萘酰亚胺类化合物诱导肿瘤细胞凋亡的机制
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摘要
恶性肿瘤是导致人类死亡的一个主要原因,据世界卫生组织(WHO)统计,在全世界50多亿人口中平均每年死于恶性肿瘤者达710万人,新发病例为870万,而且这一数字还在逐年增加。因此,肿瘤的防治已被列为全世界科学家日益关注的重要课题,寻找肿瘤药物作用的有效靶点和合成新的抗肿瘤药已经成为细胞生物学及肿瘤学等相关学科领域的重要课题。
自1961年Lerman提出平面芳香稠环结构的分子以嵌插方式与DNA相结合的模型以来,DNA作为抗肿瘤药物的靶点,日益受到人们的重视。大量的研究表明,大多数DNA损伤药物可在不同程度上诱导肿瘤细胞凋亡。嵌入剂多以嵌插方式嵌入DNA碱基对之间,或使DNA三级结构发生变化,或切断DNA或与DNA单链或双链发生共价交联,或改变DNA拓扑结构来影响其生化功能,如RNA聚合酶或拓扑结构酶Ⅱ的活性受到抑制等,致使DNA不能或不易复制和转录,最终导致细胞凋亡。
作为DNA嵌入剂之一的萘酰亚胺类化合物能嵌入DNA,抑制DNA和RNA的合成,同时也抑制拓扑异构酶。由于其在抗肿瘤方面的显著作用,已成为近几年研究的热点之一。目前研究的萘酰亚胺类化合物存在着其抗肿瘤活性弱或毒副作用大等问题。因此,人们需要合成新的具有更强的抗肿瘤活性、毒副作用更小的萘酰亚胺类化合物。
本研究采用MTT法和流式细胞术,以人宫颈癌系(Hela)、人乳腺癌细胞系(MCF-7)、人星形胶质瘤细胞系(U-251)和人肝癌细胞系(SMMC-7721)细胞为模型,从由自行设计、合成的11种萘酰亚胺为母体合成的化合物中筛选出能有效诱导肿瘤细胞凋亡的双萘酰亚胺类化合物C8。通过倒置显微镜、荧光显微镜和电镜来观察化合物C8作用下U-251细胞的形态学变化及凋亡情况;通过测定乳酸脱氢酶来判定C8的细胞毒性和对U-251细胞的细胞膜状态的影响;用流式细胞术检测C8对细胞周期的影响;通过测定线粒体膜电位、细胞色素C的释放、凋亡关键效应酶Caspase-9和Caspase-3的活性及通过实时定量PCR分析凋亡相关基因Bcl-2、Bax和p53的mRNA含量、Western Blotting分析Bcl-2蛋白表达量来研究C8诱导肿瘤细胞凋亡的凋亡通路及相关调节基因。通过这一系列的实验,较为深入地研究化合物C8诱导肿瘤细胞凋亡的机制,为进一步深入研究化合物C8的抗肿瘤作用及其未来的应用奠定理论和实验基础。
本研究的主要方法和结果:
1、MTT比色法检测细胞活力
应用MTT比色法测定不同浓度化合物(0-80μM)作用12h、18h和24h后,对MCF-7、Hela、U-251和SMMC-7721细胞体外生长的抑制作用。结果显示:用于筛选的11种化合物中,C8对四种细胞的生长均具有一定的抑制作用,但对于不同组织来源的细胞其抑制强度不尽相同,而其他10种化合物对各组细胞虽具有一定的抑制作用,但不具有显著性(P＞0.05)。化合物C8作用细胞12h后,与对照组(0.01%DMSO)相比,20μM化合物C8可显著抑制MCF-7、U-251和SMMC-7721细胞活力(p＜0.05),但对Hela细胞活力的抑制作用不具有显著性(p＞0.05);作用18h后,可显著抑制上述细胞的生长;而作用24h后,抑制作用比18h更为显著,抑制率随培养时间的延长呈增长趋势。在同一时段,随化合物C8作用浓度的增加,细胞活力逐渐下降(p＜0.05),呈现明显的剂量依赖性关系。
2、流式细胞仪检测细胞凋亡
用流式细胞仪检测终浓度为10μmol/L化合物C8,分别作用Hela、MCF-7、U-251和SMMC-7721细胞18h后的细胞凋亡结果可知,与对照组相比化合物C8对MCF-7、U-251和SMMC-7721细胞有较强的诱导凋亡能力,凋亡比率分别为12.9±1.80%、23.8±2.34%和16.1±2.84%,而对Hela细胞的作用较弱,凋亡率仅为7.7±2.55%,说明化合物C8诱导肿瘤细胞的凋亡具有一定的选择性。
3、倒置显微镜观察细胞生长状态
观察2.5μmol/L和5μmol/L化合物C8处理U-251细胞18小时后与对照组相比细胞生长缓慢,细胞密度减小,说明此浓度化合物C8对细胞的生长具有抑制作用。10μmol/L化合物C8处理18小时后,U-251细胞的形态学改变明显增强:细胞伸展不良,胞体回缩,胞质粗糙,细胞内有大量颗粒状物堆积,部分细胞不贴壁,细胞密度进一步减小:20μmol/L和40μmol/L化合物C8处理18小时后大部分细胞已看不清完整轮廓,细胞凋亡或坏死现象明显。由此可见,随着化合物C8作用浓度的增加,对U-251细胞的生长抑制作用增强。从U-251细胞的形态学改变可以看出,C8可能通过干扰细胞代谢,改变细胞的黏附性,降低细胞的黏附能力,使U-251细胞由瓶壁上脱落下来,通过改变细胞的黏附性来抑制肿瘤细胞增殖。
4、荧光显微镜观察细胞凋亡
细胞凋亡与细胞死亡不同,是一种程序性死亡(Programmed Cell Death,PCD)。细胞凋亡特征性改变包括细胞固缩、体积变小、核致密、胞质有“出泡”现象等。一般认为形态学变化是判断细胞有无发生凋亡的基础。Hoechst 33342染料进入凋亡细胞中比正常细胞更容易,而PI染料不能进入细胞膜完整的活细胞中。根据这些特性,用Hoechst 33342结合PI染料对凋亡细胞进行双染色,可在荧光显微镜或流式细胞仪上将正常细胞、凋亡细胞和坏死细胞区别开来。本研究采用Hoechst 33342和PI对细胞进行染色,在荧光倒置显微镜下观察细胞,结果发现U-251细胞萎缩,出现核固缩,染色体聚集,细胞核裂解为碎块,产生凋亡小体,核荧光强度增强等现象。
5、细胞超微结构观察
采用透射电镜观察凋亡细胞的超微结构可见,对照组细胞的细胞膜完整,细胞质分布均匀,细胞核、核膜及亚细胞结构都很完整。5μM和10μM化合物C8作用18h的实验组,可观察到细胞体积缩小,连接消失,与周围的细胞脱离,细胞核固缩、染色质核膜下聚集、胞质空泡化等凋亡特征,但细胞膜仍然保持完整;而20μM作用18h后,可见细胞核的染色质高度凝聚、边缘化,核膜核仁破碎,细胞周围有散在的核碎裂体及其它细胞内容物,此时细胞膜将要或已经破裂,细胞已发生死亡。
6、测定乳酸脱氢酶
化合物C8的细胞毒性作用可能是其抗肿瘤机制的一个方面。通过检测肿瘤细胞培养基中代谢物乳酸脱氢酶(Lactate dehydrogenase,LDH)的活性,可以在确定肿瘤细胞膜状态的同时,考察药物的细胞毒性。本研究中检测化合物C8作用U-251细胞后培养基上清中LDH释放率结果显示,实验组与对照组相比,2.5μM、5μM的化合物C8与细胞共培养12h,18h和24h后,只能检测到极少量LDH(p＞0.05)。10μM以上化合物C8对U-251细胞乳酸脱氢酶释放的作用开始明显增加,20μM化合物C8作用12h,18h和24h后在胞液中LDH活力分别为对照组的118.4±3.23%,153.2±4.51%和187.6±3.56%,40PM C8作用12h,18h和24h后在胞液中LDH活力分别为对照组的121.4±4.21%,182.2±2.24%和211.3±3.81%。可见随着化合物C8作用时间的增加,细胞外乳酸脱氢酶(LDH)活性明显增强(p＜0.05)。同时,随着化合物C8作用浓度的增加,细胞外乳酸脱氢酶(LDH)活性也明显增加(p＜0.05)。由此可见,10μM以上化合物C8对U-251细胞的细胞毒性作用表现为:在相同浓度下,化合物C8的细胞毒性随着作用时间的延长而增强;在作用时间相同条件下,化合物C8的细胞毒性则随着作用浓度的增大而增强。10μM以上化合物C8对U-251细胞的细胞毒性作用显示出较为明显的浓度依赖性和时间依赖性。
