细胞毒抗肿瘤药物的共同基因表达谱的研究
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摘要
基因表达谱芯片技术是近年来发展起来的一项前沿生物技术,可以对成千上万种基因的表达水平进行平行、快速、准确、高效的检测。基因芯片技术对于药物作用分子机制研究、药物靶标的发现、多靶点高通量药物筛选、药物活性及毒性评价等领域具有无可比拟的优势。
细胞毒抗肿瘤药物与不同的靶点结合后发挥抑制肿瘤细胞增殖、诱导细胞周期阻滞和细胞凋亡的作用。虽然目前已有大量的文献报道有关细胞增殖和凋亡的调控分子和反应通路,但是这些药物作用靶点与细胞内其它的调控分子或反应通路尚不完全清楚。由于不同的细胞毒抗肿瘤药物所致的细胞表型变化相似,因此可以推测:不同的抗肿瘤药物可能共享某些细胞内信号传导通路,而这些细胞内传导通路中的分子则可能与调控肿瘤细胞增殖和凋亡有关。因此,寻找新的细胞增殖和凋亡的调控分子和反应通路对于进一步了解药物的作用机制和发现新的药物靶点具有重要的意义。
本研究基于上述的设想,选择了喜树碱、紫杉醇、高三尖杉酯碱和放线菌素D作用于白血病HL-60细胞和肝癌QGY7703细胞,利用基因芯片技术研究药物反应的基因表达谱,并应用Genmapp分析软件和Access软件,对药物反应基因进行聚类分析和药物反应通路分析。并应用实时定量PCR对一些重要基因进行确证。结果如下:
1.为研究抗肿瘤药物早期敏感反应基因表达谱,我们首先研究4个细胞毒抗肿瘤药物对白血病HL-60和肝癌QGY7703细胞表型的影响。采用噻唑蓝(MTT)法检测紫杉醇、高三尖杉酯碱、喜树碱、放线菌素D作用于HL-60细胞和QGY-7703肝癌细胞24h的药物半数抑制浓度(IC50)分别为0.025、320、79.85、2.67、μmol/mL和2.26、52.77、19.09、0.48、μmol/mL;实验同时采用形态学观察和流式细胞仪检测细胞凋亡以及细胞周期,实验表明,两种肿瘤细胞与各药物孵育1h,细胞形态未见变化,作用24h可见细胞破碎、数目减少;流式细胞仪检测结果显示,紫杉醇和高三尖杉酯碱作用24h可加速白血病细胞和肝癌细胞通过G1/S期,导致G2/M期阻和细胞凋亡。
2. HL-60细胞和QGY-7703肝癌细胞分别用上述药物的IC50处理1h后,实验同时设加药物溶剂的对照组。抽提细胞RNA,然后用荧光试剂制备cDNA探针,再与14400型基因表达谱芯片杂交,用ScanArray 3000扫描芯片和ImaGene软件分析荧光信号的强度和比值。选择三次实验杂交比值的平均值自然对数绝对值≥0.69作为判定基因表达差异标准,筛选出各药物作用的早期反应基因。结果显示,4个抗肿瘤药物均诱发强烈的细胞基因表达改变,其中紫杉醇和高三尖杉酯碱诱导HL-60和QGY7703细胞的反应基因最多。
3.应用Genmapp分析软件对药物反应基因进行聚类分析和药物反应通路分析,发现4个抗肿瘤药物可调控细胞内许多反应通路、功能基因或细胞组分。例如,紫杉醇可调控mRNA加工过程;高三尖杉酯碱可抑制蛋白酶体组分26S亚单位PSMC3、PSMD2和PSMD11表达;喜树碱影响G蛋白信号通路中多条基因的表达;放线菌素D可调节丝裂素活化的蛋白激酶(MAPK)通路、TNF-NFkB通路等,这些结果说明药物起始靶点与许多已知的调控细胞增殖和凋亡的通路相连,而这些通路可以很好地解释药物的作用。此外,细胞毒抗肿瘤药物还调节了一群组织特异的基因,如喜树碱影响一些内脏器官、内分泌系统和中枢神经系统的基因,放线菌素D也能调节一些正常胚胎干细胞基因表达,提示这些基因的改变可能与其毒性有关。
4.应用microsoft office Access软件对药物反应基因和药物反应通路进行统计分析,发现细胞毒抗肿瘤药物之间在转录水平上可共享某些同向改变的基因或反应通路,其中2个药物之间最多,共用反应基因和通路在3个药物之间,甚至全部4个药物都可见到,例如全部4个药物在HL-60和QGY7703细胞中分别有17条和40条共用反应基因以及7条和9条反应通路。在2个细胞系中,4个抗肿瘤药物也有6条的共同反应基因,它们是GRN、MLL3、PSAP、ERP29、LRPAP1、ST3GAL4 ,也共享肌动蛋白-细胞骨架调节通路( Regulation of Actin Cytoskeleton)和粘着斑功能调节通路(Focal adhesion)。这些结果提示拓朴酶抑制、微管、核糖体和DNA模板干扰在转录水平上具有共同的下游作用,通过这些下游通路或分子可能最终导致相似的表型变化。
