Baroni A, Paoletti I, Silvestri I, Buommino E, Carriero M V
Departments of Dermatology and Experimental Medicine, II University of Naples, Naples, Italy.
Br J Dermatol. 2003 Mar;148(3):424-33. doi: 10.1046/j.1365-2133.2003.05165.x.
Recent evidence assigns the vitronectin receptors (VnRs) an important role in regulating tumour cell invasion and dissemination. In vivo and in vitro studies document that all trans-retinoid acids (ATRAs) inhibit growth-inducing apoptosis in melanomas.
We have analysed the effects of ATRA treatment on melanoma cell adhesion and motility.
Human M14 melanoma cells were treated with 10 micromol L-1 ATRA for different times and stained with rhodamine-phalloidin to analyse the effect of treatment on cytoskeleton organization. Cell adhesion and cell migration assays were performed to analyse the role of VnRs in the ATRA-induced early stages of apoptosis. VnR expression was evaluated by Western blot, immunoprecipitation and immunocytochemistry assays.
First, using an annexin V assay, we found that apoptosis was triggered by 48 h with 10 micromol L-1 ATRA exposure. At this time point, decrease in the F-actin polymerization as well as inhibition of cell adhesive ability to vitronectin (Vn) was exerted by ATRA treatment. In the presence of serum, exposure to 10 micromol L-1 ATRA for 48 h produced a dramatic inhibition of the cell adhesion ability that was comparable with that exerted by untreated cells preincubated with anti-alpha(v)beta(3) or anti-alpha(v)beta(5) VnR monoclonal antibodies. Functionally, the treatment of melanoma cells with 10 micromol L-1 ATRA for 48 h causes an inhibition of directional cell migration towards Vn-coated filters. Therefore, we analysed the effect of ATRA on the VnR expression. Both alpha(v)beta(3) and alpha(v)beta(5) VnR levels were reduced upon exposure to 10 micromol L-1 ATRA for 48 h as shown by Western blot, immunoprecipitation and immunocytochemistry assays.
Altogether, our data indicate that treatment of M14 melanoma cells with ATRA downregulates VnR expression and that this reduction is closely correlated with the ATRA-dependent inhibition of actin-fibre organization, cell adhesion and migration. Although the mechanism by which ATRA regulates the expression of VnR in M14 melanoma cells needs further elucidation, this system may represent a model for understanding the molecular basis of ATRA therapy in melanoma.
最近的证据表明，玻连蛋白受体（VnRs）在调节肿瘤细胞侵袭和扩散中起重要作用。体内和体外研究表明，全反式维甲酸（ATRAs）可抑制黑色素瘤中诱导生长的细胞凋亡。
我们分析了维甲酸治疗对黑色素瘤细胞黏附及运动能力的影响。
用10 μmol/L全反式维甲酸处理人M14黑色素瘤细胞不同时间，并用罗丹明-鬼笔环肽染色，以分析处理对细胞骨架组织的影响。进行细胞黏附及细胞迁移试验，以分析玻连蛋白受体在全反式维甲酸诱导的细胞凋亡早期阶段的作用。通过蛋白质免疫印迹法、免疫沉淀法和免疫细胞化学分析法评估玻连蛋白受体的表达。
首先，使用膜联蛋白V分析，我们发现10 μmol/L全反式维甲酸处理48小时可引发细胞凋亡。此时，全反式维甲酸处理可使F-肌动蛋白聚合减少，并抑制细胞对玻连蛋白（Vn）的黏附能力。在有血清存在的情况下，用10 μmol/L全反式维甲酸处理48小时可显著抑制细胞黏附能力，这与用抗α(v)β(3)或抗α(v)β(5)玻连蛋白受体单克隆抗体预孵育的未处理细胞所产生的抑制作用相当。在功能上，用10 μmol/L全反式维甲酸处理黑色素瘤细胞48小时会抑制细胞向包被玻连蛋白的滤膜的定向迁移。因此，我们分析了全反式维甲酸对玻连蛋白受体表达的影响。蛋白质免疫印迹法、免疫沉淀法和免疫细胞化学分析法显示，暴露于10 μmol/L全反式维甲酸48小时后，α(v)β(3)和α(v)β(5)玻连蛋白受体水平均降低。
总之，我们的数据表明，用全反式维甲酸处理M14黑色素瘤细胞可下调玻连蛋白受体表达，且这种降低与全反式维甲酸依赖性抑制肌动蛋白纤维组织、细胞黏附和迁移密切相关。尽管全反式维甲酸调节M14黑色素瘤细胞中玻连蛋白受体表达的机制需要进一步阐明，但该系统可能代表一个理解全反式维甲酸治疗黑色素瘤分子基础的模型。
