草酰腙缩乙醛酸三环己基锡酯诱导大细胞肺癌H460细胞凋亡及其机制研究
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摘要
研究背景和研究目的
据WHO统计,肺癌是当今世界上最大的癌患,其死亡率居于各种恶性肿瘤之首位,中国也已成为世界上第一肺癌大国。按组织学类型可将肺癌分为两大类:小细胞肺癌和非小细胞肺癌。非小细胞肺癌(NSCLC)占肺癌的85%以上。由于非小细胞肺癌转移后禁忌手术,且对放疗很不敏感,因此,对非小细胞肺癌化疗药物的开发与研究尤为重要。
肺癌化疗的常用药物有如下五类:直接破坏DNA药物、插入DNA模板药物、影响核酸合成药物、抑制微管装配药物、影响蛋白质合成药物五类。尽管上述药物可有效诱导肺癌细胞凋亡,但是毒副作用较大且容易引起耐药,从而导致化疗失败。因此,开发毒性小、副作用少、能克服多药耐药和有效启动肺癌细胞凋亡信号传导途径的药物是目前亟待解决的问题。
本研究选用H460、A549、H1792、H226、WI38细胞为实验细胞研究有机锡小分子化合物抑制NSCLC细胞生长的机制,原因在于:1)此四种NSCLC细胞分别来源于腺癌、大细胞癌、鳞癌,涵盖了非小细胞肺癌的三种亚型;2)此四种NSCLC细胞均可单独作为非小细胞肺癌研究领域里的模式细胞,用于细胞水平的检测;3)迄今对有机锡小分子化合物诱导NSCLC细胞凋亡的机制研究还比较少;4) WI38细胞是从人胚肺组织建立的二倍体成纤维细胞系,作为正常细胞经常被用作肺癌研究的对照细胞。因此可用以上细胞系筛选小分子化合物,并对其机制做深入研究,为NSCLC化疗药物的开发提供理论依据。
化学遗传学(chemical genetics)是利用化学工具来研究生物体系的一种新兴手段,其可以在以下两个重要方面促进新药开发:一方面是鉴定出在某种疾病形成过程中起重要作用的基因或蛋白质,从而为新药筛选提供靶点;另外一方面是发现特异性作用于某个基因或蛋白质的小分子化合物,从而为新药开发提供先导化合物。化学遗传学应用的关键之一是要有大量的可供筛选的不同结构的化合物,新兴的组合化学很好地解决了这一难题,在同一个化学反应体系中加入不同结构单元,利用这些结构单元的排列组合,就能够系统地合成大量化合物。近年来,化学遗传学在细胞凋亡研究领域得到了广泛的应用:美国哈佛大学医学院袁钧瑛教授带领课题组进行了“用小分子研究细胞死亡过程中的信号传导:从Bcl-2到程序性坏死”的研究;美国密执安大学癌症中心王少萌教授开展了“合理设计小分子拮抗剂来调控关键性细胞坏死和蛋白质-蛋白质间的相互作用”的研究;另外,美国国立癌症研究所(NCI)早在1997年启动了一项新计划,将化学遗传学应用到癌症治疗研究中,即利用细胞渗透性的小分子化合物触发肿瘤细胞死亡。这充分说明化学遗传学方法为细胞凋亡研究提供了一个可靠的平台,基于此平台,利用具有一定生物活性的小分子化合物作为研究手段来研究细胞凋亡过程中的信号传导,优势明显:小分子化合物易于进入细胞,易于引起细胞表型变化,其作用具有剂量依赖性,适用于多蛋白家族,不会引起细胞的适应性变化等,另外,这些小分子化合物还往往可以作为“先导化合物”,进而指导新药的开发研究。
近几年来,本课题组一直在从事有机小分子化合物库的构建工作,目前,库中已包含了60余种有机锡小分子化合物。研究显示,某些有机锡化合物具有抗肿瘤活性,这已经在MCF-7(乳腺癌)、WiDr(结肠癌)、P388(小鼠白血病)等抗癌活性研究中得到证实。这些研究大都停留在生长抑制实验阶段,部分研究显示有机锡化合物可以阻滞肿瘤细胞周期,但对其抑制生长、诱导凋亡的分子机制仍然不清楚。因此,探讨有机锡小分子化合诱导肿瘤细胞凋亡分子机制具有重要意义。
基于以上考量,本课题研究目的是:利用化学遗传学这一新兴技术平台,以非小细胞肺癌H460、A549、H1792和H226细胞为研究对象,对草酰腙缩乙醛酸三环己基锡酯(OGTCT)的体外抗肿瘤活性进行研究,进而探讨其诱导H460细胞凋亡的分子机制,为新型肺癌化疗药物的开发提供更加完善的理论依据。
研究内容
1.鉴定OGTCT对NSCLC细胞、耐药细胞H460/TaxR及人胚肺二倍体成纤维细胞WI38的生长抑制作用。
2.探讨OGTCT对H460细胞的凋亡诱导作用。
3.分析OGTCT对H460细胞周期分布及周期调控相关蛋白p53、p21、PCNA表达的影响。
4.分析OGTCT诱导H460细胞凋亡过程中,凋亡相关调控因子的变化及重要凋亡通路的激活情况。
研究方法
1细胞生长抑制检测:
1.1 MTT法检测细胞存活率,由此对OGTCT进行初步筛选;
1.2选择目前临床上常用抗肿瘤药物紫杉醇(Paclitaxel)为对照药物,用MTT法检测其对肿瘤细胞和正常细胞的生长抑制情况,并与OGTCT做比较。
2.细胞凋亡检测方法:
2.1倒置相差显微镜观察细胞形态变化及凋亡小体;
2.2 Hoechst 33258荧光染色,并结合荧光显微镜观察细胞核形态变化;
2.3乳酸脱氢酶(LDH)试剂盒检测细胞是否出现坏死;
2.4台盼蓝拒染法进一步检测细胞是否出现坏死;
2.5用Nucleosome ELISA试剂盒检测细胞凋亡率。
3.细胞周期及其相关调控蛋白表达变化的检测:
3.1流式细胞技术检测OGTCT对细胞周期分布的影响;
3.2酶联免疫吸附测定技术(ELISA)检测与细胞周期调控相关重要蛋白p53和p21的表达变化;
3.3免疫荧光结合激光扫描共聚焦显微镜检测增殖细胞核抗原PCNA的表达及分布;
3.4 Western blot检测p53蛋白的表达。
4.荧光探针结合激光扫描共聚焦技术检测细胞中钙离子浓度变化。
5.检测OGTCT对ICE家族蛋白酶成员激活情况,方法如下:
5.1用ApoAlert caspase-3,-8比色检测试剂盒分析caspase-3,-8酶活性变化;
5.2用ApoAlert caspase 9/6荧光检测试剂盒分析caspase-9/6酶活性变化;
5.3用caspase抑制剂检测caspase-3,8,6,9对OGTCT诱导细胞凋亡的影响。
6.免疫荧光结合激光扫描共聚焦显微镜检测抗凋亡蛋白Bcl-2的分布及表达。
7.Western blot检测细胞色素C(Cyto-C)的表达变化。
8.酶联免疫(ELISA)试剂盒检测Fas/FasL的表达变化。
研究结果
1.OGTCT抗肿瘤活性检测结果(MTT法检测细胞存活率)
1.1 OGTCT对NSCLC细胞、耐药细胞及人胚肺成纤维细胞WI38存活率的影响:在48h内,OGTCT以剂量依赖的方式,显著降低了NSCLC细胞(A549、H1792、H226、H460)和耐药细胞H460/TaxR的存活率;而相同浓度与作用时间下,WI38细胞的存活率远远高于NSCLC细胞和H460/TaxR耐药细胞的存活率。
1.2阳性药物对比检测结果:
在48h内,紫杉醇以剂量依赖的方式,降低了NSCLC细胞、H460/TaxR细胞及WI38细胞的存活率。但与OGTCT相比,紫杉醇对WI38有显著生长抑制作用,而对耐药细胞H460/TaxR的生长抑制作用极不显著。
结果表明:OGTCT与紫杉醇相比有2个明显优势,OGTCT对正常细胞WI38的毒性较小,而对耐药细胞H460/TaxR的生长抑制作用尤为明显。
2.OGTCT诱导H460细胞凋亡的研究结果
2.1倒置相差显微镜观察细胞形态变化:
0.1%DMSO溶剂对照组与对照组相比,细胞形态无明显变化;0.56μM/L的OGTCT处理H460细胞48h后发现,胞体皱缩,胞浆内空泡明显,核固缩,折光性减弱,细胞膜出芽且有脱离现象,凋亡小体形成,细胞呈现出典型的凋亡形态学特征。
