| objective:The main objective of this study is to observe the activity of diosmetin which inhibited human lung adenocarcinoma cells proliferation and to explore the possible molecular mechanism of diosmetin inducing human lung adenocarcinoma cells apoptosis.Further the animal experiment of nude mice was to verified diosmetin inhibiting human lung adenocarcinoma proliferation in vivo.It was also observed that the epigenetic changes of diosmetin regulating human lung adenocarcinoma cells.In a word,the study aimed to provide a theoretical basis for the clinical application of diosmetin in the treatment of lung cancer.Methods: The human lung adenocarcinoma cell lines HCC827,A549 were selected as major materials.The morphological changes were observed by microscope.The cell proliferation was measured by MTT and CCK-8.The cell apoptosis rate and cycles was assessed by flow cytometry.The expression level of apoptosis and cell cycle relative proteins were detected by western blotting.The cell migration ability was measured by transwell.The specific gene p53 expression was interfered by si RNA.The interfered effect was evaluated by RT-q PCR and Western bloting.The changes of P53 phosphorylation sites was observed by immunofluorescence.An orthotopic model of human lung adenocarcinoma in nude mice was developed to evaluated the therapic effect of diosmetin.The expression of m RNAs,mi RNAs and lnc RNAs on HCC827,A549 cells was detected by microarray technology.Results:1.The IC50 of diosmetin on human lung adenocarcinoma cell lines HCC827,A549 were 35?M and 43?M respectively.After diosmetin inducing,HCC827 and A549 cell morphology change was obvious and the apoptotic rate was increasing depended on diosmetin concentration.For HCC827 cell line,the control group apoptosis rate was(6.0 + 0.8)%,with increasing drug concentration,the group 5 ?mol/L?10 ?mol/L5?20?mol/L of apoptosis rate was(18.3 + 2.3)%,(32.1 + 1.5)%,(50.6 + 1.6)%respectively.Meanwhile in A549 cells the control group apoptosis rate was(6.8 +1.2)%,the apoptosis rate of 5 ?mol/L,10 ?mol/L,20?mol/L group was(15.9 +2.1)%,(28.6 + 1.8)%,(46.8 + 1.3)% respectively,compared with the control group the difference had statistical significance.(p<0.01).the result showed that diosmetin could induce human lung adenocarcinoma HCC827 and A549 cell apoptosis.20?mol/L diosmetin can inhibit human lung adenocarcinoma HCC827 and A549 cells invasion ability,control group in membrane HCC827 cells averaged21±2.7,diosmetin treatment group with an average of 9±2.3 membrane cells.As A549 membrane cells in the control group was 23±3.0.with Diosmetin treatment group was11±2.5,it has significant difference.(P < 0.01).the expression level of apoptotic relative proteins Bax,Bad cleaved-caspase-3,cleaved-caspase-9,cleaved-caspase-7and cleaved-PARP were up regulated while Bcl-2 protein was down-regulated in diosmetin treatment group(including5?mol/L,10 ?mol/L,20?mol/L group),Diosmetin may promoted cells apoptosis through caspases pathway activity.After 24 hours of diosmetin application(0?mol/L,5?mol/L,10?mol/L,20?mol/L),HCC827 cells proliferation inhibition rate was(10.9+2.5)%,(32.7+1.8)%,(41.2+2.1)%,(51.7+2.8)%respectively.and A549 cell proliferation inhibition rate was(18.9+2.3)%,(30.8+2.6)%,(42.2+1.2)%,(50.7+2.1)%.Thus,diosmetin could inhibit human lung adenocarcinoma HCC827,A549 cell proliferation activity,and had significant concentration-response relationship.Diosmetin could regulated cell cycle relative proteins cyclin B1,cdc2,CDC25 B,CDC25C,CAK,14-3-3?,p53,p21,Wee1 expression level to induce human lung adenocarcinoma cell lines A549 and HCC827 cell cycle retardation at G2/M phase.The p53 gene expression was silenced by it's specific RNAi.The relationship among diosmetin,p53/p21(waf1)signal transduction pathway and apoptosis,cell cycle retardation was observation based on p53 gene knock down.P53 phosphorylation sites had changed after diosmetin processing in human lung adenocarcinoma cell lines A549 and HCC827 cell.2.After diosmetin feeding for orthotopic model of human lung adenocarcinoma nude mice,diosmetin for 100mg/kg dose could improve the survival rate of mice,the volumn and weight of tumor in diosmetin treated groups were decreased significantly compared with control groups.The lymphatic metastasis was inhibited in diosmetin treated groups.the detailed results are as follows:HCC827 cell tumor group,the mice weight for treatment group was 28.34 ± 3.72 g,the average tumor weight0.266±0.192 g,The average tumor volume 1070.57±160.24mm3,the average survival time was 62 ± 4d.while the control group was 25.78 ± 3.63 g.0.307±0.219 g,1570.70±108.50 mm3,51±3d.in turn respectively.In addition,in A549 cell tumor group,the mice weight for treatment group was27.54±2.63 g,the average tumor weight was 0.271 ± 0.219 g,The average tumor volume was 908.52 ± 234.16mm3,the average survival time was73 ± 5d.but the control group was25.63±4.32 g,0.319±0.256 g,1497.58±310.34 mm3,49±3d respectively.3.The expression level of m RNAs,mi RNAs and lnc RNAs had significant difference after using Diosmetin in human lung adenocarcinoma cell lines A549 and HCC827 cells.Conclusions:1.The study idendified that diosmetin inhibited human lung adenocarcinoma HCC827 and A549 cells proliferation and induced them apoptosis in vitro.The possible molecular mechanism were that diosmetin promoted cells apoptosis through caspases pathway activity and p53/p21(waf1)signal pathway was induced by diosmetin and involved in cell cycle retardation.2.Nude mice animal model also confirmed that diosmetin have evidently inhibiting effect on lung adenocarcinoma.3.The diosmetin could regulated human lung adenocarcinoma cell lines A549 and HCC827 epigenetics changes.Those expression variational genes will help us better understanding the role of diosmetin on human lung adenocarcinoma.Taken together,we had roundly observed diosmetin activity on human lung adenocarcinoma and preliminarily illustrated the mechanism.It shed a beacon light on diosmetin finally clinical application. |
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