| Objective:With clinical characteristic of high malignancy, strong invasiveness, malignant melanoma is a serious life -threatening cancer, ranking third in skin cancers. At present surgical treatment of malignant melanoma is still the preferred method, but even if surgical resection is performed, the prognosis is still poor. Efficiency of chemotherapy is below 25%. Since it is highly malignant and easy to transfer, 5-year survival rate of patients is extremely low. Therefore, search for effective drug treatment has become the research focus in the field of malignant melanoma treatment.Arsenic has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Numerous studies have shown that Arsenic trioxide affects a variety of malignant tumors including induction of differentiation, inhibition of proliferation and induction of apoptosis and so on. Arsenic trioxide and INF belong to the exogenous and endogenous differentiation antileptic respectively. Combination of these drugs can elevate sensitivity of tumor, decrease dosage, avoid side effect and improve therapeutic efficacy.The mechanism of arsenic trioxide on anti-melanoma has not been fully clarified. In this study, with human malignant melanoma A375 cells as a working model, we try to explore the inhibition of arsenic trioxide, interferon at different concentrations, different time in skin melanoma cell growth and their effects on morphological differentiation, to select the best working time and dose of the combination of two drugs of time; to explore the impact of the combination of two drugs on a newly discovered oncogene PPO in human A375 cells; to study the mechanism of their synergistic effect, thus providing a foundation for the theory and experiment in the search of more effective drug to treat melanoma. Methods:1 cell cultureThe human melanoma cell line A375 were cultured in DMEM medium supplemented with 10% heat inactivated fetal bovine serum, 100u/ml penicillin and 100u/ml streptomycin and in a numidified atmosphere 5% CO2 at 37℃.2 Detection of cell growth inhibition with the MTT analysis method.2.1 Effects of different concentration of arsenic trioxide on the cell proliferation of cell line A375 at different time were measured by MTT.2.2 Effects of different concentration of INF on the cell proliferation of cell line A375 at different time were measured by MTT.2.3 Effects of arsenic trioxide with INF on the cell proliferation of cell line A375 were measured by MTT, and evaluated with iteraction index.3 The morphological changes of cells were detected by the reverse microscopy after the treatment of different groups.4 To detect the expression of PPO of the human melanoma cell line A375 in the different groups by immunohistochemical method.5 To detect the apoptosis rate and the changes of cell cycle by FCM The A375 cells were divided into four groups and treated with Arsenic trioxide in combination or not with INF in vitro. At the desired time after treatment the cell cycle and apoptosis was detected through FCM.6 Statistic analysis: Data were analyzed by statistical software Spss13.0 for windows, using single-factor analysis of variance, with significance of difference standard a=0.05Results:1 To detect growth inhibition rate of A375 cells by MTT:1.1 In group arsenic trioxide:The difference of inhibition rate among the three groups was significant(p<0.05).The results of MTT showned that Arsenic trioxide inhibited the proliferation of A375 cells in a dose-dependent and time-dependent manner.1.2 In group INF: The difference of inhibition rate among the three groups was significant(p<0.05). The results of MTT showned that arsenic trioxide inhibited the proliferation of A375 cells in a dose-dependent and time-dependent manner.1.3 In group combination of arsenic trioxide and INF: The A375 cells were divided into four groups and treated with arsenic trioxide in combination or not with INF in vitro.The results showed that the combination of arsenic trioxide with INF inhibited growth of A375 cells, inhibitory rate of combination has remarkable difference compared with Arsenic trioxide, INF and control group.The effect of Arsenic trioxide and INF were synergistic.2 To observe the differentiation of A375 cell:Cells in the negative control group grew adhesively, most in shuttle or polygon shape, moderately sized, clear nuceolus, nulear division phase found. Cells shrinkage after treatment by arsenic trioxide, the breakdown was irregular in shape; some cells appear at both ends of the pseudo-foot slender. INF-stimulated cells showed the number of bipolar cells increase. The cells treated by arsenic trioxide and INF were present in the supernatant.The density of adherence cells was diminished when compared with the control.3 To detect the expression of PPO protein by cell immunochemistry:PPO protein was expressed in the intracytoplasm of PPO cell. We can detect the expression of PPO protein diminished in arsenic trioxide -treated cells compared with the untreated controls. PPO protein in A375 cells treated with arsenic trioxide /INF were diminished significantly compared with control and arsenic trioxide, INF alone. There has difference between the control and the experiment groups(p<0.05).4 To detect the cell apoptosis rate and the cell cycle distribution by flow cytometry machine(FCM):Medications in each group will enable S phase delay, as compared with the control group decreasing G0/G1 cells (P <0.05), and increase the proportion of S phase cells. It showed that the inhibition of drugs in each group and the percentage of cells in the cell cycle change. S/G2 transformation suppression, cells were blocked in S phase, so that the ratio of S phase cells increased. Application of arsenic trioxide and interferon combined has synergistic effect; however, interferon-induced apoptosis in A375 cells is not obvious while arsenic trioxide-induced apoptosis in A375 cells is significant.Conclusions:1 Arsenic trioxide and INF could significantly inhibit the proliferation of A375 cells. All these effects were in dose-dependent and time-dependent manner within the appropriate extent.2 While Arsenic trioxide and INF act on A375 cells, with the increase in drug concentration, the proportion of S phase increase significantly. The cells were arrested in S phase, suggesting that A375 cells were arrested in S phase, which might be one reason of INF and arsenic's growth inhibition.3 The synergistic effects of a combination of arsenic trioxide and INF on inducing differentiation and growth inhibition of A375 cells is more outstanding than used alone significantly. The combination of arsenic trioxide and INF can inhibit the proliferation of A375 cell, which may relate to decreasing the expression of PPO.4 Arsenic trioxide induced apoptosis in A375 cells significantly. Although apoptosis were not observed in interferon group, it does not rule out that inter feron on human A375 cells dose not relate to apoptosis. |
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