以NHL Raji细胞株为研究对象,应用差异蛋白质组学技术分离并鉴定阿霉素处理前后Raji细胞差异表达的线粒体蛋白质,分析所鉴定蛋白的生物学特性,初步筛选与阿霉素作用机制有关的凋亡及耐药相关蛋白。
研究方法
1.阿霉素工作浓度及时间的选择。将Raji细胞分为两组,一组为对照组,一组为阿霉素处理组;阿霉素的作用浓度分别为0.2,0.5,1.0,1.5,2.0μg/ml,作用时间分别为24,48,72 h,用MTT方法测定Raji细胞在阿霉素的不同浓度及作用时间的抑制率,取IC50值作为本研究中阿霉素的工作浓度及时间的入选标准。
2.线粒体的提取及纯度验证。应用线粒体提取试剂盒分别提取对照组和阿霉素处理组Raji细胞线粒体,用Janus Green B染色确定提取线粒体的量,用western blot技术检测细胞器标志酶的表达,具体为:COX4作为线粒体的标志酶,PCNA及Cathepsin D分别作为细胞核及溶酶体的标志酶,根据电泳条带验证提取线粒体的纯度,证实本研究样本提取技术的可行性。
3.阿霉素处理前后Raji细胞线粒体的分离:采用2D-DIGE技术分离处理组及对照组线粒体蛋白:(1)应用Cy2,3,5分别标记内标、阿霉素处理组及对照组,用Ettan~(TM)DIGE荧光差异双向电泳系统和Typhoon多功能扫描仪分离待测样品蛋白,用DeCyder 6.5软件分析结果,将表达上调或下调≥1.5倍(p＜0.05)作为初步筛选差异表达蛋白的标准。
4.差异表达蛋白的质谱鉴定。应用线性离子肼四级杆-电喷雾串联质谱(LTQ-ESI-MS/MS)鉴定差异蛋白点,用Turbo SEQUEST程序及BioWorks~(TM)软件进行分析。将质谱鉴定得到的蛋白信息,检索Swiss-Prot、NCBI,、EMBL、BioInformatic Harvester、MitoP2等数据库,确认差异蛋白的亚细胞定位信息、功能分类、理论分子量、等电点等生物信息,并进行数据分析统计。
研究结果
1.MTT结果提示:1.5μg/ml的阿霉素作用48 h时,对Raji细胞的抑制率约为49.2%,因此将此剂量浓度和作用时间作为阿霉素的工作浓度及时间。
2.提取的线粒体沉淀用线粒体保存液悬起后,吸取一滴滴于载玻片上,JanusGreenB染色显示线粒体被染色成蓝绿色椭圆形或圆形小粒;western blot结果示提取的线粒体组分可以检测到明显的COX4蛋白条带,而该组分几乎检测不到细胞核的标志酶如PCNA及溶酶体的标志酶Cathepsin D,证实我们提取的线粒体具有较高的纯度,为下一步的蛋白质组学研究提供了可靠且足量的样本。
3.阿霉素处理前后Raji细胞线粒体蛋白质的分离。2D-DIGE电泳图谱分析显示:共检测到1485个蛋白点,其中63个有显著性差异表达(≥1.5-fold)。
4.差异表达蛋白的质谱鉴定。根据2D-DIGE提供的差异表达倍数(由大到小)的原则及参考三维图像,我们挑选其中26个进一步做质谱分析并鉴定出37个蛋白质及肽段信息,其中,34个蛋白表达上调,3个蛋白表达下调。差异表达的蛋白质的功能涉及多种线粒体功能如OXPHOS、细胞循环、转运和通道、DNA修复、氧化还原反应、蛋白质合成和降解等。检测到的大多数差异蛋白的等电点集中在5～9,分子量范围集中在10～60kDa,提示线粒体蛋白大多为小分子量的碱性蛋白,与前人研究结果一致。
第Ⅱ部分NHL化学耐药相关蛋白生物信息学研究及凋亡通路干扰
研究目的
通过联合应用多种生物信息学技术(如多个蛋白质数据库及蛋白质相互作用网络数据库或软件),建立包含验证后的差异表达蛋白在内的生物分子相互作用网络,并研究特异性凋亡通路抑制剂对于相关蛋白参与的凋亡通路的抑制效果,探索差异表达蛋白作为有应用前景的治疗靶标的可能性。
研究方法
1.差异蛋白验证:应用western blot技术从蛋白质水平验证挑选的感兴趣的蛋白(重点挑选与细胞凋亡及耐药有关的蛋白),分析蛋白表达的差异,确定与蛋白质组学方法得出结果的一致性。
2.应用pathway studio 5.0软件建立包含所挑选蛋白在内的生物分子相互作用网络,并做相关性分析。
3.选择与细胞凋亡密切相关的通路(PI3K/AKT),使用PI3K抑制剂(LY294002)阻断该通路,并在蛋白质及基因水平验证通路阻断前后有关蛋白及基因的表达差异,MTT法检测LY29对Raji细胞增殖的影响,流式细胞仪测定细胞周期改变。
研究结果
1.差异表达蛋白验证。我们挑选HSPA9、ABCB6、PHB三个蛋白在蛋白质水平做验证,结果提示与质谱分析结果一致。
2.凋亡相关通路的建立和验证。用pathway studio 5.0软件建立包括HSPA9、ABCB6、PHB等在内的凋亡相关通路,结果提示PHB可以作为一个共同的中间物而涉及多个生物过程如有丝分裂、防御反应、炎症、增生和凋亡等。另外,与这些蛋白有关的大多数疾病为起源于不同器官的癌症。