2.2 H33258染色结合荧光显微镜检测细胞核片段化结果:
DMSO溶剂对照组与对照组相比,细胞核形态无明显变化,均呈现均匀亮蓝色;0.56μM/L的OGTCT处理H460细胞48h后发现,细胞核固缩、染色质凝集且有明显边缘化、出现细胞核碎片,凋亡小体清晰可见,呈现典型的凋亡形态学特征。
2.3 LDH活性检测结果:
OGTCT 0.14、0.28μM/L处理H460细胞48h,与对照组细胞相比,培养液中酶活性变化不显著(p＞0.05,n=3);而OGTCT 0.56μM/L处理H460细胞48h,与对照组相比,培养液中酶活性显著升高(p＜0.01,n=3)。
2.4台盼蓝拒染实验检测表明:OGTCT 0.14和0.28μM/L处理H460细胞48h,细胞坏死率极低;而OGTCT0.56μM/L处理H460细胞48h,细胞坏死率显著增加(p＜0.01,n=3)。
2.5 Nucleosome ELISA检测发现:
用0.14、0.28、0.56μM/L的OGTCT分别处理H460细胞6h、12h、24h、48h,与对照组细胞相比,OGTCT可有效诱导H460细胞凋亡,并呈明显的剂量时间依赖性。
3.OGTCT对H460细胞周期的影响及相关分子机制研究结果
3.1 OGTCT对H460细胞周期分布的影响
分析细胞DNA变化发现,OGTCT作用24-48 h后可使H460细胞周期阻滞在G_0/G_1期,不能进入S期,并呈时间依赖性,但对M期的作用不明显。
3.2检测p53/p21蛋白表达水平
●p53、p21 ELISA检测,发现:
OGTCT可使H460细胞的p53和p21蛋白表达升高,随着作用时间的延长,表达水平显著升高,在24h时表达水平最高,此后表达水平逐步降低。
●Western Blot检测发现:OGTCT可使H460细胞中p53的表达显著升高(p＜0.01,n=3)。
3.3免疫荧光结合激光共聚焦显微技术检测PCNA蛋白表达及分布:
0.56μM/L的OGTCT作用于H460细胞48h,与对照组相比,可使PCNA的表达显著降低(p＜0.05,n=3)。
4.OGTCT诱导H460细胞凋亡的相关分子机制研究结果
4.1免疫荧光探针结合激光共聚焦显微技术检测H460细胞中钙离子变化及分布,发现:OGTCT 0.56μM/L处理48h后,可使H460细胞内的钙离子浓度显著增强(p＜0.001,n=3)。
4.2免疫细胞化学法结合激光共聚焦显微技术检测H460细胞中Bcl-2表达发现:用0.56μM/L的OGTCT处理H460细胞48h后,与对照组相比,Bcl-2表达水平显著降低(p＜0.01,n=3)。
4.3 Caspases活性检测结果:
●ApoAlert caspase-3比色检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,405 nm处光吸收值由0.246上升到0.982(p＜0.001);
●ApoAlert caspase-8比色检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,405 nm处光吸收值由0.132上升到0.787(p＜0.001);
●ApoAlert caspase-9/6荧光检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,caspase-9在460nm处的荧光强度由24.45上升到185.44,其相对活性上调了6.58倍(p＜0.001);而caspase-6在460nm处的荧光强度由18.36上升到33.55,其相对活性上调了0.82倍(p＜0.01);结果表明,在H460细胞凋亡过程中,OGTCT同时激活了caspase-3,-8,-9,6。
●Caspases抑制剂对OGTCT诱导H460细胞凋亡的影响
加caspases抑制剂后,凋亡ELISA试剂盒检测细胞凋亡率,发现:与OGTCT处理的细胞相比,pan-caspase inhibitor(Z-VAD-FMK)完全阻滞了OGTCT诱导的凋亡,caspase-3 inhibitor(Z-DEVD-FMK)、caspase-8 inhibitor(Z-IETD-FMK)和caspase-9 inhibitor(Z-LEHD-FMK)显著逆转了OGTCT诱导的凋亡,而caspase-6 inhibitor(Z-VEID-FMK)显著抑制了OGTCT诱导的凋亡,但程度稍弱。
4.4 Western blotting检测Cyto-C的表达
Western bloting检测发现:0.56μM/L OGTCT处理H460细胞48h后,可使胞浆中Cyto-C的水平显著增加(p＜0.01,n=3)。
4.5 OGTCT处理的H460细胞中Fas/FasL的表达
4.5.1 Fas/FasL ELISA assay检测结果
ELISA assay检测发现,OGTCT可使H460细胞的Fas蛋白表达升高,随着作用时间的延长,表达水平显著升高,在24h时表达水平最高,此后表达水平有所降低;而mFasL与sFasL的表达水平未发生显著变化(p＞0.05,n=3)。
4.5.2 Fas抑制剂ZB4对OGTCT诱导H460细胞凋亡的影响
用抗Fas抗体ZB4预先处理H460细胞一定时间后,再用0.56μM/L的OGTCT处理48h,MTT与nucleosome ELISA检测发现,ZB4显著逆转了OGTCT诱导的凋亡与增殖抑制。
结论:
1.OGTCT以浓度依赖的方式,显著降低NSCLC和H460/TaxR细胞的存活率。OGTCT与紫杉醇相比优势明显:对正常细胞WI38的毒性极小,且能有效克服肿瘤细胞的多药耐药性。
2.OGTCT能有效诱导H460细胞凋亡。OGTCT可能通过上调p53和p21蛋白水平、降低PCNA表达的方式将细胞周期阻滞于G_0/G_1期、下调Bcl-2蛋白表达水平、导致钙离子和Cyto-C释放、激活Fas蛋白、激活caspase-8,-9,-3,-6而发挥其凋亡诱导作用。
3.Fas信号通路和线粒体信号通路可能共同参与了OGTCT诱导的H460细胞凋亡过程。
BACKGROUND AND OBJECTIVE
According to the statistics of WHO,lung cancer is the most dangerous cancer all over the world,which mortality tops the list of all kinds of tumor.And china has become a country that has the largest number of lung cancer patient.According to histological type,lung cancer can be divided two types:small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC).NSCLC accounts for more than 85%of lung cancer.Because NSCLC become not suitable for surgery after metastasis,and not sensitive to radiotherapy,therefore,it is particularly important for development and research of chemotherapeutic drugs.