7、细胞周期分布的影响
研究显示:0-20μM的化合物C8作用24h后,U-251细胞周期分布和凋亡率的检测结果与对照组相比,随着C8作用浓度的增加,G0/G1期细胞含量增加,S期细胞含量减少,而G2/M期细胞含量无明显变化,说明C8可使U-251细胞发生G0/G1期阻滞。同时,随着C8浓度的增高,出现凋亡特征性“亚G1峰”,即sub-G1期的细胞含量逐渐增多(p＜0.05),通常认为此期细胞为凋亡细胞,说明化合物C8可诱导U-251细胞凋亡,且凋亡比率与C8的浓度成正相关。
8、测定线粒体膜电位
线粒体跨膜电位的下降,发生在细胞核凋亡特征(染色质浓缩、DNA断裂)出现之前,被认为是细胞凋亡级联反应过程中最早发生的事件,一旦线粒体膜电位崩溃,则细胞凋亡不可逆转。试验结果显示:10μM化合物C8作用1h,线粒体膜电位略有上升;之后,线粒体膜电位迅速下降,在1-6h之间下降幅度最为明显(p＜0.05)。6h后,下降速度趋于平缓(p＞0.05)。2.5μM和5μM化合物C8作用6h后,也能导致线粒体膜电位下降,但降幅均明显小于10μM C8产生的效果。说明化合物C8可导致U-251细胞线粒体膜电位的下降,使线粒体功能缺失,这是细胞凋亡的前期指征。
9、细胞色素C的释放
10μM化合物C8作用于U-251细胞4h、8h、12h、16h和20h后,采用Western blotting初步分析胞液和线粒体中Cyt C蛋白含量。结果显示:对照组细胞胞液内几乎不含Cyt C;加10μM化合物C8作用4h,胞液中Cyt C的含量升高,而线粒体中Cyt C的含量开始减少;随着作用时间的延长,线粒体中Cyt C的含量进一步减少,而胞液中Cyt C的含量则逐渐增多,说明化合物C8可能使线粒体膜通透性发生改变,导致存在于内、外膜间的CytC从外膜游离至胞液中,这一结果与线粒体膜电位的下降,在出现时间上保持一致。
10、凋亡关键效应酶Caspase-9和Caspase-3的活性
细胞色素C释放到细胞液中能导致Caspase激活。Caspase蛋白家族在细胞凋亡的执行期发挥关键作用,Caspase-9和Caspase-3为关键的执行分子,它们在凋亡信号传导的许多途径中发挥作用。检测化合物C8对Caspase-9和Caspase-3激活的影响,结果显示:与对照组相比,在10μM化合物C8作用3-6h的实验组,未检测到Caspase-9和Caspase-3的变化,但作用12h后,U251细胞内的Caspase-9和Caspase-3被显著激活,到24h和48h时,Caspase-9的活性分别增高为对照组的296.9±14.23%和326.8±12.51%,Caspase-3的活性分别增高为对照组的286.2±12.01%和308.5±13.42%。2.5μM和5μM C8作用24h,可使Caspase-9活性升高为对照组的145.5±3.22%和228.7±4.51%,使Caspase-3活性升高为对照组的138.4±4.31%和216.3±3.87%,呈明显的量效关系(p＜0.05)。
11、分析凋亡相关基因Bcl-2、Bax和p53的mRNA含量
Bcl-2蛋白家族在凋亡的线粒体途径中起着调控中心的作用,其中Bcl-2蛋白是细胞内的主要抗凋亡因子之一,直接参与线粒体膜电位的调节,而Bax等属于促凋亡蛋白。Bcl-2抑制凋亡必须通过与Bax形成异二聚体来实现。本研究采用实时定量PCR技术检测化合物C8作用U-251细胞10小时内Bcl-2基因和Bax基因mRNA含量结果显示:与对照组比较,处理组细胞的Bcl-2基因和Bax基因的mRNA含量均未见明显变化(P＞0.05),说明在较短时间内化合物C8对Bcl-2 mRNA可能没有显著影响。
采用实时定量PCR技术检测化合物C8作用U-251细胞10小时内p53基因mRNA含量结果显示:实验组细胞的p53基因mRNA含量比对照组高;化合物C8作用10小时的p53基因表达量为作用0小时(对照组)的1.5倍以上,说明化合物C8可能损伤了细胞核内DNA的固有结构,促使p53基因的表达。
12、分析凋亡相关基因Bcl-2蛋白表达量
流式细胞仪检测Bcl-2蛋白结果可见:Bcl-2蛋白的表达量对照组为97.8%,试验组为31.4%,试验组比对照组下降66%。Western Blotting结果也同样显示出明显的下降趋势,表明C8诱导U-251细胞凋亡可能与Bcl-2蛋白具有一定的相关性。
研究结论:
1.萘酰亚胺类化合物C8中对SMMC-7721、U-251和MCF-7细胞的生长均有抑制作用和诱导凋亡作用。
2.化合物C8具有细胞毒性作用可能是其抗肿瘤机制的一个方面。
3.化合物C8能够阻滞细胞周期于G_0/G_1期,抑制肿瘤细胞S期的DNA合成,诱导U-251细胞凋亡。
4.化合物C8能够嵌入细胞核DNA,使核DNA的构型发生改变,迫使肿瘤细胞进入凋亡程序。
5.本研究初步明确了化合物C8诱导癌细胞凋亡的线粒体途径:即通过降低线粒体膜电位和提高线粒体膜通透性,促使细胞色素C从线粒体向胞液中释放;抑制Bcl-2蛋白表达:激活Caspase级联反应,最终导致细胞凋亡。
Malignant tumor is one of the main causes of human beings' death.The WHO's statistics suggests that there are 7.1 million persons dying from malignant tumor every year on average among the world population of 5 billion odd,and the number of new cases is 8.7 million,which is still increasing year by year.Therefore,the prevention and treatment of tumor has been regarded as the important subject attracting more and more attention of all the scientists in the world,and to look for the effective target spot of the effect of tumor drugs and to compound the new anti-tumor drugs has become the important subjects in the fields of cell biology and oncology as well as others.