5.共用反应基因在功能上可分为:生长增殖相关基因、凋亡相关基因、糖代谢相关基因及其它功能基因4类,这些共用基因所编码的蛋白生物功能多样,分别参与了细胞周期调节,DNA、RNA和蛋白质合成,能量代谢,泛素(类泛素)蛋白酶体依赖的蛋白质降解,信号传导通路、抗凋亡或促凋亡等重要的细胞生命活动以及内质网以及线粒体等构成。(1)抗肿瘤药物能显著抑制细胞表达细胞周期相关基因,如蛋白磷酸酶2A、翻译延长因子1、HnRNPB1等,这些基因参与了细胞有丝分裂、核酸合成、DNA复制和修复以及RNA加工和蛋白质合成;此外,还能抑制具有促增殖作用的肿瘤相关蛋白基因(如GRN、MLL3 )的表达,表明抗肿瘤药物可通过调节细胞增殖基因的表达,来抑制肿瘤细胞的生长甚至导致死亡;(2)抗肿瘤药物能诱导细胞表达促凋亡基因PMAIP1以及抑制抗凋亡基因PSAP的表达,说明调节凋亡相关基因的表达也是抗肿瘤药物诱导细胞凋亡的一个重要机制;(3)抗肿瘤药物能调节内质网以及线粒体构成诱导细胞凋亡,如ERP29是内质网腔膜蛋白,作为氧化还原酶和分子伴侣,参与蛋白质折叠和运输。ARF5是囊泡转运包被复合体形成所必需,参与了内质网和高尔基体间的蛋白转运。抗肿瘤药物抑制两者表达,可能阻断蛋白在内质网与高尔基体的转运,从而诱导内质网应激而使细胞凋亡。mitochondrial ribosomal protein S18A是线粒体核糖体组分,衰老细胞线粒体核糖体蛋白合成的准确性下降。isocitrate dehydrogenase 2参与三羧酸循环, SLC25A6为ADP/ATP转运子,因此,抑制参与能量代谢的基因表达可能会引起线粒体呼吸链功能缺陷或者ATP耗竭而导致细胞死亡。(4)抗肿瘤药物能抑制泛素(类泛素)蛋白体蛋白降解系统相关基因PSMC3、VCP和SAE1表达。PSMC3是蛋白酶体26S蛋白酶体19S调节亚基的核心组分,VCP/p97是一种AAA ATP酶,在辅助因子协助下,可使错误折叠的蛋白从内质网膜脱位进入胞浆,经泛素蛋白酶体系统降解。SAE1/SAE2是小泛素相关修饰剂(Small Ubiquitin-related Modifier, SUMO-1)活化酶,参与类泛素途径调节,由于蛋白酶体参与细胞周期、增殖和凋亡调节,因此抑制泛素蛋白酶体途径可能也是抗肿瘤药物的一个重要机制(5)抗肿瘤药物能抑制唾液酸转移酶4C和糖苷转运子SLC35D1表达,而这二者均在多种肿瘤中表达增加,参与肿瘤的转移。因此,抑制蛋白糖基化可能与药物的抗转移作用有关。
文献检索发现,有些共用基因已作为抗肿瘤药物筛选靶点,例如,蛋白酶体抑制剂是一种新型的抗肿瘤药物,已进入临床实验;基于蛋白磷酸酶PP2A分子模建,目前已设计了系列蛋白磷酸酶抑制剂,体内外结果显示蛋白磷酸酶抑制剂具有广谱的抗肿瘤效果。此外,唾液酸转移酶抑制剂也能显著抑制肿瘤生长和抗肿瘤转移的作用。这些结果提示通过进一步的研究有望从这些共用反应基因中发现一些新药靶。此外,以共用反应基因为基础制成芯片,可以建立针对基因群的多靶标高效筛药和评价模型。
综上所述,应用基因芯片技术和多个细胞毒抗肿瘤药物寻找得到的共同反应基因对于阐明抗肿瘤药物作用机制和发现新药靶具有重要的意义。
Gene expression microarray is a newly-developed biotechnology in recent years, permits the simultaneous and rapid measurement of the expression levels of thousands of genes, and allows the analysis of molecular events of drug response and potentially identifying novel drug targets. It is also suitable for high-throughput drug screening with multi-targets and evaluation of drug activity and toxicology.