The classifications of frequently-used drugs for lung cancer chemotherapy are five categories as bellows:directly damage DNA drugs,inserted DNA template drugs,affecting nucleic acid synthesis drugs,repressing microtubule assembly drugs and impacting protein synthesis drugs.Although the above drugs can induce apoptosis of lung cancer cells,but strong toxicity side effect and easily leading to drug resistance cause frequently failure of chemotherapy.Therefore developments of drug which has minor toxicity,less side effect,ability to overcome multi-drug resistance,and able to start apoptosis signal transduction pathway,are the urgent problems to be settled.
In this study,reasons that H460,A549,H1792,H226 and WI38 were selected as experimental cells to investigate mechanisms of growth-inhibition in NSCLC cells are as follows:1) These four kinds of NSCLC cells were derived from adenocarcinoma,large cell carcinoma,squamous cell carcinoma,covering three subtypes of NSCLC;2) These four kinds of NSCLC cells can be used seperately in detection on the cellular level in the field of lung cancer study as model cell;3) To date,investigation of mechanism of small molecular organotin-induced apoptosis in NSCLC cells remains relative few.4) WI38 is a diploid fibroblast cell line which derived from human embryonic lung tissue and frequently used as control cell in the study of lung cancer.Thus the above cell lines are available for screening small molecular compound,and make in-depth study of its mechanism aim to provide a theoretical basis for development of chemotherapeutic drug of NSCLC.
Chemical genetics is a new means that utilizing chemical tools for investigating biological system,and it could promote the developments of new drugs in the following two important aspects:on the one hand,identifying gene or protein which plays an important role in formation of a disease,so as to provide targets for the new drug screening;The other hand is finding small molecular compound which act with a gene or protein,so as to provide lead compounds for developing new drugs.One of the key of chemical genetics researches is to have a large number of compounds with different structures for screening.The emerging combined chemistry is the core technology accessing to numerous small molecular compounds.The principle of the combined chemistry is to add different structural units into the same chemical reaction system,and to synthesize systematically numerous compounds utilizing permutations and combinations of these structural units.
Over the years,the chemical genetics has been widely applied in the field of cell apoptosis research:Professor Yuan Junying from Harvard Medical School lead the research team carrying out the investigation named as "using small molecules to study on the signal transduction in the process of cell death:from Bcl-2 to the programmed necrosis";Professor Wang Shaomeng from the cancer research center of U.S.Michigan University launched study named as "rationally design small molecular antagonist to regulate key cell necrosis and protein-protein interactions"; Additionally,the U.S.National Cancer Institute(NCI) launched a new plan in 1997, the application chemical genetics to research of cancer treatment,namely the use of cell permeable small molecular compound to trigger death of tumor cell.The above examples fully prove that chemical genetics method provides a reliable platform for research of cell apoptosis.In this platform,utilizing small molecular compound with a certain biological activity as a tool to investigate signal transduction in the process of cell apoptosis has many advantages:small molecular compounds to enter cells easily,easy to cause changes in the cellular phenotype,its role in a dose-dependent relationship,applicable to multi-proteins family as well as not cause adaptive changes in cells,etc.In addition,these small molecular compounds often serve as "lead compounds" to further drug development and research.
In recent years,our team has been engaged in construction the "library" of small organic compounds,at present,the library already contains more than 60 species of small molecular organotin.Studies have shown that some organotin have anti-tumor activities which are already confirmed in MCF-7,WiDr and P388 cells. Most of these studies still remained in the experimental phase of growth inhibition. Some studies indicated that organotin could block cell cycle of tumor cell.However, the molecular mechanisms of inhibiting growth and inducing apoptosis remain unclear.So investigation of molecular mechanism of apoptosis induced by small molecular organotin is of great significance.
Based on the above considerations,aims of this research are as follows: applying chemical genetics methods,to investigate In vitro anti-tumor activity of OGTCT in H460,A549,H1792,H226 cells,and then,to explore molecular mechanisms of OGTCT-induced apoptosis in H460 cells.This study can also provide a more complete theoretical basis for the development of new chemotherapeutic agents for the treatment of lung cancer.
STUDY CONTENTS
1 Identification growth inhibition to NSCLC,H460/TaxR and WI38 of OGTCT.
2 Exploration of apoptosis induction in H460 cells by OGTCT.
3 Analysis of the influences of OGTCT on cycle distribution and p53,p21,PCNA protein levels in H460 cells.
4 To analyze changes of apoptosis-related factors and the important apoptosis pathways activated by OGTCT in H460 cells.
METHODS
1.Cell growth inhibition Detection:
1.1 MTT assay for cell viability,by which OGTCT was screened initially;
1.2 Select the current clinically used anticancer drug(Paclitaxel) as control drug, using MTT assay for growth inhibition to tumor cells and normal cells,and make comparison to OGTCT.
2.Methods for apoptosis identification:
2.1 Observation of cell morphological changes and apoptosis bodies by Phase Contrast Microscope;
2.2 Observation of nuclear morphological changes by Hoechst33258 staining combined with fluorescence microscopy;
2.3 LDH(Lactate dehydrogenase) assay to determine cell toxicity induced by OGTCT;
2.4 Trypan blue dye exclusion assay to confirm whether the cells treated by OGTCT undergo necrosis;
2.5 Detection of apoptosis rates by Nucleosome ELISA assay kits.
3.Detection of changes in cell cycle distribution and related regulatory proteins expression:
3.1 Analysis of the changes in cell cycle distribution by Flow cytometry;
3.2 ELISA methods for analysis of expression changes in p53/p21 proteins associated to regulation of cell cycle;
3.3 Detection of expression and distribution of PCNA by immuno-fluorescence combined with Laser Scanning Confocal Microscopy(LSCM);
3.4 Confirmation of the changes in p53 expression by Western blot.
4.Detection changes of Ca~(2+) concentration in H460 cells by immuno-fluorescence probe combined with LSCM.
5.Methods for determining whether caspases are activated by OGTCT:
5.1 Analyzing caspase-3,-8 activities by ApoAlert caspase-3,-8 colorimetric assay kits;
5.2 Analyzing caspase-9/6 activity by ApoAlert caspase-9/6 fluoremetric assay kits;
5.3 Identification the influences of caspase-3,-8,-6,-9 on apoptosis induced by OGTCT with caspase inhibitors.
6.To analyze distribution and expression of anti-apoptotic protein Bcl-2 by Immuno-fluorescence combined with LSCM.
7.Detection expression changes of Cytochrome C(Cyto-C) by Western blot.
8.Detection changes of expression in Fas/FasL proteins by ELISA kits.