Since Lerman proposed the model that the molecule in the plate condensed-ring aromatic structure combines with DNA in the mode of intercalating in 1961,that DNA works as the target spot of anti-tumor drugs has attracted more and more attention.A large amount of research suggests that most of the drugs for DNA damage can induce the tumor cell apoptosis to different degree.The intercalator is usually inserted between the DNA base pair by means of intercalating,to make the DNA tertiary structure change, cut off the DNA,enable the covalent cross-linking with DNA single strand or double strands,or change the DNA topology structure to influence the biochemical function.For example,the activity of the RNA polymerase or enzymeⅡin topology structure will be inhibited so that the DNA could not be replicated or transcribed,and consequently the cell apoptosis is caused.
As one of the DNA intercalators,Naphthalimide compound can intercalate in the DNA,inhibiting the compound of DNA and RNA synthesis,and the topoismerase as well. In view of its obvious anti-tumor effect,it has been one of the hot topics in these years. The current Naphthalimide compounds have several problems,such as the weak antineoplastic activity,high toxic and side effect,etc.Therefore,the Naphthalimide compound with strong antineoplastic activity and slighter toxic and side effect are demanded.
This research adopts the MTT method and flow cytometry,taking the Hela,MCF-7, U-251 and SMMC-7721 as the models,and select double-Naphthalimide compound C8 which can effectively induce the apoptosis of tumor cells from the compounds taking eleven kinds of Naphthalimide designed and synthesized by own as the parent substance.Observe shape change and apoptosis situation of U-251 cells under the action of C8 by inverted microscope,fluorescence microscope and electron microscope; judge the toxicity of C8 and the influence on the cell membrane of U-251 by determining lactic dehydrogenase;detect the influence on the cell cycle by flow cytometry;study the apoptosis pathway of C8 inducing the apoptosis of tumor cell and relevant regulatory genes by determining the mitochondria membrane potential,the release of Cytlchrome C and activities of Caspase-9 and Caspase-3,analyzing the mRNA content of Bcl-2,Bax and p53 by real-time quantitative analysis and analyzing Bcl-2 protein expression by Western Blotting.The series of experiments can deeply research on the mechanism of C8 inducing tumor cell apoptosis,laying the foundation for further research on antineoplastic effects of compound C8 and its application in the future.
Main methods and result of this research:
1.Determine the cell viability with MTT colorimetric method
Make use of the MTT colorimetric method to determine the inhibition of compounds in different concentrations(0-80μM) on the MCF-7,Hela,U-251 and SMMC-7721 cell in vitro growth after the action of 12h,18h and 24h.The result shows that among the 11 kinds of compounds,C8 has the inhibition effect on all of the four kinds of cells.However, its inhibition effects on different cells are different.The other 10 kinds of compounds have some inhibition effect on the cells,but the effect is not so obvious(P＞0.05)(See Fig. 2-1,2-2,2-3,2-4,2-5,2-6,2-7,2-8,2-9,2-10,2-11 and 2-12).After the action of 12h, compared with the contrast group(0.01%DMSO),20μM C8 can obviously inhibit the cell viability of MCF-7,U-251 and SMMC-7721(p＜0.05),but its inhibition on the cell viability of Hela is not so significant(p＞0.05).After 18 h,compared with the compounds in smaller concentration,C8 can obviously inhibit the growth of foresaid cells.After 24h,the inhibition effect is more obviously than that after 18h,and the inhibition rate tends to increase with the time of culture passing.In the same period,with the concentration of C8 increasing,the cell viability gradually reduces(p＜0.05),and the obvious dependence on the dosage appears.
2.Result of determination with flow cytometer
Use the flow cytometer to determine the cell apoptosis of Hela,MCF-7,U-251 and SMMC-7721 after the C8 whose final concentration is 10μmol/L acting on them respectively for 18h.The result is as follows:compared with the contrast group,C8 has stronger inducement of apoptosis to MCF-7,U-251 and SMMC-7721,at the apoptosis percentage of 12.9±1.80%,23.8±2.34%and 16.1±2.84%respectively.But its effect on the Hela is weaker,only at 7.7±2.55%,which demonstrates that the compound has certain selectivity to the tumor cell.
3.Use the microscope to observe the state of cell growth
In this research,we observe that compared with the contrast group,the cell grows slower and the cell density is smaller after the 18h's action of 2.5μmol/L and 5μmol/L C8,which proves that the C8 in this concentration has the inhibition on the cell growth. The morphology of U-251 changes greatly after the 18h's action of 10μmol/L C8.That is, the unsatisfactory cell spreading,cell body retraction,rough cytoplasm,a large amount of accumulation of particles in the cell,some cells not adhering to the wall,and the cell density further reduced.After the 18h's action of 20μmol/L and 40μmol/L C8,the complete contour of most of the cells is fuzzy and the phenomenon of cell apoptosis or necrosis is obvious.Thus it can be seen that with the increase of concentration of C8, inhibition of growth of U-251 intensifies.The morphological change of U-251 shows that C8 can make U-251 peel from wall by means of disturbance of cell metabolism,change of cell adhesion and reduction of adhesive power,thus inhibit the cell proliferation through the change of cell adhesion.
4.Use the fluorescence microscope to observe the cell apoptosis.
Different from the cell death,the cell apoptosis is a kind of programmed cell death (PCD).The characteristic change of cell apoptosis includes the cell pycnosis,volume shrinkage,karyopycnosis and cytoplasm bubble etc.The morphological change is usually deemed to be the basis to determine whether there is any cell apoptosis. Hoechst 33342 dye is easier to enter to the apoptosis cell than to the normal cells,and the PI dye can not enter to the living cell with complete cell membrane.According to these characteristics,use the two dyes Hoechst 33342 and PI to the apoptosis cell,and the normal cell,apoptosis cell and necrosis cell can be distinguished by the fluorescence microscope or the flow cytometer.This research uses the Hoechst 33342 and PI to dye the cells and observes the cells under the fluorescence inverted microscope.The following phenomena are found to the U-251 in the experiment group:cellular atrophy, karyopycnosis,chromosome accumulation,karyon cracking to fragments,apoptotic body appearance and increase of karyon fluorescence strength.
5.Observe the cell ultramicrostructure
Observe the ultramicrostructure of the apoptotic cells with the transmission electron microcopy,the membrane of the cells in the contrast group is complete,the cytoplasm is distributed evenly and the karyon,karyolemma and subcellular structure are all complete. In the experiment group where the 5μM and 10μM C8 act for 18h,the cell volume shrinks and the connection disappears,away from the surrounding cells;the apoptosis characteristics including the karyopycnosis,chromatin accumulation under the karyolemma and the cytoplasm bubbling etc appear;but the cell membrane keeps complete.After the 18h action of 20μM C8,the chromatin of karyon highly condenses and tends to edge.The karyolemma and core break and there are dispersed fragments and other cell content surrounding the cell.At this moment,the karyolemma is to break or has broken and the cell has been dead.
