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Iso-suillin Isolated From Suillus Luteus, Induces G1phase Arrest And Apoptosis In Human Hepatoma SMMC-7721Cells

Posted on:2015-03-08Degree:MasterType:Thesis
Country:ChinaCandidate:Z Q JiaFull Text:PDF
GTID:2254330428974454Subject:Cell biology
Abstract/Summary:PDF Full Text Request
Objective: Hepatocellular carcinoma (HCC) is a primary malignancy ofthe liver. The incidence of HCC is highest in Asia, particularly in China andJapan. In China,50people per100,000are affected annually. HCC is now thesecond leading cause of cancer death in China. Traditional surgical resection,and assisted by means of radiotherapy, chemotherapy is still the maintreatment measures. At present, the clinical use of chemotherapy drugs to killcancer cells, at the same time, also kill the normal cells and lymphocytes,caused the patient much pain. Therefore, looking for high efficiency, lowtoxicity of drugs is the enormity of the task for every medical staff. Fromnatural plants and large fungal metabolites is the choice of the drugs.We isolated a suillin isoform from a petroleum ether extract of Suillusflavus, called iso-suillin, which also belonged to the prenylphenol class. Weget the relative molecular weight as440.29. In present study, we foundiso-suillin has the activity of antioxidant and antitumor, so we investigated theeffects of iso-suillin on cell proliferation and apoptosis and examined theexpression of the proteins associated with cell cycle regulation and apoptosisin SMMC-7721cells.Methods:1. Cell cultureSMMC-7721cells were cultured in RPMI-1640medium supplementedwith10%heat-inactivated fetal bovine serum, penicillin100U/mL,streptomycin100μg/mL at37℃in an incubator, containing5%CO2.2. Cell viability measured by MTT assay in vitroThe exponential growth phase of SMMC-7721was planted into96-wellmultiplates and after adhesion the cells were treated with series concentrationsof iso-suillin, Cisplatin, or5-fluorouracil. The cells were cultured for different time periods (24,48, or72h). We also conducted a test for normalhuman lymphocytes after iso-suillin or cisplatin incubation for24h. Thesupernatant was discarded and20μl MTT (1mg/mL) was added to each well.The cells were cultured for another4h and then centrifuged. After thesupernatant was discarded,150μl DMSO was added by shaking for10min inthe dark. The absorbance at490nm was measured using a microplate reader(BioTek, USA). The50%inhibitory concentration (IC50) was calculated.3. The distribution of cell cycle and apoptosis rate detected by flow cytometry(FCM)The cells were collected, fixed with70%ethanol for24hours at4℃, thenstained using EB and an Annexin V-FITC/PI kit which according to themanufacturer’s instructions for30minutes respectively. The cells weremeasured by flow cytometry.4. Colony formation assaySMMC-7721cells were treated with series concentrations of iso-suillin for48h. After treatment, the cells were suspended and re-seeded into6-wellplates at a density of200cells per well. After two weeks, cells were fixedusing4%paraformaldehyde (PFA) and stained with Crystal Violet StainingSolution. The visible colonies (≥50cells) were counted and typical imageswere photographed using a common Nikon camera.5. Observation of morphologic changesSMMC-7721cells were seeded into6-well plates and exposed to theindicated concentration of iso-suillin for48h. The cells were stained with4’,6-diamidino-2-phenylindole (DAPI [Sigma, USA]). Cellular morphology wasobserved using a fluorescence microscope (Nikon, Tokyo, Japan). Apoptosisdetection was completed in the Hebei Medical University ElectronMicroscope Room using transmission electron microscopy (H-7500, Hitachi).6. Analysis of the mitochondrial membrane potentialThe change in mitochondrial membrane potential (MMP) after iso-suillintreatment was analyzed by FCM using rhodamine (Rh)-123(Sigma, USA)staining. Then the cells were stained in PBS containing3μg/mL Rh-123at 37°C in the dark for30min. The stained cells were then washed with ice-coldPBS, and the Rh-123fluorescence was detected by FCM.7. Protein extraction and Western blottingThe protein was abstracted on ice from SMMC-7721cells lysed in buffer.Protein concentration was determined using Coomassie brilliant blue G250protein assay kit. Samples (50μg) were separated by SDS-PAGE and weretransferred to a nitrocellulose filter (NC filter). The membrane was blocked for1hour at room temperature with blocking solution containing5%nonfat milkand then incubated with primary antibody overnight at4℃. The membranewas washed and incubated with secondary antibody (goat antimouseIgG-horseradish peroxidase) at37℃for1hour and then washed againanddeveloped using the ECL kit according to the supplier’s instructions. Themembrane was photographed and then analysed with gel image analysissystem expressed as the ratio of the integrated optical density (IOD) to that ofβ-actin acting as an internal standard.8. Statistical analysesThe results were expressed as mean±standard