RESULTS
1.Detected results of anti-tumor activity of OGTCT(MTT assay for cell viability)
1.1 Influences of OGTCT on viability of NSCLC,H460/TaxR and WI38 cell:
Within 48h,OGTCT reduced significantly viabilities of NSCLC cells (H460,A549,H1792,H226) and drug-resistant cell H460/TaxR in a dose-dependent manner;but at the same concentration and time,viabilities of WI38 cell were much higher than that of NSCLC and H460/TaxR cells.
1.2 Results of positive drug comparison test:
Within 48h,paclitaxel reduced viabilities of NSCLC,H460/TaxR and WI38 cells in a dose-dependent manner.But compared to OGTCT,paclitaxel inhibited obviously growth of WI38 cells,and its growth inhibition to H460/TaxR was not extremely significant.
The results indicated:OGTCT had two advantages compared with paclitaxel, OGTCT had a minor toxicity to normal cell WI38,and its growth inhibition to drug-resistant cell H460/TaxR was particularly obvious.
2.Results of apoptosis induced by OGTCT in H460 cells
2.1 Cell morphological changes observed under Phase Contrast Microscope: Treated with 0.1%DMSO,cell morphology has no obvious change;Exposed to 0.56μM/L OGTCT for 48h,morphological changes are as follows:cell body shrinkage,obvious cytoplasmic vacuolization,karyopyknosis,refractive index decreased,membrane blebbing,formed apoptotic bodies,and cells showed typical morphological characteristics of apoptosis.
2.2 Results of detection cell nuclear fragmentation by Hoechst33258 staining combined with Fluorescence Microscopy:
There had no obvious change in cell nuclear morphology between DMSO group and control group,and cell nuclei showed a uniform light blue;When H460 cells were treated with 0.56μM/L OGTCT for 48h,typical apoptotic features such as cell shrinkage,chromatin condensation and marginalization,emerging nuclei fragmentation,apoptotic bodies clearly visible occurred.
2.3 Detected results of activity of LDH:
When H460 cells were respectively incubated in 0.14,0.28μM/L OGTCT for 48h,compared with the control group,there was no significant change of activity of LDH in culture medium(p>0.05,n=3);While LDH activity in culture medium containing 0.56μM/L OGTCT was significantly enhanced(p<0.01,n=3).
2.4 Results of Trypan blue dye exclusion assay:
Cell necrosis rate of 0.14 and 0.28μM/L test groups were extremely low, compared with the control group;While the necrosis rate of 0.56μM/L test group increased obviously(p<0.01,n=3).
2.5 Results of nucleosome ELISA assay:
When H460 cells were respectively treated with 0.14,0.28,0.56μM/L OGTCT for 6,12,24 and 48h,compared with the control group,OGTCT induced effectively apoptosis of H460 cells in dose- and time-dependent manners.
3.Influence of OGTCT on H460 cell cycle and assay of related proteins
3.1 Influence of OGTCT on H460 cell cycle distribution
After changes of DNA were analyzed,we found that cell cycles of H460 cells witch treated by OGTCT for 24-48h were blocked at G_0/G_1 phase in a time-dependent manner.Furthermore,H460 cells could not enter S phase. However,OGTCT has a minor effect on M phase.
3.2 Distribution and expression of p53/p21 protein
·p53/p21 ELISA assay
The levels of p53 and p21 proteins were up-regulated by OGTCT and increased significantly with the extension of time,reached the maximum at 24h,since then, the levels of p53 and p21 gradually reduced.
·Western blot assay confirmed that OGTCT significantly up-regulated the expression of p53 protein(p<0.01,n=3).
3.3 Immuno-fluorescence combined with LSCM detection for expression and distribution of PCNA protein in H460 cells:
Expression level of PCNA in H460 cells was significantly reduced after treated with 0.56μM/L OGTCT for 48h(p<0.05,n=3).
4.Research findings of OGTCT-induced apoptosis in H460 cells and its related molecular mechanisms
4.1 Detection changes and distribution of Ca~(2+) in H460 cells by immuno-fluorescence probe combined with LSCM:
Calcium concentration was significantly enhanced in H460 cells treated with 0.56μM/L OGTCT for 48h(p<0.001,n=3).
4.2 To detect expressions of protein Bcl-2 in H460 cells by immuno-fluorescence assay combined with LSCM:
When cells were treated with OGTCT 0.56μM/L for 48h,in comparison to control group,the expression of protein Bcl-2 was down-regulated significantly(p<0.01,n=3).
4.3 Assay results of activities of caspases:
·ApoAlert caspase-3 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.982 vs 0.246,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.
·ApoAlert caspase-8 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.787 vs 0.132,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.
·ApoAlert caspase-9/6 fluoremetric assay showed that fluorescence intensity of caspase-9 at 460nm was dramatically up-regulated from 24.45 to 185.44 by OGTCT 0.56μM/L(p<0.001),in other words,its activity increased by 6.58 folds. And activity of capase-6 increased by 0.82 fold with the fluorescence intensity enhanced from 18.36 to 33.55(p<0.01).
·Detection influences of caspases inhibitors on apoptosis induced by OGTCT: Compared to 0.56μM/L OGTCT treatment group,pan-caspase inhibitor (Z-VAD-FMK) completely blocked the apoptosis induced by OGTCT,caspase-3 inhibitor(Z-DEVD-FMK),caspase-8 inhibitor(Z-IETD-FMK) and caspase-9 inhibitor(Z-LEHD-FMK) significantly reversed the OGTCT-induced apoptosis in H460 cells.And caspase-6 inhibitor(Z-VEID-FMK) reversed partially the OGTCT-induced apoptosis.
4.4 Detection expression of Cytochrome C(Cyto-C) by Western blot assay Compared with the control group,the level of Cyto-C was significantly up-regulated by 0.56μM/L OGTCT at 48h in cytoplasm of H460 cells (p<0.01,n=3).
4.5 ELISA assay detected expressions of Fas/FasL in H460 cells treated with OGTCT:
4.5.1 Results of Fas/FasL ELISA assay
The expression of Fas protein was up-regulated by OGTCT and significantly increased with the extension of time,reached the maximum at 24h,since then,the level of Fas reduced to some extent.However,the expressions of mFasL and sFasL did not change significantly(p>0.05,n=3).
4.5.2 Influences of Fas inhibitor(anti-Fas Ab,ZB4) on apoptosis induced by OGTCT in H460 cells
Cells were treated with 0.56μM/L OGTCT after ZB4 pre-treatment for 48h, MTT and ELISA assay indicated:apoptosis induced by OGTCT and proliferation inhibition were reversed by ZB4.
CONCLUSIONS
1 OGTCT reduced significantly viabilities of NSCLC cells and drug-resistant cell H460/TaxR in a dose-dependent manner;OGTCT had two advantages compared with paclitaxel:OGTCT had a minor toxicity to normal cell WI38,and it could overcome the multi drug-resistance of tumor cells.
2 OGTCT could effectively induce apoptosis in H460 cells.It may well exert it's function of apoptosis induction through the following factors:up-regulation of levels of p53 and p21 proteins,reducing expression of PCNA,blocking cell cycle at G_0/G_1 phase,down-regulation expression level of Bcl-2,induction release of Ca~(2+) and Cyto-C into cytosol,activations of capase-3,-8,-9,-6.
3 Fas signaling pathway and mitochondrial signaling pathway might jointly participate in the process of apoptosis induced by OGTCT in H460 cells.
