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Anti-angiogenic action of hyperthermia by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro.

Sawaji Y, Sato T, Takeuchi A, Hirata M, Ito A - Br. J. Cancer (2002)

Bottom Line: The gene expression of alternative splicing variants for vascular endothelial growth factor, VEGF121, VEGF165 and VEGF189, was constitutively detected in HT-1080 cells, but the VEGF189 transcript was less abundant than VEGF121 and VEGF165.On the other hand, HT-1080 cell-conditioned medium showed vascular endothelial growth factor-dependent cell proliferative activity and the augmentation of pro-matrix metalloproteinase-1 production in human umbilical vein endothelial cells.The augmentation of endothelial-cell proliferation and pro-matrix metalloproteinase-1 production was poor when human umbilical vein endothelial cells were treated with conditioned medium from heat-shocked HT-1080 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

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HT-1080 cell-derived VEGF stimulates HUVECs to produce proMMP-1. (A) Conditioned medium from HT-1080 cells and the culture medium supplemented with human recombinant VEGF165 (20 ng ml−1) were pretreated with or without an antibody against VEGF (50 μg ml−1) and then HUVECs were treated with these conditioned media for 24 h. The harvested culture medium was subjected to Western blot analysis for proMMP-1 as described in Materials and Methods. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium; lane 3, HUVECs treated with the HT-1080 cell-conditioned medium pretreated with VEGF antibody; lane 4, HUVECs treated with recombinant human VEGF165 and lane 5, HUVECs treated with recombinant human VEGF165 pretreated with VEGF antibody. (B) Confluent HUVECs were treated with or without conditioned medium from untreated or heat-shocked HT-1080 cells. Three independent experiments were reproducible and typical data were shown. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium and lane 3, HUVECs treated with the heat-shocked HT-1080 cell-conditioned medium. The relative amounts of proMMP-1 production were quantified by densitometric scanning and expressed taking the untreated HUVECs as 100.
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fig7: HT-1080 cell-derived VEGF stimulates HUVECs to produce proMMP-1. (A) Conditioned medium from HT-1080 cells and the culture medium supplemented with human recombinant VEGF165 (20 ng ml−1) were pretreated with or without an antibody against VEGF (50 μg ml−1) and then HUVECs were treated with these conditioned media for 24 h. The harvested culture medium was subjected to Western blot analysis for proMMP-1 as described in Materials and Methods. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium; lane 3, HUVECs treated with the HT-1080 cell-conditioned medium pretreated with VEGF antibody; lane 4, HUVECs treated with recombinant human VEGF165 and lane 5, HUVECs treated with recombinant human VEGF165 pretreated with VEGF antibody. (B) Confluent HUVECs were treated with or without conditioned medium from untreated or heat-shocked HT-1080 cells. Three independent experiments were reproducible and typical data were shown. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium and lane 3, HUVECs treated with the heat-shocked HT-1080 cell-conditioned medium. The relative amounts of proMMP-1 production were quantified by densitometric scanning and expressed taking the untreated HUVECs as 100.

Mentions: The augmentation of proteolytic activity is crucial for extracellular matrix (ECM) remodelling in order to provide a permissive environment in which activated endothelial cells can proliferate and form new vessels (Moses, 1997; Liotta et al, 1991). In addition, endothelial MMPs such as MMP-1 and MT1-MMP have been shown to participate in ECM remodelling in the perivascular environment (Fisher et al, 1994; Hiraoka et al, 1998). We therefore investigated the effect of conditioned medium from HT-1080 cells on the production of proMMP-1 in HUVECs. Western blot analysis showed that the production of proMMP-1 in HUVEC was augmented by the conditioned medium from HT-1080 cells (6.2-fold) as well as by recombinant human VEGF165 (4.2-fold) (Figure 7AFigure 7


Anti-angiogenic action of hyperthermia by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro.