3.选择PI3K/AKT通路作为研究对象,用PI3K特异性抑制剂LY29004抑制该通路,western blot及real-time PCR结果证实:HSPA9、PHB、AKT及p-AKT表达下调,TP53表达上调,ABCB6表达无显著差异。
4.MTT法检测LY294002对Raji细胞增殖的影响:阿霉素联合应用LY294002能显著抑制Raji细胞增殖,细胞周期分析对照组、阿霉素处理组、LY29&-阿霉素处理组的Raji细胞大多分别位于S、G2/M、G0/G1期。
第Ⅲ部分X射线对Raji细胞线粒体蛋白质组的影响
研究目的
应用蛋白质组学技术分离鉴定射线处理前后的Raji细胞差异表达的线粒体蛋白质,寻找并分析与放射抵抗性有关的生物靶点。
研究方法
1.细胞照射:取对数生长期的Raji细胞,细胞悬液浓度调整为1×10~6/ml,在室温下采用Varian2100C/D直线加速器产生的16MVX线照射细胞,剂量率为200cGy/min,放射源至细胞面的距离为100cm,照射剂量分别为2Gy,4Gy,6Gy,另设立对照组。照射完毕后Raji细胞被置于与照射前同等条件下继续分别培养24h,48h,72h。
2.MTT法测细胞抑制率:细胞分组及处理同上,方法同第Ⅰ部分。
3.Annexin V/PI双染法检测Raji细胞受照射后细胞凋亡率:在培养48h后,分别收集对照组、2Gy-、4Gy-、6Gy-射线照射组Raji细胞,按Annexin V-FITC凋亡检测试剂盒说明,用流式细胞仪检测Raji细胞凋亡率。
4.2Gy-射线处理前后Raji细胞差异表达的线粒体蛋白质的分离、鉴定:方法同第Ⅰ部分。
5.差异表达蛋白验证:应用western blot技术从蛋白质水平验证挑选的感兴趣的蛋白(重点挑选与细胞凋亡及放射抵抗有关的蛋白),分析蛋白表达的差异,确定与蛋白质组学方法得出结果的一致性。
研究结果
1.MTT结果提示:不同射线剂量及培养时间条件下,对Raji细胞的抑制率呈剂量依赖性及时间依赖性,2Gy-X线对Raji细胞的增殖抑制效应不显著。
2.流式细胞术检测Raji细胞凋亡:各组细胞培养48h后,对照组、2Gy-、4Gy-、6Gy-射线处理组的Raji细胞凋亡率逐渐增高,且与对照组相比,2Gy-射线诱导的细胞凋亡不明显。
3.射线对Raji细胞线粒体蛋白质组的影响:2D-DIGE电泳图谱分析显示:共检测到1391个蛋白点,其中34个有显著性差异表达(≥1.5-fold),根据2D-DIGE提供的差异表达倍数(由大到小)的原则及参考三维图像,我们挑选其中13个蛋白点进一步做质谱分析并鉴定出23个蛋白质及肽段信息,其中,19个蛋白表达上调,4个蛋白表达下调。差异表达的蛋白质的功能涉及多种线粒体功能如OXPHOS、脂肪酸β-氧化、细胞循环调控、DNA修复、蛋白质合成等。
4.差异表达蛋白验证。我们挑选RECQL4、ATAD3B两个蛋白在蛋白质水平做验证,结果提示与质谱分析结果一致
结论
1.联合应用多种蛋白质组学技术及生物分子相互作用软件如Pathway Studio是从整体研究线粒体蛋白质组的高效、高通量手段。
2.发现的与阿霉素耐药相关的蛋白如HSPA9、PHB,ABCB6可以作为有潜在应用价值的研究化学耐药性的靶蛋白。
3.在PI3K/AKT通路中,HSPA9,PHB均与细胞凋亡及化学耐药性密切相关,并可能存在相互作用,LY29可有效阻断Raji细胞中PI3K/AKT途径,并影响HSPA9、PHB、AKT及TP53等蛋白的表达,而对ABCB6的影响不显著。
4.2Gy-X线对Raji细胞的细胞凋亡增殖抑制效应不显著,细胞凋亡不明显。
5.GAPDH,RECQL4,MKI67,AAA-TOB3等蛋白有可能成为有潜在应用价值的逆转放射抵抗的生物靶标。
Background
Much progress in conventional therapy for NHL have been achieved over the past decades,but there are still 40%to 70%of patients with intermediate and high grade NHL failing to achieve long-term disease-free survival(DFS),and no curative treatment strategies have been established for patients with low grade NHL yet.In this study,the reason for selecting Raji cells is that the Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression,which are commonly present in patients with