Experimental Study On Anti-proliferation And Apoptosis-induction Of Gastric Cancer Cell Lines Induced By Parthenolide

ObjectiveParthenolide(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.Methods1.The inhibition of parthenolide on the proliferation of gastric cancer cells was analyzed by MTT assayFor 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 cytometryAfter 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 cytometryAfter 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 cytometryAfter 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 transwellGastric 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 matrigelGastric 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 microscopeGastric 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 microscopyAfter 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 assayAfter 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-8After treatment with 7.5μmol/L parthenolide for 24,48,72 hours,RNA was collected for assay.11.Statistical analysisData 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.Results1.Effect of parthenolide on proliferation of gastric cancer cell linesParthenolide 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 linesThe percentage of cells in G₀/G₁,S,G₂/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 G₀/G₁ 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 G₂/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 G₀/G₁,S,G₂/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 G₂/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 G₀/G₁ 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 G₂/M phase of the cell cycle in BGC823 cells.3.Effect of parthenolide on apoptosis of gastric cancer cell linesThe 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 vitroThe 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 vitroThe 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 morphousCell 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 morphologyNuclear 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-2The 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-8The 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.Conclusions1.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 G₀/G₁ phase and attenuation in S phase of the cell cycle in SGC7901 cells,and induced attenuation in G₂/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.