6.Determination of Lactate dehydrogenase(LDH)
The cytotoxicity of C8 may be one aspect of its anti-tumor mechanism.The determination of the activity of the metabolite LDH in the culture medium of tumor cell can not only determine the state of tumor cell membrane but also check the cytotoxicity of drugs.This research chooses the U-251 as the model in vitro to determine the influence of C8 on the release rate of LDH in the supernatant of culture medium of U-251.The result shows that,comparing the experiment group with the contrast group, only a minute quantity of LDH(p＞0.05) can be detected after the 2.5μM and 5μM C8 cultured with the cell for 12h,18h and 24h.The effect of C8 in the concentration over 10μM on the release of U-251 LDH starts to increase significantly.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 20μM C8 for 12h, 18h and 24h are 118.4±3.23%,153.2±4.51%and 187.6±3.56%respectively.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 40μM C8 for 12h,18h and 24h are 121.4±4.21%,182.2±2.24%and 211.3±3.81%respectively.It shows that with the increase of acting time of C8,the viability of outside LDH obviously strengthens(p＜0.05).In the meantime,with the increase of concentration of C8,the viability of outside LDH obviously strengthens(p＜0.05).Thus it can be seen that the cytotoxicity of C8 in the concentration over 10μM on the U-251 is as follows:in the same concentration,the cytotoxicity of C8 intensifies with the increase of acting time.In the same acting time,the cytotoxicity of C8 intensifies with the increase of concentration.The cytotoxicity of C8 in concentration over 10μM on the U-251 shows the obvious dependence on concentration and time.
7.Influence of cell cycle distribution
The research shows that comparing the test results of the U-251 cell cycle distribution and apoptosis percentage after the 24h action of C8 in the concentration within 0-20μM with the contrast group,with the increase of C8 concentration,the cell content in G0/G1 cycle increases,decreases in S cycle,and without obvious change in the G2/M cycle,which demonstrates that C8 can make the U-251 encounter with the blockage in G0/G1 cycle.In the meantime,with the increase of C8 concentration,the "sub G1 peak" mirroring the apoptosis characteristics appears.That is,the cell content in the sub-Gl cycle gradually increases(p＜0.05),and the cells in this cycle is usually deemed to be the apoptotic cells.This shows that the C8 can induce the U-251cell apoptosis and the apoptosis rate is in positive correlation to the C8 concentration.
8.Determination of mitochondria membrane potential
The reduction of mitochondria membrane potential occurs before the appearance of the karyon apoptosis characteristics(chromatin concentration,DNA fracture).It is deemed to be the earliest event in the cascade reaction of the cell apoptosis.Once the mitochondria membrane potential collapses,the cell apoptosis will not be reversed.The experiment result suggests that the mitochondria membrane potential rises a little after 1h action of 10μM C8.Then the mitochondria membrane potential rapidly decreases, which is the most obvious within 1-6h(p＜0.05).After 6h,the decreasing speed tends to be stable(p＞0.05).The 6h action of 2.5μM and 5μM C8 can also lead to the decreasing of mitochondria membrane potential,but the decreasing amplitudes are smaller than those of 10μM C8.This demonstrates that C8 may lead to the decrease of mitochondria membrane potential of U-251 and enable the afunction of mitochondria which is the early-state characteristic of cell apoptosis.
9.The release of Cytochrome C
After 10μM C8 acts on U-251 for 4h,8h,12h,16h and 20h,adopt Western Blotting to make preliminary analysis of cell sap and Cyt C protein content in mitochondria.The results show that:the cell sap of contrast group is hardly without Cyt C;add 10μM C8 and act for 4h,the Cyt C content in the cell sap increases,but the Cyt C content in the mitochondria begins to decrease;with the prolongation of acting time,the Cyt C content in the mitochondria will further decrease,but the Cyt C content in the cell sap will gradually increase,which prove that C8 may change the permeability of mitochondria membrane and cause the Cyt C existing between inner membrane and outer membrane to dissociate from outer membrane to the cell sap.This result keeps consistent with the occurrence time of electric potential descending of mitochondria membrane.
10.The activities of Caspase-9 and Caspase-3
The Cytochrome C released to the cell sap can result in Caspase activation.The Caspase protein family plays the key role during the executing period of cell apoptosis, Caspase-9 and Caspase-3 are the key executing molecules and they play roles on many ways of the apoptosis signal transduction.Detect the influences of Caspase-9 and Caspase-3 activation and the results show that:compared with the contrast group,after 3-6h of application of C8(10μM),the changes of Caspase-9 and Caspase-3 are not detected,but after 12h,the Caspase-9 and Caspase-3 in U251 are obviously activated, after 24h and 48h,the activities of Caspase-9 respectively increase to 296.9±14.23% and 326.8±12.51%of the contrast group,and the activities of Caspase-3 respectively increase to 286.2±12.01%and 308.5±13.42%.2.5μM and 5μM C8 acting for 24h can make the activities of Caspase-3 increase to 138.4±4.31%and 216.3±3.87%of the contrast group,displaying obvious dose-effect relationship(p＜0.05).
11.Analyze the mRNA contents of Bcl-2,Bax and p53
The Bcl-2 protein family acts as the regulation and control center in the mitochondria pathway of apoptosis,in which Bcl-2 is one of the main anti-apoptosis factors in the cell and directly participates in the regulation of mitochondria membrane potential;and Bax and etc are the apoptosis promoting proteins.The apoptosis inhibition of Bcl-2 must be realized through its formation of Hetero-dimers together with the Bax.This research adopts the real-time quantitative PCR technology to determine the mRNA contents in Bcl-2 and Bax when C8 acting on U-251 in 10 h and the result shows that:compared with the contrast group,the mRNA contents of Bcl-2 and Bax in the cells of the treatment group have no obvious change(p＞0.05),which demonstrates that C8 is likely to have no obvious influence on Bcl-2 mRNA in short time.
Adopt the real-time quantitative PCR technology to determine the p53 mRNA content when C8 acting on U-251 in 10 h and the result shows that:the p53 mRNA content of the experiment group is higher than that of the contrast group;the p53 expression of C8 acting in 10 h is 1.5 times above than that of C8 acting in 0 h,which demonstrates that C8 is likely to damage DNA inherent structure in the cell nucleus, causing the p53 expression.
12.Analyze Bcl-2 protein expression
The detecting result of Bcl-2 protein by the flow cytometer shows that:the contrast group of Bcl-2 protein expression is 97.8%and the experiment group of that is 31.4%, which is less than the contrast group by 66%.The result of Western Blotting also displays the same decline trend,showing that C 8 inducing U-251 cell apoptosis maybe have certain relationship with Bcl-2 protein.
Conclusions of this research:
1.The Naphthalimide compound C8 has inhibitory action and inducing apoptosis action on cell growth of SMMC-7721、U-251和MCF-7.
2.Maybe the cell toxicity of compound C8 is one factor of its antineoplastic mechanism.
3.Compound C8 can block cell cycle in G0/G1 cycle,inhibit the DNA synthesis of tumor cell in S cycle and induce the cell apoptosis of U-251.
4.Compound C8 can embed in nucleus DNA,change the structure of the nucleus DNA and cause the tumor cell into apoptosis procedures.