NHL and are considered a source of chemotherapy failure in these patients associated with chemoresistance.Chemotherapy is commonly used as part of the treatment against non-Hodgkin's lymphoma(NHL) and can be advantageous for some time.Adriamycin(ADR) is widely used in drug combination strategy like CHOP(Cyclophosphamide+Adriamycin+Vincristine+Prednisolone) against NHL as a classic anthracycline agent,while some patients have shown resistance to it.The chemoresistance and anticipated dose-related cardiotoxicity associated with ADR are critical obstacles to a successful outcome. Multidrug-resistance(MDR) is a common pattem of anti-chemotherapy in tumor cells. The known mechanisms of MDR included:1) Outflow of chemotherapeutic drug decrease concentration in cells.MDR1,MRP and LRP are the mostly involved resistant genes and proteins;2) Conversion and detoxification of anti-tumor drug by carcinoma cells is enhanced,including GSTs and P450;3) Chemotherapeutic drugs result in modification of target molecule on cells,including TOPⅡand reductase of dihydrofolic acid;4) Enhancement of DNA repair,including MGMT;5) Chemotherapeutic drug was unable to induce apoptosis of cancer cells.
Lymphoma is a category of radiosensitive malignance and radiation therapy(RT) alone may be used as a curative treatment for stageⅠandⅡin patients with indolent non-Hodgldn's lymphoma(NHL).For more extensive and aggressive NHLs, RT is used in combination with chemotherapeutic agents,while 50-70%of these patients relapse.The aim of radiotherapy on NHL is to induce apoptosis with the endpoint of proliferative cell death(PCD,also known as "secondary apoptosis") and lead to unrepaired DNA injury.NHL is more radiosensitive than most solid malignancies while resistance to radiotherapy is a major obstacle to a successful outcome.The cellular radiation response is complex and apoptosis is the main purpose for radiotherapy to suppress the proliferation of NHL cells,so mitochondrial proteins play crucial role in the radiation signal transduction pathway due to their vital function in apoptosis.
