小白菊内酯对胃癌细胞增殖抑制和诱导凋亡作用的研究
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摘要
目的
小白菊内酯(parthenolide,Par)作为一种西方传统药物,除了免疫调节功效,对于来自肝脏,乳腺,前列腺,血液等的多种肿瘤细胞,在其发生发展过程中发挥良好的抑制效应,可以显著抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡。小白菊内酯的抗肿瘤机制与其作为NF-κB通路抑制剂息息相关,通过对NF-κB的阻断,调节细胞周期蛋白,由TNF-a通路调控众多下游基因如Bcl-2,Caspase及影响线粒体功能,显示了其在肿瘤治疗中的应用价值,但目前,国际上对Par在胃癌的研究方面还远不深入,为了探讨Par在胃癌治疗中的应用价值,我们研究了Par对胃癌细胞株SGC7901,BGC823细胞增殖,细胞周期和凋亡的影响;对胃癌细胞体外迁移和侵袭能力的影响,通过检测Par在处理细胞后线粒体膜电位(△Ψm)及凋亡检测蛋白Bcl-2和Caspase基因的改变,探讨在Par诱导胃癌细胞凋亡中线粒体途径和凋亡调节蛋白Bcl-2和Caspase-8的作用,进一步阐明Par抗肿瘤作用的机制。
方法
1、MTT法检测不同浓度Par对肿瘤细胞的抑制作用
取人胃癌细胞株SGC7901、BGC823细胞株,加入96孔板,实验组加入不同浓度的Par,对照组则加等量不含药物的培养液。Par处理24、48、72小时后,MTT法检测各孔吸光度(OD)值,计算细胞生长抑制率。
2、流式细胞仪分析细胞周期
消化收集各浓度Par处理24h的细胞,PI复合染液染色后,流式细胞仪检测,分析细胞周期。
3、流式细胞仪检测细胞凋亡
消化收集各浓度Par处理24h的细胞,Annexin V/FITC染色后,流式细胞仪检测,分析细胞凋亡百分比。
4、流式细胞仪检测Par对细胞△Ψm的影响
消化收集各浓度Par处理24h的细胞,罗丹明123染色后,流式细胞仪检测,分析线粒体膜电位降低细胞所占百分比。
5、transwell小室检测Par对细胞体外迁移的影响
以不同浓度Par处理细胞后,加入到transwell小室中,作用48h后计数穿膜细胞数。
6、被覆Matrigel胶的transwell小室检测Par对细胞体外侵袭的影响
以不同浓度Par处理细胞后,加入到transwell小室中,小室上层被覆Matrigel胶,作用48h后计数穿膜细胞数。
7、光镜下细胞形态观察
细胞接种于6孔板,以不同浓度Par处理细胞24h后,后倒置显微镜下用100×摄片,观察各组细胞形态。
8、荧光显微镜下观察细胞凋亡
收集以不同浓度Par处理24h的细胞,吖啶橙溶液染色,在荧光显微镜下用400×摄片,观察凋亡细胞形态。
9、western blot方法检测Bcl-2蛋白的表达
收集7.5μmol/L作用24,48,72h后的细胞蛋白,用western blot方法检测Bcl-2蛋白的表达。
10、RT-PCR方法检测Caspase-8 mRNA的表达
收集7.5μmol/L作用24,48,72h后的细胞蛋白,用RT-PCR方法检测Caspase-8基因的表达。
11、统计学处理
本实验所有数据用均数±标准差表示,利用SPSS11.5分析软件,采用单因素方差分析法进行差异性分析,P＜0.05被认为有显著差异。
结果
1、Par对胃癌细胞生长的抑制作用
Par在20μmol/L以下对SGC7901、BGC823细胞生长无显著影响,60μmol/LPar分别处理SGC7901、BGC823细胞48h后对细胞生长有抑制作用(P＜0.05),在100～200μmol/L浓度范围内,Par作用24h后,对胃癌细胞增殖有抑制作用。随着浓度的增加,对细胞的抑制率也逐渐增加。同一浓度的Par对胃癌细胞的抑制作用随着时间的延长也逐渐增强。
2、Par对胃癌细胞周期分布的影响
对照组SGC7901细胞的G_0/G_1期、S期和G_2/M期比例分别为49.60%±2.45%,30.80%±2.50%,19.6%±1.37%。在经140、160、180、200μmol/L处理24h后,G_0/G_1期比例逐渐增高,分别为63.56%±3.28%(p＜0.05),65.88%±1.60%(p＜0.01),74.27%±1.13%(p＜0.01),79.33%±3.21%(p＜0.01),S期比例逐渐下降,分别为21.56%±0.71%,16.81%±1.82%(p＜0.01),13.96%±0.35%(p＜0.05),6.40%±5.56%(p＜0.05),G_2/M期比例分别为14.88%±2.78%,17.31%±0.71%,11.78%±0.97%(p＜0.01),16.25%±1.45%。对照组BGC823细胞的G_0/G_1期、S期和G_2/M期比例分别为63.55%±1.03%,24.88%±1.01%,11.57%±0.54%。在经140、160、180、200μmol/L处理24h后,G_2/M期逐渐减少,分别为8.49%±0.12%(p＜0.05),8.11%±0.16%(p＜0.05),0.17%±0.04%(p＜0.01),0.06%±0.02%(p＜0.01),G_0/G_1期比例无显著变化,S期比例升高,分别为25.90%±0.70%,28.05%±13.77%,37.48%±0.95%(p＜0.01),35.14%±0.88%(p＜0.01)。在SGC7901细胞中,160～200μmol/L的Par处理后G_0/G_1期比例增高、S期比例下降有统计学意义。在BGC823细胞中160～200μmol/L的Par处理后G_2/M期比例下降,S期比例升高有统计学意义。
3、Par对胃癌细胞凋亡的影响
对照组SGC7901细胞早期凋亡率和晚期凋亡及坏死率分别为3.03%±0.75%和1.37%±0.57%,与对照组比较,经80、100、180μmol/L的Par处理24h后,,SGC7901细胞的早期凋亡率逐渐增加,分别为6.93%±2.55%,25.47%±4.93%(p＜0.05),50.16%±2.11%(p＜0.01),晚期凋亡和坏死率也逐渐增加,分别为6.26%±4%,11.67%±3.07%,18.95%±1.46%(p＜0.01)。对照组BGC823细胞的早期凋亡率和晚期凋亡率及坏死率分别为4.34%±0.73%,3.54%±1.41%,与对照组比较,经80、100、180μmol/L的Par处理24h后,BGC823细胞的早期凋亡率逐渐增加,分别为8.64%±1.52%,19.65%±2.89%(p＜0.05),32.78%±3.19(p＜0.01),晚期凋亡率及坏死率也逐渐增加,分别为6.78%±1.09%,21.56%±3.19%(p＜0.05),31.55%±3.78%(p＜0.01),上述结果表明Par以浓度依赖方式诱导胃癌细胞产生明显凋亡,包括早期和晚期凋亡。