5.This research preliminarily determines the mitochondria pathway of the compound C8 inducing the cancer cell apoptosis:cause the release of Cytochrome C from the mitochondria to the cell sap by reducing the potential of mitochondria membrane and improving the permeability of mitochondria membrane;inhibit Bcl-2 protein expression;activate Caspase cascade reaction,and finally leading to the cell apoptosis.
自1961年Lerman提出平面芳香稠环结构的分子以嵌插方式与DNA相结合的模型以来,DNA作为抗肿瘤药物的靶点,日益受到人们的重视。大量的研究表明,大多数DNA损伤药物可在不同程度上诱导肿瘤细胞凋亡。嵌入剂多以嵌插方式嵌入DNA碱基对之间,或使DNA三级结构发生变化,或切断DNA或与DNA单链或双链发生共价交联,或改变DNA拓扑结构来影响其生化功能,如RNA聚合酶或拓扑结构酶Ⅱ的活性受到抑制等,致使DNA不能或不易复制和转录,最终导致细胞凋亡。
作为DNA嵌入剂之一的萘酰亚胺类化合物能嵌入DNA,抑制DNA和RNA的合成,同时也抑制拓扑异构酶。由于其在抗肿瘤方面的显著作用,已成为近几年研究的热点之一。目前研究的萘酰亚胺类化合物存在着其抗肿瘤活性弱或毒副作用大等问题。因此,人们需要合成新的具有更强的抗肿瘤活性、毒副作用更小的萘酰亚胺类化合物。
本研究采用MTT法和流式细胞术,以人宫颈癌系(Hela)、人乳腺癌细胞系(MCF-7)、人星形胶质瘤细胞系(U-251)和人肝癌细胞系(SMMC-7721)细胞为模型,从由自行设计、合成的11种萘酰亚胺为母体合成的化合物中筛选出能有效诱导肿瘤细胞凋亡的双萘酰亚胺类化合物C8。通过倒置显微镜、荧光显微镜和电镜来观察化合物C8作用下U-251细胞的形态学变化及凋亡情况;通过测定乳酸脱氢酶来判定C8的细胞毒性和对U-251细胞的细胞膜状态的影响;用流式细胞术检测C8对细胞周期的影响;通过测定线粒体膜电位、细胞色素C的释放、凋亡关键效应酶Caspase-9和Caspase-3的活性及通过实时定量PCR分析凋亡相关基因Bcl-2、Bax和p53的mRNA含量、Western Blotting分析Bcl-2蛋白表达量来研究C8诱导肿瘤细胞凋亡的凋亡通路及相关调节基因。通过这一系列的实验,较为深入地研究化合物C8诱导肿瘤细胞凋亡的机制,为进一步深入研究化合物C8的抗肿瘤作用及其未来的应用奠定理论和实验基础。
本研究的主要方法和结果:
1、MTT比色法检测细胞活力
应用MTT比色法测定不同浓度化合物(0-80μM)作用12h、18h和24h后,对MCF-7、Hela、U-251和SMMC-7721细胞体外生长的抑制作用。结果显示:用于筛选的11种化合物中,C8对四种细胞的生长均具有一定的抑制作用,但对于不同组织来源的细胞其抑制强度不尽相同,而其他10种化合物对各组细胞虽具有一定的抑制作用,但不具有显著性(P＞0.05)。化合物C8作用细胞12h后,与对照组(0.01%DMSO)相比,20μM化合物C8可显著抑制MCF-7、U-251和SMMC-7721细胞活力(p＜0.05),但对Hela细胞活力的抑制作用不具有显著性(p＞0.05);作用18h后,可显著抑制上述细胞的生长;而作用24h后,抑制作用比18h更为显著,抑制率随培养时间的延长呈增长趋势。在同一时段,随化合物C8作用浓度的增加,细胞活力逐渐下降(p＜0.05),呈现明显的剂量依赖性关系。
2、流式细胞仪检测细胞凋亡
用流式细胞仪检测终浓度为10μmol/L化合物C8,分别作用Hela、MCF-7、U-251和SMMC-7721细胞18h后的细胞凋亡结果可知,与对照组相比化合物C8对MCF-7、U-251和SMMC-7721细胞有较强的诱导凋亡能力,凋亡比率分别为12.9±1.80%、23.8±2.34%和16.1±2.84%,而对Hela细胞的作用较弱,凋亡率仅为7.7±2.55%,说明化合物C8诱导肿瘤细胞的凋亡具有一定的选择性。
3、倒置显微镜观察细胞生长状态
观察2.5μmol/L和5μmol/L化合物C8处理U-251细胞18小时后与对照组相比细胞生长缓慢,细胞密度减小,说明此浓度化合物C8对细胞的生长具有抑制作用。10μmol/L化合物C8处理18小时后,U-251细胞的形态学改变明显增强:细胞伸展不良,胞体回缩,胞质粗糙,细胞内有大量颗粒状物堆积,部分细胞不贴壁,细胞密度进一步减小:20μmol/L和40μmol/L化合物C8处理18小时后大部分细胞已看不清完整轮廓,细胞凋亡或坏死现象明显。由此可见,随着化合物C8作用浓度的增加,对U-251细胞的生长抑制作用增强。从U-251细胞的形态学改变可以看出,C8可能通过干扰细胞代谢,改变细胞的黏附性,降低细胞的黏附能力,使U-251细胞由瓶壁上脱落下来,通过改变细胞的黏附性来抑制肿瘤细胞增殖。
4、荧光显微镜观察细胞凋亡
细胞凋亡与细胞死亡不同,是一种程序性死亡(Programmed Cell Death,PCD)。细胞凋亡特征性改变包括细胞固缩、体积变小、核致密、胞质有“出泡”现象等。一般认为形态学变化是判断细胞有无发生凋亡的基础。Hoechst 33342染料进入凋亡细胞中比正常细胞更容易,而PI染料不能进入细胞膜完整的活细胞中。根据这些特性,用Hoechst 33342结合PI染料对凋亡细胞进行双染色,可在荧光显微镜或流式细胞仪上将正常细胞、凋亡细胞和坏死细胞区别开来。本研究采用Hoechst 33342和PI对细胞进行染色,在荧光倒置显微镜下观察细胞,结果发现U-251细胞萎缩,出现核固缩,染色体聚集,细胞核裂解为碎块,产生凋亡小体,核荧光强度增强等现象。
5、细胞超微结构观察
采用透射电镜观察凋亡细胞的超微结构可见,对照组细胞的细胞膜完整,细胞质分布均匀,细胞核、核膜及亚细胞结构都很完整。5μM和10μM化合物C8作用18h的实验组,可观察到细胞体积缩小,连接消失,与周围的细胞脱离,细胞核固缩、染色质核膜下聚集、胞质空泡化等凋亡特征,但细胞膜仍然保持完整;而20μM作用18h后,可见细胞核的染色质高度凝聚、边缘化,核膜核仁破碎,细胞周围有散在的核碎裂体及其它细胞内容物,此时细胞膜将要或已经破裂,细胞已发生死亡。
6、测定乳酸脱氢酶
化合物C8的细胞毒性作用可能是其抗肿瘤机制的一个方面。通过检测肿瘤细胞培养基中代谢物乳酸脱氢酶(Lactate dehydrogenase,LDH)的活性,可以在确定肿瘤细胞膜状态的同时,考察药物的细胞毒性。本研究中检测化合物C8作用U-251细胞后培养基上清中LDH释放率结果显示,实验组与对照组相比,2.5μM、5μM的化合物C8与细胞共培养12h,18h和24h后,只能检测到极少量LDH(p＞0.05)。10μM以上化合物C8对U-251细胞乳酸脱氢酶释放的作用开始明显增加,20μM化合物C8作用12h,18h和24h后在胞液中LDH活力分别为对照组的118.4±3.23%,153.2±4.51%和187.6±3.56%,40PM C8作用12h,18h和24h后在胞液中LDH活力分别为对照组的121.4±4.21%,182.2±2.24%和211.3±3.81%。可见随着化合物C8作用时间的增加,细胞外乳酸脱氢酶(LDH)活性明显增强(p＜0.05)。同时,随着化合物C8作用浓度的增加,细胞外乳酸脱氢酶(LDH)活性也明显增加(p＜0.05)。由此可见,10μM以上化合物C8对U-251细胞的细胞毒性作用表现为:在相同浓度下,化合物C8的细胞毒性随着作用时间的延长而增强;在作用时间相同条件下,化合物C8的细胞毒性则随着作用浓度的增大而增强。10μM以上化合物C8对U-251细胞的细胞毒性作用显示出较为明显的浓度依赖性和时间依赖性。