Mitochondria are eukaryotic organdies that play a crucial role in several cellular processes,including energy production,fatty acid metabolism,oxidative stress,ion homeostasis and programmed cell death.The main chemical components of mitochondrion are protein and lipide,accounting for 65-70%and 25-30%of the total dry weight of mitochondia respectively.Mitochondria is wrapped by an outer and inner membrane,having four domains including outer-membrane,inner-membrane, intermembrane space and ground substance.In mitochondria of a liver cell,the content of protein of the above-mentioned four parts are 8%,21%,4%and 67% respectively.The inner membrane is especially rich in proteins,e.g.the high molecular weight multi-protein-complexes of the respiratory chain are located at the inner mitochondrial membrane.The total number of different proteins or polypeptides making up a mitochondrion is estimated to be around 2000～2500.Mammalian mtDNA encodes for 13 essential polypeptide components of the oxidative phosphorylation system,small and large rRNA's,and 22 tRNA's.The remaining mitochondrial proteins of the oxidative phosphorylation system,metabolic enzymes, membrane channel proteins,and any other protein regulating mitochondrial function are derived from the nuclear genome.Import of these proteins into the mitochondria is highly regulated through the function of protein chaperones and the inner and outer tr nsmembrane peptide import complexes(TIM and TOM).
In recent years,with the development of proteomics technology,it becomes more and more achievable to have a thorough investagtion of comparative proteomics.For instance,many new significant proteins have been discovered with diverse physiological and pathophysiological effects.Two-dimensional gel electrophoresis (2-DE) is currently the mainstay of proteomic analysis as it allows the simultaneous visualization of thousands of proteins and their post translational modifications (PTM's)(12).It separates a complex protein mixture based upon 2 intrinsic protein characteristics,net charge(isoelectric point,pI) and molecular mass.First,proteins are separated according to their net charge though isoelectrie focusing(IEF) and they are subsequently separated orthogonally by molecular mass using sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Although theoretically 2-DE has no difficulty in separating all the proteins,it still has many disadvantages,such as time-consuming,sensitivity,linear dynamic range, repeatability,quantitation truth(precision/accuracy) and compatibility with mass spectra.In addition,2-DE is not good at detecting proteins with low abundance,high molecular weight(＞200KDa) or the minimum molecular weight(＜10KDa) and extreme acid and basylous proteins.Two-dimensional fluorescence different gel electrophoresis(2D-DIGE) is the new development of 2-DE,it also allows for the direct comparation of protein abundance changes which is less than 10%across multiple samples simultaneously with 95%statistics reliability coefficient without interference due to gel-to-gel variation.Now many new data were added into forthcoming databases such as SWISS-PROT、NCBI and MitoPeoteome,ect. MitoP2 is the most comprehensive database about mitochondrial proteome which includes the.explanation of moiety on human,mouse and yeast.The proteins above-mentioned of the three parts are 624,615,522 respectively.However,due to the spurt of the emergement of this new subject,the databases are still very insufficient and require more exploration to enrich them.
PartⅠComparative Mitoehondrial Proteomie Analysis of Raji Cells Exposed to Adriamycin
Objective
In this study,we used Raji cells as a model of human NHL.We aimed to investigate ADR about its antineoplastic activity,drug resistance and unexpected toxicity on non-Hodgkin's lymphoma(NHL) Raji cells at mitochondrial proteomic level.
Methods
1.Selection of the dose and time exposure of ADR.NHL Raji cells(4×10~(5)) were exposed to ADR(0.2,0.5,1.0,1.5,2.0μg/ml respectively) for the various incubation times indicated(24 h,48 h,and 72 h).The result of the cell proliferation evaluated by the MTT was exhibited as the inhibition rate.The IC50 values were determined directly from the semi-logarithmic dose-response curves.
2.Mitochondria isolation and purification validation.ADR(1.5μg/ml) was added to the 2×10~(7) Raji cells at the initiation of the 48 hour culture period.The selection of the dose and time of exposure was based on the MTT results.At this stage, the mitochondria of Raji cells were isolated using a mitochondrial isolation kit according to the manufacturer's instructions.Separated mitochondria were mounted on glass slides,incubated with 0.1%Janus green B solution for 10 min,and then visualized under a light microscope.Purified mitochondrial pellets were preserved at -80℃until further analysis.The purity of the isolated mitochondria was validated by western blot.COXIV,PCNA,and Cathepsin D were used as mitochondria,nuclear and lysosomal markers,respectively.
3.Two-dimensional differential in-gel electrophoresis and imagine.Protein concentrations determined by Bio-Rad protein assay for the control and ADR-treated groups were used to normalize the quantities of protein loaded in each sample. Aliquots of 100μg protein from each of the two samples and labeled with 200 pmol of either Cy3(treated) or Cy5(control) respectively.The Biological Variation Analysis(BVA) module of Decyder 6.5 was used to compare the control and test sample to generate list of differentially expressed proteins.Taking cut-off of 1.5-fold up/down-regulation with a t-test score p＜0.05 as an initial threshold for significance.