Cytotoxic anticancan drugs inhibit cell proliferation, disturb cellcycle and induce cell apoptosis after binding to diverse molecular targets. The growing numbers of documents have shown the key regulatory molecules and cellular pathways control cell apoptosis and the cell cycle. However, the connectivity of known mechanisms with other regulators or pathways remains unclear. Although the anticancer drugs have diverse initial molecular targets, cellular phenotypic outcomes are similar for different drug treatments. Therefore, it appears reasonable to assume that some of genetic alternations among the different drug responses might be likely shared as common key effects of the drug-target interactions. Analysis of these intersection genes may provide new insights into the molecular mechanisms of the antitumor agents and discover new potential target molecules for the development of novel antineoplastic drugs.
This study was designed to test above hopothesis with microarray analysis of the sensitive early gene expression profiles in QGY-7703 hepatoma cells and HL-60 leukemia cells stimulated with the chosen representative drug paclitaxel (Taxol), dactinomycin (ACD), camptothecin (CPT), and homoharringtonine (HHRT), based on their identical cell growth inhibition actions. Drug response genes were then clustered and cellular pathways were analyzed by Genmapp anlysis software and Microsoft Ascess software. Realtime PCR was also conducted for some interest genes to comfirm our microarray results. Results are shown as follows:
1. To analyze the sensitive early gene expression profiles, cell phenotypes of growth inhibition, apoptosis and cell cycle in response to drug treatment were determined by MTT assay and flow cytometry. 50% of inhibitory concentration (IC50) of Taxol, HHRT, CPT and ACD was 0.025、320、79.85、2.67、μmol/mL and 2.26、52.77、19.09、0.48、μmol/mL respectively in HL-60 leukemia cells and QGY-7703 hepatoma cells after 24 cluture. Morphological changes were not observed by microscope after 1 h incubation of drugs with both cell lines, wheras the decreased cell numbers and cell pieces were seen in both cells treated with the chosen drugs for 24 h. Flow cytometry results showed that treatment of paclitaxel and homoharringtonine for 24 h markedly accelerated leukaemia and heptoma cells through G1/S interface resulting G2/M arrest and apoptotic cell death. These results are consitent with the reported actions of these drugs.
2. Total RNA was isolated from drug treated cells and from control (equal volume of drug solvent) cells after 1 h incubation. Labeling of cDNA, microarray hybrization and fluorescence detection was performed as standard protocol of Unite Gene Corpoation. Genes were identified as differentially expressed if the absolute value of the natural logarithm of the average ratios of three independent experiments was > 0.69. The number of regulated genes differed significantly among drugs, with paclitaxel and homoharringtonine causing the strongest responses in HL-60 and QGY7703 cells respectively.
3. Genmapp software was used to gene cluster and pathway analysis of the drug response genes. A number of cellular pathways, functional molecules and cellular components were found as classied by the used software. For example, paclitaxel reglulated mRNA processing; homoharringtonine inhibited the expression of protesome component PSMC3、PSMD2 and PSMD11;camptothecin affected G protein signaling pathways and dactinomycin modulated MAPK pathways and TNF-NFkB pathways. These results indicate initial tagets are connected with many known pathways responsible for cell proliferation and apoptosis, which may clearly explain actions of these antitumor agents. In addition, cytotoxic agents also regulated several groups of genes specific to internal organs, endocrine and CNS as well as embryo stem cells, suggesting these genes may be related to the cytotoxicities of antitumor drugs.
4. Microsoft office Access software was used to sort common drug response genes and common cellular pathways. Our results demonstrated that antitumor agents shared many cellular pathways and genes with the same regulated direction in the transcriptional levels. The largest overlap was found between two agents. Common response genes and pathways were also identified among three drugs, even in all four four drugs. For exaple, 17 and 40 common response genes and 7 and 9 pathways were obtained in HL-60 cell and in QGY7703 cells respectively. There were 6 common response genes including granulin (GRN)、myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3)、prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy, PSAP)、endoplasmic reticulum protein 29 (ERP29)、low density lipoprotein receptor-related protein associated protein 1 (LRPAP1)、ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) and 2 pathways (regulation of Actin Cytoskeleton and focal adhesion) shared by all drugs in both cell lines. These results provided evidence for common down stream effects of topoisomerase inhibition and microtubular dynamics or ribosome function interference as well as DNA template corruption, which may result in final similar phenotypic outcomes.
