Intracellular targets of RGDS peptide in melanoma cells.
BACKGROUND
RGD-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.


RESULTS
In 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 muM) 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.


CONCLUSIONS
RGDS 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.
Aguzzi
Maria Simona
MS
Laboratorio Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Roma, Italy.
Fortugno
Paola
P
Giampietri
Claudia
C
Ragone
Gianluca
G
Capogrossi
Maurizio C
MC
Facchiano
Antonio
A
D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002469:Cell Separation; D004789:Enzyme Activation / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D051034:Inhibitor of Apoptosis Proteins; D008545:Melanoma / Q000378:metabolism*; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009842:Oligopeptides; D034741:RNA, Small Interfering; D000077022:Survivin; C046617:arginyl-glycyl-aspartyl-serine; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9