据WHO统计,肺癌是当今世界上最大的癌患,其死亡率居于各种恶性肿瘤之首位,中国也已成为世界上第一肺癌大国。按组织学类型可将肺癌分为两大类:小细胞肺癌和非小细胞肺癌。非小细胞肺癌(NSCLC)占肺癌的85%以上。由于非小细胞肺癌转移后禁忌手术,且对放疗很不敏感,因此,对非小细胞肺癌化疗药物的开发与研究尤为重要。
肺癌化疗的常用药物有如下五类:直接破坏DNA药物、插入DNA模板药物、影响核酸合成药物、抑制微管装配药物、影响蛋白质合成药物五类。尽管上述药物可有效诱导肺癌细胞凋亡,但是毒副作用较大且容易引起耐药,从而导致化疗失败。因此,开发毒性小、副作用少、能克服多药耐药和有效启动肺癌细胞凋亡信号传导途径的药物是目前亟待解决的问题。
本研究选用H460、A549、H1792、H226、WI38细胞为实验细胞研究有机锡小分子化合物抑制NSCLC细胞生长的机制,原因在于:1)此四种NSCLC细胞分别来源于腺癌、大细胞癌、鳞癌,涵盖了非小细胞肺癌的三种亚型;2)此四种NSCLC细胞均可单独作为非小细胞肺癌研究领域里的模式细胞,用于细胞水平的检测;3)迄今对有机锡小分子化合物诱导NSCLC细胞凋亡的机制研究还比较少;4) WI38细胞是从人胚肺组织建立的二倍体成纤维细胞系,作为正常细胞经常被用作肺癌研究的对照细胞。因此可用以上细胞系筛选小分子化合物,并对其机制做深入研究,为NSCLC化疗药物的开发提供理论依据。
化学遗传学(chemical genetics)是利用化学工具来研究生物体系的一种新兴手段,其可以在以下两个重要方面促进新药开发:一方面是鉴定出在某种疾病形成过程中起重要作用的基因或蛋白质,从而为新药筛选提供靶点;另外一方面是发现特异性作用于某个基因或蛋白质的小分子化合物,从而为新药开发提供先导化合物。化学遗传学应用的关键之一是要有大量的可供筛选的不同结构的化合物,新兴的组合化学很好地解决了这一难题,在同一个化学反应体系中加入不同结构单元,利用这些结构单元的排列组合,就能够系统地合成大量化合物。近年来,化学遗传学在细胞凋亡研究领域得到了广泛的应用:美国哈佛大学医学院袁钧瑛教授带领课题组进行了“用小分子研究细胞死亡过程中的信号传导:从Bcl-2到程序性坏死”的研究;美国密执安大学癌症中心王少萌教授开展了“合理设计小分子拮抗剂来调控关键性细胞坏死和蛋白质-蛋白质间的相互作用”的研究;另外,美国国立癌症研究所(NCI)早在1997年启动了一项新计划,将化学遗传学应用到癌症治疗研究中,即利用细胞渗透性的小分子化合物触发肿瘤细胞死亡。这充分说明化学遗传学方法为细胞凋亡研究提供了一个可靠的平台,基于此平台,利用具有一定生物活性的小分子化合物作为研究手段来研究细胞凋亡过程中的信号传导,优势明显:小分子化合物易于进入细胞,易于引起细胞表型变化,其作用具有剂量依赖性,适用于多蛋白家族,不会引起细胞的适应性变化等,另外,这些小分子化合物还往往可以作为“先导化合物”,进而指导新药的开发研究。
近几年来,本课题组一直在从事有机小分子化合物库的构建工作,目前,库中已包含了60余种有机锡小分子化合物。研究显示,某些有机锡化合物具有抗肿瘤活性,这已经在MCF-7(乳腺癌)、WiDr(结肠癌)、P388(小鼠白血病)等抗癌活性研究中得到证实。这些研究大都停留在生长抑制实验阶段,部分研究显示有机锡化合物可以阻滞肿瘤细胞周期,但对其抑制生长、诱导凋亡的分子机制仍然不清楚。因此,探讨有机锡小分子化合诱导肿瘤细胞凋亡分子机制具有重要意义。
基于以上考量,本课题研究目的是:利用化学遗传学这一新兴技术平台,以非小细胞肺癌H460、A549、H1792和H226细胞为研究对象,对草酰腙缩乙醛酸三环己基锡酯(OGTCT)的体外抗肿瘤活性进行研究,进而探讨其诱导H460细胞凋亡的分子机制,为新型肺癌化疗药物的开发提供更加完善的理论依据。
研究内容
1.鉴定OGTCT对NSCLC细胞、耐药细胞H460/TaxR及人胚肺二倍体成纤维细胞WI38的生长抑制作用。
2.探讨OGTCT对H460细胞的凋亡诱导作用。
3.分析OGTCT对H460细胞周期分布及周期调控相关蛋白p53、p21、PCNA表达的影响。
4.分析OGTCT诱导H460细胞凋亡过程中,凋亡相关调控因子的变化及重要凋亡通路的激活情况。
研究方法
1细胞生长抑制检测:
1.1 MTT法检测细胞存活率,由此对OGTCT进行初步筛选;
1.2选择目前临床上常用抗肿瘤药物紫杉醇(Paclitaxel)为对照药物,用MTT法检测其对肿瘤细胞和正常细胞的生长抑制情况,并与OGTCT做比较。
2.细胞凋亡检测方法:
2.1倒置相差显微镜观察细胞形态变化及凋亡小体;
2.2 Hoechst 33258荧光染色,并结合荧光显微镜观察细胞核形态变化;
2.3乳酸脱氢酶(LDH)试剂盒检测细胞是否出现坏死;
2.4台盼蓝拒染法进一步检测细胞是否出现坏死;
2.5用Nucleosome ELISA试剂盒检测细胞凋亡率。
3.细胞周期及其相关调控蛋白表达变化的检测:
3.1流式细胞技术检测OGTCT对细胞周期分布的影响;
3.2酶联免疫吸附测定技术(ELISA)检测与细胞周期调控相关重要蛋白p53和p21的表达变化;
3.3免疫荧光结合激光扫描共聚焦显微镜检测增殖细胞核抗原PCNA的表达及分布;
3.4 Western blot检测p53蛋白的表达。
4.荧光探针结合激光扫描共聚焦技术检测细胞中钙离子浓度变化。
5.检测OGTCT对ICE家族蛋白酶成员激活情况,方法如下:
5.1用ApoAlert caspase-3,-8比色检测试剂盒分析caspase-3,-8酶活性变化;
5.2用ApoAlert caspase 9/6荧光检测试剂盒分析caspase-9/6酶活性变化;
5.3用caspase抑制剂检测caspase-3,8,6,9对OGTCT诱导细胞凋亡的影响。
6.免疫荧光结合激光扫描共聚焦显微镜检测抗凋亡蛋白Bcl-2的分布及表达。
7.Western blot检测细胞色素C(Cyto-C)的表达变化。
8.酶联免疫(ELISA)试剂盒检测Fas/FasL的表达变化。
研究结果
1.OGTCT抗肿瘤活性检测结果(MTT法检测细胞存活率)
1.1 OGTCT对NSCLC细胞、耐药细胞及人胚肺成纤维细胞WI38存活率的影响:在48h内,OGTCT以剂量依赖的方式,显著降低了NSCLC细胞(A549、H1792、H226、H460)和耐药细胞H460/TaxR的存活率;而相同浓度与作用时间下,WI38细胞的存活率远远高于NSCLC细胞和H460/TaxR耐药细胞的存活率。
1.2阳性药物对比检测结果:
在48h内,紫杉醇以剂量依赖的方式,降低了NSCLC细胞、H460/TaxR细胞及WI38细胞的存活率。但与OGTCT相比,紫杉醇对WI38有显著生长抑制作用,而对耐药细胞H460/TaxR的生长抑制作用极不显著。
结果表明:OGTCT与紫杉醇相比有2个明显优势,OGTCT对正常细胞WI38的毒性较小,而对耐药细胞H460/TaxR的生长抑制作用尤为明显。
2.OGTCT诱导H460细胞凋亡的研究结果
2.1倒置相差显微镜观察细胞形态变化:
0.1%DMSO溶剂对照组与对照组相比,细胞形态无明显变化;0.56μM/L的OGTCT处理H460细胞48h后发现,胞体皱缩,胞浆内空泡明显,核固缩,折光性减弱,细胞膜出芽且有脱离现象,凋亡小体形成,细胞呈现出典型的凋亡形态学特征。
2.2 H33258染色结合荧光显微镜检测细胞核片段化结果:
