ABSTRACT
Matrix metalloproteinase-9 (MMP-9) plays a crucial role in both angiogenesis and tumor invasion. Vascular endothelial growth factor (VEGF) has been shown to up-regulate the expression of MMP-9 in vascular smooth muscle cells. We recently reported that hemoglobin (Hb) enhances the expression of tissue factor (TF) and VEGF on TF-positive human malignant cells. Therefore, to explore the relationship between tumor cell angiogenic protein VEGF and MMP-9, we studied the effect of Hb on MMP-9 production in human A375 malignant melanoma and J82 bladder carcinoma (TF+) cells and in KG1 myeloid leukemia (TF-) cells. Malignant cells were incubated with varying concentrations (0-1.0 mg/ml) of Hb and analyzed for released MMP-9 by gelatin zymography, dot immunoblotting, enzyme-linked immunosorbent assay, and Western blotting. Hb (0.50 mg/ml) induced an almost two-fold increase of MMP-9 in both A375 malignant melanoma (398 +/- 62 versus 233 +/- 61.0 ng/ml, P = 0.027) and J82 bladder carcinoma cells (1.55 +/- 0.12 versus 0.80 +/- 0.004 ng/ml, P = 0.004), compared with cells incubated without Hb. This release of MMP-9 was significantly inhibited by cycloheximide (95%) and by the specific inhibitors of protein tyrosine kinase, genistein (70 +/- 3.0%, P = 0.00027 and 67 +/- 1.0%, P = 0.00005) and mitogen-activated protein (MAP)-kinase, PD98059 (56 +/- 2.0%, P = 0.0001 and 62 +/- 1.0%, P = 0.00003) in A375 and J82 cells, respectively. In contrast, Hb (2.0 mg/ml) did not increase MMP-9 in KG1 cells. We conclude that Hb-induced synthesis of active MMP-9 in TF-bearing malignant cells is due to de novo synthesis of newly formed protein and is mediated by protein tyrosine kinase and by mitogen-activated protein kinase pathways.
Subject(s)
Hemoglobins/pharmacology , Matrix Metalloproteinase 9/biosynthesis , Neoplasm Proteins/biosynthesis , Neoplastic Stem Cells/drug effects , Carcinoma/pathology , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Genistein/pharmacology , Humans , Leukemia, Myeloid/pathology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Matrix Metalloproteinase 9/metabolism , Melanoma/pathology , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/metabolism , Protein Synthesis Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/physiology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology , Tumor Cells, Cultured/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
Vascular endothelial growth factor (VEGF) is an angiogenic hormone that increases the growth of many malignant tumors. Tissue factor (TF), the initiator of blood coagulation, is implicated in VEGF regulation. We recently reported that hemoglobin (Hb) upregulates TF on malignant cells. Therefore, to explore the role of Hb in angiogenesis, we examined its effect on VEGF production in A375 melanoma and J82 bladder carcinoma (TF+) and KG1 myeloid leukemia (TF-) cells. Hb (0.50 mg/ml) induced VEGF expression and secretion in TF+ malignant cells. VEGF secretion was inhibited by cycloheximide (85%) and the specific inhibitors of protein tyrosine kinase, genistein (71+/-0.74 and 55+/-4.90%) and mitogen-activated protein (MAP)-kinase, PD098059 (82+/-2.0 and 59+/-6.7%) in A375 and J82 cells respectively. In contrast, Hb (2.0 mg/ml) did not increase VEGF in KG1 cells. Hb-induced VEGF was purified from the culture medium of J82 cells using immunoaffinity chromatography and two isoforms (46 and 30 kd) identified. We conclude that Hb-induced synthesis of VEGF in TF-bearing malignant cells is mediated by protein tyrosine kinase and by MAP-kinase pathways.
