The Mechanism Of Acitretin Combined INF On Malignant Melanoma B16 Cells

Objective:Melignant malanoma is one of the skin cancer which lead to death.It is the third of the malignant skin tumor with clinical characteristic of high malignancy,strong invasiveness.At present,surgical operation is still a preferred method of treatment of melignant melanoma.But the prognosis remains poor even if surgery.So to find effective drugs treatment of melignant melanoma have become a hot research field.Retinoids are structural and functional analogs of vitamin A that display a wide range of biologic activity.As retinoids are critical for the maintenance of epithelial growth and differentiation,they are effective therapeutic and chemopreventive agents for some skin diseases and epithelial cancer.Numerous studies have shown that retinoic acid affects a variety of malignant tumors including induction of differentiation, inhibition of proliferation and induction of apoptosis and so on.Acitretin and INF belong to the exogenous and endogenous differentiation atileptic respectively.Combination of these drugs can elevate sensitivity of tumor,decrease dosage,avoid side effect and improve therapeutic efficacy.To date,retinol and the retinoids are the only compounds proved chemoproventive for melignant melanoma,but the mechanisms have not been fully clarfied.In this study,we take the mouse melanoma cell line B16 as our study object watch the growth inhibiting effect and the morphological changes of acitretin combined with INF by different concentration or at different time,to screen the best drug dose and time,study the mechanism of their synergistic effect.thus to provide the experimence for searching for more effective drug to treat melanoma.Methods:1 cell cultureThe mouse melanoma cell line B16 were cultured in RPMI-1640 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 inhition with the MTT analysis method.2.1 Effects of different concentration of acitretin on the cell proliferation of cell line B16 at different time were measured by MTT.2.2 Effects of different concentration of INF on the cell proliferation of cell line B16 at different time were measured by MTT.2.3 Effects of acitretin with INF on the cell proliferation of cell line B16 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 PTEN of the mouse melanoma cell line B16 in the different groups by immunohistochemical method.5 To detect the apoptosis rate and the changes of cell cycle by FCMThe B16 cells were divided in four groups and treated with acitretin in combination or not with INF in vitro.At the desired time after treatment the cell cycle and apoptosis was detected through FCM.6 The FCM was used to detect the changes of PTEN in different groups.7 Statistic analysis: Data wre analyzed by 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 B16 cells by MTT:1.1 In group acitretin:The difference of inhibition rate among the three groups was significant(p<0.05).The results of MTT showned that acitretin inhibited the proliferation of B16 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 acitretin inhibited the proliferation of B16 cells in a dose-dependent and time-dependent manner. 1.3 In group combination of acitretin and INF: The B16 cells were divided in four groups and treated with acitretin in combination or not with INF in vitro.The results showned that the combination of acitretin with INF inhibited growth of B16 cells,inhibitory rate of combination has remarkable difference compared with acitretin,INFand control group.The effect of acitretin and INF were synergistic.2 To observe the differentiation of B16 cell:Cells in the negative control group well grew adhesively,most in shuttle shape,moderately sized,clear nuceolus,nulear division phase found.Cells shrinkage after treatment by acitretin,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 acitretin 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 PTEN protein by cell immunochemistry:PTEN protein was expressed in the intracytoplasm of B16 cell.We can detect the expression of PTEN protein increased in acitretin-treated cells compared with the untreated controls.PTEN protein in B16 cells treated with acitretin/INF were activated significantly compared with control and acitretin,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): The difference of the cell cycle distribution among the four groups were signifcant(p<0.05).Acitretin and INF all could increased G1-phase population and reduced the S-phase population in B16 cells. But the early apoptosis of B16 cells was not observed significantly.5 To detect the expreesion of PTEN protein by FCM:The expreesion of PTEN protein of B16 cells in the experiment groups after 48 hours were significantly higher than that in the control group.The FI-value of PTEN in the experiment groups was 1.1233﹑1.2033和1.6300 respectively.There has significantly difference among the experiment groups(p<0.05).Conclusions:1 Acitretin and INF could significantly inhibit the proliferation of B16 cells.All these effects were in dose-dependent and time-dependent manner within the appropriate extent.2 The synergistic effects of a combination of acitretin with INF on inducing differentiation,apoptosis and growth inhibition of B16 cells which is more outstanding than used alone significantly.3 The experiment groups all could increase the proportion of G0/G1 period and decrease the proportion of S period,but the apoptosis of B16 cells was not observed.4 The combination of acitretin and INF can inhibit the proliferation of B16 cell,which may be through increasing the expression of PTEN.