4、Par对胃癌细胞△Ψm的影响
流式细胞仪结果表明SGC7901细胞正常对照组△Ψm降低的比例为10.38%±0.73%,经80、100、160、200μmol/L的Par处理24h后,△Ψm下降的比例分别为41.33%±11.06%,73.86%±5.66%(p＜0.01),98.8%±1.13%(p＜0.01),99.13%±0.77%(p＜0.01)。BGC823细胞正常对照组△Ψm降低的比例为15.43%±1.24%,经80、100、160、200μmol/L的Par处理24h后,△Ψm下降的比例分别为25.5%±7.56%,75.38%±1.29%(p＜0.01),89.23%±2.33%(p＜0.01),91.6%±1.74%(p＜0.01),结果表明Par以浓度依赖方式引起胃癌细胞发生线粒体膜电位去极化,提示Par可能通过降低线粒体膜电位引起细胞凋亡。
5、P2ur对胃癌细胞体外迁移的影响
Transwell结果表明SGC7901细胞正常对照组48h后穿膜细胞数为51±3.61,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为46±5.57,25.67±4.04(p＜0.01),9.67±2.08(p＜0.01)。BGC823细胞正常对照组48h后穿膜细胞数为116±9,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为95.33±7.02,46±6.56(p＜0.01),29±4(p＜0.01),结果表明Par以浓度依赖方式抑制胃癌细胞体外迁移活性。
6、Par对胃癌细胞体外侵袭的影响
transwell结果表明SGC7901细胞正常对照组48h后穿膜细胞数为95.33±8.74,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为76±6.56,33±6(p＜0.01),12.67±3.21(p＜0.01)。BGC823细胞正常对照组48h后穿膜细胞数为85.33±11.23,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为69.33±3.06,47.33±5.51(p＜0.05),23±6.56(p＜0.01),结果表明Par以浓度依赖方式抑制胃癌细胞体外侵袭活性。
7、光镜下细胞形态观察
对照组胃癌细胞其贴壁生长良好,细胞轮廓清晰,细胞间结构紧密,细胞生长旺盛,经80、100、200μmol/LPar作用后,逐渐脱落,悬浮于培养液中,贴壁细胞数量减少,细胞形态变圆,体积缩小,核出现固缩。从形态学上证实Par对胃癌细胞的生长抑制。
8、荧光显微镜下观察细胞凋亡
对照组的胃癌细胞核染色质着绿色,细胞核完整,经80、100、200μmol/L的Par作用后见凋亡细胞,细胞体积缩小,核染色质着橘黄色,核浓缩,有部分碎裂,呈大小不等、形态不规则的碎片,可找到凋亡小体。从形态学上证实Par诱导胃癌细胞凋亡的作用。
9、Par对胃癌细胞Bcl-2蛋白表达的影响
两株细胞的对照组经western blot检测可见明显的Bcl-2蛋白条带,SGC7901细胞对照组蛋白表达强度为2.65±0.08,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐减低,分别为2.57±0.029,2.18±0.95(p＜0.05),1.35±0.15(p＜0.01)。BGC823细胞对照组蛋白表达强度为2.75±0.08,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐减低,分别为2.44±0.16,2.14±0.1,1.42±0.11(p＜0.01)。提示Par在胃癌细胞中引起Bcl-2表达下调。
10、Par对胃癌细胞Caspase-8 mRNA表达的影响
SGC7901细胞对照组基因表达强度为0.23±0.046,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐增加,分别为0.36±0.046,0.53±0.035(p＜0.01),0.85±0.08(p＜0.01)。BGC823细胞对照组基因表达强度为0.23±0.055,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐增加,分别为0.35±0.092,0.55±0.073(p＜0.05),0.82±0.035(p＜0.01)。提示Par在胃癌细胞中引起Caspase-8表达上调。
结论
Par具有抑制SGC7901、BGC823细胞生长,诱导其凋亡的作用,可能是通过以下的机制发挥作用的。
1、Par能以剂量和时间依赖的方式抑制SGC7901、BGC823细胞生长,以剂量依赖的方式使SGC7901细胞周期阻止在G_0/G_1-S期,使BGC823细胞周期阻止在S-G_2/M期。
2、Par以剂量依赖方式降低SGC7901、BGC823细胞的线粒体膜电位,从而诱导细胞凋亡。
3、Par以剂量依赖方式抑制SGC7901、BGC823细胞体外迁移和侵袭活性。
4、Par以时间依赖方式下调SGC7901、BGC823细胞的Bcl-2蛋白表达水平。
5、Par以时间依赖方式上调SGC7901、BGC823细胞的Caspase-8 mRNA表达水平。
6、Bcl-2和Caspase-8两个基因参与了Par对SGC7901、BGC823细胞诱导凋亡的作用。
Objective
Parthenolide(Par) is a traditional medicine in western world.Besides its immunomodulating effect,it is believed to play a role in the treatment of human cancer,including hepatic cancer,breast cancer,leukemia,et al.Parthenolide regulates multiple cellular and molecular events in order to induce tumor cells apoptosis.