7、细胞周期分布的影响
研究显示:0-20μM的化合物C8作用24h后,U-251细胞周期分布和凋亡率的检测结果与对照组相比,随着C8作用浓度的增加,G0/G1期细胞含量增加,S期细胞含量减少,而G2/M期细胞含量无明显变化,说明C8可使U-251细胞发生G0/G1期阻滞。同时,随着C8浓度的增高,出现凋亡特征性“亚G1峰”,即sub-G1期的细胞含量逐渐增多(p＜0.05),通常认为此期细胞为凋亡细胞,说明化合物C8可诱导U-251细胞凋亡,且凋亡比率与C8的浓度成正相关。
8、测定线粒体膜电位
线粒体跨膜电位的下降,发生在细胞核凋亡特征(染色质浓缩、DNA断裂)出现之前,被认为是细胞凋亡级联反应过程中最早发生的事件,一旦线粒体膜电位崩溃,则细胞凋亡不可逆转。试验结果显示:10μM化合物C8作用1h,线粒体膜电位略有上升;之后,线粒体膜电位迅速下降,在1-6h之间下降幅度最为明显(p＜0.05)。6h后,下降速度趋于平缓(p＞0.05)。2.5μM和5μM化合物C8作用6h后,也能导致线粒体膜电位下降,但降幅均明显小于10μM C8产生的效果。说明化合物C8可导致U-251细胞线粒体膜电位的下降,使线粒体功能缺失,这是细胞凋亡的前期指征。
9、细胞色素C的释放
10μM化合物C8作用于U-251细胞4h、8h、12h、16h和20h后,采用Western blotting初步分析胞液和线粒体中Cyt C蛋白含量。结果显示:对照组细胞胞液内几乎不含Cyt C;加10μM化合物C8作用4h,胞液中Cyt C的含量升高,而线粒体中Cyt C的含量开始减少;随着作用时间的延长,线粒体中Cyt C的含量进一步减少,而胞液中Cyt C的含量则逐渐增多,说明化合物C8可能使线粒体膜通透性发生改变,导致存在于内、外膜间的CytC从外膜游离至胞液中,这一结果与线粒体膜电位的下降,在出现时间上保持一致。
10、凋亡关键效应酶Caspase-9和Caspase-3的活性
细胞色素C释放到细胞液中能导致Caspase激活。Caspase蛋白家族在细胞凋亡的执行期发挥关键作用,Caspase-9和Caspase-3为关键的执行分子,它们在凋亡信号传导的许多途径中发挥作用。检测化合物C8对Caspase-9和Caspase-3激活的影响,结果显示:与对照组相比,在10μM化合物C8作用3-6h的实验组,未检测到Caspase-9和Caspase-3的变化,但作用12h后,U251细胞内的Caspase-9和Caspase-3被显著激活,到24h和48h时,Caspase-9的活性分别增高为对照组的296.9±14.23%和326.8±12.51%,Caspase-3的活性分别增高为对照组的286.2±12.01%和308.5±13.42%。2.5μM和5μM C8作用24h,可使Caspase-9活性升高为对照组的145.5±3.22%和228.7±4.51%,使Caspase-3活性升高为对照组的138.4±4.31%和216.3±3.87%,呈明显的量效关系(p＜0.05)。
11、分析凋亡相关基因Bcl-2、Bax和p53的mRNA含量
Bcl-2蛋白家族在凋亡的线粒体途径中起着调控中心的作用,其中Bcl-2蛋白是细胞内的主要抗凋亡因子之一,直接参与线粒体膜电位的调节,而Bax等属于促凋亡蛋白。Bcl-2抑制凋亡必须通过与Bax形成异二聚体来实现。本研究采用实时定量PCR技术检测化合物C8作用U-251细胞10小时内Bcl-2基因和Bax基因mRNA含量结果显示:与对照组比较,处理组细胞的Bcl-2基因和Bax基因的mRNA含量均未见明显变化(P＞0.05),说明在较短时间内化合物C8对Bcl-2 mRNA可能没有显著影响。
采用实时定量PCR技术检测化合物C8作用U-251细胞10小时内p53基因mRNA含量结果显示:实验组细胞的p53基因mRNA含量比对照组高;化合物C8作用10小时的p53基因表达量为作用0小时(对照组)的1.5倍以上,说明化合物C8可能损伤了细胞核内DNA的固有结构,促使p53基因的表达。
12、分析凋亡相关基因Bcl-2蛋白表达量
流式细胞仪检测Bcl-2蛋白结果可见:Bcl-2蛋白的表达量对照组为97.8%,试验组为31.4%,试验组比对照组下降66%。Western Blotting结果也同样显示出明显的下降趋势,表明C8诱导U-251细胞凋亡可能与Bcl-2蛋白具有一定的相关性。
研究结论:
1.萘酰亚胺类化合物C8中对SMMC-7721、U-251和MCF-7细胞的生长均有抑制作用和诱导凋亡作用。
2.化合物C8具有细胞毒性作用可能是其抗肿瘤机制的一个方面。
3.化合物C8能够阻滞细胞周期于G_0/G_1期,抑制肿瘤细胞S期的DNA合成,诱导U-251细胞凋亡。
4.化合物C8能够嵌入细胞核DNA,使核DNA的构型发生改变,迫使肿瘤细胞进入凋亡程序。
5.本研究初步明确了化合物C8诱导癌细胞凋亡的线粒体途径:即通过降低线粒体膜电位和提高线粒体膜通透性,促使细胞色素C从线粒体向胞液中释放;抑制Bcl-2蛋白表达:激活Caspase级联反应,最终导致细胞凋亡。
Malignant tumor is one of the main causes of human beings' death.The WHO's statistics suggests that there are 7.1 million persons dying from malignant tumor every year on average among the world population of 5 billion odd,and the number of new cases is 8.7 million,which is still increasing year by year.Therefore,the prevention and treatment of tumor has been regarded as the important subject attracting more and more attention of all the scientists in the world,and to look for the effective target spot of the effect of tumor drugs and to compound the new anti-tumor drugs has become the important subjects in the fields of cell biology and oncology as well as others.
