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Induction of thrombospondin-1 partially mediates the anti-angiogenic activity of dexrazoxane.

Maloney SL, Sullivan DC, Suchting S, Herbert JM, Rabai EM, Nagy Z, Barker J, Sundar S, Bicknell R - Br. J. Cancer (2009)

Bottom Line: The differentially expressed genes were validated using real-time RT-PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology.The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge.Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Angiogenesis Group, Institute for Biomedical Research, College of Medicine and Dentistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

ABSTRACT

Background: Considerable interest lies in the identification of novel anti-angiogenic compounds for cancer therapy. We have investigated whether dexrazoxane has anti-angiogenic properties and if so, the mechanism of the inhibition.

Methods: The phenotypic effects of dexrazoxane on endothelial cell behaviour was investigated both in vitro using human umbilical vein endothelial cells (HUVECs) in cell proliferation, migration, cell cycle and aortic ring assays; and in vivo using the mouse angiogenesis subcutaneous sponge assay. Custom angiogenesis pathway microarrays were used to identify differentially expressed genes in endothelial cells after treatment with dexrazoxane vs a control. The differentially expressed genes were validated using real-time RT-PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology.

Results: Treatment of endothelial cells with dexrazoxane resulted in a dose-response inhibition of cell growth lasting for up to 5 days after a single dose of the drug. Dexrazoxane was inhibitory in the aortic ring tube forming assay and strongly anti-angiogenic in vivo in the rodent subcutaneous sponge model. The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge. Treatment of microvascular endothelial cells in vitro with subtoxic doses of dexrazoxane stimulated thrombospondin-1 (THBS-1) secretion. Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1.

Conclusion: We show that dexrazoxane administered in small repeated doses is strongly anti-angiogenic and that this activity is mediated by induction of the anti-angiogenic THBS-1 in endothelial cells.

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Upregulation of thrombospondin-1 by dexrazoxane in vitro. (A) Western blotting of HUVEC whole cell lysate and medium after treatment with 50 μ Dexrazoxane for 2, 5, 9 and 24 h displayed an increase in THBS-1 protein expression over the time course. (B) This result was confirmed by ELISA using conditioned medium of cells treated with Dexrazoxane at different time points.
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fig3: Upregulation of thrombospondin-1 by dexrazoxane in vitro. (A) Western blotting of HUVEC whole cell lysate and medium after treatment with 50 μ Dexrazoxane for 2, 5, 9 and 24 h displayed an increase in THBS-1 protein expression over the time course. (B) This result was confirmed by ELISA using conditioned medium of cells treated with Dexrazoxane at different time points.

Mentions: Arrays of genes involved in processes relating to angiogenesis were used to screen for differential gene expression after treatment of endothelial cells with 50 μ dexrazoxane. Of most interest in view of its known anti-angiogenic activity was a 3.7-fold upregulation of THBS-1 expression. To confirm this result, cells were treated for 2, 5, 9 and 24 h with 50 μ dexrazoxane, followed by western blotting of the total cell lysate to measure bound THBS-1 protein and the medium to measure secreted THBS-1 protein (Figure 3A). The THBS-1 level in the cell lysates was found to increase with 50 μ dexrazoxane and continued to increase for up to 24 h after drug treatment. The levels of secreted THBS-1 increased sharply 2 h after treatment and showed a plateau when measured by western blotting. This result was confirmed using a human THBS-1 ELISA (Figure 3B). When measured by ELISA, THBS-1 secretion increases for up to 5 h after dexrazoxane treatment and then remained steady for up to 24 h.


Induction of thrombospondin-1 partially mediates the anti-angiogenic activity of dexrazoxane.

Maloney SL, Sullivan DC, Suchting S, Herbert JM, Rabai EM, Nagy Z, Barker J, Sundar S, Bicknell R - Br. J. Cancer (2009)

Upregulation of thrombospondin-1 by dexrazoxane in vitro. (A) Western blotting of HUVEC whole cell lysate and medium after treatment with 50 μ Dexrazoxane for 2, 5, 9 and 24 h displayed an increase in THBS-1 protein expression over the time course. (B) This result was confirmed by ELISA using conditioned medium of cells treated with Dexrazoxane at different time points.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743367&req=5

fig3: Upregulation of thrombospondin-1 by dexrazoxane in vitro. (A) Western blotting of HUVEC whole cell lysate and medium after treatment with 50 μ Dexrazoxane for 2, 5, 9 and 24 h displayed an increase in THBS-1 protein expression over the time course. (B) This result was confirmed by ELISA using conditioned medium of cells treated with Dexrazoxane at different time points.
Mentions: Arrays of genes involved in processes relating to angiogenesis were used to screen for differential gene expression after treatment of endothelial cells with 50 μ dexrazoxane. Of most interest in view of its known anti-angiogenic activity was a 3.7-fold upregulation of THBS-1 expression. To confirm this result, cells were treated for 2, 5, 9 and 24 h with 50 μ dexrazoxane, followed by western blotting of the total cell lysate to measure bound THBS-1 protein and the medium to measure secreted THBS-1 protein (Figure 3A). The THBS-1 level in the cell lysates was found to increase with 50 μ dexrazoxane and continued to increase for up to 24 h after drug treatment. The levels of secreted THBS-1 increased sharply 2 h after treatment and showed a plateau when measured by western blotting. This result was confirmed using a human THBS-1 ELISA (Figure 3B). When measured by ELISA, THBS-1 secretion increases for up to 5 h after dexrazoxane treatment and then remained steady for up to 24 h.

Bottom Line: The differentially expressed genes were validated using real-time RT-PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology.The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge.Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Angiogenesis Group, Institute for Biomedical Research, College of Medicine and Dentistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

ABSTRACT

Background: Considerable interest lies in the identification of novel anti-angiogenic compounds for cancer therapy. We have investigated whether dexrazoxane has anti-angiogenic properties and if so, the mechanism of the inhibition.

Methods: The phenotypic effects of dexrazoxane on endothelial cell behaviour was investigated both in vitro using human umbilical vein endothelial cells (HUVECs) in cell proliferation, migration, cell cycle and aortic ring assays; and in vivo using the mouse angiogenesis subcutaneous sponge assay. Custom angiogenesis pathway microarrays were used to identify differentially expressed genes in endothelial cells after treatment with dexrazoxane vs a control. The differentially expressed genes were validated using real-time RT-PCR and western blotting; and the functional effect of one induced gene was confirmed using siRNA technology.

Results: Treatment of endothelial cells with dexrazoxane resulted in a dose-response inhibition of cell growth lasting for up to 5 days after a single dose of the drug. Dexrazoxane was inhibitory in the aortic ring tube forming assay and strongly anti-angiogenic in vivo in the rodent subcutaneous sponge model. The anti-angiogenic effect in the sponge was seen after systemic injection into the tail vein as well as after direct injection of dexrazoxane into the sponge. Treatment of microvascular endothelial cells in vitro with subtoxic doses of dexrazoxane stimulated thrombospondin-1 (THBS-1) secretion. Knockdown of THBS-1 with siRNA removed the angiogenesis inhibition effect of dexrazoxane, which is consistent with the anti-angiogenic and vascular normalising properties of the drug being principally mediated by THBS-1.

Conclusion: We show that dexrazoxane administered in small repeated doses is strongly anti-angiogenic and that this activity is mediated by induction of the anti-angiogenic THBS-1 in endothelial cells.

Show MeSH