Early vitronectin receptor downregulation in a melanoma cell line during all-trans retinoic acid-induced apoptosis

Background: 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. Objectives: We have analysed the effects of ATRA treatment on melanoma cell adhesion and motility. Methods: Human M14 melanoma cells were treated with 10 μmol 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. Results: First, using an annexin V assay, we found that apoptosis was triggered by 48 h with 10 μmol 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 μmol 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-α_vβ₃ or anti-α_vβ₅ VnR monoclonal antibodies. Functionally, the treatment of melanoma cells with 10 μmol 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 α_vβ₃ and α_vβ₅ VnR levels were reduced upon exposure to 10 μmol L^-1 ATRA for 48 h as shown by Western blot, immunoprecipitation and immunocytochemistry assays. Conclusions: 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.