Matrix metalloproteinase-9 (MMP-9) plays a crucial role in both angiogenesis and tumor invasion. Vascular endothelial growth factor (VEGF) has been shown to upregulate 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. (C) 2003 Lippincott Williams Wilkins.