Role of Hypoxia-Inducible Factor 1 in Gastric Cancer Cell Growth, Angiogenesis, and Vessel Maturation

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Abstract
Background: Hypoxia-inducible factor 1 (HIF-1), a heterodimer comprising the oxygen-regulated subunit, HIF-1α, and HIF-1β, mediates transcription of the gene for vascular endothelial growth factor (VEGF). Overexpression of HIF-1α is associated with tumor angiogenesis and tumor cell proliferation and invasion. We examined the effects of inhibiting HIF-1α activity on angiogenesis and human gastric cancer growth in vivo . Methods: Human gastric cancer TMK-1 cells were stably transfected with pHIF-1αDN, an expression plasmid encoding a dominant-negative form of HIF-1α that dimerizes with endogenous HIF-1β to produce HIF-1 complexes that cannot activate transcription, or with the empty expression vector (pCEP4). Two clones of pHIF-1αDN–transfected cells, DN2 and DN3, were tested in all experiments. We used an enzyme-linked immunosorbent assay to measure VEGF secretion by transfected cells cultured in hypoxic (1% O 2 ) or nonhypoxic (20% O 2 ) conditions. We used subcutaneous and orthotopic mouse tumor models to examine the growth of tumors derived from injected pHIF-1αDN–or pCEP4-transfected cells. Tumor cell proliferation, vessel area (a measure of functional vascular volume), and tumor endothelial cell association with pericyte-like cells (a measure of vessel maturation) were analyzed by immunohistochemical or immunofluorescent staining. All statistical tests were two-sided. Results: DN2 cells and DN3 cells secreted less VEGF than pCEP4-transfected TMK-1 cells when cultured in nonhypoxic or hypoxic conditions (e.g., DN2 versus pCEP4 in nonhypoxic conditions: 645 pg of VEGF/10 6 cells versus 1591 pg of VEGF/10 6 cells, difference = 946 pg of VEGF/10 6 cells [95% confidence interval {CI} = 640 to 1251 pg of VEGF/10 6 cells; P = .006]; DN2 versus pCEP4 in hypoxic conditions: 785 pg of VEGF/10 6 cells versus 2807 pg of VEGF/10 6 cells, difference = 2022 pg of VEGF/10 6 cells [95% CI = 1871 to 2152 pg of VEGF/10 6 cells; P <.001]). In the subcutaneous tumor model, tumors derived from DN2 or DN3 cells had lower final volumes, weights, and vessel areas, less tumor endothelial cell association with desmin-positive cells, and fewer proliferating tumor cells than tumors derived from pCEP4-transfected cells. In the orthotopic tumor model, tumors derived from DN2 cells had smaller volumes and less vessel area and maturation than tumors derived from pCEP4-transfected cells. Conclusions: Inhibition of HIF-1α activity impairs gastric tumor growth, angiogenesis, and vessel maturation.