At the molecular level,these effect corresponded with the inhibition of nuclear factor-kappa B pathway,by which parthenolide regulates cycilns.Through regulation of tumor necrosis factor alpha parthenolide up-regulates or down-regulates downstream genes such as B-cell CLL/Lymphoma 2-associated X(Bcl-2) family of proteins and cysteinyl aspartate specific proteinase(caspase),in addition,parthenolide promotes the loss of mitochondrial lunction.These findings show the value of parthenolide in tumor therapy.However,the effect of parthenolide on gastric cancer is not understood.In order to know the value of parthenolide in treatment on gastric cancer,this study was aimed to gain further sight into the pathways mediating the anticancer proliferation and induction apoptosis of parthenolide.In this study,we examinated the effect of parthenolide on growth and apoptosis of the gastric cancer lines SGC7901,BGC823,the change of cell cycle,mitochondrial potential(ΔΨm),migration and invasion in vitro,and expression of bcl-2 in protein level and caspase-8 in mRNA level.
Methods
1.The inhibition of parthenolide on the proliferation of gastric cancer cells was analyzed by MTT assay
For MTT assay,gastric cancer cells were plated in 96-well plates,cells were treated with parthenolide for various concentration and various time periods.After drug treatment,attached cells were incubated with MTT and subsequely solubilized in DMSO.The absorbency at 490nm was then measured using a micoplate reader.
2.Cell cycle analysis was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with propidium iodide(PI) solution and analyzed by flow cytometry.Cell cycle was analyzed.
3.Cell apoptosis assays was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with annexin and V-FITC,then cells were analyzed by flow cytometry.
4.Measurement of mitochondrial membrane potential was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with rhodamine123 solution,then cells were analyzed by flow cytometry.
5.Migration capacity in vitro assays was performed by transwell
Gastric cancer cells were plated in transwell rooms,cells were treated with parthenolide for various concentration,after 48 hours we counted number of cells through the basement membrance.
6.Invasion capacity in vitro assays was performed by transwell covered with matrigel
Gastric cancer cells were plated in transwell rooms which covered with matrigel in advance,cells were treated with parthenolide for various concentration,after 48 hours we counted number of cells through the basement membrance.
7.Cell Morphous was observated with light microscope
Gastric cancer cells were plated in 6-well plates,cells were treated with parthenolide for various concentration,after 24 hours,we observated cell morphous with light microscope and took pictures with 100 photographic rate.
8.Apoptotic morphology of cells was observated with fluorescence microscopy
After parthenolide treatment for 24 hours,cells were collected and labeled with acridine orange,then we observated Apoptotic morphology of cells with 1 fluorescence microscopy and took pictures with 400 photographic rate.
9.Expression of Bcl-2 protein were detected by Western blot assay
After treatment with 7.5μmol/L parthenolide for 24,48,72 hours,cell protein was collected for assay.
10.Reverse transcription-PCR was used to detect transcriptional regulation of Caspase-8
After treatment with 7.5μmol/L parthenolide for 24,48,72 hours,RNA was collected for assay.
11.Statistical analysis
Data was presented as means±SD.Single factor analysis of variance was used for analyzing differences between groups with SPSS11.5,P value＜0.05 was considered significant.
Results
1.Effect of parthenolide on proliferation of gastric cancer cell lines
Parthenolide of 60μmol/L inhibited the proliferation of SGC7901 and BGC823 cells after treating cells for 48 hours(P＜0.05).After exposed to 100-200μmol/L parthenolide for 24 hours,the growth of two gastric cancer lines were significantly inhibited in dose-dependent manner.When cells were incubated with parthenolide for from 24 hours to 72 hours,cell viability was decreased in a time-dependent manner.
2.Effect of parthenolide on cell cycle status of gastric cancer cell lines
The percentage of cells in G_0/G_1,S,G_2/M phases of the cell cycle in untreated SGC7901 cells were 49.60%±2.45%,30.80%±2.50%and19.6%±1.37%.After exposed to 140,160,180,200μmol/L parthenolide for 24 hours,the rates of cells in the G_0/G_1 phase were 63.56%±3.28%(p＜0.05),65.88%±1.60%(p＜0.01),74.27%±1.13%(p＜0.01), and 79.33%±3.21%(p＜0.01),respectively,the rates of cells in the S phase were 21.56%±0.71%,16.81%±1.82%(p＜0.01),13.96%±0.35%(p＜0.05),6.40%±5.56% (p＜0.05),the rates of cells in the G_2/M phase were 14.88%±2.78%,17.31%±0.71%, 11.78%±0.97%(p＜0.01),16.25%±1.45%,respectively.The percentage of cells in G_0/G_1,S,G_2/M phases of the cell cycle in untreated BGC823 cells were 63.55%±1.03%, 24.88%±1.01%,11.57%±0.54%,respectively..After exposed to 140,160,180,200μmol/L parthenolide for 24 hours,the rates of cells in the G_2/M phase were 8.49%±0.12%(p＜0.05),8.11%±0.16%(p＜0.05),0.17%±0.04%(p＜0.01),0.06%±0.02% (p＜0.01),the rates of cells in the S phase were 25.90%±0.70%,28.05%±13.77%, 37.48%±0.95%(p＜0.01),35.14%±0.88%(p＜0.01),respectively.
Parthenolide in concentration of 160-200μmol/L induced accumulation in G_0/G_1 phase and attenuation in S phase of the cell cycle in SGC7901 cells.Parthenolide in concentration of 160-200μmol/L induced accumulation in S phase and attenuation in G_2/M phase of the cell cycle in BGC823 cells.
