Apoptosis Induction Of Resveratrol In Human Lung Adnocarcinoma A549 Cells

【Backgroud and Objectives】Resveratrol, a natural phytoestrogen found in grape and a variety of plants, was reported tohave many potential effects, for example, anti-aging, anti-inflammation and inducing tumorapoptosis and so on. In this paper we only study inducing-apoptosis effects of resveratrol, and weknow that resveratrol can inhibit the growth of A549 cells, and also can lead A549 cells toapoptosis. Promyelocytic leukaemia（PML）gene is located in the NO.15 chromatin body，which could code PML protein. PML protein can inhibit the growth of tumor, and induce tumorapoptosis. Before this experiment, A549 cells knocked down PML gene were constructed. So weused the MTT assay as a method to detect inhibiting effects of resveratrol on the experimentgroup and the control group. In this study, we confirmed resveratrol could inhibit the growth ofboth cells, and the effects of resveratrol on the experiment group and the control group weredifferential.The resveratrol treatment resulted in a concentration-depedent inhibition of A549 cellgrowth ,as compared to untreated controls.【Methods】A549 cells were cultured in the medium containing different concentration ofresveratrol,and the morphological changes of A549 cells were observed under phase contrastmicroscope and fluorescence microscopy then photographed.MTT assay was used to measurethe inhibitory effect of resveratrol on the growth of A549 cells.Apoptosis features were observedwith stained with DAPI.The effect of resveratrol on cell cycle was analyzed by flowcytometry.The different expression of whole protein after treated with resveratrol were identifiedby 2D.The expression changes of P53,PML,Bcl-2 and Bax were detected by westernblotting.Also the expression of P53,PML and Wrap53 were detected by immunofluorescence.The protein and mRNA expression of P53 and Wrap53 were detected afterA549 cells treated by PML siRNA.【ResultResults】1. MTT assay was used to measure the inhibitory effect of resveratrol on growth of A549 cellsand found the IC50 (100μM).2. Nuclei with chromatin condensation and formation of apoptotic bodies could be observed bymorphological analysis with DAPI staining ,when A549 cells had been treated with 100μMresveratrol for 24h.3. The effect of resveratrol on cell cycle perturbations was detected by flow cytometry,thetreatment caused an arrest of 69.38%, 73.82%, 80.33%，73.28% cells in G1 phase of cell cycleat 25,50,100,200μM of resveratrol(compared to 54.53% of control).4. The amout of P53 was markedly increased after resveratrol treatment,compared with thisprotein,the expression of PML was inhibited.And the Bcl-2/Bax ratio did not changesignificantly.5. Gene transcript levels were measured by real-time PCR after treatment withresveratrol,reseratrol (25,50,100μM )leaded to increase mRNA of pml ,but pml gene levelwas very low when treated with resveratrol of 200μM.Resveratrol leaded to increase mRNAof p53 when treated with low concentration of resveratrol,but decrease in highconcentration.And resveratrol leaded to a does-dependent increase of wrap53 mRNA.6. The expression of PML,P53 and Wrap53 were deteced by immunofluorescence,and foundthat PML was inhibited in does-dependent.【Conclusions】1. Resveratrol significantly inhibited the growth and proliferation of A549 cells in time anddoes dependent manners and the IC50 value was 100μM .2. Resveratrol of certain concentration can effectively induce apoptosis of A549 cells, change the distribution of the cell cycle, and cause A549 cells G1 / S arrest.3. In this research,we found the mechanism may be up-regulation of the p53,down-regulationof the PML,but the Bcl-2/Bax ratio was unchanged.4. In protein levels, PML were inhibited, but in RNA level it had been first activated butinhibited later, so we presumed the inhibition effects may not be through genetic level, maybe at the transcription, or in the translation level.5. After the treatment of with low concentration resveratrol,Wrap53 and p53 were up-regulatedin RNA level,which suggested that wrap53 could regulate p53 ,and siRNA konckdown ofWrap53 result in a significant dcrease in p53 RNA.6. The PML protein was fixed in nucleus,and with the increase of concentration ofresveratrol ,the expression of PML was inhibited.Meanwhile we observed that PML and p53were positioning together,PML and Wrap53,Wrap53 and p53 were partial positioningtogether.