4.LTQ-ESI-MS/MS analysis and database searching.Total 26 protein spots were cut out of 2D-DIGE gels using a Gelpix Spot-Excision Robot and the digested pieces were analyzed via LTQ-ESI-MS/MS using a surveyor high-performance liquid chromatography(HPLC) system.All identified proteins were determined to be mitochondrial-associated proteins by inquesting several protein databases such as Swiss-Prot,NCBI,EMBL,Biolnformatic Harvester,WOLF PROST,Target P, MitoSub,and MitoP2.
Results
1.The result of MTT indicated that_incubation of Raji cells with ADR showed that inhibition rate was in a dose-and time-dependent manner.It reached 49.82%with 1.5μg/ml ADR treatment for 48 h.Based on these result,we used 1.5μg/ml of ADR for 48 h for further experiments.
2.Mitochondria Isolation and Purity Validation.To obtain mitochondria of Raji cells with high purity for the reliabilities of proteomic analysis,Janus green B staining was carried out to identify the mitochondrial fraction and western blot analysis was applied to validate its purity.The isolated mitochondria were stained as bluish-green round particles.COXIV was specially detected in the purified mitochondrial fraction and this fraction lacked any detectable contamination of abundant nuclear and lysosome proteins such as PCNA and Cathepsin D.Our result demonstrated a high purity of mitochondria with the use of our subcellular isolation method.
3.Isolation of mitochondrial proteins exposed to ADR.Total 1485 spots were detected,and 63 were differentially expressed(≥1.5-fold).
4.Mass spectrometry identification of differentially expressed mitochondrial proteins.We chose 26 spots for further analysis by mass spectrometry and identified 37 unique proteins.Among them,34 proteins decreased and 3 proteins increased.The altered proteins identified in this study encompass a range of mitochondrial functions including OXPHOS,cell cycle regulation,transporters and channels,DNA repair, redox,protein synthesis and degradation,ect.A relative large percentage of the proteins have slightly basic pI value range of 5-9(32 out of 37,86.49%).About 75.68%(28 out of 37) proteins identified have masses between 10-60kDa.
PartⅡBioinformatics Analysis of Proteins Associated with Chemoresistance of NHL and Apoptotic Pathway Interference
Objective
In this part,we aimed to establish biomolecular interactive networks including several identified mitochondrial proteins with multi-informatics techniques.By using the specific apoptotic pathway inhibitor,the mitochondrial proteins associated with the chemoresistance will be explored as potential anti-tumor targets in NHL treatment.
Methods
1.Validation of selected identified proteins by western blot.To further validate the alterations in protein abundance derived from 2D-DIGE/MS,we examined the variance of three enzymes of interest(HSP70,PHB,and ABCB6) by western blot. These proteins were chosen due to their crucial functions in the action of ADR on Raji cells and the commercial availability of the corresponding antibodies.
2.To study functional interactions and possible pathways of the HSP70,PHB,and ABCB6,Pathway Studio 5.0 software(Ariadne Genomics,Rockville,MD,USA) was used.Briefly,we firstly import a protein list including HSP70,PHB,and ABCB6 into a new pathway diagram,and build a pathway using the option "Find all entities connected to selected entities(Expand Pathway)".The program will search the current pathway database and ResNet for interactions with the selected entities and add them to the pathway.
3.LY294002 was added to Raji cells prior to ADR.The inhibited effect of LY294002 was evaluated by analyzing several proteins and genes closely associated with chemoresistance such as HSPA9,PHB,P53 and AKT.The proliferation of Raji cells treated with LY294002 was evaluated by MTT analysis.Cell cycle of the treated Raji cells and the control were determined by flow cytometry using PI staining.
Results
1.Validation of HSP70,PHB,and ABCB6.Comparing mitochondrial lysates from control and ADR-treated Raji cells by Western blot,the expressions of HSP70 and PHB were increased whereas the expression of ABCB6 was decreased.The results were in conformity with 2D-DIGE.