3.Effect of parthenolide on apoptosis of gastric cancer cell lines
The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control SGC7901 cells were 3.03%±0.75%and 1.37%±0.57%.Compared with the control group,after exposed to 80,100,180μmol/L parthenolide for 24hours,the percentage of SGC7901 cells undergoing apoptotic cell death gradually increased,the percentage of cells undergoing apoptotic cell death were 6.93%±2.55%,25.47%±4.93%(p＜0.05),50.16%±2.11%(p＜0.01),respectively.the percentage of cells undergoing necrotic cell death were 6.26%±4%,11.67%±3.07%, 18.95%±1.46%,respectively.The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control BGC823 cells were 4.34%±0.73%and3.54%±1.41%.Compared with the control group,after exposed to 80,100,180μmol/L parthenolide for 24hours,the percentage of BGC823 cells undergoing apoptotic cell death gradually increased,the percentage of cells undergoing apoptotic cell death were 8.64%±1.52%,19.65%±2.89%(p＜0.05),32.78±3.19(p＜0.01), respectively,the percentage of cells undergoing necrotic cell death were 6.78%±1.09%,21.56%±3.19%(p＜0.05),31.55%±3.78%(p＜0.01),respectively.Results showed that parthenolide induced dose-dependent apoptosis in both cell lines.
4.Effect of parthenolide on mitochondrial membrane potential(ΔΨm)
The percentage of cells with reducedΔΨm were 10.38%±0.73%in untreated SGC7901 cells.After exposed to 80,100,160,200μmol/L parthenolide for 24h,the percentage of cells with reducedΔΨm were 41.33%±11.06%,73.86%±5.66%(p＜0.01), 98.8%±1.13%(p＜0.01),99.13%±0.77%(p＜0.01),respectively.The percentage of cells with reducedΔΨm were 15.43%±1.24%in untreated BGC823 cells.After exposed to 80,100,160,200μmol/L parthenolide for 24h,the percentage of cells with reducedΔΨm were 25.5%±7.56%,75.38%±1.29%(p＜0.01),89.23%±2.33%(p＜0.01), 91.6%±1.74%(p＜0.01),respectively.Results showed that parthenolide induced a dose-dependent loss ofΔΨm in both cell lines.
5.Effect of parthenolide on migration capacity in vitro
The number of cells through the basement membrance were 51±3.61in untreated SGC7901 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 46±5.57,25.67±4.04(p＜0.01),9.67±2.08 (p＜0.01).The number of cells through the basement membrance were 116±9 in untreated BGC823 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 95.33±7.02,46±6.56(p＜0.01), 29±4(p＜0.01).Results showed that parthenolide induced a dose-dependent loss of migration capacity in both cell lines.
6.Effect of parthenolide on invasion capacity in vitro
The number of cells through the basement membrance were 95.33±8.74 in untreated SGC7901 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 76±6.56,33±6(p＜0.01), 12.67±3.21(p＜0.01).The number of cells through the basement membrance were 85.33±11.23 in untreated BGC823 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 69.33±3.06, 47.33±5.51(p＜0.05),23±6.56(p＜0.01).Results showed that parthenolide induced a dose-dependent loss of invasion capacity in both cell lines.
7.Effect of parthenolide on cell morphous
Cell adherenting wall growed well,close and strong in untreated cells.After exposed to 80,100,200μmol/L parthenolide for 24h,cell in smaller size exfoliated,then floated in water,decreased in number,karyopyknosis could be found.Results support from the morphology that parthenolide decreased proliferation in both cell lines.
8.Effect of parthenolide on apoptotic morphology
Nuclear remained integrity,nuclear chromatin stained green in untreated cells. After exposed to 80,100,200μmol/L parthenolide for 24h,nuclear chromatin stained organge in smaller size cells,karyopyknosis,fragmentation,and apoptotic bodies could be found.Results support from the morphology that apoptosis induction of parthenolide in both cell lines.
9.Effect of parthenolide on the expression of Bcl-2
The express intensity of Bcl-2 was 2.65±0.08 in untreated SGC7901 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of Bcl-2 were 2.57±0.029,2.18±0.95(p＜0.05),1.35±0.15(p＜0.01).The express intensity of Bcl-2 was 2.75±0.08 in untreated BGC823 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of Bcl-2 were 2.44±0.16, 2.14±0.1,1.42+0.11(p＜0.01).Results showed that parthenolide induced dose-dependent decrease of expression of Bcl-2 in both cell lines.
10.Effect of parthenolide on the expression of Caspase-8
The express intensity of caspase-8 was 0.23±0.046 in untreated SGC7901 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of caspase-8 were 0.36±0.046,0.53±0.035(p＜0.01),0.85±0.08(p＜0.01).The express intensity of caspase-8 was 0.23±0.055 in untreated BGC823 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of caspase-8 were 0.35±0.092,0.55±0.073(p＜0.05),0.82±0.035(p＜0.01).Results showed that parthenolide induced dose-dependent increase of expression of Caspase-8 in both cell lines.
Conclusions
1.Parthenolide inhibited proliferation of SGC7901 and BGC823 cells in dose-and time-dependent manners,cell morphous also support these points.
2.Parthenolide induced accumulation in G_0/G_1 phase and attenuation in S phase of the cell cycle in SGC7901 cells,and induced attenuation in G_2/M phase and accumulation in S phase of the cell cycle in BGC823 cells.
3.Parthenolide induced dose-dependent apoptosis in SGC7901 and BGC823 cells.,apoptotic morphology also support these points.
4.Parthenolide induced a dose-dependent loss ofΔΨm in SGC7901 and BGC823 cells.
5.Parthenolide induced a dose-dependent loss of migration capacity in SGC7901 and BGC823 cells.
6.Parthenolide induced a dose-dependent loss of invasion capacity in SGC7901 and BGC823 cells.
7.Parthenolide induced dose-dependent decrease of expression of Bcl-2 in SGC7901 and BGC823 cells.
8.Parthenolide induced dose-dependent increase of expression of Caspase-8 in SGC7901 and BGC823 cells.