5. The common response genes may be classied into four categrories including growth and proliferation related genes, apoptosis related genes, carbohydrate metabolism related genes and unkown function genes. The biochemical functions of intersection response genes are diverse and include cell cycle regulators, proapoptotic and antiapoptotic factors, growth factors, signal mediators, metabolic regulators, proteases and glycosyltransferases, and proteins of miscellaneous or unknown function. (1) Chemotherapeutical agents-treated cells decreased expression of a cluster of genes involved in cell proliferation. For example, a broad range of genes is associated with cell cycle and mitosis (QSCN6,PPP2R1A),gene transcription [general transcription factor IIIC (GTF3C5) , activating transcription factor 5 (ATF5)],RNA processing[heterogeneous nuclear ribonucleoprotein A/B (HNRPAB),PRP6 pre-mRNA processing factor 6 homolog (S. cerevisiae) (PRPF6),nucleolin (NCL)] and protein synthesis[eukaryotic translation elongation factor 1 delta EEF1D, Eukaryotic translation initiation factor 4A, isoform 1,EIF4A1]。In addition, tumor associated genes granulin、myeloid/lymphoid or mixed-lineage leukemia 3 were also inhibited upon multiple treatment.The alterations in these specific genes functionally related to cell proliferation might determine tumor cell growth arrest, even ultimate cell death, correlating well with the cytotoxic agents-mediated cytotoxicity. (2) Treatment of antitumor agents induced proapototic genes phorbol-12-myristate- 13-acetate-induced protein 1(PMAIP1) and decreased antipoptotic gene prosaposin (PSAP), suggesting the involvement of cell apoptotic mechanisms in these drug actions. (3) Regulation of endoplasmic reticulum (ER) and mitochondrial components was shown by drug treatment, for example, ERP29, a disulfide isomerase (PDI)-like proteina, is involved in oxidative stress and regulates protein unfolding and facilitates the transport of secretory proteins. ARF5 is a member of ADP-ribosylation factors necessary for the formation of clathrin- and coatomer protein (COP) I-coated vesiclesare, and isivovled in protein transport between ER and Golgi compartments. Inhibition of tese ER associated genes by antitumor agents may result in ER stress leading to cell apoptosis. Moreover, the expression of mitochondrial components was also observed in response to antitumor drugs. Isocitrate dehydrogenase 2 participates in tricarboxylic acid cycle and solute carrier family 25 (SLC25A6) is adenine nucleotide translocator. Thus the decresed energy production and transport may be responsible for cytotoxic agents-mediated apoptosis. (4) Cells exposed to cytotoxic agents resulted in the down regulation of a cluster of Ubiquitin/proteasome degradation pathways (UPDP) related genes, such as PSMC3, VCP and SAE1. PSMC3 is a core component of the 19S regulatory particle of the 26S proteasome, VCP helps dislocateing misfolded proteins from the ER membrane into the cytosol for proteasomal degradation, and SAE1/SAE2 is the activating enzyme E1 of small ubiquitin-related modifier (SUMO) responsible for post-translational modifications of proteins. Since ubiquitin/proteasome degradation pathways have function in regulating cell proliferation and apoptosis, the decraesed expression of UPDP related genes may be contributed to cytotoxic effects of antitumor drugs. (5) Genes functioned in the intracellular glycoprotein glycosylation pathway such as sialyltransferase 4C and SLC35D1expression were reduced in drug responses, which may imply the modulating effects of these antitumor drugs on the tumor invasion and metastasis due to the involvement of specific carbohydrate structures in cellular recognition processes and cancer metastasis.
6. Some common response genes have been documented to be the targets for drug design and screening. For example, proteasome becomes a new target for novel drug therapy. Based on a modelled structure of PP2A, a new class of protein phosphatase inhibitors, cantharimides, are designed and exhibit broad-spectrum anti-cancer activity. Suppression of sialyltransferases by chemicals has been shown to reduce the potentiality of growth and metastasis of various tumors. These suggest that another unexplored drug targets might be discovered from these intersection genes. In addition, these common response genes may also be used for screening and evaluation of antitumor agents as made of multiple-target microarray chip.