deviation and evaluated withSPSS13.0solftware by analysis of variance (One-Way ANOVA). P<0.05wasconsided statistically significant.Results:1. Proliferation inhibition caused by iso-suillin treatment in SMMC-7721cellsThe cells were treated with different concentrations of iso-suillin for24,48,or72h, and MTT assay showed iso-suillin significantly suppressedSMMC-7721cell proliferation and viability in a concentration-dependentmanner, with time-dependent inhibition. IC50values were26.1,2.8and1.2μM for24,48and72h respectively. Cisplatin and5-fluorouracil are commonchemotherapy drugs, so we also investigated cell viability after iso-suillin,cisplatin and5-fluorouracil incubation for48h. The results showed that allthree drugs could significantly inhibit SMMC-7721cell viability. For normalhuman lymphocytes, there was no obvious inhibitory effect observed afteriso-suillin or cisplatin incubation for24h. 2. The distribution of cell cycle and apoptosis rate by FCMThe G0/G1phase of SMMC-7721cells after iso-suillin treated48h are51±2.00%、64.03±3.00%、69.53±0.97%及73.64±1.06%; S phase cells are26.07±2.18%、17.30±1.30%、14.67±1.54%and11.2±0.98%; the G2/M phasecells are22.93±0.59%、18.67±1.86%、15.80±2.39%、15.20±1.35%. TheG0/G1phase cells of SMMC-7721were increased as compared with control(P<0.05). This result suggests that G0/G1cell cycle arrest exists inSMMC-7721cells. The early apoptosis rates are1.27±0.12%、3.61±0.15%、4.79±0.08%、2.41±0.08%; the later apoptosis rates are3.85±0.44%、35.64±1.73%、38.82±0.72%、46.76±0.97%, there were both significant inSMMC-7721cells compared with control (P<0.05).3. Colony formation inhibition caused by iso-suillin treatment in SMMC-7721cellsThe number of colony foci was100±0、77.33±8.02、49.33±7.09and26.67±9.61, which showed a dose-dependent decrease after treatment with0,1.4,2.8and5.6μM iso-suillin.4. Morphological changes in iso-suillin-treated SMMC-7721cellsWe conducted DAPI staining and electron microscopy to observemorphologic changes. When SMMC-7721cells were treated with differentconcentrations of iso-suillin for48h, followed by DAPI staining, iso-suillininduced cell morphological changes and decreased cell numbers. Cells alsobecame smaller, round and blunt and showed nuclear fragmentation whencompared with control SMMC-7721cells. After treatment of cells withiso-suillin for48h, we observed the cells using transmission electronmicroscopy. We found that in normal cells, the plasma membrane, nuclearenvelope and nucleolus were complete and distinct, while the treated cellsexhibited shrinkage, chromatic agglutination, nuclear and plasma membraneconvolution. The results indicated that the cytotoxic action of iso-suillin wasdue to its ability to induce apoptosis.5. The effect of iso-suillin on mitochondrial membrane potentialCells were treated with0,1.4,2.8and5.6μM iso-suillin for48h,then the cells were stained with rhodamine123and analyzed by FCM, themitochondrial membrane potential decreased remarkably when treated with1.4μM iso-suillin and this became more pronounced when treated with2.8and5.6μM iso-suillin.6. Protein extraction and Western blottingThe ratios of IOD between Caspase-3and β-actin protein were0.53±0.03、1.14±0.16、1.66±0.33and1.43±0.02; the ratio of Caspase-8were1.11±0.13、1.22±0.09、1.34±0.20and1.59±0.56; the ratio of Caspase-9were1.22±0.11、1.21±0.01、1.44±0.21and1.79±0.06; the ratio of Cyto c were0.13±0.01、0.16±0.01、0.18±0.02and0.89±0.03; the ratio of FADD were0.14±0.01、0.21±0.04、0.22±0.18and0.32±0.01; the ratio of p53were0.87±0.01、1.26±0.16、1.82±0.34and1.56±0.45; the ratio of p21were0.14±0.01、0.16±0.02、0.21±0.01and0.87±0.30; the ratio of p-Rb were1.64±0.07、1.41±0.10、1.46±0.47and0.94±0.06; the ratio of E2F-1were1.22±0.14、0.44±0.01、0.42±0.04and0.31±0.04; the ratio of CDK2were1.03±0.03、0.98±0.05、0.80±0.07and0.59±0.09; the ratio of CDK4were3.39±0.38、2.30±0.41、1.26±0.28and1.00±0.09; the ratio of Cyclin E1were0.87±0.11、0.25±0.03、0.24±0.01and0.23±0.02; the ratio of Cyclin D1were0.91±0.04、0.78±0.04、0.73±0.01'0.74±0.02; the results showed that after iso-suillintreated for48h, Caspase-3、-8、-9、Cytochrome C、FADD、p53and p21proteinexpression was up-regulated; p-RB、E2F-1、CyclinE1、CyclinD1、CDK2、CDK4protein expression was down-regulated. There was significancebetween experimental group and control group (P<0.05).Conclusions:1Iso-suillin significantly suppressed SMMC-7721cells proliferation andviability and there was no obvious inhibitory effect for normal humanlymphocytes.2Iso-suillin induced apoptosis in SMMC-7721cells.3Iso-suillin induced G0/G1cell cycle arrest in SMMC-7721cells.4Iso-suillin can induce G0/G1cell cycle arrest, which may be closely relatedto the expression of p53/p21, cyclin E1, cyclinD1, CDK2, CKD4. 5Iso-suillin induced apoptosis by the death receptor pathway and themitochondrial pathway in SMMC-7721cells.
Keywords/Search Tags:Iso-suillin, G0/G1phase arrest, apoptosis, SMMC-7721cells, cell proliferation, mitochondrial pathway, death-receptor pathway
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