小白菊内酯(parthenolide,Par)作为一种西方传统药物,除了免疫调节功效,对于来自肝脏,乳腺,前列腺,血液等的多种肿瘤细胞,在其发生发展过程中发挥良好的抑制效应,可以显著抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡。小白菊内酯的抗肿瘤机制与其作为NF-κB通路抑制剂息息相关,通过对NF-κB的阻断,调节细胞周期蛋白,由TNF-a通路调控众多下游基因如Bcl-2,Caspase及影响线粒体功能,显示了其在肿瘤治疗中的应用价值,但目前,国际上对Par在胃癌的研究方面还远不深入,为了探讨Par在胃癌治疗中的应用价值,我们研究了Par对胃癌细胞株SGC7901,BGC823细胞增殖,细胞周期和凋亡的影响;对胃癌细胞体外迁移和侵袭能力的影响,通过检测Par在处理细胞后线粒体膜电位(△Ψm)及凋亡检测蛋白Bcl-2和Caspase基因的改变,探讨在Par诱导胃癌细胞凋亡中线粒体途径和凋亡调节蛋白Bcl-2和Caspase-8的作用,进一步阐明Par抗肿瘤作用的机制。
方法
1、MTT法检测不同浓度Par对肿瘤细胞的抑制作用
取人胃癌细胞株SGC7901、BGC823细胞株,加入96孔板,实验组加入不同浓度的Par,对照组则加等量不含药物的培养液。Par处理24、48、72小时后,MTT法检测各孔吸光度(OD)值,计算细胞生长抑制率。
2、流式细胞仪分析细胞周期
消化收集各浓度Par处理24h的细胞,PI复合染液染色后,流式细胞仪检测,分析细胞周期。
3、流式细胞仪检测细胞凋亡
消化收集各浓度Par处理24h的细胞,Annexin V/FITC染色后,流式细胞仪检测,分析细胞凋亡百分比。
4、流式细胞仪检测Par对细胞△Ψm的影响
消化收集各浓度Par处理24h的细胞,罗丹明123染色后,流式细胞仪检测,分析线粒体膜电位降低细胞所占百分比。
5、transwell小室检测Par对细胞体外迁移的影响
以不同浓度Par处理细胞后,加入到transwell小室中,作用48h后计数穿膜细胞数。
6、被覆Matrigel胶的transwell小室检测Par对细胞体外侵袭的影响
以不同浓度Par处理细胞后,加入到transwell小室中,小室上层被覆Matrigel胶,作用48h后计数穿膜细胞数。
7、光镜下细胞形态观察
细胞接种于6孔板,以不同浓度Par处理细胞24h后,后倒置显微镜下用100×摄片,观察各组细胞形态。
8、荧光显微镜下观察细胞凋亡
收集以不同浓度Par处理24h的细胞,吖啶橙溶液染色,在荧光显微镜下用400×摄片,观察凋亡细胞形态。
9、western blot方法检测Bcl-2蛋白的表达
收集7.5μmol/L作用24,48,72h后的细胞蛋白,用western blot方法检测Bcl-2蛋白的表达。
10、RT-PCR方法检测Caspase-8 mRNA的表达
收集7.5μmol/L作用24,48,72h后的细胞蛋白,用RT-PCR方法检测Caspase-8基因的表达。
11、统计学处理
本实验所有数据用均数±标准差表示,利用SPSS11.5分析软件,采用单因素方差分析法进行差异性分析,P＜0.05被认为有显著差异。
结果
1、Par对胃癌细胞生长的抑制作用
Par在20μmol/L以下对SGC7901、BGC823细胞生长无显著影响,60μmol/LPar分别处理SGC7901、BGC823细胞48h后对细胞生长有抑制作用(P＜0.05),在100～200μmol/L浓度范围内,Par作用24h后,对胃癌细胞增殖有抑制作用。随着浓度的增加,对细胞的抑制率也逐渐增加。同一浓度的Par对胃癌细胞的抑制作用随着时间的延长也逐渐增强。
2、Par对胃癌细胞周期分布的影响
对照组SGC7901细胞的G_0/G_1期、S期和G_2/M期比例分别为49.60%±2.45%,30.80%±2.50%,19.6%±1.37%。在经140、160、180、200μmol/L处理24h后,G_0/G_1期比例逐渐增高,分别为63.56%±3.28%(p＜0.05),65.88%±1.60%(p＜0.01),74.27%±1.13%(p＜0.01),79.33%±3.21%(p＜0.01),S期比例逐渐下降,分别为21.56%±0.71%,16.81%±1.82%(p＜0.01),13.96%±0.35%(p＜0.05),6.40%±5.56%(p＜0.05),G_2/M期比例分别为14.88%±2.78%,17.31%±0.71%,11.78%±0.97%(p＜0.01),16.25%±1.45%。对照组BGC823细胞的G_0/G_1期、S期和G_2/M期比例分别为63.55%±1.03%,24.88%±1.01%,11.57%±0.54%。在经140、160、180、200μmol/L处理24h后,G_2/M期逐渐减少,分别为8.49%±0.12%(p＜0.05),8.11%±0.16%(p＜0.05),0.17%±0.04%(p＜0.01),0.06%±0.02%(p＜0.01),G_0/G_1期比例无显著变化,S期比例升高,分别为25.90%±0.70%,28.05%±13.77%,37.48%±0.95%(p＜0.01),35.14%±0.88%(p＜0.01)。在SGC7901细胞中,160～200μmol/L的Par处理后G_0/G_1期比例增高、S期比例下降有统计学意义。在BGC823细胞中160～200μmol/L的Par处理后G_2/M期比例下降,S期比例升高有统计学意义。
3、Par对胃癌细胞凋亡的影响
对照组SGC7901细胞早期凋亡率和晚期凋亡及坏死率分别为3.03%±0.75%和1.37%±0.57%,与对照组比较,经80、100、180μmol/L的Par处理24h后,,SGC7901细胞的早期凋亡率逐渐增加,分别为6.93%±2.55%,25.47%±4.93%(p＜0.05),50.16%±2.11%(p＜0.01),晚期凋亡和坏死率也逐渐增加,分别为6.26%±4%,11.67%±3.07%,18.95%±1.46%(p＜0.01)。对照组BGC823细胞的早期凋亡率和晚期凋亡率及坏死率分别为4.34%±0.73%,3.54%±1.41%,与对照组比较,经80、100、180μmol/L的Par处理24h后,BGC823细胞的早期凋亡率逐渐增加,分别为8.64%±1.52%,19.65%±2.89%(p＜0.05),32.78%±3.19(p＜0.01),晚期凋亡率及坏死率也逐渐增加,分别为6.78%±1.09%,21.56%±3.19%(p＜0.05),31.55%±3.78%(p＜0.01),上述结果表明Par以浓度依赖方式诱导胃癌细胞产生明显凋亡,包括早期和晚期凋亡。
