Thrombin up-regulates cathepsin D which enhances angiogenesis, growth, and metastasis.
Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by approximately 2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3- to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2- to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9.
Departments of Medicine and Radiation Oncology and Cell Biology, New York University School of Medicine, New York, New York.
Liang
L
Hu
Jennifer M
JM
Roth
Peter
P
Brooks
Joanna
J
Luty
Simon
S
Karpatkin
D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000098:blood supply* / Q000188:drug therapy*; D009389:Neovascularization, Pathologic / Q000209:etiology* / Q000473:pathology; D010436:Pepstatins / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation
D006629:Hirudins; D010436:Pepstatins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013917:Thrombin; D002402:Cathepsin D; D020780:Matrix Metalloproteinase 9; C031375:pepstatin