Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells.
To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the 'RGD loop' (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin's interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.
McLane
Mary Ann
MA
Department of Medical Technology, University of Delaware, Newark, Del. 19716, USA. mclane@udel.edu
Zhang
Xiaoming
X
Tian
Jing
J
Zelinskas
Claire
C
Srivastava
Apoorva
A
Hensley
Brett
B
Paquette-Straub
Carrie
C
D000409:Alanine / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism*; D045744:Cell Line, Tumor; D019483:Disintegrins; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D014757:Viper Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects*
D019483:Disintegrins; D016023:Integrins; D010455:Peptides; D014757:Viper Venoms; C084060:eristostatin; D000409:Alanine