4、Par对胃癌细胞△Ψm的影响
流式细胞仪结果表明SGC7901细胞正常对照组△Ψm降低的比例为10.38%±0.73%,经80、100、160、200μmol/L的Par处理24h后,△Ψm下降的比例分别为41.33%±11.06%,73.86%±5.66%(p＜0.01),98.8%±1.13%(p＜0.01),99.13%±0.77%(p＜0.01)。BGC823细胞正常对照组△Ψm降低的比例为15.43%±1.24%,经80、100、160、200μmol/L的Par处理24h后,△Ψm下降的比例分别为25.5%±7.56%,75.38%±1.29%(p＜0.01),89.23%±2.33%(p＜0.01),91.6%±1.74%(p＜0.01),结果表明Par以浓度依赖方式引起胃癌细胞发生线粒体膜电位去极化,提示Par可能通过降低线粒体膜电位引起细胞凋亡。
5、P2ur对胃癌细胞体外迁移的影响
Transwell结果表明SGC7901细胞正常对照组48h后穿膜细胞数为51±3.61,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为46±5.57,25.67±4.04(p＜0.01),9.67±2.08(p＜0.01)。BGC823细胞正常对照组48h后穿膜细胞数为116±9,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为95.33±7.02,46±6.56(p＜0.01),29±4(p＜0.01),结果表明Par以浓度依赖方式抑制胃癌细胞体外迁移活性。
6、Par对胃癌细胞体外侵袭的影响
transwell结果表明SGC7901细胞正常对照组48h后穿膜细胞数为95.33±8.74,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为76±6.56,33±6(p＜0.01),12.67±3.21(p＜0.01)。BGC823细胞正常对照组48h后穿膜细胞数为85.33±11.23,经30、50、80μmol/L的Par处理48h后,穿膜细胞数逐渐减少,分别为69.33±3.06,47.33±5.51(p＜0.05),23±6.56(p＜0.01),结果表明Par以浓度依赖方式抑制胃癌细胞体外侵袭活性。
7、光镜下细胞形态观察
对照组胃癌细胞其贴壁生长良好,细胞轮廓清晰,细胞间结构紧密,细胞生长旺盛,经80、100、200μmol/LPar作用后,逐渐脱落,悬浮于培养液中,贴壁细胞数量减少,细胞形态变圆,体积缩小,核出现固缩。从形态学上证实Par对胃癌细胞的生长抑制。
8、荧光显微镜下观察细胞凋亡
对照组的胃癌细胞核染色质着绿色,细胞核完整,经80、100、200μmol/L的Par作用后见凋亡细胞,细胞体积缩小,核染色质着橘黄色,核浓缩,有部分碎裂,呈大小不等、形态不规则的碎片,可找到凋亡小体。从形态学上证实Par诱导胃癌细胞凋亡的作用。
9、Par对胃癌细胞Bcl-2蛋白表达的影响
两株细胞的对照组经western blot检测可见明显的Bcl-2蛋白条带,SGC7901细胞对照组蛋白表达强度为2.65±0.08,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐减低,分别为2.57±0.029,2.18±0.95(p＜0.05),1.35±0.15(p＜0.01)。BGC823细胞对照组蛋白表达强度为2.75±0.08,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐减低,分别为2.44±0.16,2.14±0.1,1.42±0.11(p＜0.01)。提示Par在胃癌细胞中引起Bcl-2表达下调。
10、Par对胃癌细胞Caspase-8 mRNA表达的影响
SGC7901细胞对照组基因表达强度为0.23±0.046,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐增加,分别为0.36±0.046,0.53±0.035(p＜0.01),0.85±0.08(p＜0.01)。BGC823细胞对照组基因表达强度为0.23±0.055,经7.5μmol/L的Par作用24h、48h、72h后,表达强度逐渐增加,分别为0.35±0.092,0.55±0.073(p＜0.05),0.82±0.035(p＜0.01)。提示Par在胃癌细胞中引起Caspase-8表达上调。
结论
Par具有抑制SGC7901、BGC823细胞生长,诱导其凋亡的作用,可能是通过以下的机制发挥作用的。
1、Par能以剂量和时间依赖的方式抑制SGC7901、BGC823细胞生长,以剂量依赖的方式使SGC7901细胞周期阻止在G_0/G_1-S期,使BGC823细胞周期阻止在S-G_2/M期。
2、Par以剂量依赖方式降低SGC7901、BGC823细胞的线粒体膜电位,从而诱导细胞凋亡。
3、Par以剂量依赖方式抑制SGC7901、BGC823细胞体外迁移和侵袭活性。
4、Par以时间依赖方式下调SGC7901、BGC823细胞的Bcl-2蛋白表达水平。
5、Par以时间依赖方式上调SGC7901、BGC823细胞的Caspase-8 mRNA表达水平。
6、Bcl-2和Caspase-8两个基因参与了Par对SGC7901、BGC823细胞诱导凋亡的作用。
Objective
Parthenolide(Par) is a traditional medicine in western world.Besides its immunomodulating effect,it is believed to play a role in the treatment of human cancer,including hepatic cancer,breast cancer,leukemia,et al.Parthenolide regulates multiple cellular and molecular events in order to induce tumor cells apoptosis.
