The Chemoprevention Effects Of ARTS And DHA On NSCLC By Demethylating Axin1

ObjectiveLung cancer is the most common malignancy worldwide and is one of the most common leading causes of cancer-related deaths due to the highest incidence rate and mortality rate among all types of cancer.About 85%of the lung cancer cases are non-small cell lung cancer(NSCLC).Chemotherapy remains to be the effective treatment for cancer because most patients are diagnosed with advanced cancer.Recent studies have revealed that the epigenetic alterations,including DNA methylation,histone methylation,histone acetylation,etc,in tumor suppressor genes and the resulting abnormal alterations of cellular functions contribute to lung cancer' progression and metastasis greatly.Artemisia annua L.is a traditional medicine herb well known for its predominant anti-malaria activity,taking artemisinin as its main effective gredient.Dihydroartemisinin(DHA)and artesunate(ARTS),two semi synthet ic derivates of artemisinin,have been demonstrated to exhibi t inhibitory effects against various human cancers,i ncluding lung cancer,hepatocarcinoma,etc Epidemiological studies have shown that ARTS and DHA intake is correlated to the incidence of multiple chronic cliseases negatively,including cancer.In vitro studies have demonstrated that ARTS and DHA regulate a number of coll signaling pathways,which are associated with cell prol i feration,differentiation,apoptosis,angiogenesis,inflammation,and so on.Many reported research have discovered that ARTS and DHA could induce tumor cell apoptosis and protect normal cells,indicating that ARTS and DHA are potential resources for tumor chemoprevention and chemotherapy drugs.However,the effects and mechanisms of ARTS and DHA on the prevention of lung cancer have not been reported in the literature.Based on previous studies we have performed,we evaluate the chemoprevention effects of ARTS and DHA on lung cancer via interfering benzopyrene-induced A/J mice lung cancer with ARTS and DHA.Furthermore,we determined the effect on DNA methylation of ARTS and DHA to put insight into the epigenetic mechanisms by which ARTS and DHA inhibit the progression of lung cancer.Methods1.Effects of ARTS and DHA on lung cancer cell viabilities and Axial mRNA expression levels.The commercial lung cancer cell lines,A549 and H1299,were treated with different concentration of ARTS and DHA(0-64 ?M)for 3 days to verified treatment concentration and time for following experiments by MTT assay.The mRNA and protein expression level of Axinl in the two cell lines were determined by RT-PCR,Western blot and immunofluorescence.2.Effects on Axinl epigenetic alterations in lung cancer cell by ARTS and DHA.Based on the drug concentration determined by MTT assay,cells were treated with optimal concentrations of ARTS and DHA for 3 days,and the expression of methyl-transferase and histone deacetylase were detected by Western blot and RT-PCR in A549 and H1299 lung cancer cells.The demethylation effect of CpG island in Axinl promoter in the two cell lines by ARTS and DHA were verified by MSP.3.Effects of ARTS and DHA on Axinl upstream Akt signaling.The key genes and protei ns expression levels at Akt signaling pathway were determined by RT-PCR and Western blot assay in A549 and H1299 cell lines after ARTS and DHA treatment.4.Effects of ARTS and DHA on Axinl downstream Wnt/?-catenin and EMT signaling.The genes and proteins expression of wnt5a,LRp6,?-catenin,Naked1/2,Dv12/3(in Wnt/?-catenin signaling pathway)and ZO-1,E-cadherin,N-cadherin,vimentin,snail,slug(in EMT signaling pathway)were determined by RT-PCR and Western blot assay in A549 and H1299 cell lines after ARTS and DHA treatment.We further detect cell proliferation,cell cycle and cell apoptosis proportion of ARTS and DHA untreated and treated groups using EdU,colony formation and flow cytometry.In addition,migration and invasion of two cell lines were tested by wound healing and transwell.5.Chemoprevention effect of ARTS and DHA on A/J induced lung cancer animal model.Female A/J mice,aged four to six weeks,were supplied from Jackson Laboratory.Record baseline weights and all groups received a single dose of BP(100 mg/kg)in 0.2 mL of tricapryl in through intraperitoneal injection.Two week after the initiation of BP,all mice were randomized into 3 groups of chemical and herbal treated mice and gavaged with drug 5 times a week for 30 weeks and at the meantime.The gavage control group was also gaveged with 50%propanediol.The body weights of mice were measured every week for the duration of the study.After 30 weeks,all mice in different groups will be sacrificed for further analysis.The lung tumor number were count,then lung were fixed in the paraformaldehyde for HE staining and immunohistochemistry of DNMT1 and Axinl protein.The protein expressions of DNMT1,Axinl and other key proteins of Akt and Wnt signaling pathway were detected