Taken togethe, determination of the common response genes with microarrays and multiple tool agents is of great value for our deciphering complex regulatory pathways and discovering new potential target molecules.
细胞毒抗肿瘤药物与不同的靶点结合后发挥抑制肿瘤细胞增殖、诱导细胞周期阻滞和细胞凋亡的作用。虽然目前已有大量的文献报道有关细胞增殖和凋亡的调控分子和反应通路,但是这些药物作用靶点与细胞内其它的调控分子或反应通路尚不完全清楚。由于不同的细胞毒抗肿瘤药物所致的细胞表型变化相似,因此可以推测:不同的抗肿瘤药物可能共享某些细胞内信号传导通路,而这些细胞内传导通路中的分子则可能与调控肿瘤细胞增殖和凋亡有关。因此,寻找新的细胞增殖和凋亡的调控分子和反应通路对于进一步了解药物的作用机制和发现新的药物靶点具有重要的意义。
本研究基于上述的设想,选择了喜树碱、紫杉醇、高三尖杉酯碱和放线菌素D作用于白血病HL-60细胞和肝癌QGY7703细胞,利用基因芯片技术研究药物反应的基因表达谱,并应用Genmapp分析软件和Access软件,对药物反应基因进行聚类分析和药物反应通路分析。并应用实时定量PCR对一些重要基因进行确证。结果如下:
1.为研究抗肿瘤药物早期敏感反应基因表达谱,我们首先研究4个细胞毒抗肿瘤药物对白血病HL-60和肝癌QGY7703细胞表型的影响。采用噻唑蓝(MTT)法检测紫杉醇、高三尖杉酯碱、喜树碱、放线菌素D作用于HL-60细胞和QGY-7703肝癌细胞24h的药物半数抑制浓度(IC50)分别为0.025、320、79.85、2.67、μmol/mL和2.26、52.77、19.09、0.48、μmol/mL;实验同时采用形态学观察和流式细胞仪检测细胞凋亡以及细胞周期,实验表明,两种肿瘤细胞与各药物孵育1h,细胞形态未见变化,作用24h可见细胞破碎、数目减少;流式细胞仪检测结果显示,紫杉醇和高三尖杉酯碱作用24h可加速白血病细胞和肝癌细胞通过G1/S期,导致G2/M期阻和细胞凋亡。
2. HL-60细胞和QGY-7703肝癌细胞分别用上述药物的IC50处理1h后,实验同时设加药物溶剂的对照组。抽提细胞RNA,然后用荧光试剂制备cDNA探针,再与14400型基因表达谱芯片杂交,用ScanArray 3000扫描芯片和ImaGene软件分析荧光信号的强度和比值。选择三次实验杂交比值的平均值自然对数绝对值≥0.69作为判定基因表达差异标准,筛选出各药物作用的早期反应基因。结果显示,4个抗肿瘤药物均诱发强烈的细胞基因表达改变,其中紫杉醇和高三尖杉酯碱诱导HL-60和QGY7703细胞的反应基因最多。
3.应用Genmapp分析软件对药物反应基因进行聚类分析和药物反应通路分析,发现4个抗肿瘤药物可调控细胞内许多反应通路、功能基因或细胞组分。例如,紫杉醇可调控mRNA加工过程;高三尖杉酯碱可抑制蛋白酶体组分26S亚单位PSMC3、PSMD2和PSMD11表达;喜树碱影响G蛋白信号通路中多条基因的表达;放线菌素D可调节丝裂素活化的蛋白激酶(MAPK)通路、TNF-NFkB通路等,这些结果说明药物起始靶点与许多已知的调控细胞增殖和凋亡的通路相连,而这些通路可以很好地解释药物的作用。此外,细胞毒抗肿瘤药物还调节了一群组织特异的基因,如喜树碱影响一些内脏器官、内分泌系统和中枢神经系统的基因,放线菌素D也能调节一些正常胚胎干细胞基因表达,提示这些基因的改变可能与其毒性有关。
4.应用microsoft office Access软件对药物反应基因和药物反应通路进行统计分析,发现细胞毒抗肿瘤药物之间在转录水平上可共享某些同向改变的基因或反应通路,其中2个药物之间最多,共用反应基因和通路在3个药物之间,甚至全部4个药物都可见到,例如全部4个药物在HL-60和QGY7703细胞中分别有17条和40条共用反应基因以及7条和9条反应通路。在2个细胞系中,4个抗肿瘤药物也有6条的共同反应基因,它们是GRN、MLL3、PSAP、ERP29、LRPAP1、ST3GAL4 ,也共享肌动蛋白-细胞骨架调节通路( Regulation of Actin Cytoskeleton)和粘着斑功能调节通路(Focal adhesion)。这些结果提示拓朴酶抑制、微管、核糖体和DNA模板干扰在转录水平上具有共同的下游作用,通过这些下游通路或分子可能最终导致相似的表型变化。