At the molecular level,these effect corresponded with the inhibition of nuclear factor-kappa B pathway,by which parthenolide regulates cycilns.Through regulation of tumor necrosis factor alpha parthenolide up-regulates or down-regulates downstream genes such as B-cell CLL/Lymphoma 2-associated X(Bcl-2) family of proteins and cysteinyl aspartate specific proteinase(caspase),in addition,parthenolide promotes the loss of mitochondrial lunction.These findings show the value of parthenolide in tumor therapy.However,the effect of parthenolide on gastric cancer is not understood.In order to know the value of parthenolide in treatment on gastric cancer,this study was aimed to gain further sight into the pathways mediating the anticancer proliferation and induction apoptosis of parthenolide.In this study,we examinated the effect of parthenolide on growth and apoptosis of the gastric cancer lines SGC7901,BGC823,the change of cell cycle,mitochondrial potential(ΔΨm),migration and invasion in vitro,and expression of bcl-2 in protein level and caspase-8 in mRNA level.
Methods
1.The inhibition of parthenolide on the proliferation of gastric cancer cells was analyzed by MTT assay
For MTT assay,gastric cancer cells were plated in 96-well plates,cells were treated with parthenolide for various concentration and various time periods.After drug treatment,attached cells were incubated with MTT and subsequely solubilized in DMSO.The absorbency at 490nm was then measured using a micoplate reader.
2.Cell cycle analysis was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with propidium iodide(PI) solution and analyzed by flow cytometry.Cell cycle was analyzed.
3.Cell apoptosis assays was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with annexin and V-FITC,then cells were analyzed by flow cytometry.
4.Measurement of mitochondrial membrane potential was performed by flow cytometry
After parthenolide treatment for 24 hours,cells were collected and labeled with rhodamine123 solution,then cells were analyzed by flow cytometry.
5.Migration capacity in vitro assays was performed by transwell
Gastric cancer cells were plated in transwell rooms,cells were treated with parthenolide for various concentration,after 48 hours we counted number of cells through the basement membrance.
6.Invasion capacity in vitro assays was performed by transwell covered with matrigel
Gastric cancer cells were plated in transwell rooms which covered with matrigel in advance,cells were treated with parthenolide for various concentration,after 48 hours we counted number of cells through the basement membrance.
7.Cell Morphous was observated with light microscope
Gastric cancer cells were plated in 6-well plates,cells were treated with parthenolide for various concentration,after 24 hours,we observated cell morphous with light microscope and took pictures with 100 photographic rate.
8.Apoptotic morphology of cells was observated with fluorescence microscopy
After parthenolide treatment for 24 hours,cells were collected and labeled with acridine orange,then we observated Apoptotic morphology of cells with 1 fluorescence microscopy and took pictures with 400 photographic rate.
9.Expression of Bcl-2 protein were detected by Western blot assay
After treatment with 7.5μmol/L parthenolide for 24,48,72 hours,cell protein was collected for assay.
10.Reverse transcription-PCR was used to detect transcriptional regulation of Caspase-8
After treatment with 7.5μmol/L parthenolide for 24,48,72 hours,RNA was collected for assay.
11.Statistical analysis
Data was presented as means±SD.Single factor analysis of variance was used for analyzing differences between groups with SPSS11.5,P value＜0.05 was considered significant.
Results
1.Effect of parthenolide on proliferation of gastric cancer cell lines
Parthenolide of 60μmol/L inhibited the proliferation of SGC7901 and BGC823 cells after treating cells for 48 hours(P＜0.05).After exposed to 100-200μmol/L parthenolide for 24 hours,the growth of two gastric cancer lines were significantly inhibited in dose-dependent manner.When cells were incubated with parthenolide for from 24 hours to 72 hours,cell viability was decreased in a time-dependent manner.
2.Effect of parthenolide on cell cycle status of gastric cancer cell lines
The percentage of cells in G_0/G_1,S,G_2/M phases of the cell cycle in untreated SGC7901 cells were 49.60%±2.45%,30.80%±2.50%and19.6%±1.37%.After exposed to 140,160,180,200μmol/L parthenolide for 24 hours,the rates of cells in the G_0/G_1 phase were 63.56%±3.28%(p＜0.05),65.88%±1.60%(p＜0.01),74.27%±1.13%(p＜0.01), and 79.33%±3.21%(p＜0.01),respectively,the rates of cells in the S phase were 21.56%±0.71%,16.81%±1.82%(p＜0.01),13.96%±0.35%(p＜0.05),6.40%±5.56% (p＜0.05),the rates of cells in the G_2/M phase were 14.88%±2.78%,17.31%±0.71%, 11.78%±0.97%(p＜0.01),16.25%±1.45%,respectively.The percentage of cells in G_0/G_1,S,G_2/M phases of the cell cycle in untreated BGC823 cells were 63.55%±1.03%, 24.88%±1.01%,11.57%±0.54%,respectively..After exposed to 140,160,180,200μmol/L parthenolide for 24 hours,the rates of cells in the G_2/M phase were 8.49%±0.12%(p＜0.05),8.11%±0.16%(p＜0.05),0.17%±0.04%(p＜0.01),0.06%±0.02% (p＜0.01),the rates of cells in the S phase were 25.90%±0.70%,28.05%±13.77%, 37.48%±0.95%(p＜0.01),35.14%±0.88%(p＜0.01),respectively.
Parthenolide in concentration of 160-200μmol/L induced accumulation in G_0/G_1 phase and attenuation in S phase of the cell cycle in SGC7901 cells.Parthenolide in concentration of 160-200μmol/L induced accumulation in S phase and attenuation in G_2/M phase of the cell cycle in BGC823 cells.
