Limits...
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.

Show MeSH
Downregulation of THBS-1 by siRNA. (A) Western blot showing the downregulation of THBS-1 in HUVEC at 24 and 48 h posttransfection with THBS-1 targeting siRNA. (B) Real-time PCR showing the downregulation (P-value <0.001) of THBS-1 in HUVEC, targeted by THBS-1 siRNA at 24 and 48 h posttransfection. (C and D) Real-time PCR showing no significant upregulation of the interferon response genes ISG 20 and OAS I in HUVEC targeted with THBS-1 siRNA (duplex 1) at 24 and 48 h posttransfection.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2743367&req=5

fig4: Downregulation of THBS-1 by siRNA. (A) Western blot showing the downregulation of THBS-1 in HUVEC at 24 and 48 h posttransfection with THBS-1 targeting siRNA. (B) Real-time PCR showing the downregulation (P-value <0.001) of THBS-1 in HUVEC, targeted by THBS-1 siRNA at 24 and 48 h posttransfection. (C and D) Real-time PCR showing no significant upregulation of the interferon response genes ISG 20 and OAS I in HUVEC targeted with THBS-1 siRNA (duplex 1) at 24 and 48 h posttransfection.

Mentions: We investigated whether THBS-2, which is also anti-angiogenic, could mediate the THBS-1 insensitive inhibition. Analysis of HUVEC mRNA and protein lysates showed that they do not express THBS-2 (data not shown). To determine the contribution of THBS-1 secretion towards dexrazoxane-mediated anti-angiogenesis, THBS-1-specific siRNAs were designed. Western blotting of HUVEC lysates showed that THBS-1 protein was reduced at 24 and 48 h posttreatment with siRNA compared with controls (Figure 4A). RT–PCR of RNA extracted from HUVEC treated with THBS-1 siRNAs showed downregulation of THBS-1 using two specific siRNA duplexes (data not shown). This was confirmed using real-time RT–PCR and showed a 95% knockdown at 24 h and 82% knockdown at 48 h posttransfection with 50 n siRNA duplex 1 (Figure 4B). This result was sustained on titration down to 12 n siRNA (data not shown). Levels of THBS-1 started to return to baseline both at the RNA and protein level from 72 h posttransfection with siRNA (data not shown). Introduction of siRNA into cells can give artifactual results attributable to induction of INF secretion from the transfected cell (the so-called INF response) (Reynolds et al, 2006). To rule out the possibility of an INF response being generated by siRNA duplexes, the INF response genes ISG20 and OASI were examined by real-time RT–PCR post-siRNA transfection. Duplex 1 at a concentration of 50 nmol l−1 failed to illicit an INF response and so this duplex was taken forward in subsequent experiments (Figure 4C and D).


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)

Downregulation of THBS-1 by siRNA. (A) Western blot showing the downregulation of THBS-1 in HUVEC at 24 and 48 h posttransfection with THBS-1 targeting siRNA. (B) Real-time PCR showing the downregulation (P-value <0.001) of THBS-1 in HUVEC, targeted by THBS-1 siRNA at 24 and 48 h posttransfection. (C and D) Real-time PCR showing no significant upregulation of the interferon response genes ISG 20 and OAS I in HUVEC targeted with THBS-1 siRNA (duplex 1) at 24 and 48 h posttransfection.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Downregulation of THBS-1 by siRNA. (A) Western blot showing the downregulation of THBS-1 in HUVEC at 24 and 48 h posttransfection with THBS-1 targeting siRNA. (B) Real-time PCR showing the downregulation (P-value <0.001) of THBS-1 in HUVEC, targeted by THBS-1 siRNA at 24 and 48 h posttransfection. (C and D) Real-time PCR showing no significant upregulation of the interferon response genes ISG 20 and OAS I in HUVEC targeted with THBS-1 siRNA (duplex 1) at 24 and 48 h posttransfection.
Mentions: We investigated whether THBS-2, which is also anti-angiogenic, could mediate the THBS-1 insensitive inhibition. Analysis of HUVEC mRNA and protein lysates showed that they do not express THBS-2 (data not shown). To determine the contribution of THBS-1 secretion towards dexrazoxane-mediated anti-angiogenesis, THBS-1-specific siRNAs were designed. Western blotting of HUVEC lysates showed that THBS-1 protein was reduced at 24 and 48 h posttreatment with siRNA compared with controls (Figure 4A). RT–PCR of RNA extracted from HUVEC treated with THBS-1 siRNAs showed downregulation of THBS-1 using two specific siRNA duplexes (data not shown). This was confirmed using real-time RT–PCR and showed a 95% knockdown at 24 h and 82% knockdown at 48 h posttransfection with 50 n siRNA duplex 1 (Figure 4B). This result was sustained on titration down to 12 n siRNA (data not shown). Levels of THBS-1 started to return to baseline both at the RNA and protein level from 72 h posttransfection with siRNA (data not shown). Introduction of siRNA into cells can give artifactual results attributable to induction of INF secretion from the transfected cell (the so-called INF response) (Reynolds et al, 2006). To rule out the possibility of an INF response being generated by siRNA duplexes, the INF response genes ISG20 and OASI were examined by real-time RT–PCR post-siRNA transfection. Duplex 1 at a concentration of 50 nmol l−1 failed to illicit an INF response and so this duplex was taken forward in subsequent experiments (Figure 4C and D).

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