| ObjectiveIn this study,we mainly investigated the regulation effect of EVO and its mechanism on Notch signaling.We used two kinds of NSCLC cell lines,A549 and H1299,to examine the effect and mechanism of EVO on Notch signaling in vitro.Human embryonic lung fibroblasts cell line,MRC-5,were used to observe the toxicity of EVO on human normal cell line.In vivo,lung cancer animal model in FVB mice induced by Urethane was established to further explore the role of EVO in NSCLC.Methods1.Effect of Evodiamine on cell viabilityA549,H1299,H1975 and MRC-5 were used to detect the effect of EVO on cell viability in 48 hours.IC50 value was calculated according to the experimental procedure of MTT assay.The appropriate cell lines and drug concentration were selected as the cell model in vitro.2.Effect of Evodiamine on Proliferation and cell cycleA549 and H1299 were screened by MTT assay as the follow-up cell model with evodiamine treatment for 48 h.EdU assay was used to detect the effect of EVO on the proliferation.The effect of EVO on cell cycle was detected by flow cytometry.The protein expression related to cell cycle was inspected by western blot.3.Effects of Evodiamine on tumor malignancyTumor malignancy can be evaluated by tumor cell migration,invasion,cell stemness,etc.Epithelial-mesenchymal transition(EMT)promotes tumor cell migration and metastasis.Wound healing is a commom method to evaluate the effect on cell migration.Cell stemness was detected by soft agarose cloning assay.CD44 and CD133 are biomarkers of stem cell,they were detected by flow cytometry.The EMT-related protein such as E-cadherin,N-cadherin and Vimentin,were analyzed by western blot.4.Effect of Evodiamine on the key protein of Notch signalingThe Notch pathway-related protein expression level was detected by western blot.While mRNA expression level of Notch pathway downstream was analyzed by RT-PCR.5.The mechanism of Evodiamine regulating Notch signalingThe methylation effect in Notch3 promoter region was confirmed by MSP assay.The degree of methylation in each CG site was detected by methylation mass spectrometry.Western blot was used to detect the effect of EVO on DNA Methyltransferase(DNMTs)protein expression6.Chemopreventive effect of evodiamine on lung cancer animal modelThe chemopreventive effect of EVO on the lung tumorigenesis and tumor progression was investigated by EVO 0.5%CMC-Na,5 mg/kg and 10 mg/kg for 22 weeks.The expression of Notch3 protein in lung tissue was detected by immunohistochemical.7.Statistical analysisWe use the SPSS 20.0 statistical software for data analysis,multi-group comparison using One way ANOVA,p<0.05 shous that has significant differences.GraphPad 5.0 was used for data analysis mapping,the data shows as Mean ąSD.Results1.Evodiamine significantly inhibited the cell viability of A549 and H1299After 48 hours of administration of evodiamine,20 ?M and 5 ?M evodiamine showed significant inhibitory effects on A549 and H1299,respectively.According to the MTT test results,determine the concentration of evodiamine administration:0,4,8,16 ?M in A549;H1299 administration 0,1,2,4 ?M.2.EVO suppressed the proliferation of A549 and H1299,and the cell cycle was arrested in G2/M phaseThe results of EDU showed that evodiamine significantly inhibited the proliferation of A549 and H1299 cells.Flow cytometry results showed that EVO could arrest cell cycle in G2/M phase,especially on A549 cell line.At the same time,the expression of cyclinBl/cdc2 was decreased,detecting by western blot.3.EVO prohibited the malignance of A549 and H1299Wound healing assay showed that EVO could inhibit the migration of A549 and H1299.Soft agarose clone assay showed that EVO could reduce the malignant degree of tumor cells.Western blot showed that EVO up-regulated the protein expression of E-cadherin and decreased the expression of N-cadherin and Vimentin,thus EMT process was inhibited.4.EVO markedly inhibited Notch signalingThe results of western blot showed that Notchl,Notch2,Notch2 and Notch3 showed a decreasing trend after administration of EVO.Notch3 was especially obvious.Immunofluorescence assay of Notch3 showed that Notch3 localized on the membrane and decreased after EVO administration.The levels of Notch ligands such as jagged1 were decreased,as well as the second cleavage catalyzing enzyme ADAM9 and the third cleavage catalyzing enzyme ?-secretase complex.RT-PCR results showed that notch signaling downstream genes such as hes family,HER-2,Myc mRNA levels also showed a downward trend.5.EVO epigenetic regulation of Notch signaling pathways via promoting the methyl at ion level in Notch3 promoter regionMSP assay showed that evodiamine increased the methylation level of Notch3 DNA promoter region,which resulted in the decrease of Notch3 protein expression.The results of methylation mass spectrometry showed that 15 CG sites of NOTCH3 DNA promoter region were methylated in A549.Among those,methylation degree increased to 2-fold in 5 CG sites compared with control group.The maximum methylation effect increased by 3.25-fold in 3 CG sites.Analogously,16 CG sites were methylated in H1299.Among those,methylation degree increased to 2-fold in 7 CG sites compared with control group.The maximum methylation effect increased by 5.7-fold in 3 CG sites.Western blot analysis showed that EVO up-regulated the expression of DNMT3A and DNMT3B.6.Chemopreventive effect of evodiamine on urethane-induced lung carsinogenes is in FVB miceIn the urethane-induced lung adenocarcinoma FVB mice model,evodiamine significantly reduced the incidence of pulmonary nodules.The incidence of lung cancer was decreased by 28.57%in the 5 mg/kg group and 34.50%in the 10 mg/kg group,with no significant difference in the body weight,organ index.This indicated that Evodiamine did not show apparent toxicity in vivo.In addition,HE staining showed that the lung nodules of the administration group were smaller than the control group.Immunohistochemistry showed that the expression of Notch3 was down-regulated.The protein expression levels of Notch3,ADAM9 and ?-secretase complex were also decreased,consistent with in vitro studies.These results suggested that evodiamine could inhibit the progress of Notch signaling in vitro and in vivo.ConclusionIn this paper,we investigated the chemical preventive effect of evodiamine on urethane-induced lung carsinogenesis.The results show that evodiamine has a potential treatment for lung cancer.In vitro,Evodiamine up-ragulated the methylation level of Notch3 DNA promoter region through elevating the DNMTs expression.Thus,Notch3 protein expression was decreased.As the following,Notch signaling was suppressed,thus suppressing cell cycle,inhibiting cell proliferation,reducing tumor malignancy.In conclusion,evodiamine is a promising agent for the chemoprevention of NSCLC. |
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