5.共用反应基因在功能上可分为:生长增殖相关基因、凋亡相关基因、糖代谢相关基因及其它功能基因4类,这些共用基因所编码的蛋白生物功能多样,分别参与了细胞周期调节,DNA、RNA和蛋白质合成,能量代谢,泛素(类泛素)蛋白酶体依赖的蛋白质降解,信号传导通路、抗凋亡或促凋亡等重要的细胞生命活动以及内质网以及线粒体等构成。(1)抗肿瘤药物能显著抑制细胞表达细胞周期相关基因,如蛋白磷酸酶2A、翻译延长因子1、HnRNPB1等,这些基因参与了细胞有丝分裂、核酸合成、DNA复制和修复以及RNA加工和蛋白质合成;此外,还能抑制具有促增殖作用的肿瘤相关蛋白基因(如GRN、MLL3 )的表达,表明抗肿瘤药物可通过调节细胞增殖基因的表达,来抑制肿瘤细胞的生长甚至导致死亡;(2)抗肿瘤药物能诱导细胞表达促凋亡基因PMAIP1以及抑制抗凋亡基因PSAP的表达,说明调节凋亡相关基因的表达也是抗肿瘤药物诱导细胞凋亡的一个重要机制;(3)抗肿瘤药物能调节内质网以及线粒体构成诱导细胞凋亡,如ERP29是内质网腔膜蛋白,作为氧化还原酶和分子伴侣,参与蛋白质折叠和运输。ARF5是囊泡转运包被复合体形成所必需,参与了内质网和高尔基体间的蛋白转运。抗肿瘤药物抑制两者表达,可能阻断蛋白在内质网与高尔基体的转运,从而诱导内质网应激而使细胞凋亡。mitochondrial ribosomal protein S18A是线粒体核糖体组分,衰老细胞线粒体核糖体蛋白合成的准确性下降。isocitrate dehydrogenase 2参与三羧酸循环, SLC25A6为ADP/ATP转运子,因此,抑制参与能量代谢的基因表达可能会引起线粒体呼吸链功能缺陷或者ATP耗竭而导致细胞死亡。(4)抗肿瘤药物能抑制泛素(类泛素)蛋白体蛋白降解系统相关基因PSMC3、VCP和SAE1表达。PSMC3是蛋白酶体26S蛋白酶体19S调节亚基的核心组分,VCP/p97是一种AAA ATP酶,在辅助因子协助下,可使错误折叠的蛋白从内质网膜脱位进入胞浆,经泛素蛋白酶体系统降解。SAE1/SAE2是小泛素相关修饰剂(Small Ubiquitin-related Modifier, SUMO-1)活化酶,参与类泛素途径调节,由于蛋白酶体参与细胞周期、增殖和凋亡调节,因此抑制泛素蛋白酶体途径可能也是抗肿瘤药物的一个重要机制(5)抗肿瘤药物能抑制唾液酸转移酶4C和糖苷转运子SLC35D1表达,而这二者均在多种肿瘤中表达增加,参与肿瘤的转移。因此,抑制蛋白糖基化可能与药物的抗转移作用有关。
文献检索发现,有些共用基因已作为抗肿瘤药物筛选靶点,例如,蛋白酶体抑制剂是一种新型的抗肿瘤药物,已进入临床实验;基于蛋白磷酸酶PP2A分子模建,目前已设计了系列蛋白磷酸酶抑制剂,体内外结果显示蛋白磷酸酶抑制剂具有广谱的抗肿瘤效果。此外,唾液酸转移酶抑制剂也能显著抑制肿瘤生长和抗肿瘤转移的作用。这些结果提示通过进一步的研究有望从这些共用反应基因中发现一些新药靶。此外,以共用反应基因为基础制成芯片,可以建立针对基因群的多靶标高效筛药和评价模型。
综上所述,应用基因芯片技术和多个细胞毒抗肿瘤药物寻找得到的共同反应基因对于阐明抗肿瘤药物作用机制和发现新药靶具有重要的意义。
Gene expression microarray is a newly-developed biotechnology in recent years, permits the simultaneous and rapid measurement of the expression levels of thousands of genes, and allows the analysis of molecular events of drug response and potentially identifying novel drug targets. It is also suitable for high-throughput drug screening with multi-targets and evaluation of drug activity and toxicology.
