| Purpose: 1.To investigate the effects of Chitin oligosaccharide alone or combined with cisplatin on the proliferation,migration,apoptosis of lung adenocarcinoma A549 cells and implant tumor of nude mice.To explore that chitin oligosaccharide plus cisplatin had positive synergistic effects on anti-tumor.It will provide experimental and theoretical basis for the future development,application and clinical transformation of Chitin oligosaccharide in anti-tumor therapy.Material and method: Chitin oligosaccharide,cisplatin,Chitin oligosaccharide plus cisplatin were used to observe the effects of biological behaviors on human lung adenocarcinoma cell line A549 by MTS assay,cell scratch test,transwell chamber migration assay and flow cytometry apoptosis assay.Then the nude mice were transplanted subcutaneously of A549 tumor cells.According to different administration,the nude mice were randomly divided into control group,Chitin oligosaccharide group,cisplatin group and Chitin oligosaccharide plus cisplatin group.The morphological changes of tumor tissues were observed by HE staining method.Immunohistochemical method was used to detect the expression of Ki67,P53 and TTF-1,and investigate the effects of drugs on tumor prognosis.Results: 1.Tranditional Chinese medicine metabolites chitin oligosaccharide could inhibit the proliferation and migration of A549 cells,and has an increased effect depend on concentration.The 24 h IC50 of chitin oligosaccharide was 6.840mg/mL,and the concentration of 7mg/mL was used in the following experiments.2.Chitin oligosaccharide's inhibition rates on cell proliferation of 24 hours,48 hours,72 hours and 96 hours were 49.80%,55.90%,71.30% and 73.50%,respectively.Cisplatin were 25%,47.4%,61.7% and 71.2% respectively,and the inhibition rates of the combination group were 52.4%,73.7%,86.8% and 87.4%,respectively.The difference of the combination group after 24 hours was not statistically significant compared with the chitin oligosaccharide group(P>0.05),while compared with the cisplatin group,the difference was statistically significant(P<0.05).After drugs acted for 48 hours,72 hours and 96 hours,the difference of the combination group were statistically significant compared with other two groups(P<0.05).Chitin oligosaccharide combined with cisplatin could significantly inhibit the proliferation of lung adenocarcinoma A549 cells,and shown a higher efficacy than Chitin oligosaccharide or cisplatin.3.Cell scratch test showed that when the concentration of chitin oligosaccharide was 1mg/mL,the relative scratch width of 24 h and 48 h were 82.33% and 42.68%,respectively.When the concentration of chitin oligosaccharide was 3mg/mL,the relative scratch width of 24 h and 48 h were 83.46% and 60.52% respectively,while in the control group were 84.78% and 21.79% respectively,the difference was statistically significance(P<0.05).When the concentration of chitin oligosaccharide was 7mg/mL,the relative scratch width of 24 h and 48 h were 82.49%,64.07%,respectively.The concentration of cisplatin was 3?g/mL,the relative scratch width of 24 h and 48 h were 83.23%,55.91%,respectively.The relative scratch width of the combination group were 83.91%,69.67%,and the control group were 84.31%,23.09%,respectively.The difference of 48 hours were statistically significant(P<0.05).Compared with single drug groups,chitin oligosaccharide combined with cisplatin could significantly reduce the migration of A549 cells.4.The calculated number of transwell migration experimental of A549 cells were control group 578±26,chitin oligosaccharide group 472±21,cisplatin group 423±28,combination group 236±23.There was a significant difference between the combination group and other groups(P<0.05).Chitin oligosaccharide combined with cisplatin can significantly inhibit the migration of lung adenocarcinoma A549 cells,and shown a higher efficacy than Chitin oligosaccharide or cisplatin.5.Flow cytometry apoptosis experiments showed that chitin oligosaccharide act on A549 cells 24 hours,the early and late apoptosis rate were 11.8% and 5.3% respectively(total apoptosis rate: 17.1%),cisplatin group were 5.3% and 4.5% respectively(total apoptosis rate: 9.8%),the early and late apoptosis rate of the combination group were 6.7% and 8% respectively(total apoptosis rate: 14.7%),while in the control group were 3.4% and 2.8% respectively(total apoptosis rate: 6.2%).There was no significantly difference between the combination group and chitin oligosaccharide group(P>0.05),but the difference was statistically significant between the combination group and the cisplatin and the control group(P < 0.05).Both chitin oligosaccharide and Chitin oligosaccharide plus cisplatin could induce apoptosis of A549 cells.6.Through the HE staining of tumor tissue of nude mice,we found that the control group,chitin oligosaccharide group and cisplatin group had the same morphological features,cancer cells exihibited diffuse distribution,obviously heteromorphism,pathological mitosis,and there were punctiform necrosis in tumor tissue.In addition to the above performance,the combination group also exihibited focal necrosis,nuclear fragmentation and pyknosis,chromatin margination.7.Immunohistochemistry staining showed that the integral median of Ki67 among the control group,chitin oligosaccharide group,cisplatin group and combination group were 9,5,5,1,expression levels were strongly positive(+ + +),positive(+ +),positive(+ +)and weakly positive(+),the expression of the treatment groups decreased significantly compared with the control group,the difference was statistically significant(P<0.05);The integral medians of P53 were 2,1,1,1,expression levels were all weakly positive(+).There was no significantly difference between the combination group and other groups(P>0.05).The integral medians of TTF-1 were 3,1,1,0,expression levels were weakly positive(+),weakly positive(+),weakly positive(+)and negative(-),the expression of the treatment groups decreased significantly compared with that of other groups,the difference was statistically significant(P<0.05).The expression of Ki67 and TTF-1 in the transplanted tumor of human lung adenocarcinoma cell line A549 could be decreased by Chitin oligosaccharide combined with cisplatin.The effect is significantly lower than chitin oligosaccharide or cisplatin.Conclusion: 1.Through the cellular level in vitro and animal models in vivo experiments,this study proved that tranditional Chinese medicine metabolites chitin oligosaccharide plus cisplatin had positive synergistic effects on the inhibition of proliferation,migration and induction of apoptosis of A549 cells.It showed a higher efficacy than Chitin oligosaccharide or cisplatin on the inhibition of proliferation and migration.2.The combination of chitin oligosaccharide and cisplatin may promote tumor cells necrosis by decreasing the expression of Ki67 and TTF-1,especially Ki67. |
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