| Objective: As one of the most important meaning, radiotherapy have been widely used in the treatment of lung carcinoma which possess the highest morbidity and mortality of all malignancy. However, considering that not only does radiotherapy bring serious injure to normal tissue, but parts of malignant cells will produce some effect of treatment resistant in various degree after this approach was employed. It become an urgent statement to search new antineoplastic drugs and radio-sensitizers which own low toxicity and high effectiveness. Some study indicate that dihydromyricetin(DMY) which was discovered in recent years as a new flavonoids can exert an antineolastic effect whether in vivo or vitro, and furthermore, it has some advantage such as protecting liver, decreasing lipid level in blood, antioxidant and improving immunity. Additionally, There are a great number of research show that flavonoids can play some role in radio sensibilization. As one kind of flavonoids, it is possible that DMY will become a low-toxic and high-effective antineoplastic drug and tumor radiotherapy sensitizer. According to this point, it may provide a new thought for the treatment of lung carcinoma and further materials for the development and application of natural plant medicines which are used in anticarcinoma in our country.Methods:1 A549 cells were cultured and steadily subcultured in vitro, then Cells were selected for the experiment during the logarithmic phase.2 The cells in the logarithmic phase were used for preparation of single-cell suspension. According to the standard of 5000 per pore, the cells were seeded in 96 well plates. 24 hours after incubation, when the cells attached well and the attachment rate is about 30-40%, the DMY mediums of different concentration(0, 20, 40, 60, 80, 100, 140, 200μg/ml) were respectively applied to continue to incubate, and the MTT assay was used to detect the proliferation activity of different DMY concentration group after 48 hours, then count the IC50.3 According to the standard of 5000 per pore, the cells were seeded in 96 well plates and cultured 24 h, then incubated by DMY of different concentration(0, 20, 40, 60, 80, 140μg/ml). After 48 hours, the cells were placed under the inverted microscope with the purpose of observing and detecting the impact of DMY in different concentration on the shape and growth of them.4 According to the different initial seeding density(1×103, 5×103, 10×103, 50×103, 100×103, 500×103 per pore), the cells were respectively seeded into the 6 well plate. When the cell attached and stretched well, the 40μg/ml DMY complete medium replace for continuous incubation and these cells were taken under the inverted microscope for observing the effect of the same concentration DMY on the different seeding density cells.5 According to 400 per pore, the cells were seeded into the 6 well plates for culture. When the cells attached and stretched well, the DMY of different concentration(0, 6, 7, 8, 9, 10, 12, 15μg/ml) were employed in 48 hours and continuous culture was made. The clone formation was observed and the long-term inhibition of DMY on the A549 cells was detected, then count cell survival fraction and IC50.6 Flow cytometry(FCM) apoptosis kit was used to detect the effect of DMY of different concentration(0, 20, 40, 80μg/ml) on the inducing apoptosis of A549 cells after 48 hours.7 Western Blotting was used to detect the effect of DMY of different concentration(0, 20, 40, 80μg/ml) on the Bax and Bcl-2 protein expression of apoptosis pathway after 48 hours.8 FCM was used to detect the change of A549 cell cycle phase impacted by DMY of IC20 during different time(6h, 12 h, 24 h, 48h).9 After pre-intervention were employed by using DMY of IC20 concentration during 24 hours, the cells were radiated under the different dose(0, 2, 4, 6, 8Gy)and succeed to be cultured. The clone formation were observed and the impact of DMY on the sensibility of radiotherapy with A549 cells in vitro.10 Applied statistics processing software SPSS19.0 was used for one-way analysis of variance and the data shows in the form of x ±s. according to the difference of the aim and type of the material, univariate analysis of variance and one-way analysis of variance was used to dispose the data. SNK-q test was applied on comparison between any two means. With P<0.05 for significance test standard.Results:1 MTT assay indicate that different concentration DMY produce a obvious inhibition on the proliferation of A549 after it affected 48 hours(P<0.05), and the proliferation of A549 cells was inhibited in a dose-dependent manner. The IC50 is 64.45μg/ml.2 As the concentration of DMY increase, it can be seen that cell pyknosis become more evident and the growing ability decrease and even lost.3 After action of the same concentration of DMY on the different density of A549 cells, it can be seen under the microscope that the lower density the more serious phenomenon pyknosis.4 The result of Clone formation assay indicate that the higher DMY concentration is, the more obvious antineoplastic effect they have. IC50 is in the position about 8.46 μg/ml.5 FCM apoptosis assay show that the higher DMY concentration is the higher apoptosis rate A549 cells possess.6 The result of western blotting assay reveal that the Bax protein expression increase and Bcl-2 protein expression decrease in the high DMY concentration.7 FCM cycle experiment show that the rate of G2/M phase ratio obviously increased when A549 cells were incubated 24 hours with DMY.8 A significant sensibilization leading by the effect of radiotherapy sensibilization on A549 cells after DMY pre-intervention 24 hours was not detected by Clone formation sensibilization assay.Conclusion:1 DMY possesses an antineoplastic effect on A549 and the effect become more obvious as the drug concentration increase.2 The antineoplastic effect of DMY on A549 cells has a relationship with the cells density when the drug was given. The lower the cells density is, the stronger antineoplastic effect of DMY on A549 cells they have. It indicate that the cells survival ability relates to cells density and it should be taken more attention to the impact of the effect of drug that produced by the initial seeding density when different experiment methods were used.3 The mechanism of the anti-proliferation and inducing apoptosis effect with DMY on A549 cells in vitro may relate to that the apoptosis pathway in which Bax/Bcl-2 involve for regulating is activated.4 The rate of G2/M phase ratio obviously increased when A549 cells were incubated 24 hours with DMY.5 In our study the cells were pre-intervened by DMY and radiated after 24 hours, but we have not seen any obvious sensibilization by now.Discussion: Whether there is other mechanism of the antineoplastic effect of DMY in vitro on A549 cells, Whether there is some antineoplastic effect of DMY in vivo and whether there is radiotherapy sensibilization, further study should be made for solving all of these questions. |
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