探討RXD Motif 在 Rhodostomin 中對於整合蛋白辨識以及抑制黑色素細胞瘤的活性所扮演的角色

Integrins are involved in many biological processes such as angiogenesis, thrombosis, inflammation, osteoporosis, and thus play a key role in many severe human diseases. The arginine-glycine-aspartate (RGD) motif is found in several important extracellular matrix proteins, which serve as adhesive integrin ligands. It was also reported that the motifs other than RGD can be recognized by integrins. For example, an RHD motif found in the Alzheimer's-disease causing beta peptides can interact with integrin alpha5beta1, and an RYD motif is found in an integrin alphaIIbbeta3-binding antibody. In this study I used rhodostomin, a disintegrin containing 68 amino acid residues with a 48PRGDMP53 motif, as the scaffold to study the role of the middle residue in the RXD motif in recognizing integrins. According to the results of cell adhesion and platelet aggregation assays, I found that the middle residues in the RXD motif of Rho play an important role in recognizing integrins. Rho containing the RGD motif has the highest activity toward integrins alphaIIbbeta3, alphaVbeta3, and alpha5beta1. In contrast, mutations on the middle residue in the RXD motif caused 2.8-35.4-, 5.1-695.3-, and 8.0-1989.1-folds deceases in inhibiting integrins alphaVbeta3, alpha5beta1, and alphaIIbbeta3. We also found that the Rho mutants containing L, V, I, E, and D in the middle residue of the RXD motif can selectively inhibit integrin alphaVbeta3. It is reported that integrinalphaVbeta3 increases 50- to 100-fold as human melanoma cells progress to a more metastatic phenotype. It was consistent with our flow cytometric analysis that integrin alphaVbeta3 was highly expressed on the surface of A375 cells. Besides, A375 cells also expressed integrin alphaVbeta5 andalpha5beta1. I then used integrin alphaVbeta3-specific Rho mutants to study the activities of integrin alphaVbeta3-specific Rho mutants in inhibiting the proliferation, migration, invasion, and apoptosis of human melanoma cells. The cell adhesion analysis showed that Rho, RLD and ARLDDL mutants inhibited the adhesion of A375 to fibrinogen with the IC50 values of 4.0, 13.1, and 13.1 nM, to vitronectin with the IC50 values of 135.5, 1556, 1768 nM, but not to fibronectin. The migration analysis showed that Rho, RLD and ARLDDL mutants effectively inhibited cell migration with IC50 values of 9.2, 37.4, and 45.1 nM, respectively. The result of matrigel invasion assay showed that Rho, RLD and ARLDDL mutants effectively inhibited cell invasion with IC50 values of 18.0, 50.5, and 63.2 nM, respectively. They also reduced cell growth by changing cell cycle and changed cell morphology of A375 melanoma cell. In contrast, RLD and ARLDDL mutants cannot induce cell apoptosis and inhibit the formation of colonies in soft agar. In conclusion, we designed integrin alphaVbeta3-specific disintegrins by mutating the RXD motif of Rho, and found they can inhibit the adhesion, migration, invasion, and growth of human melanoma cells in the nanomolar concentration range. This study demonstrated that integrin alphaVbeta3-specific Rho mutants designed by this study are potential drug candidates for melanoma treatment.