3.Effect of parthenolide on apoptosis of gastric cancer cell lines
The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control SGC7901 cells were 3.03%±0.75%and 1.37%±0.57%.Compared with the control group,after exposed to 80,100,180μmol/L parthenolide for 24hours,the percentage of SGC7901 cells undergoing apoptotic cell death gradually increased,the percentage of cells undergoing apoptotic cell death were 6.93%±2.55%,25.47%±4.93%(p＜0.05),50.16%±2.11%(p＜0.01),respectively.the percentage of cells undergoing necrotic cell death were 6.26%±4%,11.67%±3.07%, 18.95%±1.46%,respectively.The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control BGC823 cells were 4.34%±0.73%and3.54%±1.41%.Compared with the control group,after exposed to 80,100,180μmol/L parthenolide for 24hours,the percentage of BGC823 cells undergoing apoptotic cell death gradually increased,the percentage of cells undergoing apoptotic cell death were 8.64%±1.52%,19.65%±2.89%(p＜0.05),32.78±3.19(p＜0.01), respectively,the percentage of cells undergoing necrotic cell death were 6.78%±1.09%,21.56%±3.19%(p＜0.05),31.55%±3.78%(p＜0.01),respectively.Results showed that parthenolide induced dose-dependent apoptosis in both cell lines.
4.Effect of parthenolide on mitochondrial membrane potential(ΔΨm)
The percentage of cells with reducedΔΨm were 10.38%±0.73%in untreated SGC7901 cells.After exposed to 80,100,160,200μmol/L parthenolide for 24h,the percentage of cells with reducedΔΨm were 41.33%±11.06%,73.86%±5.66%(p＜0.01), 98.8%±1.13%(p＜0.01),99.13%±0.77%(p＜0.01),respectively.The percentage of cells with reducedΔΨm were 15.43%±1.24%in untreated BGC823 cells.After exposed to 80,100,160,200μmol/L parthenolide for 24h,the percentage of cells with reducedΔΨm were 25.5%±7.56%,75.38%±1.29%(p＜0.01),89.23%±2.33%(p＜0.01), 91.6%±1.74%(p＜0.01),respectively.Results showed that parthenolide induced a dose-dependent loss ofΔΨm in both cell lines.
5.Effect of parthenolide on migration capacity in vitro
The number of cells through the basement membrance were 51±3.61in untreated SGC7901 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 46±5.57,25.67±4.04(p＜0.01),9.67±2.08 (p＜0.01).The number of cells through the basement membrance were 116±9 in untreated BGC823 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 95.33±7.02,46±6.56(p＜0.01), 29±4(p＜0.01).Results showed that parthenolide induced a dose-dependent loss of migration capacity in both cell lines.
6.Effect of parthenolide on invasion capacity in vitro
The number of cells through the basement membrance were 95.33±8.74 in untreated SGC7901 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 76±6.56,33±6(p＜0.01), 12.67±3.21(p＜0.01).The number of cells through the basement membrance were 85.33±11.23 in untreated BGC823 cells.After exposed to 30,50,80μmol/L parthenolide for 48h,the number of cells through the basement membrance were 69.33±3.06, 47.33±5.51(p＜0.05),23±6.56(p＜0.01).Results showed that parthenolide induced a dose-dependent loss of invasion capacity in both cell lines.
7.Effect of parthenolide on cell morphous
Cell adherenting wall growed well,close and strong in untreated cells.After exposed to 80,100,200μmol/L parthenolide for 24h,cell in smaller size exfoliated,then floated in water,decreased in number,karyopyknosis could be found.Results support from the morphology that parthenolide decreased proliferation in both cell lines.
8.Effect of parthenolide on apoptotic morphology
Nuclear remained integrity,nuclear chromatin stained green in untreated cells. After exposed to 80,100,200μmol/L parthenolide for 24h,nuclear chromatin stained organge in smaller size cells,karyopyknosis,fragmentation,and apoptotic bodies could be found.Results support from the morphology that apoptosis induction of parthenolide in both cell lines.
9.Effect of parthenolide on the expression of Bcl-2
The express intensity of Bcl-2 was 2.65±0.08 in untreated SGC7901 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of Bcl-2 were 2.57±0.029,2.18±0.95(p＜0.05),1.35±0.15(p＜0.01).The express intensity of Bcl-2 was 2.75±0.08 in untreated BGC823 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of Bcl-2 were 2.44±0.16, 2.14±0.1,1.42+0.11(p＜0.01).Results showed that parthenolide induced dose-dependent decrease of expression of Bcl-2 in both cell lines.
10.Effect of parthenolide on the expression of Caspase-8
The express intensity of caspase-8 was 0.23±0.046 in untreated SGC7901 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of caspase-8 were 0.36±0.046,0.53±0.035(p＜0.01),0.85±0.08(p＜0.01).The express intensity of caspase-8 was 0.23±0.055 in untreated BGC823 cells.After exposed to 7.5μmol/L parthenolide for 24,48,72 hours,the express intensity of caspase-8 were 0.35±0.092,0.55±0.073(p＜0.05),0.82±0.035(p＜0.01).Results showed that parthenolide induced dose-dependent increase of expression of Caspase-8 in both cell lines.
Conclusions
1.Parthenolide inhibited proliferation of SGC7901 and BGC823 cells in dose-and time-dependent manners,cell morphous also support these points.
2.Parthenolide induced accumulation in G_0/G_1 phase and attenuation in S phase of the cell cycle in SGC7901 cells,and induced attenuation in G_2/M phase and accumulation in S phase of the cell cycle in BGC823 cells.
3.Parthenolide induced dose-dependent apoptosis in SGC7901 and BGC823 cells.,apoptotic morphology also support these points.
4.Parthenolide induced a dose-dependent loss ofΔΨm in SGC7901 and BGC823 cells.
5.Parthenolide induced a dose-dependent loss of migration capacity in SGC7901 and BGC823 cells.
6.Parthenolide induced a dose-dependent loss of invasion capacity in SGC7901 and BGC823 cells.
7.Parthenolide induced dose-dependent decrease of expression of Bcl-2 in SGC7901 and BGC823 cells.
8.Parthenolide induced dose-dependent increase of expression of Caspase-8 in SGC7901 and BGC823 cells.
引文
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