| ObjectiveTo study the mechanism of reversing drug resistance effect of noscapine in A549/DDP drug-resistant cell lines,the impact of noscapine on apoptosis and apoptosis-related proteins of human lung adenocarcinoma A549 and A549/DDP drug-resistant cell lines was observed.MethodsIn this study,we cultured human lung adenocarcinoma A549 and A549/DDP drug-resistant cell lines in vitro,which were treated with cisplatin at concentrations of 2.5,5,10,20,40 and 80 mg/L,respectively.Then,cell proliferation was detected by CCK-8 assay after 24 h incubation.Similarly,A549/DDP drug-resistant cell lines were treated with noscapine at concentrations of 2.5,5,10,20,40 and 80 ?mol/L.Then,cell proliferation was detected by CCK-8 assay after 24 h and 48 h incubation,respectively.Next,A549/DDP drug-resistant cell lines were treated with 2.5,5,10,20,40,80 mg/L cisplatin combined with 10 ?mol/L noscapine.Cell proliferation was detected by CCK-8 assay after 24 h incubation.Second,the study was divided into four groups: blank control group,20 mg/L cisplatin group,10 ?mol/L noscapine group and 10 mg/L cisplatin combined with 10 ?mol/L noscapine group.Next,observing the apoptosis of each group by fluorescence microscopy using Hoechst-33342 staining,detecting the effect of different treatments on the apoptosis of A549/DDP drug-resistant cell lines by flow cytometry,and studying the expression of apoptosis-related proteins bcl-2,bax and bag-1 of A549/DDP drug-resistant cell lines in each group by Western blot.The statistical software used for analysis and statistics was IBM SPSS 17.0 software package.Expressing the measurement data by mean(),testing the two comparisons by t-test and completing the comparison between groups by one-way ANOVA.A P value of < 0.05 was considered as statistically significant.Results1.Effects of cisplatin on the proliferation inhibition of A549 and A549/DDP drug-resistant cells: 24 h survival rates of cells after treatment with 2.5,5,10,20,40 and 80 mg/L cisplatin for 24 hours were 78.41 ± 4.32%,89.19 ± 5.22%;46.24 ± 5.61%,77.23 ± 4.42%;41.15 ± 4.56%,73.86 ± 4.98%;33.29 ± 5.01%,59.38 ± 5.17%;23.76 ± 4.98%,41.19 ± 4.83%;15.18 ± 5.11%,32.19 ± 4.76%,respectively.According to the formula,the IC50 values of cisplatin on A549 and A549/DDP cells were calculated(4.13 ± 0.89)mg/L and(24.11 ± 1.42)mg/L,respectively,with statistically significant difference(P < 0.01),and obviously significant resistance coefficient(RI = 5.8).2.IC50 values of noscapine on A549/DDP cells at 24 h and 48 h were 38.74 ?mol/L and 24.53 ?mol/L respectively,indicating that cell proliferation was inhibited over time.After 24 h treatment,the cell survival rates of 2.5,5,10 ?mol/L noscapine were 95.7%,94.2%,and 93.5%,respectively,with no statistical difference(P > 0.05).The cell survival rate was more than 90% at 10 ?mol/L of noscapine,that is,the cell survival rate decreased significantly with the increase of the concentration of narcotine.3.The IC50 of cisplatin combined with 10 ?mol/L noscapine for 24 hours was 12.56 mg/L,which was significantly different from cisplatin alone(P < 0.01).The reversal multiple of drug resistance was 2.1.4.The results of Hoechst-33342 staining showed that apoptotic cells in combination group were significantly higher than those with statistical difference(P < 0.01).(the percentage of apoptotic cells was 6% in group A,14% in group B,27% in group C and 58% in group D,respectively.)Compared with the other three groups,the changes of apoptosis were more obvious in the combination group,with the nuclear aggregation and pyknosis.5.The results of flow cytometry showed that the apoptotic rates of each group were as follows: blank control group(3.9 ± 1.84)%,cisplatin group(23.9 ± 2.15)%,noscapine group(5.6 ± 1.44)%,combined drug group(56.9 ± 2.04)%.Compared with other groups,the apoptosis rate increased in the combination group with statistical difference(P<0.05).Among them,compared with cisplatin group,the apoptosis rate in the combination group was significantly higher,with statistically significant difference(P < 0.01).6.Western blotting results showed that compared with control group and cisplatin group,the expression of bcl-2 and bag-1 in noscapine combined with cisplatin group decreased significantly(P < 0.05),while the expression of apoptotic protein increased significantly with statistical difference(P < 0.05).The bcl-2/bax ratio showed a downward trend.ConclusionsNoscapine could inhibit the proliferation of human lung adenocarcinoma A549/DDP drug-resistant cells.Studies have shown that noscapine of 10 ?mol/L concentration treats it alone without obvious toxicity.The combination of noscapine and cisplatin could enhance the sensitivity of cisplatin to A549/DDP drug-resistant cells,which may reverse the resistance of cisplatin.The mechanism may be related to the regulation of the expression of bcl-2,bax and bag-1 proteins.Therefore,noscapine could be used as a potential drug to reverse cisplatin resistance in lung adenocarcinoma,providing a theoretical basis for clinical treatment. |
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