2.The software Pathway Studio 5.0 was used to search possible protein-protein interactions,common regulators,cell processes,and related diseases for HSPA9,PHB, and ABCB6.By this approach,we found that proteins belonged to different structural and functional families had PHB as common target and were involved in mitogenesis, defense response,germination,inflammation,proliferation,and apoptosis,ect. Furthermore,most of the diseases associated with these three proteins were cancer derived from various organs.
3.The pathway studio software construct interactive network within the AKT pathway.Then two proteins associated with chemoresistance including HSPA9 and PHB were investigated in detail at mRNA level and protein level using real-time PCR and western blot analysis.Both HSPA9 and PHB were up-regulated in ADR-treated Raji cells.After treated with LY294002,an inhibitor for PI3K,the expressions of these two proteins were down-regulated with a significant alteration in cell cycle and up-regulation of p53.
Objective
The aim of this present investigation is to get a comprehensive overview of the protein turnover reactions in mitochondria of Raji and discover molecular biomarkers in radiation response.
Methods
1.Irradiation procedures:Exponentially-growing Raji ceils were suspended at a concentration of 1×106 cells/ml in 1640 and irradiated at room temperature using a X-ray source(16 MV,CLINAC2100C/D Linear Accelerator,Varian,USA) at a dose rate of 200 cGy/min.Single doses of 2,4,and 6 Gy were given and the cells were subsequently cultured at various times(24,48,and 72 h) for analysis.
2.MTT assay:Cell proliferation was evaluated by MTT.
3.Apopotosis assessment of 2-Gy X-ray on Raji ceils by Annexin V/PI double staining:To assess apoptosis,Raji ceils exposed to 2 Gy X-ray for 48 h and its control partner were stained with FITC-conjugated Annexin-V protein along with PI according to the recommendations of the manufacturer and analyzed by flow cytometry.
4.Comparative mitochondfial proteomic analysis of Raji cells exposed to radiation: 2D-DIGE in combination with LTQ-ESI-MS/MS were utilized as a non-biased approach to evaluate mitochondfial protein alterations of NHL Raji cells after exposed to radiation.
5.Validation of Selected identified Proteins by Western Blot:To further validate the alterations in protein abundance derived from 2D-DIGE/MS,we examined the variance of two interested enzymes(RECQL4,and ATAD3B) by Western blot.These proteins were chosen clue to their crucial function in the action of radiation on Raji ceils and the commercial availability of correspondent antibodies.The expression levels of all tested proteins were in conformity with 2D-DIGE(Figure 4).
Results
1.The result of MTT indicated that_incubation of Raji ceils with various dose radiation showed that inhibition rate was in a dose-and time-dependent manner.
2.Apoptosis analysis of Raji cells exposed to various dose radiation:the apoptotic response to radiation treatment is lower if compared to the control Raji cells.
3.Isolation and identification of mitochondrial proteins exposed to radiation.Total 1391 spots were detected,and 34 were differentially expressed(≥1.5-fold).We chose 26 spots for further analysis by mass spectrometry and identified 23 unique proteins. Among them,19 proteins decreased and 4 proteins increased.The altered proteins identified in this study encompass a range of mitochondrial functions including OXPHOS,energy metabolism,cell cycle regulation,DNA repair,protein synthesis and degradation,ect.
4.Validation of RECQL4,and ATAD3B:Comparing mitochondrial lysates from control and radiation-treated Raji cells by Western blot,the expressions of RECQL4 increased whereas the expression of ATAD3B was decreased.The results were in conformity with 2D-DIGE.
Conclusions
1.In this study,2D-DIGE combined with mass spectrometry and database interrogation were used to simultaneously screen the mitochondrial protein abundance changes of Raji cells exposed to ADR and radiation.The results of our proteomic analysis indicated that our study strategy was suitable and high performance.
2.A series proteins associated with chemoresistance relevant to ADR including HSP70,PHB,and ABCB6 were found to be altered in their expressions.However, such markers require functional studies before being introduced to the clinic.
4.In the PI3K/AKT pathway,HSPA9 and PHB were closely associated with chemoresistance.The expression of these two proteins could be influenced by LY29.
5.Proliferation of Raji cells,shown in our model by the MTT,Annexin V/PI analysis and proteomic study,was not significantly slowed down and the apoptotie response to radiation treatment is lower if compared to the control Raji cells.
6.GAPDH,RECQL4,MKI67,and ATAD3B could be used as promising biomarkers for radioresistance.
引文
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