Anti-tumor Effect Of Tms On A549 Cells And Molecular Mechanisms

Background:Lung cancer is one of malignant tumors that usually occur in China. Resveratrol is a polyphenol found in different plant species and is synthesized by plants in response to pathogen attack.Resveratrol is present in high concentration in the skin of grapes and in wines but essentially nonexistent in white wine.Because of this,resveratrol has been considered to be one of the candidate molecules to explain the French Paradox,a phenomenon that the French have relatively low rates of ischemic heart diseases despite a large consumption of saturated fats and high serum cholesterol levels. Resveratrol displays numerous properties related to very different mechanisms of action.Major activities of resveratrol include:modulation of lipid metabolism,inhibition of platelet aggregation, free-radical scavenging,anti-inflammatory activity,antitumor activity. Researches in antituomr effect of resveratrol are very popular in the recent years,but there is no systemic reports about its antitumor activity in vivo and in vitro.At the same time,there are a variety of viewpoints about its antitumor metabolism.Resveratrol derivatives and analogs which including hydroxyl, methyl, esterderivatives and soon were reviewed classifiedly. These compounds have so multiple biological and pharmacological activities as resveratrol,of which some are superior in activity,selectivity and stability to resveratrol and have become a focus of research in recent years.NF-κB P65 is one of significant cell nuclear factors.Previous studies indicate that NF-κB P65 is over-expressed in most of human malignant tumors, but it can not be detected or weak-expressed in terminal differentiation normal tissue.NF-κB P65 becomes a new hot spot in researchs of carcinoma diagnosis and treatment because of its characteristic of relativity to tumor.To understand its antitumor activity and to reveal its antitumor metabolism,we carried out this experiment. In our study, we observe whether TMS inhibits proliferation, induce apoptosis of A549 cells and explore its molecular mechanisms. Part I Identificat chemical structure of trihydroxystilbeneObjectiveSynthesized TMS by Methylation of Res from Polygonum cuspidatum5and identificat chemical structure of that.MethodsIdentificat chemical structure of TMS by UV absorption spectra,1H-NMR spectroscopy,Infrared absorption spectra,13C-NMR and Mass Spectrometry.ResultThe UV map of compounds isλmax (MeOH):318nm,306.2nm, 217.8nm; analysis map shows IRvmax(cm-1):2999,2935,2836,1591, 1511,1456;1H-NMR map shows it contents three Methoxy,while 13C-NMR map shows it's contents 17 carbon signals and some symmetrical structure fragment,.Mass Spectrometry analysis of this compounds shows ESI-MS m/z:271[M+H]+,256[M+H-CH3]+, 241[256-CH3]+, Molecular Weight suggested was 270, and Molecular Formula is supposed to be C17H18O3;. So we can confirm this compounds is 3.5.4' - trihydroxystilbene.ConclusionTMS, trihydroxystilbene;whose Molecular Formula is supposed to be C17H18O3 Part II Proliferation-inhibiting effect of TMS on A549 cellsObjectiveTo determine proliferation-inhibiting capability of TMS on A549 cells.MethodsA549 cells were treated with different concentrations of TMS, and then cell viability and formation of colony were analyzed with MTT' assay and colony culture test.Results(1) TMS effectively inhibited the proliferation of A549 in a concentration-dependent way, and the IC50 was 86/μmol/L. (2) TMS significantly inhibited colonic formation of A549 cells in a dose-dependent way.ConclusionTMS can inhibit proliferation of A549 cells effectively on a concentration-dependent manner. Part III Apoptosis-inducing effect of TMS on A549 cellsObjectiveTo ensure whether TMS can induce the apoptosis of A549 cells.MethodsAfter A549 cells were incubated with differentconcentrations of TMS, (1) morphologic character of apoptosis was evaluated by Hochest33258 staining; (2) the molecular character of apoptosis was analyzed by Western blot detecting Caspase-3 expression.Results(1) Hochest33258 staining and flow cytometry detection testify TMS can induce the apoptosis of A549 cells in a concentration-dependent way; (2) The apoptosis of A549 cells, which was induced by TMS, kept company with up-regulation and cleavage of Caspase-3.ConclusionTMS can induce the apoptosis of A549 cells with a concentration-dependent manner; The molecular mechanisms of apoptosis induction was related to up-regulated caspase-3 expression and activation. PartⅣMolecular mechanisms of the proliferation-inhibiting and apoptosis-inducing effects of TMS on A549 cellsObjectiveTo explore the mechanism of proliferation- inhibiting and apoptosis-inducing effects of TMS on A549 cells.MethodsAfter A549 cells were incubated with different concentrations of TMS, (1) The protein expression of NF-κB, IκB, STAT3, STAT5b, JAK2 in A549 cells were detected by Western blot; (2) The subcellular distribution and changes of NF-κB in A549 cells were detected with indirect immunofluorescence technique.Results(1) TMS down-regulated NF-κB, STAT3, STAT5b proteins,and up-regulated IκB protein in a concentration-dependent way in A549 cells; but no JAK2 protein changes were observed; (2) NF-κB was located in cytoplasm of A549 cells.ConclusionThe anti- NSCLC effect of TMS is related with inhibition of signal transduction pathway of NF-κB and STATs.