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Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell
Wei-Hong Hou1, Tian-Yun Wang, Bao-Mei Yuan
1Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China.
Abstract:
Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.
Insights
Recombinant mouse canstatin effectively inhibits angiogenesis and endothelial cell proliferation. This study demonstrates its potential as an anti-cancer agent by suppressing tumor blood vessel formation and growth.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Canstatin, a fragment of type IV collagen, is a known inhibitor of angiogenesis and tumor growth.
- Investigating recombinant mouse canstatin's anti-angiogenic properties is crucial for developing novel cancer therapies.
Purpose of the Study:
- To assess the in vivo anti-angiogenesis activity of recombinant mouse canstatin.
- To evaluate the in vitro effects of recombinant mouse canstatin on endothelial cell proliferation.
Main Methods:
- Constructed a prokaryotic expression vector (pQE-mCan) for mouse canstatin.
- Expressed and purified recombinant mouse canstatin from E. coli.
- Assessed anti-angiogenesis using chicken embryo chorioallantoic membranes (CAM) assay.
- Determined endothelial cell proliferation inhibition in vitro.
Main Results:
- Recombinant mouse canstatin was successfully expressed and purified to approximately 93% purity.
- Significant regression of newly formed blood vessels was observed in chicken embryos treated with mouse canstatin.
- Mouse canstatin potently inhibited endothelial cell proliferation in a dose-dependent manner without affecting non-endothelial cells.
Conclusions:
- Recombinant mouse canstatin exhibits significant in vivo anti-angiogenesis activity.
- Mouse canstatin effectively suppresses in vitro proliferation of human umbilical vein endothelial cells.
- These findings support the potential of recombinant mouse canstatin as an anti-cancer therapeutic agent.