by Western blot.6.Statistical analysisData were expressed as the mean±standard deviation(SD)of at least three independent experiments.Statistical significance of the data was determined by one-way ANO VA using SPSS 19.0 software.Stati stical significance was considered at P?0.05.Results1.ARTS and DHA significantly inhibited the activity of lung cancer cells and up-regulated the expression of Axin mRNA and protein.Three days after ARTS and DHA administration,8 ?M ARTS and DHA showed a significant inhibitory effect on A549,2 ?M ARTS and DHA showed a significant inhibitory effect on H1299.According to the MTT test results,to ensure that more than 80%of cells survive,so to determine the concentration of ARTS and DHA 0,1,2,8 ? M,administration time 3 days in A549;determine the concentration of ARTS and DHA 0,0.5,1,2 ?M,administration time 3 days in H1299.2.ARTS and DHA up-regulate Axinl gene expression in lung cancer cells at epigenetic level.The results of RT-PCR showed that 5-AZA methylation inhibitor up-regulated the expression of Axinl mRNA in A549 cells,which was up-regulated by 104%±3.65%(P<0.05),while that high-dose ARTS group was significantly increased by 262%±24.34%(P<0.001)times.In A549,the DHA dose-dependently increased the Axinl mRNA expression levels by 144.5%±2.46%(P<0.05),255%±21.38%(P<0.001),545%±33.7%(P<0.001).And it is similar to H1299 cells.According to the results of Western blot and cofocal,we found that the protein of Axinl was significant enhanced in the ARTS and DHA groups compared with control,which indicated that ARTS and DHA could promote the production and enrichment of Axinl protein in the cytoplasm.In addition,the MSP and Mass Array results showed that ARTS and DHA could inhibit the expression of methyltransferase and histone deacetylase in both cells.Meanwhile,ARTS and DHA decreased the methylation level of Axin gene in A549 and H1299.3.ARTS and DHA inhibit Axinl upstream Akt signaling pathway.The results showed that the ARTS and DHA significantly dose-dependent]y suppressed the expression of p-Akt,P-GSK3 ? and DNMT1,which were in the Axinl upstream pathway,in A549 and H1299 lung cancer cells.The down-regulation of DNMT1 protein could mean the methylation was decreasing,which provide the basis for upregulation of Axinl gene.In addition,we used SC79 to activate Akt signaling pathway in A549 and H1299 cells.The expression of p-Akt,p-GSK3 p and DNMT1 were significantly up-regulated by 162%±14.7%(P<0.001),155%±18.35%(P<0.05)and 153%± 15.79%(P<0.001).Compared with the group of SC79,the groups of ARTS+SC79 and DHA+SC79 could significantly inhibit the expression of p-Akt,p-GSK3 p and DNMT1.Consequently,ARTS and DHA could up-regulate the gene expression of Axinl through Akt signaling pathway.4.ARTS and DHA inhibit Axinl downstream Wnt/?-catenin,EMT signaling pathway.The results showed that Wnt/?-catenin,EMT pathway were ?-catenin,Dv13,LRP6,Wnt5a/b,slug and snail in the downstream of Axinl was inhibited in a dose-dependent manner.Edu and plate clony formation experiments showed that ARTS and DHA could inhibit the proliferation of A549 and H1299 in a concentration-dependent manner.ARTS and DHA could inhibit the migration and invasion of cells in a dose-dependent manner,in which the high concentration group was the most significant.Flow cytometry detection of ARTS and DHA can block A549 and H1299 cells in G1 phase,thereby inducing apoptosis.5.ARTS and DHA in vivo prevention of lung cancer and its mechanism.In the A/J mice lung cancer model induced by benzopyrene,ARTS and DHA could reduce the incidence of lung cancer.The tumor inhibition rate was 6d.12%and 81.25%respectively.Thc re was no significant difference in the body weight of each group,which indicated that the drug toxicity was low.In addition,the results of MR staining showed that ARTS and DHA could decrease t he BF-induced pulmonary nodule production and tumor eell infiltration in A/J mice.Immunohistochemical results showed that ARTS and DHA could reduce the expression of DNMT1 protein in tumor cells and promote the expression of Axinl in normal cell s.Western blot showd that ARTS and DHA could significantly inhibit the expression of DNMT1 protein by 38%±5.16%(P<0.05)and 95%± 7.69%(P<0.05),compared with model control group.The expression of Axin1 protein was upregulated by 425%±57.14%(P<0.01)and 268%±14.4%(P<0.01).ConclusionIn this study,we investigated the inhibitory effects of ARTS and DHA on lung cancer both in vitro and in vivo.It was found that ARTS and DHA could upregulate the expression of Axin1 gene by regulating the expression of DNMTs and HDACs protein.With the recovery of Axin1 gene expression,Wnt signaling pathways and EMT signaling pathways were inactivated,thereby inhibiting cell proliferation,invasion and metastasis.Taking together,this study can provide an experimental basis for chemoprevention and chemotherapy of lung cancer by ARTS and DHA.