DMSO溶剂对照组与对照组相比,细胞核形态无明显变化,均呈现均匀亮蓝色;0.56μM/L的OGTCT处理H460细胞48h后发现,细胞核固缩、染色质凝集且有明显边缘化、出现细胞核碎片,凋亡小体清晰可见,呈现典型的凋亡形态学特征。
2.3 LDH活性检测结果:
OGTCT 0.14、0.28μM/L处理H460细胞48h,与对照组细胞相比,培养液中酶活性变化不显著(p＞0.05,n=3);而OGTCT 0.56μM/L处理H460细胞48h,与对照组相比,培养液中酶活性显著升高(p＜0.01,n=3)。
2.4台盼蓝拒染实验检测表明:OGTCT 0.14和0.28μM/L处理H460细胞48h,细胞坏死率极低;而OGTCT0.56μM/L处理H460细胞48h,细胞坏死率显著增加(p＜0.01,n=3)。
2.5 Nucleosome ELISA检测发现:
用0.14、0.28、0.56μM/L的OGTCT分别处理H460细胞6h、12h、24h、48h,与对照组细胞相比,OGTCT可有效诱导H460细胞凋亡,并呈明显的剂量时间依赖性。
3.OGTCT对H460细胞周期的影响及相关分子机制研究结果
3.1 OGTCT对H460细胞周期分布的影响
分析细胞DNA变化发现,OGTCT作用24-48 h后可使H460细胞周期阻滞在G_0/G_1期,不能进入S期,并呈时间依赖性,但对M期的作用不明显。
3.2检测p53/p21蛋白表达水平
●p53、p21 ELISA检测,发现:
OGTCT可使H460细胞的p53和p21蛋白表达升高,随着作用时间的延长,表达水平显著升高,在24h时表达水平最高,此后表达水平逐步降低。
●Western Blot检测发现:OGTCT可使H460细胞中p53的表达显著升高(p＜0.01,n=3)。
3.3免疫荧光结合激光共聚焦显微技术检测PCNA蛋白表达及分布:
0.56μM/L的OGTCT作用于H460细胞48h,与对照组相比,可使PCNA的表达显著降低(p＜0.05,n=3)。
4.OGTCT诱导H460细胞凋亡的相关分子机制研究结果
4.1免疫荧光探针结合激光共聚焦显微技术检测H460细胞中钙离子变化及分布,发现:OGTCT 0.56μM/L处理48h后,可使H460细胞内的钙离子浓度显著增强(p＜0.001,n=3)。
4.2免疫细胞化学法结合激光共聚焦显微技术检测H460细胞中Bcl-2表达发现:用0.56μM/L的OGTCT处理H460细胞48h后,与对照组相比,Bcl-2表达水平显著降低(p＜0.01,n=3)。
4.3 Caspases活性检测结果:
●ApoAlert caspase-3比色检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,405 nm处光吸收值由0.246上升到0.982(p＜0.001);
●ApoAlert caspase-8比色检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,405 nm处光吸收值由0.132上升到0.787(p＜0.001);
●ApoAlert caspase-9/6荧光检测试剂盒检测分析发现:0.56μM/L OGTCT处理H460细胞48h后,与对照组相比较,caspase-9在460nm处的荧光强度由24.45上升到185.44,其相对活性上调了6.58倍(p＜0.001);而caspase-6在460nm处的荧光强度由18.36上升到33.55,其相对活性上调了0.82倍(p＜0.01);结果表明,在H460细胞凋亡过程中,OGTCT同时激活了caspase-3,-8,-9,6。
●Caspases抑制剂对OGTCT诱导H460细胞凋亡的影响
加caspases抑制剂后,凋亡ELISA试剂盒检测细胞凋亡率,发现:与OGTCT处理的细胞相比,pan-caspase inhibitor(Z-VAD-FMK)完全阻滞了OGTCT诱导的凋亡,caspase-3 inhibitor(Z-DEVD-FMK)、caspase-8 inhibitor(Z-IETD-FMK)和caspase-9 inhibitor(Z-LEHD-FMK)显著逆转了OGTCT诱导的凋亡,而caspase-6 inhibitor(Z-VEID-FMK)显著抑制了OGTCT诱导的凋亡,但程度稍弱。
4.4 Western blotting检测Cyto-C的表达
Western bloting检测发现:0.56μM/L OGTCT处理H460细胞48h后,可使胞浆中Cyto-C的水平显著增加(p＜0.01,n=3)。
4.5 OGTCT处理的H460细胞中Fas/FasL的表达
4.5.1 Fas/FasL ELISA assay检测结果
ELISA assay检测发现,OGTCT可使H460细胞的Fas蛋白表达升高,随着作用时间的延长,表达水平显著升高,在24h时表达水平最高,此后表达水平有所降低;而mFasL与sFasL的表达水平未发生显著变化(p＞0.05,n=3)。
4.5.2 Fas抑制剂ZB4对OGTCT诱导H460细胞凋亡的影响
用抗Fas抗体ZB4预先处理H460细胞一定时间后,再用0.56μM/L的OGTCT处理48h,MTT与nucleosome ELISA检测发现,ZB4显著逆转了OGTCT诱导的凋亡与增殖抑制。
结论:
1.OGTCT以浓度依赖的方式,显著降低NSCLC和H460/TaxR细胞的存活率。OGTCT与紫杉醇相比优势明显:对正常细胞WI38的毒性极小,且能有效克服肿瘤细胞的多药耐药性。
2.OGTCT能有效诱导H460细胞凋亡。OGTCT可能通过上调p53和p21蛋白水平、降低PCNA表达的方式将细胞周期阻滞于G_0/G_1期、下调Bcl-2蛋白表达水平、导致钙离子和Cyto-C释放、激活Fas蛋白、激活caspase-8,-9,-3,-6而发挥其凋亡诱导作用。
3.Fas信号通路和线粒体信号通路可能共同参与了OGTCT诱导的H460细胞凋亡过程。
BACKGROUND AND OBJECTIVE
According to the statistics of WHO,lung cancer is the most dangerous cancer all over the world,which mortality tops the list of all kinds of tumor.And china has become a country that has the largest number of lung cancer patient.According to histological type,lung cancer can be divided two types:small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC).NSCLC accounts for more than 85%of lung cancer.Because NSCLC become not suitable for surgery after metastasis,and not sensitive to radiotherapy,therefore,it is particularly important for development and research of chemotherapeutic drugs.