Since Lerman proposed the model that the molecule in the plate condensed-ring aromatic structure combines with DNA in the mode of intercalating in 1961,that DNA works as the target spot of anti-tumor drugs has attracted more and more attention.A large amount of research suggests that most of the drugs for DNA damage can induce the tumor cell apoptosis to different degree.The intercalator is usually inserted between the DNA base pair by means of intercalating,to make the DNA tertiary structure change, cut off the DNA,enable the covalent cross-linking with DNA single strand or double strands,or change the DNA topology structure to influence the biochemical function.For example,the activity of the RNA polymerase or enzymeⅡin topology structure will be inhibited so that the DNA could not be replicated or transcribed,and consequently the cell apoptosis is caused.
As one of the DNA intercalators,Naphthalimide compound can intercalate in the DNA,inhibiting the compound of DNA and RNA synthesis,and the topoismerase as well. In view of its obvious anti-tumor effect,it has been one of the hot topics in these years. The current Naphthalimide compounds have several problems,such as the weak antineoplastic activity,high toxic and side effect,etc.Therefore,the Naphthalimide compound with strong antineoplastic activity and slighter toxic and side effect are demanded.
This research adopts the MTT method and flow cytometry,taking the Hela,MCF-7, U-251 and SMMC-7721 as the models,and select double-Naphthalimide compound C8 which can effectively induce the apoptosis of tumor cells from the compounds taking eleven kinds of Naphthalimide designed and synthesized by own as the parent substance.Observe shape change and apoptosis situation of U-251 cells under the action of C8 by inverted microscope,fluorescence microscope and electron microscope; judge the toxicity of C8 and the influence on the cell membrane of U-251 by determining lactic dehydrogenase;detect the influence on the cell cycle by flow cytometry;study the apoptosis pathway of C8 inducing the apoptosis of tumor cell and relevant regulatory genes by determining the mitochondria membrane potential,the release of Cytlchrome C and activities of Caspase-9 and Caspase-3,analyzing the mRNA content of Bcl-2,Bax and p53 by real-time quantitative analysis and analyzing Bcl-2 protein expression by Western Blotting.The series of experiments can deeply research on the mechanism of C8 inducing tumor cell apoptosis,laying the foundation for further research on antineoplastic effects of compound C8 and its application in the future.
Main methods and result of this research:
1.Determine the cell viability with MTT colorimetric method
Make use of the MTT colorimetric method to determine the inhibition of compounds in different concentrations(0-80μM) on the MCF-7,Hela,U-251 and SMMC-7721 cell in vitro growth after the action of 12h,18h and 24h.The result shows that among the 11 kinds of compounds,C8 has the inhibition effect on all of the four kinds of cells.However, its inhibition effects on different cells are different.The other 10 kinds of compounds have some inhibition effect on the cells,but the effect is not so obvious(P＞0.05)(See Fig. 2-1,2-2,2-3,2-4,2-5,2-6,2-7,2-8,2-9,2-10,2-11 and 2-12).After the action of 12h, compared with the contrast group(0.01%DMSO),20μM C8 can obviously inhibit the cell viability of MCF-7,U-251 and SMMC-7721(p＜0.05),but its inhibition on the cell viability of Hela is not so significant(p＞0.05).After 18 h,compared with the compounds in smaller concentration,C8 can obviously inhibit the growth of foresaid cells.After 24h,the inhibition effect is more obviously than that after 18h,and the inhibition rate tends to increase with the time of culture passing.In the same period,with the concentration of C8 increasing,the cell viability gradually reduces(p＜0.05),and the obvious dependence on the dosage appears.
2.Result of determination with flow cytometer
Use the flow cytometer to determine the cell apoptosis of Hela,MCF-7,U-251 and SMMC-7721 after the C8 whose final concentration is 10μmol/L acting on them respectively for 18h.The result is as follows:compared with the contrast group,C8 has stronger inducement of apoptosis to MCF-7,U-251 and SMMC-7721,at the apoptosis percentage of 12.9±1.80%,23.8±2.34%and 16.1±2.84%respectively.But its effect on the Hela is weaker,only at 7.7±2.55%,which demonstrates that the compound has certain selectivity to the tumor cell.
3.Use the microscope to observe the state of cell growth
In this research,we observe that compared with the contrast group,the cell grows slower and the cell density is smaller after the 18h's action of 2.5μmol/L and 5μmol/L C8,which proves that the C8 in this concentration has the inhibition on the cell growth. The morphology of U-251 changes greatly after the 18h's action of 10μmol/L C8.That is, the unsatisfactory cell spreading,cell body retraction,rough cytoplasm,a large amount of accumulation of particles in the cell,some cells not adhering to the wall,and the cell density further reduced.After the 18h's action of 20μmol/L and 40μmol/L C8,the complete contour of most of the cells is fuzzy and the phenomenon of cell apoptosis or necrosis is obvious.Thus it can be seen that with the increase of concentration of C8, inhibition of growth of U-251 intensifies.The morphological change of U-251 shows that C8 can make U-251 peel from wall by means of disturbance of cell metabolism,change of cell adhesion and reduction of adhesive power,thus inhibit the cell proliferation through the change of cell adhesion.
4.Use the fluorescence microscope to observe the cell apoptosis.
Different from the cell death,the cell apoptosis is a kind of programmed cell death (PCD).The characteristic change of cell apoptosis includes the cell pycnosis,volume shrinkage,karyopycnosis and cytoplasm bubble etc.The morphological change is usually deemed to be the basis to determine whether there is any cell apoptosis. Hoechst 33342 dye is easier to enter to the apoptosis cell than to the normal cells,and the PI dye can not enter to the living cell with complete cell membrane.According to these characteristics,use the two dyes Hoechst 33342 and PI to the apoptosis cell,and the normal cell,apoptosis cell and necrosis cell can be distinguished by the fluorescence microscope or the flow cytometer.This research uses the Hoechst 33342 and PI to dye the cells and observes the cells under the fluorescence inverted microscope.The following phenomena are found to the U-251 in the experiment group:cellular atrophy, karyopycnosis,chromosome accumulation,karyon cracking to fragments,apoptotic body appearance and increase of karyon fluorescence strength.
5.Observe the cell ultramicrostructure
Observe the ultramicrostructure of the apoptotic cells with the transmission electron microcopy,the membrane of the cells in the contrast group is complete,the cytoplasm is distributed evenly and the karyon,karyolemma and subcellular structure are all complete. In the experiment group where the 5μM and 10μM C8 act for 18h,the cell volume shrinks and the connection disappears,away from the surrounding cells;the apoptosis characteristics including the karyopycnosis,chromatin accumulation under the karyolemma and the cytoplasm bubbling etc appear;but the cell membrane keeps complete.After the 18h action of 20μM C8,the chromatin of karyon highly condenses and tends to edge.The karyolemma and core break and there are dispersed fragments and other cell content surrounding the cell.At this moment,the karyolemma is to break or has broken and the cell has been dead.
