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

• Published in: British journal of dermatology (1951) Vol. 148; no. 3; pp. 424 - 433
• Main Authors: Baroni, A., Paoletti, I., Silvestri, I., Buommino, E., Carriero, M.V.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-03-2003; Blackwell; Oxford University Press
• Subjects: all-trans retinoic acid; Antineoplastic Agents - pharmacology; Apoptosis; Biological and medical sciences; Blotting, Western - methods; cell adhesion; Cell Adhesion - drug effects; cell migration; Cell Movement - drug effects; Cytoskeleton - drug effects; Dermatology; DNA, Neoplasm - analysis; Down-Regulation - drug effects; Humans; Integrin alphaVbeta3 - metabolism; M14 cells; Medical sciences; melanoma; Melanoma, Experimental - metabolism; Neoplasm Proteins - metabolism; Precipitin Tests; Tretinoin - pharmacology; Tumor Cells, Cultured - drug effects; Tumors of the skin and soft tissue. Premalignant lesions; vitronectin receptor; all-trans retinoic acid; Retinoid; Mobility; Vitronectin; melanoma; Malignant tumor; Adhesion; M14 cells; Cell line; Cell death; vitronectin receptor; cell adhesion; Malignant melanoma; cell migration; Tumor cell; Apoptosis; Biological receptor; Tretinoin
• ISSN: 0007-0963; 1365-2133
• DOI: 10.1046/j.1365-2133.2003.05165.x

Summary 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‐αvβ3 or anti‐αvβ5 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 αvβ3 and αvβ5 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.