The classifications of frequently-used drugs for lung cancer chemotherapy are five categories as bellows:directly damage DNA drugs,inserted DNA template drugs,affecting nucleic acid synthesis drugs,repressing microtubule assembly drugs and impacting protein synthesis drugs.Although the above drugs can induce apoptosis of lung cancer cells,but strong toxicity side effect and easily leading to drug resistance cause frequently failure of chemotherapy.Therefore developments of drug which has minor toxicity,less side effect,ability to overcome multi-drug resistance,and able to start apoptosis signal transduction pathway,are the urgent problems to be settled.
In this study,reasons that H460,A549,H1792,H226 and WI38 were selected as experimental cells to investigate mechanisms of growth-inhibition in NSCLC cells are as follows:1) These four kinds of NSCLC cells were derived from adenocarcinoma,large cell carcinoma,squamous cell carcinoma,covering three subtypes of NSCLC;2) These four kinds of NSCLC cells can be used seperately in detection on the cellular level in the field of lung cancer study as model cell;3) To date,investigation of mechanism of small molecular organotin-induced apoptosis in NSCLC cells remains relative few.4) WI38 is a diploid fibroblast cell line which derived from human embryonic lung tissue and frequently used as control cell in the study of lung cancer.Thus the above cell lines are available for screening small molecular compound,and make in-depth study of its mechanism aim to provide a theoretical basis for development of chemotherapeutic drug of NSCLC.
Chemical genetics is a new means that utilizing chemical tools for investigating biological system,and it could promote the developments of new drugs in the following two important aspects:on the one hand,identifying gene or protein which plays an important role in formation of a disease,so as to provide targets for the new drug screening;The other hand is finding small molecular compound which act with a gene or protein,so as to provide lead compounds for developing new drugs.One of the key of chemical genetics researches is to have a large number of compounds with different structures for screening.The emerging combined chemistry is the core technology accessing to numerous small molecular compounds.The principle of the combined chemistry is to add different structural units into the same chemical reaction system,and to synthesize systematically numerous compounds utilizing permutations and combinations of these structural units.
Over the years,the chemical genetics has been widely applied in the field of cell apoptosis research:Professor Yuan Junying from Harvard Medical School lead the research team carrying out the investigation named as "using small molecules to study on the signal transduction in the process of cell death:from Bcl-2 to the programmed necrosis";Professor Wang Shaomeng from the cancer research center of U.S.Michigan University launched study named as "rationally design small molecular antagonist to regulate key cell necrosis and protein-protein interactions"; Additionally,the U.S.National Cancer Institute(NCI) launched a new plan in 1997, the application chemical genetics to research of cancer treatment,namely the use of cell permeable small molecular compound to trigger death of tumor cell.The above examples fully prove that chemical genetics method provides a reliable platform for research of cell apoptosis.In this platform,utilizing small molecular compound with a certain biological activity as a tool to investigate signal transduction in the process of cell apoptosis has many advantages:small molecular compounds to enter cells easily,easy to cause changes in the cellular phenotype,its role in a dose-dependent relationship,applicable to multi-proteins family as well as not cause adaptive changes in cells,etc.In addition,these small molecular compounds often serve as "lead compounds" to further drug development and research.
In recent years,our team has been engaged in construction the "library" of small organic compounds,at present,the library already contains more than 60 species of small molecular organotin.Studies have shown that some organotin have anti-tumor activities which are already confirmed in MCF-7,WiDr and P388 cells. Most of these studies still remained in the experimental phase of growth inhibition. Some studies indicated that organotin could block cell cycle of tumor cell.However, the molecular mechanisms of inhibiting growth and inducing apoptosis remain unclear.So investigation of molecular mechanism of apoptosis induced by small molecular organotin is of great significance.
Based on the above considerations,aims of this research are as follows: applying chemical genetics methods,to investigate In vitro anti-tumor activity of OGTCT in H460,A549,H1792,H226 cells,and then,to explore molecular mechanisms of OGTCT-induced apoptosis in H460 cells.This study can also provide a more complete theoretical basis for the development of new chemotherapeutic agents for the treatment of lung cancer.
STUDY CONTENTS
1 Identification growth inhibition to NSCLC,H460/TaxR and WI38 of OGTCT.
2 Exploration of apoptosis induction in H460 cells by OGTCT.
3 Analysis of the influences of OGTCT on cycle distribution and p53,p21,PCNA protein levels in H460 cells.
4 To analyze changes of apoptosis-related factors and the important apoptosis pathways activated by OGTCT in H460 cells.
METHODS
1.Cell growth inhibition Detection:
1.1 MTT assay for cell viability,by which OGTCT was screened initially;
1.2 Select the current clinically used anticancer drug(Paclitaxel) as control drug, using MTT assay for growth inhibition to tumor cells and normal cells,and make comparison to OGTCT.
2.Methods for apoptosis identification:
2.1 Observation of cell morphological changes and apoptosis bodies by Phase Contrast Microscope;
2.2 Observation of nuclear morphological changes by Hoechst33258 staining combined with fluorescence microscopy;
2.3 LDH(Lactate dehydrogenase) assay to determine cell toxicity induced by OGTCT;
2.4 Trypan blue dye exclusion assay to confirm whether the cells treated by OGTCT undergo necrosis;
2.5 Detection of apoptosis rates by Nucleosome ELISA assay kits.
3.Detection of changes in cell cycle distribution and related regulatory proteins expression:
3.1 Analysis of the changes in cell cycle distribution by Flow cytometry;
3.2 ELISA methods for analysis of expression changes in p53/p21 proteins associated to regulation of cell cycle;
3.3 Detection of expression and distribution of PCNA by immuno-fluorescence combined with Laser Scanning Confocal Microscopy(LSCM);
3.4 Confirmation of the changes in p53 expression by Western blot.
4.Detection changes of Ca~(2+) concentration in H460 cells by immuno-fluorescence probe combined with LSCM.
5.Methods for determining whether caspases are activated by OGTCT:
5.1 Analyzing caspase-3,-8 activities by ApoAlert caspase-3,-8 colorimetric assay kits;
5.2 Analyzing caspase-9/6 activity by ApoAlert caspase-9/6 fluoremetric assay kits;
5.3 Identification the influences of caspase-3,-8,-6,-9 on apoptosis induced by OGTCT with caspase inhibitors.
6.To analyze distribution and expression of anti-apoptotic protein Bcl-2 by Immuno-fluorescence combined with LSCM.
7.Detection expression changes of Cytochrome C(Cyto-C) by Western blot.
8.Detection changes of expression in Fas/FasL proteins by ELISA kits.
RESULTS
1.Detected results of anti-tumor activity of OGTCT(MTT assay for cell viability)
1.1 Influences of OGTCT on viability of NSCLC,H460/TaxR and WI38 cell:
Within 48h,OGTCT reduced significantly viabilities of NSCLC cells (H460,A549,H1792,H226) and drug-resistant cell H460/TaxR in a dose-dependent manner;but at the same concentration and time,viabilities of WI38 cell were much higher than that of NSCLC and H460/TaxR cells.