6.Determination of Lactate dehydrogenase(LDH)
The cytotoxicity of C8 may be one aspect of its anti-tumor mechanism.The determination of the activity of the metabolite LDH in the culture medium of tumor cell can not only determine the state of tumor cell membrane but also check the cytotoxicity of drugs.This research chooses the U-251 as the model in vitro to determine the influence of C8 on the release rate of LDH in the supernatant of culture medium of U-251.The result shows that,comparing the experiment group with the contrast group, only a minute quantity of LDH(p＞0.05) can be detected after the 2.5μM and 5μM C8 cultured with the cell for 12h,18h and 24h.The effect of C8 in the concentration over 10μM on the release of U-251 LDH starts to increase significantly.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 20μM C8 for 12h, 18h and 24h are 118.4±3.23%,153.2±4.51%and 187.6±3.56%respectively.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 40μM C8 for 12h,18h and 24h are 121.4±4.21%,182.2±2.24%and 211.3±3.81%respectively.It shows that with the increase of acting time of C8,the viability of outside LDH obviously strengthens(p＜0.05).In the meantime,with the increase of concentration of C8,the viability of outside LDH obviously strengthens(p＜0.05).Thus it can be seen that the cytotoxicity of C8 in the concentration over 10μM on the U-251 is as follows:in the same concentration,the cytotoxicity of C8 intensifies with the increase of acting time.In the same acting time,the cytotoxicity of C8 intensifies with the increase of concentration.The cytotoxicity of C8 in concentration over 10μM on the U-251 shows the obvious dependence on concentration and time.
7.Influence of cell cycle distribution
The research shows that comparing the test results of the U-251 cell cycle distribution and apoptosis percentage after the 24h action of C8 in the concentration within 0-20μM with the contrast group,with the increase of C8 concentration,the cell content in G0/G1 cycle increases,decreases in S cycle,and without obvious change in the G2/M cycle,which demonstrates that C8 can make the U-251 encounter with the blockage in G0/G1 cycle.In the meantime,with the increase of C8 concentration,the "sub G1 peak" mirroring the apoptosis characteristics appears.That is,the cell content in the sub-Gl cycle gradually increases(p＜0.05),and the cells in this cycle is usually deemed to be the apoptotic cells.This shows that the C8 can induce the U-251cell apoptosis and the apoptosis rate is in positive correlation to the C8 concentration.
8.Determination of mitochondria membrane potential
The reduction of mitochondria membrane potential occurs before the appearance of the karyon apoptosis characteristics(chromatin concentration,DNA fracture).It is deemed to be the earliest event in the cascade reaction of the cell apoptosis.Once the mitochondria membrane potential collapses,the cell apoptosis will not be reversed.The experiment result suggests that the mitochondria membrane potential rises a little after 1h action of 10μM C8.Then the mitochondria membrane potential rapidly decreases, which is the most obvious within 1-6h(p＜0.05).After 6h,the decreasing speed tends to be stable(p＞0.05).The 6h action of 2.5μM and 5μM C8 can also lead to the decreasing of mitochondria membrane potential,but the decreasing amplitudes are smaller than those of 10μM C8.This demonstrates that C8 may lead to the decrease of mitochondria membrane potential of U-251 and enable the afunction of mitochondria which is the early-state characteristic of cell apoptosis.
9.The release of Cytochrome C
After 10μM C8 acts on U-251 for 4h,8h,12h,16h and 20h,adopt Western Blotting to make preliminary analysis of cell sap and Cyt C protein content in mitochondria.The results show that:the cell sap of contrast group is hardly without Cyt C;add 10μM C8 and act for 4h,the Cyt C content in the cell sap increases,but the Cyt C content in the mitochondria begins to decrease;with the prolongation of acting time,the Cyt C content in the mitochondria will further decrease,but the Cyt C content in the cell sap will gradually increase,which prove that C8 may change the permeability of mitochondria membrane and cause the Cyt C existing between inner membrane and outer membrane to dissociate from outer membrane to the cell sap.This result keeps consistent with the occurrence time of electric potential descending of mitochondria membrane.
10.The activities of Caspase-9 and Caspase-3
The Cytochrome C released to the cell sap can result in Caspase activation.The Caspase protein family plays the key role during the executing period of cell apoptosis, Caspase-9 and Caspase-3 are the key executing molecules and they play roles on many ways of the apoptosis signal transduction.Detect the influences of Caspase-9 and Caspase-3 activation and the results show that:compared with the contrast group,after 3-6h of application of C8(10μM),the changes of Caspase-9 and Caspase-3 are not detected,but after 12h,the Caspase-9 and Caspase-3 in U251 are obviously activated, after 24h and 48h,the activities of Caspase-9 respectively increase to 296.9±14.23% and 326.8±12.51%of the contrast group,and the activities of Caspase-3 respectively increase to 286.2±12.01%and 308.5±13.42%.2.5μM and 5μM C8 acting for 24h can make the activities of Caspase-3 increase to 138.4±4.31%and 216.3±3.87%of the contrast group,displaying obvious dose-effect relationship(p＜0.05).
11.Analyze the mRNA contents of Bcl-2,Bax and p53
The Bcl-2 protein family acts as the regulation and control center in the mitochondria pathway of apoptosis,in which Bcl-2 is one of the main anti-apoptosis factors in the cell and directly participates in the regulation of mitochondria membrane potential;and Bax and etc are the apoptosis promoting proteins.The apoptosis inhibition of Bcl-2 must be realized through its formation of Hetero-dimers together with the Bax.This research adopts the real-time quantitative PCR technology to determine the mRNA contents in Bcl-2 and Bax when C8 acting on U-251 in 10 h and the result shows that:compared with the contrast group,the mRNA contents of Bcl-2 and Bax in the cells of the treatment group have no obvious change(p＞0.05),which demonstrates that C8 is likely to have no obvious influence on Bcl-2 mRNA in short time.
Adopt the real-time quantitative PCR technology to determine the p53 mRNA content when C8 acting on U-251 in 10 h and the result shows that:the p53 mRNA content of the experiment group is higher than that of the contrast group;the p53 expression of C8 acting in 10 h is 1.5 times above than that of C8 acting in 0 h,which demonstrates that C8 is likely to damage DNA inherent structure in the cell nucleus, causing the p53 expression.
12.Analyze Bcl-2 protein expression
The detecting result of Bcl-2 protein by the flow cytometer shows that:the contrast group of Bcl-2 protein expression is 97.8%and the experiment group of that is 31.4%, which is less than the contrast group by 66%.The result of Western Blotting also displays the same decline trend,showing that C 8 inducing U-251 cell apoptosis maybe have certain relationship with Bcl-2 protein.
Conclusions of this research:
1.The Naphthalimide compound C8 has inhibitory action and inducing apoptosis action on cell growth of SMMC-7721、U-251和MCF-7.
2.Maybe the cell toxicity of compound C8 is one factor of its antineoplastic mechanism.
3.Compound C8 can block cell cycle in G0/G1 cycle,inhibit the DNA synthesis of tumor cell in S cycle and induce the cell apoptosis of U-251.
4.Compound C8 can embed in nucleus DNA,change the structure of the nucleus DNA and cause the tumor cell into apoptosis procedures.
5.This research preliminarily determines the mitochondria pathway of the compound C8 inducing the cancer cell apoptosis:cause the release of Cytochrome C from the mitochondria to the cell sap by reducing the potential of mitochondria membrane and improving the permeability of mitochondria membrane;inhibit Bcl-2 protein expression;activate Caspase cascade reaction,and finally leading to the cell apoptosis.
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