The Effect Of Acitretin Combined TNF-a On Human Malignant Melanoma A375Cells And Its Mechanism

Objective：Malignant melanoma is derived from the melanin cell ofmalignant tumor, occurs more often in the skin, accounting for all skin ofmalignant tumor third.Its highiy malignant and prone to early lymphatic andhematogenous metastasis, the prognosis poor. Surgical resection, biologicaltreatment, radiotherapy and chemotherapy are commonly used treatment ofmalignant melanoma, but the results are not satisfactory. With early diagnosisand surgical intervention and the development of the auxiliary treatment,patients with early by more than surgery can be cured,5years of survival rateof about95%, but in patients with adanced surial only2to8months,5yearsof survival rate to less than5%. Traditional chemotherapy for malignantmelanoma curative effect is poorer, therefore, seeks good effect and sideeffects of small new treatment and drugs are hotspot oncolosts.Retinoids includes vitamin A, natural and synthetic derivatives. It has theantiproliferation and inducing differentiation effects on a variety of tumor cells.It is also a kind of effective prevention of skin cancer drugs, but the resistanceof skin melanoma mechanism has not been fully elucidated. TNF-a has a widerang of biological activity, with a strong anti-tumor effect, is found so fa, thestrongest anti-tumor effect of cytokines, but its side effects. They mayimprove the sensitivity of the tumor indecer, enhanced anti-tumor effect,which erduces the signle drug dose, and to erduce the toxic side effects, butalso can improve treatment. The aim of this study to the human melanoma cellline A375to investigate retinoid, TNF-a in different concentrations anddifferent time of skin melanoma cells inhibit the proliferation andmorphological differentiation of the screening of the two drugs combined bestthe role of time and dose.Explore the acitretin, and the combination of twodrug to the expression of MMP-1and TIMP-1in human A375cells.TNF-a, Retinoids for skin tumor cell apoptosis in basic research and clinicalapplication provide an experimental basis.Method:1Cell cultureThe human melanoma cell line A375were cultured in DMEM mediumsupplemented with10%heat inactivated fetal bovine serum,100u/mlpenicillin and100u/ml streptomycin and in a numidified atmosphere5%CO2at37℃.2Detection of cell growth inhibition with the MTT analysis method.3The morphological and differentiation changes of cells were detected by thereverse microscopy after the treatment of different drugs.4To detect the expression of MMP-1, TIMP-1of the human melanoma cellline A375in the different groups by immunohistochemical method.5Statistic analyses: Data were analyzed by statistical software Spss13.0forwindows. Based on the One-way ANOVA with a=0.05as significant differences standards.Results:1To detect growth inhibition rate of A375cells by MTT:1.1In group acitretin: The difference of inhibition rate among the three groupswas significant(p＜0.05).The results of MTT showed that acitretin inhibitedthe proliferation of A375cells ina dose-dependent and time-dependentmanner.1.2In group combination of acitretin and TNF-a: The A375cells were dividedin there groups and treated with acitretin in combination or not with TNF-a invitro. The results showed that the combination of acitretin with TNF-ainhibited growth of A375cells, inhibitory rate of combination has remarkabledifference compared with acitretin and control group. The effect of acitretinand TNF-a were synergistic.2To observe the differentiation and morphology of A375cell by differentdrugs.Cells in the negative control group grow well stick wall, uniform distribution, moderately sized, cytoplasm full, most in shuttle or polygon shap,transparent, refractive index of proliferation exuberant. Cells go round, shrinks,rupture after treatment by acitretin after24hours. Refraction weakened andfloating cells appeared, along with the role the extension of time of the abovephenpmenon more apparent. The cells treated by acitretin and TNF-a werepresent in the supernatant. The density of adherence cells was diminishedwhen compared with the control.3To detect the exepression of MMP-1, TIMP-1protein by cellimmunochemistry.MMP-1, TIMP-1protein was expressed in the intracytoplasm of A375cell. We can detect the expression of MMP-1, TIMP-1protein diminished inarsenic acitretin-treated cells compared with the untreated controls. MMP-1,TIMP-1protein in A375cells treated with acitretin/TNF-a was diminishedsignificantly compared with control and acitretin, TNF-a alone. There hasdifference between the control and the exeperiment groups (p<0.05).4To detect the cell apoptosis rate and the cell cycle distribution by flowcytometry machine (FCM):Compared with the control group, medication in each group couldincreased G1-phase population and reduced the S-phase population in A375cells. Application of acitretin and TNF-a combined has synergistic effect;However, TNF-a and combination therapy could induce the apoptosis of A375cells. The combination therapy-induced apoptosis of A375cells moresignificant.Conclusion:1Acitretin and TNF-a could significantly inhibit the proliferation of A375cells. All these effects were in dose-dependent and time-dependent mannerwithin the appropriate extent.2The synergistic effects of a combination of acitretin with TNF-a on inducingdifferentiation, apoptosis and growth inhibition of A375cells which is moreoutstanding than used alone significantly.3The experiment groups all could increase the proportion of G0/G1period and decrease the proportion of S period, and the apoptosis of A375cells wasmore observed. But combination therapy-induced apoptosis of A375cells ismore obvious.4The combination of acitretin and TNF-a can inhibit the proliferation of A375cell, which may relate to decreasing the expression of MMP-1, TIMP-1.Thismay be related to its anti-tumor mechanism.