| Objective:To observe the effects of 5-aza-2'-deoxycytidine(5-Aza-CdR)on the methylation status of RAR-?,WIF1 and RASSF2A in lung adenocarcinoma cell line SPC-A-1,and to investigate the regulatory mechanism of 5-Aza-CdR on the defective expression of RAR-?,WIF1 and RASSF2A gene.Methods:The human lung adenocarcinoma cell line SPC-A-1 was treated by 5-Aza-CdR with different concentration and different time.The cell line SPC-A-1 was divided into group A(untreated group)and group B,C,D(add 3,6,12 ?mol/L 5-Aza-CdR,respectively)according to different concentration,and were all cultured in vitro for 72h.In addition,the cell line was divided into 24h,48h,72h and 96h groups according to different time,and were all added with 12 ?mol/L concentration of 5-Aza-CdR.Differences in RAR-?,WIF1 or RASSF2A gene methylation status among the four groups(A,B,C,D)were detected by methylation-specific PCR(MSP).Differences in mRNA expression and protein expression of RAR-?,WIF1 or RASSF2A gene among the different concentrations or different time groups were detected by RT-PCR and Western blot,respectively.Results:(1)RAR-P,WIF1 and RASSF2A gene methylation was observed in group A,while demethylation of RAR-?,WIF1 and RASSF2A gene was observed in the other 3 groups.(2)Among the different concentrations groups,mRNA and protein expression levels of RAR-?,WIF1 or RASSF2A gene in the group A were the lowest,while the mRNA and protein expression levels in the group D were the highest(p<0.05).The expression level of mRNA and protein increased with the concentration of 5-Aza-CdR(p<0.05),and mRNA or protein expression had a certain concentration dependence.Furthermore,there was no significant difference in mRNA and protein expression between 72h group and 96h group(p>0.05).However,the expression level of mRNA and protein increased gradually with the prolongation of time in 72h(p<0.05),and mRNA or protein expression had a certain time dependence.Conclusion:The expression of RAR-?,WIF1 and RASSF2A gene in lung cancer was defective due to hypermethylation of DNA,and methylation is a reversible process,which can be used as a target for tumor specific therapy.5-Aza-CdR can reactivate the expression of defective RAR-?,WIF1 and RASSF2A gene via its demethylation effect,and cause the expression of mRNA and protein be up-regulated,and thus plays a role in tumor inhibition.Objective:To investigate the effects of 5-Aza-CdR on cell proliferation and apoptosis of lung adenocarcinoma cell line SPC-A-1 and its possible mechanism.Methods:The human lung adenocarcinoma cell line SPC-A-1 was treated by 5-Aza-CdR with different concentration.The cell line SPC-A-1 was divided into group A(untreated group)and group B,C,D(add 3,6,12 ?mol/L 5-Aza-CdR,respectively)according to different concentration,and were all cultured in vitro for 72h.Differences in protein expression of Bcl-2 and Bax among the four groups were detected by Western blot.Then differences in cell cycle and apoptosis rate among the four groups were detected by flow cytometry.Moreover,Hoechst33258 staining was used to observe the effect of 5-Aza-CdR with different concentration on the apoptosis of lung adenocarcinoma cell line SPC-A-1.Results:(1)Among the four groups,the expression level of Bcl-2 protein decreased gradually with the increase of concentration of 5-Aza-CdR,while the expression level of Bax protein increased gradually with the concentration of 5-Aza-CdR(p<0.05).The doses of 5-Aza-CdR were correlated with the effects.(2)Overall apoptosis rates of in 5-Aza-CdR treatment groups were significantly increased compared with group A(P<0.01),and the overall apoptosis rate in the group D was the highest among the four groups(17.45±1.82)%(P<0.05).The proportion of cells in G0/G1 phase was significantly increased with the concentration of 5-Aza-CdR,while the proportion of cells in S phase or G2/M phase decreased significantly with the increase of concentration of 5-Aza-CdR(p<0.05).The effect of 5-Aza-CdR on the ratio of cell cycle and apoptosis was concentration dependent.(3)The expression levels of RAR-P,WIFI1 RASSF2A and Bax protein in group D were positively correlated with the apoptosis rate(p<0.05).Meanwhile,the expression level of Bcl-2 protein was negatively correlated with the apoptosis rate(p<0.05).Conclusion:5-Aza-CdR can interfere with the cell cycle of lung cancer and arrest the cell cycle in G0/G1 phase,thus it can inhibit cell proliferation of lung cancer.5-Aza-CdR can down-regulate the expression of Bcl-2 and up-regulate the expression of Bax,and up-regulate the expression of RAR-?,WIF1 and RASSF2A by demethylation,therefore it can induce apoptosis and exert anticancer effect.Objective:To investigate the antitumor effect of 5-Aza-CdR on human lung cancer xenograft in nude mice and its possible mechanism.Methods:Nude mice model with human lung adenocarcinoma cell line SPC-A-1 transplanted was established.A total of 40 nude mice with lung adenocarcinoma xenografted tumor were randomly divided into the model group,treatment ?,? and? groups with 10 mice in each group.After model establishment,mice in the model group were given normal saline by lavage,qod.Meanwhile,according to the nude mice body weight,treatment ?,? and ? groups were injected intraperitoneally with 0.5?g/g,1?g/g,and 2?g/g unit dose of 5-Aza-CdR injection,respectively,qod.The treatment lasted for 4 weeks.The tumor volume and body weight were measured in 4 groups,and the tumor inhibition rate was calculated.We observed the effects of different doses of 5-Aza-CdR on the growth of human lung cancer xenograft in nude mice and its side effects.Differences in RAR-?,WIF1 or RASSF2A gene methylation status among the four groups were detected by methylation-specific PCR(MSP).Then,differences in protein expression of RAR-?,WIF1 or RASSF2A in vivo among the four groups were detected by Western blot.Results:(1)After 4 weeks of 5-Aza-CdR treatment,the mean tumor volume and weight of ?,? and ? groups were significantly decreased compared with the model group(P<0.05).Meanwhile,the mean tumor volume and weight decreased significantly with the increase of 5-Aza-CdR dose(P<0.05),and the tumor inhibition rate was significantly increased with the increase of 5-Aza-CdR dose.Tumor inhibition effect was dose dependent.(2)RAR-?,WIFI and RASSF2A gene methylation was observed in the model group,while demethylation of RAR-?,WIF1 and RASSF2A gene was observed in the other 3 groups.(3)The protein expression levels of RAR-?,WIF1 and RASSF2A in the model group were the lowest among the 4 groups,while the levels of protein expression in treatment III group were the highest(p<0.05).The expression levels of protein increased with the dose of 5-Aza-CdR(p<0.05),and protein expression had a certain dose dependence.Conclusion:5-Aza-CdR can suppress the growth of human lung cancer xenograft in nude mice,furthermore it plays a role in tumor inhibition via up-regulation of the expression of RAR-?,WIF1 and RASSF2A and apoptosis inducement.It may provide a new method and theoretical basis for clinical treatment of lung cancer in the future to explore the mechanism of anti-tumor effect of 5-Aza-CdR in vivo and in vitro. |
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