1.2 Results of positive drug comparison test:
Within 48h,paclitaxel reduced viabilities of NSCLC,H460/TaxR and WI38 cells in a dose-dependent manner.But compared to OGTCT,paclitaxel inhibited obviously growth of WI38 cells,and its growth inhibition to H460/TaxR was not extremely significant.
The results indicated:OGTCT had two advantages compared with paclitaxel, OGTCT had a minor toxicity to normal cell WI38,and its growth inhibition to drug-resistant cell H460/TaxR was particularly obvious.
2.Results of apoptosis induced by OGTCT in H460 cells
2.1 Cell morphological changes observed under Phase Contrast Microscope: Treated with 0.1%DMSO,cell morphology has no obvious change;Exposed to 0.56μM/L OGTCT for 48h,morphological changes are as follows:cell body shrinkage,obvious cytoplasmic vacuolization,karyopyknosis,refractive index decreased,membrane blebbing,formed apoptotic bodies,and cells showed typical morphological characteristics of apoptosis.
2.2 Results of detection cell nuclear fragmentation by Hoechst33258 staining combined with Fluorescence Microscopy:
There had no obvious change in cell nuclear morphology between DMSO group and control group,and cell nuclei showed a uniform light blue;When H460 cells were treated with 0.56μM/L OGTCT for 48h,typical apoptotic features such as cell shrinkage,chromatin condensation and marginalization,emerging nuclei fragmentation,apoptotic bodies clearly visible occurred.
2.3 Detected results of activity of LDH:
When H460 cells were respectively incubated in 0.14,0.28μM/L OGTCT for 48h,compared with the control group,there was no significant change of activity of LDH in culture medium(p>0.05,n=3);While LDH activity in culture medium containing 0.56μM/L OGTCT was significantly enhanced(p<0.01,n=3).
2.4 Results of Trypan blue dye exclusion assay:
Cell necrosis rate of 0.14 and 0.28μM/L test groups were extremely low, compared with the control group;While the necrosis rate of 0.56μM/L test group increased obviously(p<0.01,n=3).
2.5 Results of nucleosome ELISA assay:
When H460 cells were respectively treated with 0.14,0.28,0.56μM/L OGTCT for 6,12,24 and 48h,compared with the control group,OGTCT induced effectively apoptosis of H460 cells in dose- and time-dependent manners.
3.Influence of OGTCT on H460 cell cycle and assay of related proteins
3.1 Influence of OGTCT on H460 cell cycle distribution
After changes of DNA were analyzed,we found that cell cycles of H460 cells witch treated by OGTCT for 24-48h were blocked at G_0/G_1 phase in a time-dependent manner.Furthermore,H460 cells could not enter S phase. However,OGTCT has a minor effect on M phase.
3.2 Distribution and expression of p53/p21 protein
·p53/p21 ELISA assay
The levels of p53 and p21 proteins were up-regulated by OGTCT and increased significantly with the extension of time,reached the maximum at 24h,since then, the levels of p53 and p21 gradually reduced.
·Western blot assay confirmed that OGTCT significantly up-regulated the expression of p53 protein(p<0.01,n=3).
3.3 Immuno-fluorescence combined with LSCM detection for expression and distribution of PCNA protein in H460 cells:
Expression level of PCNA in H460 cells was significantly reduced after treated with 0.56μM/L OGTCT for 48h(p<0.05,n=3).
4.Research findings of OGTCT-induced apoptosis in H460 cells and its related molecular mechanisms
4.1 Detection changes and distribution of Ca~(2+) in H460 cells by immuno-fluorescence probe combined with LSCM:
Calcium concentration was significantly enhanced in H460 cells treated with 0.56μM/L OGTCT for 48h(p<0.001,n=3).
4.2 To detect expressions of protein Bcl-2 in H460 cells by immuno-fluorescence assay combined with LSCM:
When cells were treated with OGTCT 0.56μM/L for 48h,in comparison to control group,the expression of protein Bcl-2 was down-regulated significantly(p<0.01,n=3).
4.3 Assay results of activities of caspases:
·ApoAlert caspase-3 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.982 vs 0.246,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.
·ApoAlert caspase-8 colorimetric assay showed a higher light absorption at 405 nm compared to control group(0.787 vs 0.132,p<0.001) by OGTCT 0.56μM/L in H460 cells at 48 h.
·ApoAlert caspase-9/6 fluoremetric assay showed that fluorescence intensity of caspase-9 at 460nm was dramatically up-regulated from 24.45 to 185.44 by OGTCT 0.56μM/L(p<0.001),in other words,its activity increased by 6.58 folds. And activity of capase-6 increased by 0.82 fold with the fluorescence intensity enhanced from 18.36 to 33.55(p<0.01).
·Detection influences of caspases inhibitors on apoptosis induced by OGTCT: Compared to 0.56μM/L OGTCT treatment group,pan-caspase inhibitor (Z-VAD-FMK) completely blocked the apoptosis induced by OGTCT,caspase-3 inhibitor(Z-DEVD-FMK),caspase-8 inhibitor(Z-IETD-FMK) and caspase-9 inhibitor(Z-LEHD-FMK) significantly reversed the OGTCT-induced apoptosis in H460 cells.And caspase-6 inhibitor(Z-VEID-FMK) reversed partially the OGTCT-induced apoptosis.
4.4 Detection expression of Cytochrome C(Cyto-C) by Western blot assay Compared with the control group,the level of Cyto-C was significantly up-regulated by 0.56μM/L OGTCT at 48h in cytoplasm of H460 cells (p<0.01,n=3).
4.5 ELISA assay detected expressions of Fas/FasL in H460 cells treated with OGTCT:
4.5.1 Results of Fas/FasL ELISA assay
The expression of Fas protein was up-regulated by OGTCT and significantly increased with the extension of time,reached the maximum at 24h,since then,the level of Fas reduced to some extent.However,the expressions of mFasL and sFasL did not change significantly(p>0.05,n=3).
4.5.2 Influences of Fas inhibitor(anti-Fas Ab,ZB4) on apoptosis induced by OGTCT in H460 cells
Cells were treated with 0.56μM/L OGTCT after ZB4 pre-treatment for 48h, MTT and ELISA assay indicated:apoptosis induced by OGTCT and proliferation inhibition were reversed by ZB4.
CONCLUSIONS
1 OGTCT reduced significantly viabilities of NSCLC cells and drug-resistant cell H460/TaxR in a dose-dependent manner;OGTCT had two advantages compared with paclitaxel:OGTCT had a minor toxicity to normal cell WI38,and it could overcome the multi drug-resistance of tumor cells.
2 OGTCT could effectively induce apoptosis in H460 cells.It may well exert it's function of apoptosis induction through the following factors:up-regulation of levels of p53 and p21 proteins,reducing expression of PCNA,blocking cell cycle at G_0/G_1 phase,down-regulation expression level of Bcl-2,induction release of Ca~(2+) and Cyto-C into cytosol,activations of capase-3,-8,-9,-6.
3 Fas signaling pathway and mitochondrial signaling pathway might jointly participate in the process of apoptosis induced by OGTCT in H460 cells.
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