Anoikis and metastatic potential of Cloudman S91 melanoma cells

Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whethe r tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability, In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the pl astic, Prevention of their adherence by rocking or by covering culture plat es with polyhydroxyethylmethacrylate resulted in induction of anoikis and d eath of almost all cells. Their death was prevented in the presence of casp ase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistan t cells, S91 cells floating in the culture medium were sequentially isolate d and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected, These S91 no nadherent (S91Nadh) cells were completely resistant to anoikis and manifest ed higher metastatic ability than S91Adh cells, Anoikis resistance of S91Na dh cells was not attributable to their resistance to other apoptotic signal s in vitro, and they showed no increase in their survival in vivo in the lu ngs after i.v. inoculation. Increased metastatic potential of the anoikis-r esistant S91Nadh cells was associated with various phenotypic changes, incl uding increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49 alpha (CD49d) gene. In parallel, they sho wed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 a nd matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohy drates, such as appearance of alpha -galactosyl epitopes as a result of up- regulation of the alpha1,3-galactosyltransferase gene and concomitant reduc tion in cell membrane sialylation, Thus, selection of S91 melanoma cells fo r anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.