Cytotoxic anticancan drugs inhibit cell proliferation, disturb cellcycle and induce cell apoptosis after binding to diverse molecular targets. The growing numbers of documents have shown the key regulatory molecules and cellular pathways control cell apoptosis and the cell cycle. However, the connectivity of known mechanisms with other regulators or pathways remains unclear. Although the anticancer drugs have diverse initial molecular targets, cellular phenotypic outcomes are similar for different drug treatments. Therefore, it appears reasonable to assume that some of genetic alternations among the different drug responses might be likely shared as common key effects of the drug-target interactions. Analysis of these intersection genes may provide new insights into the molecular mechanisms of the antitumor agents and discover new potential target molecules for the development of novel antineoplastic drugs.
This study was designed to test above hopothesis with microarray analysis of the sensitive early gene expression profiles in QGY-7703 hepatoma cells and HL-60 leukemia cells stimulated with the chosen representative drug paclitaxel (Taxol), dactinomycin (ACD), camptothecin (CPT), and homoharringtonine (HHRT), based on their identical cell growth inhibition actions. Drug response genes were then clustered and cellular pathways were analyzed by Genmapp anlysis software and Microsoft Ascess software. Realtime PCR was also conducted for some interest genes to comfirm our microarray results. Results are shown as follows:
1. To analyze the sensitive early gene expression profiles, cell phenotypes of growth inhibition, apoptosis and cell cycle in response to drug treatment were determined by MTT assay and flow cytometry. 50% of inhibitory concentration (IC50) of Taxol, HHRT, CPT and ACD was 0.025、320、79.85、2.67、μmol/mL and 2.26、52.77、19.09、0.48、μmol/mL respectively in HL-60 leukemia cells and QGY-7703 hepatoma cells after 24 cluture. Morphological changes were not observed by microscope after 1 h incubation of drugs with both cell lines, wheras the decreased cell numbers and cell pieces were seen in both cells treated with the chosen drugs for 24 h. Flow cytometry results showed that treatment of paclitaxel and homoharringtonine for 24 h markedly accelerated leukaemia and heptoma cells through G1/S interface resulting G2/M arrest and apoptotic cell death. These results are consitent with the reported actions of these drugs.
2. Total RNA was isolated from drug treated cells and from control (equal volume of drug solvent) cells after 1 h incubation. Labeling of cDNA, microarray hybrization and fluorescence detection was performed as standard protocol of Unite Gene Corpoation. Genes were identified as differentially expressed if the absolute value of the natural logarithm of the average ratios of three independent experiments was > 0.69. The number of regulated genes differed significantly among drugs, with paclitaxel and homoharringtonine causing the strongest responses in HL-60 and QGY7703 cells respectively.
3. Genmapp software was used to gene cluster and pathway analysis of the drug response genes. A number of cellular pathways, functional molecules and cellular components were found as classied by the used software. For example, paclitaxel reglulated mRNA processing; homoharringtonine inhibited the expression of protesome component PSMC3、PSMD2 and PSMD11;camptothecin affected G protein signaling pathways and dactinomycin modulated MAPK pathways and TNF-NFkB pathways. These results indicate initial tagets are connected with many known pathways responsible for cell proliferation and apoptosis, which may clearly explain actions of these antitumor agents. In addition, cytotoxic agents also regulated several groups of genes specific to internal organs, endocrine and CNS as well as embryo stem cells, suggesting these genes may be related to the cytotoxicities of antitumor drugs.
4. Microsoft office Access software was used to sort common drug response genes and common cellular pathways. Our results demonstrated that antitumor agents shared many cellular pathways and genes with the same regulated direction in the transcriptional levels. The largest overlap was found between two agents. Common response genes and pathways were also identified among three drugs, even in all four four drugs. For exaple, 17 and 40 common response genes and 7 and 9 pathways were obtained in HL-60 cell and in QGY7703 cells respectively. There were 6 common response genes including granulin (GRN)、myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3)、prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy, PSAP)、endoplasmic reticulum protein 29 (ERP29)、low density lipoprotein receptor-related protein associated protein 1 (LRPAP1)、ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) and 2 pathways (regulation of Actin Cytoskeleton and focal adhesion) shared by all drugs in both cell lines. These results provided evidence for common down stream effects of topoisomerase inhibition and microtubular dynamics or ribosome function interference as well as DNA template corruption, which may result in final similar phenotypic outcomes.
