Effect Of PAB On The Apoptosis, Mitochondrial Membrane Potential And COX-2 Expression In Hepatocellular Carcinoma Line BEL-7402

ObjectiveThe tree Pseudolarix kaempferi Gorden(Pinaceae)is indigenous to central China. The bark of this tree has been processed into the medicinal herb 'tujinpi'.Subsequent phytochemical studies led to the isolation and identification of the diterpenoid pseudolaric acid B(PAB)as the major biologically active components.Recently,it was discovered that PAB exhibited considerable cytotoxicity toward several cancer cell lines,including those of stomach,lung,colon,cervix and melanoma.PAB could inhibit these tumor cells' proliferation and induced apoptosis which may be associated with the change of activity of cyclin,cyclin-dependent kinase,p53,protein kinase C, Caspase,Bcl-2 family and some important signal transmitting paths.But the related mechanism was not completely clear and may be different because of the various cell types.At present people pay close attention to the role of mitochodria in apoptosis following deeper studies and mitochodria are thought to be the controller of apoptosis. Mitochodria play two important roles in apoptosis.First,they provide the necessary energy to cells in the form of ATP.Second,mitochodria have initiate a series of events,eg.The decrease of mitochondria membrane potential,the reversal of membrane permeability,the release of apoptosis specific enzyme activator cytochrome C and apoptosis inducing factor(AIF),which will decide the occurrence of apoptosis.Thus mitochondria are thought to be the controller of apoptosis. Cycloxygenase(COX)-2 is inducible.COX-2 can't be detected in the normal and physiological status and increases when cells are stimulated by inflammatory signal,some cytokines,carcinogenic promoting agents and so on.The recent studies show that COX-2 is closely associated with the genesis of hepatoma and colon cancer,except its important role in inflammation.The hepatocellular carcinoma line BEL-7402 express COX-2 strongly.This study observed the effect of PAB on the proliferation and apoptosis of hepatocellular carcinoma line BEL-7402 and discussed the changes of mitochodria membrane potential and COX-2 during apoptosis in order to understand the antitumor mechanism of PAB.Methods1.cell cultureThe cells were inoculated with medium RPMI-1640 which contained 10%fetal bovine serum and 100U/L penicillin and streptomycin.The incubator was set to 37℃,5 %CO₂ and 95%humidity.The cells grew in the way of monolayer and adherence and the passage were went on every 48 or 72 hours.2.The proliferation of BEL-7402 was analyazed by MTT assayThe density of BEL-7402 was regulated to 5×10⁴/L,then inocubated in 96-well plates with various concentration of PAB for 24,48,72h.The optical density of every well was detected by MTT assay and the cell growth inhibiting ratio was calculated.3.The cell cycle analysis was performed by flow cytometryThe cells treated with various concentration of PAB for 24h were collected and the number was modulated to 1×10⁶.The cells were fixed overnight in 70%ethanol at 4℃and stained with PI compound staining solution for 20 minutes away from light before analyzed by flow cytometry.CellQuest soft gets 10⁴single cell and the cell cycle and the rate of apoptosis was analyzed by ModFitLT soft.4.The acridine orange staining The cells treated with various concentration of PAB for 24h were collected and made to cell suspension.After stained with 0.1mg/mL acridine orange the cells were observed in fluorescence microscope and taken pictures.5.The detection of mitochodria membrane potential(Δψm)The cells treated with various concentration of PAB for 24h were collected and stained with JC-1solution.The mitochodria membrane potential was detected by flow cytometry(Ex=488nm;Em=530nm)and the cells were observed in fluorescence microscope and taken pictures.6.Expression of COX-2 protein was detected by Western blot7.Statistical analysisAll data were demonstrated as mean±SD and Students't test was performed by SPSS 13.0 soft.Results1.Growth inhibition of PAB on BEL-7402Various concentration of PAB(0.5,1.0,2.0,4.0μmol/L)inhibited cell growth in a time-dependent and dose-dependent manner.The inhibition ratio approach to 100% after the cells were treated with 2.0μmol/L PAB for 72h.2.The effect of PAB on cell cycle and apoptosisAfter the cells were treated with different concentration of PAB for 24h,the ratio of G0/G1 phase and S phase decreased,the ratio of G2/M phase increased gradually.After the cells were treated with 4.0μmol/L PAB for 24h,the ratio of G0/G1,S and G2/M phase was(0.61±0.12)%,(1.96±0.07)%,(97.43±1.46)%, suggesting that PAB could induce cell cycle arrest at the G2/M phase;Compared with control group,2.0,4.0μmol/L PAB could induce more cell apoptosis and the rate was (19.06±3.87)%,(31.19±1.46)%respectively(P＜0.05).3.The change of morphology The cells of control group showed as green fluorescence with normal nuclear structure.After the cells were treated with 1.0μmol/L PAB for 24h we observed the typical and characteristic change in flurescence microscope:the nuclear of apoptosis cells presented as condensated or half-moon shape with orange flurescence.4.The change of mitochodria membrane potentialThe normal mitochondria is flavo-green.We found that mitochondfia turned into green after the cells were treated by 0.5μmol/LPAB.Mitochodria membrane potential decreased and was(33.46±0.87)%,(51.97±1.42)%,(63.34±2.67)%,(70.78±7.39) %,(77.62±8.13)respectively after the cells were treated with various concentration of PAB which suggest the depolarization of mitochondria.5.Effect of PAB on the expression of COX-2 proteinAfter the cells were treated by various concentration of PAB(0,0.5,1.0,2.0, 4.0μmol/L)for 24h the expression of COX-2 decreased with the increase of the dose of PAB.The effect of 2.0,4.0μmol/L PAB is especially obvious.Conclusions1.PAB inhibited hepatocellular carcinoma line BEL-7402 growth in a dose-dependent and time-dependent manner;PAB induced apoptosis and the cell cycle arrest at G2/M phase in a dose-dependent manner2.PAB induced the depolarization of mitochodria membrane potential in a dose-dependent manner.3.PAB inhibited the expression of COX-2 protein in a dose-dependent manner.