Abstract
BackgroundRGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment.ResultsIn the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 μM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA.ConclusionsRGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma.
Highlights
RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties
Previously investigated by us and Others, involves RGD peptides cell-internalization, intracellular targeting and direct activation of caspase-3, caspase-8 or caspase-9 in lymphocytes, cardiomyocytes, endothelial cells and chondrocytes, leading to apoptosis most-likely via an integrin-independent mechanism [3,22,23,24]. These findings suggest that RGD motif, in addition to targets exposed onto the external surface of cell membrane, recognizes intracellular targets, leading to procaspase auto-processing and activation
Melanoma cells proliferation induced by human Fibroblast Growth Factor-2 (FGF-2) was significantly reduced in the presence of RGDS (Figure 1A top) (46 ± 16% inhibition, p < 0.005) at 500 μg/ml, indicating that, despite the presence of the strong survival factor FGF-2, RGDS exerts a potent anti-proliferative effect on SK-MEL-110 cells, independently of its anti-adhesive properties
Summary
RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment. RGD (Arg-Gly-Asp) motif is largely investigated as mediator of cell adhesion to extracellular matrix and to cells, via cell-surface receptors named integrins. These receptors belong to a large family of twenty-four heterodimeric members. Integrins control the interaction of tumor cells with the surrounding environment, with a direct effect on cell proliferation, migration, metastatic dissemination, invasion, cell survival [10,11]. RGD peptides are involved in tumor and endothelial cells-targeting via the αvβ receptors [16,17,18,19] as well as in noninvasive tumor imaging, targeting and radio-treatment [20]
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