The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways.
Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATP pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 degrees C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.
Hwang
Hyun Sook
HS
Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Republic of Korea.
Kim
Soung Soo
SS
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002196:Capillaries / Q000166:cytology*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002738:Chloroquine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D042783:Endothelial Cells / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D018082:Kringles; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015739:Nocodazole / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry*; D015398:Signal Transduction; D013696:Temperature; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D010455:Peptides; D013778:Tetrazolium Salts; D013844:Thiazoles; D016222:Fibroblast Growth Factor 2; D002738:Chloroquine; D000255:Adenosine Triphosphate; D011516:Prothrombin; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue; D015739:Nocodazole