| ObjectiveChemosensitization induced by nitric oxide is a hot topic in current research.High concentration of NO can enhance the sensitivity of tumor cells to chemotherapeutic drugs through various mechanisms.In this study,we observed the growth inhibition of nasopharyngeal carcinoma CNE-2 cell line treated by different concentrations of sodium nitroprusside(SNP),DDP and both chemical to investigate whether exogenous NO could increase the chemosensitization of cis-platinum(DDP)on CNE2 cells and the mechanism,and provide experimental and theoretical basis for improving chemotherapy of nasopharyngeal carcinoma.Methods(1)Cell culture: The nasopharyngeal carcinoma CNE-2 cell line was selected as the object.They were cultured in medium containing 10% fetal bovine serum(FBS)and placed at incubation with 37℃ and 5% CO2.The logarithmic growth cells were chose for experiment.(2)The morphological changes of cells were observed by inverted phase contrast microscope: CNE-2 cells were treated by groups of SNP1(SNP200umol/L),SNP2(SNP600umol/L),SNP3(SNP1000umol/L),DDP(DDP20000nmol/L),SNP1+DDP(SNP200umol/L+DDP20000nmol/L),SNP2+DDP(SNP600umol/L+DDP20000n mol/L)and SNP3+DDP(SNP1000umol/L+DDP20000nmol/L).They were placed at incubation with 37℃ and 5% CO2.The morphological changes of cells were observed by inverted phase contrast microscope(×100)after 24 hours.(3)CCK-8 was used to examine the viability of cells: the logarithmic growth cells were chose to be treated by different medicine groups of SNP1,SNP2,SNP3,DDP,SNP1+DDP,SNP2+DDP and SNP3+DDP.Equal amounts of media were used as blank controls.CCK-8 was used to detect optical density for calculating the inhibitory rate of cells in each group [inhibitory rate=(1-experimental group/control group)× 100%].(4)Nitrate reductase method was used to detect the concentration of NO: we take out the supernatant from each group described above in step(3)and to detect the concentration of NO.(5)AnnexinV-FITC/PI double-staining flow cytometry method was applied to detect the apoptosis rate of CNE-2 cells in every group: the logarithmic growth cells were chose for experiment and intervened by different measures.CNE-2 cells were treated with groups of SNP1(SNP600umol/L),DDP(DDP 20000nmol/L),both chemicals SNP2+P(SNP600umol/L+DDP20000nmol/L)and equal amount of medium respectively for 24 hours,and flow cytometry method was used to detect.Quest software was applied to analyze the experimental date and calculate the apoptosis rate.(6)Statistical analysis: the experimental date in this research showed as mean±standard deviation(?χ±s).SPSS 19.0 for windows and Microcoft Excel statistical software were used for statistical analyze and charting.Indepentent-Sample T Test was applied for compare means between the two groups.One-way ANOVA and Kruskal-Wallis H Test were used for compare means among multi groups.All tests were bilateral and the test level of α=0.05.Results(1)The morphological changes of cells were observed by inverted phase contrast microscope: With the increase of SNP concentration,the cell survival rate decreased significantly;Compared with SNP group,DDP group and control group,CNE-2 cells in SNP2+DDP and SNP3+DDP groups showed more morphological changes and significantly decreased survival rate.(2)The concentration of NO was associated with the concentration of SNP: Compare with the control group without SNP,the differences of concentrations of NO in groups of SNP1,SNP2 and SNP3 groups were statistically significant(215.5432±0.553908 VS 88.1777±0.422719,t=-192.070,P<0.05;323.6549±1.375172 VS 88.1777±0.422719,t=-243.526,P<0.05;387.1118±1.69286 VS88.1777±0.422719,t=-292.482,P<0.5);Moreover,compare with each other of the groups of SNP1,SNP2 and SNP3,the differences of concentrations of NO was statistically significant(387.1118±1.69286 VS 323.6549±1.375172 VS 215.5432±0.553908,F=8104.661,P=0.000<0.05).Concentration of NO in supernatant was positively correlated with the concentration of SNP(r=0.961,P=0.039<0.05).(3)SNP inhibit proliferation of CNE-2 cell in a concentration-dependent manner: The inhibitory rates of CNE-2 cells treated with SNP1,SNP2 and SNP3 groups for 24 hours were(-7.342±0.6047)%,(3.3899±1.0622)% and(52.3524±1.0239)%respectively,the differences of inhibitory rates of each groups was statistically significant(statistic of test=7.322,P=0.026<0.05);The inhibitory rates of CNE-2 cells treated with DDP20000nmol/L group for 24 hours was(43.6473±1.0348)%.The inhibitory rates of CNE-2 cells treated with Group of SNP1+P,SNP2+P and SNP3+P for 24 hours were(52.0065±1.4293)%,(78.5137±0.2429)% and(95.1610±1.0439)%respectively.The inhibition rate of cell proliferation was significant between different groups of combinations above(statistic of test = 7.385,P = 0.025 <0.05).Compared with DDPgroup,the inhibition rate of the cells treated by groups of SNP1+P was increased,but the difference was not significant(t=1.220,p=0.251>0.05),SNP2+DDP and SNP3+DDP were more significant higher(t =9.049,P=0.00 < 0.05;t=3.536,P=0.005 <0.05).Compared with SNP2+DDPgroup,the inhibition rate of the cells treated by groups of SNP3+DDP were more significant higher(t=-5.287,P=0.000<0.05).(4)Apoptosis of CNE-2 cells : The percentage of spontaneous apoptosis of CNE-2 cells was(1.21±0.28)%;The percentage of apoptosis in the SNP group and DDP group were(3.44±0.14)% and(33.37±1.74)% respectively;Compared with the group using SNP or DDP individually,flow cytometry analysis showed that the apoptosis rate of CNE-2 cells on the group treated by both SNP and DDP was significantly increased(t=-26.930,P=0.022<0.05;t=-5.242,P=0.035<0.05).Conclusion(1)Exogenous nitric oxide can inhibit CNE-2 proliferation,and the inhibitory effect was positively correlated with the concentration of NO;(2)Exogenous nitric oxide can significantly enhance the chemosensitivity of cisplatin on CNE-2 cells,which was also positively correlated with the concentration of NO. |
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