5. The common response genes may be classied into four categrories including growth and proliferation related genes, apoptosis related genes, carbohydrate metabolism related genes and unkown function genes. The biochemical functions of intersection response genes are diverse and include cell cycle regulators, proapoptotic and antiapoptotic factors, growth factors, signal mediators, metabolic regulators, proteases and glycosyltransferases, and proteins of miscellaneous or unknown function. (1) Chemotherapeutical agents-treated cells decreased expression of a cluster of genes involved in cell proliferation. For example, a broad range of genes is associated with cell cycle and mitosis (QSCN6,PPP2R1A),gene transcription [general transcription factor IIIC (GTF3C5) , activating transcription factor 5 (ATF5)],RNA processing[heterogeneous nuclear ribonucleoprotein A/B (HNRPAB),PRP6 pre-mRNA processing factor 6 homolog (S. cerevisiae) (PRPF6),nucleolin (NCL)] and protein synthesis[eukaryotic translation elongation factor 1 delta EEF1D, Eukaryotic translation initiation factor 4A, isoform 1,EIF4A1]。In addition, tumor associated genes granulin、myeloid/lymphoid or mixed-lineage leukemia 3 were also inhibited upon multiple treatment.The alterations in these specific genes functionally related to cell proliferation might determine tumor cell growth arrest, even ultimate cell death, correlating well with the cytotoxic agents-mediated cytotoxicity. (2) Treatment of antitumor agents induced proapototic genes phorbol-12-myristate- 13-acetate-induced protein 1(PMAIP1) and decreased antipoptotic gene prosaposin (PSAP), suggesting the involvement of cell apoptotic mechanisms in these drug actions. (3) Regulation of endoplasmic reticulum (ER) and mitochondrial components was shown by drug treatment, for example, ERP29, a disulfide isomerase (PDI)-like proteina, is involved in oxidative stress and regulates protein unfolding and facilitates the transport of secretory proteins. ARF5 is a member of ADP-ribosylation factors necessary for the formation of clathrin- and coatomer protein (COP) I-coated vesiclesare, and isivovled in protein transport between ER and Golgi compartments. Inhibition of tese ER associated genes by antitumor agents may result in ER stress leading to cell apoptosis. Moreover, the expression of mitochondrial components was also observed in response to antitumor drugs. Isocitrate dehydrogenase 2 participates in tricarboxylic acid cycle and solute carrier family 25 (SLC25A6) is adenine nucleotide translocator. Thus the decresed energy production and transport may be responsible for cytotoxic agents-mediated apoptosis. (4) Cells exposed to cytotoxic agents resulted in the down regulation of a cluster of Ubiquitin/proteasome degradation pathways (UPDP) related genes, such as PSMC3, VCP and SAE1. PSMC3 is a core component of the 19S regulatory particle of the 26S proteasome, VCP helps dislocateing misfolded proteins from the ER membrane into the cytosol for proteasomal degradation, and SAE1/SAE2 is the activating enzyme E1 of small ubiquitin-related modifier (SUMO) responsible for post-translational modifications of proteins. Since ubiquitin/proteasome degradation pathways have function in regulating cell proliferation and apoptosis, the decraesed expression of UPDP related genes may be contributed to cytotoxic effects of antitumor drugs. (5) Genes functioned in the intracellular glycoprotein glycosylation pathway such as sialyltransferase 4C and SLC35D1expression were reduced in drug responses, which may imply the modulating effects of these antitumor drugs on the tumor invasion and metastasis due to the involvement of specific carbohydrate structures in cellular recognition processes and cancer metastasis.
6. Some common response genes have been documented to be the targets for drug design and screening. For example, proteasome becomes a new target for novel drug therapy. Based on a modelled structure of PP2A, a new class of protein phosphatase inhibitors, cantharimides, are designed and exhibit broad-spectrum anti-cancer activity. Suppression of sialyltransferases by chemicals has been shown to reduce the potentiality of growth and metastasis of various tumors. These suggest that another unexplored drug targets might be discovered from these intersection genes. In addition, these common response genes may also be used for screening and evaluation of antitumor agents as made of multiple-target microarray chip.
Taken togethe, determination of the common response genes with microarrays and multiple tool agents is of great value for our deciphering complex regulatory pathways and discovering new potential target molecules.
引文
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