Sawaji Y, Sato T, Takeuchi A, Hirata M, Ito A - Br. J. Cancer (2002)

HT-1080 cell-derived VEGF stimulates HUVECs to produce proMMP-1. (A) Conditioned medium from HT-1080 cells and the culture medium supplemented with human recombinant VEGF165 (20 ng ml−1) were pretreated with or without an antibody against VEGF (50 μg ml−1) and then HUVECs were treated with these conditioned media for 24 h. The harvested culture medium was subjected to Western blot analysis for proMMP-1 as described in Materials and Methods. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium; lane 3, HUVECs treated with the HT-1080 cell-conditioned medium pretreated with VEGF antibody; lane 4, HUVECs treated with recombinant human VEGF165 and lane 5, HUVECs treated with recombinant human VEGF165 pretreated with VEGF antibody. (B) Confluent HUVECs were treated with or without conditioned medium from untreated or heat-shocked HT-1080 cells. Three independent experiments were reproducible and typical data were shown. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium and lane 3, HUVECs treated with the heat-shocked HT-1080 cell-conditioned medium. The relative amounts of proMMP-1 production were quantified by densitometric scanning and expressed taking the untreated HUVECs as 100.
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Related In: Results  -  Collection

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fig7: HT-1080 cell-derived VEGF stimulates HUVECs to produce proMMP-1. (A) Conditioned medium from HT-1080 cells and the culture medium supplemented with human recombinant VEGF165 (20 ng ml−1) were pretreated with or without an antibody against VEGF (50 μg ml−1) and then HUVECs were treated with these conditioned media for 24 h. The harvested culture medium was subjected to Western blot analysis for proMMP-1 as described in Materials and Methods. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium; lane 3, HUVECs treated with the HT-1080 cell-conditioned medium pretreated with VEGF antibody; lane 4, HUVECs treated with recombinant human VEGF165 and lane 5, HUVECs treated with recombinant human VEGF165 pretreated with VEGF antibody. (B) Confluent HUVECs were treated with or without conditioned medium from untreated or heat-shocked HT-1080 cells. Three independent experiments were reproducible and typical data were shown. Lane 1, untreated HUVECs; lane 2, HUVECs treated with the HT-1080 cell-conditioned medium and lane 3, HUVECs treated with the heat-shocked HT-1080 cell-conditioned medium. The relative amounts of proMMP-1 production were quantified by densitometric scanning and expressed taking the untreated HUVECs as 100.
Mentions: The augmentation of proteolytic activity is crucial for extracellular matrix (ECM) remodelling in order to provide a permissive environment in which activated endothelial cells can proliferate and form new vessels (Moses, 1997; Liotta et al, 1991). In addition, endothelial MMPs such as MMP-1 and MT1-MMP have been shown to participate in ECM remodelling in the perivascular environment (Fisher et al, 1994; Hiraoka et al, 1998). We therefore investigated the effect of conditioned medium from HT-1080 cells on the production of proMMP-1 in HUVECs. Western blot analysis showed that the production of proMMP-1 in HUVEC was augmented by the conditioned medium from HT-1080 cells (6.2-fold) as well as by recombinant human VEGF165 (4.2-fold) (Figure 7AFigure 7

Bottom Line: The gene expression of alternative splicing variants for vascular endothelial growth factor, VEGF121, VEGF165 and VEGF189, was constitutively detected in HT-1080 cells, but the VEGF189 transcript was less abundant than VEGF121 and VEGF165.On the other hand, HT-1080 cell-conditioned medium showed vascular endothelial growth factor-dependent cell proliferative activity and the augmentation of pro-matrix metalloproteinase-1 production in human umbilical vein endothelial cells.The augmentation of endothelial-cell proliferation and pro-matrix metalloproteinase-1 production was poor when human umbilical vein endothelial cells were treated with conditioned medium from heat-shocked HT-1080 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Show MeSH
Related in: MedlinePlus