E xperimental Cell Research 3 1 4 ( 2 0 0 8 ) 3 2 9 2 – 3 3 0 5

1. Characterization of the anti-angiogenic properties of arresten,an α1β1 integrin-dependent collagen-derived tumor suppressor E xperimental Cell Research 3 1 4 ( 2 0 0 8 ) 3 2 9 2 – 3 3 0 5 陈洪栋 2011.9.14

2. Angiogenesis, the formation of newblood vessels, is a critical event in tumor growth and metastasis Arresten ★a 26-kDa anti-angiogenic fragment from the α1chain of type IV collagen. ★ inhibits endothelial cell proliferation, migration, tube formation and Matrigel neovascularization ★ inhibits the growth of human tumors in nude mice and the development of tumor metastasis ★ shown to bind to α1β1 integrin and heparan sulphate proteoglycans(HSPG) ★ many of the anti-angiogenic properties of arresten are mediated through α1β1 integrin

3. Methods and Results Calf pulmonary aortic endothelial (C-PAE) cells HT1080 fibrosarcomacells renal cell carcinoma cells (786-0) Human prostate adenocarcinoma cells Human tongue squamous cell carcinoma cells (HSC-3) Human umbilical vein endothelial cells HUVEC (ATCC CRL-1730) human primary microvascular endothelial (HMVEC,Lonza) cells CT26 colon carcinoma cells

4. Production of recombinant arresten and arrestendeletion mutants arresten Arr-1 (the first 115amino acids) Arr-2 (the last 115 amino acids)

5. Arr-1 Arr-2 TNF-α arresten PBS C-PAEcells Annexin V-FITC assay apoptosis Annexin V-FITC assay

6. increase the amount ofapoptotic cells 2.4-fold in 2 h, and 3.9-fold in 4 h

7. TNF-α various concentrations of arresten (350, 700and 1400 nM) HMVEC endothelial cells, HSC-3 oral carcinomacells, PC-3 prostate adenocarcinoma cells, 789-0 renal cellcarcinoma cells, HT1080 fibrosarcoma cells and primary gingivalfibroblasts whether the apoptosis-inducingproperty of arresten is endothelial cell specific Caspase-3 assay

9. the pro-apoptotic effect ofarresten is endothelial cell specific in vitro.

10. the active anti-angiogenic site is localized within thelast 113 amino acids of the arresten molecule.

11. TUNEL assay to study apoptotic activity of arresten in vitroand in vivo in vitro arresten or TNF-α C-PAE cells in vivo tumor-bearing mice weresacrificed after 16 days of arresten or PBS treatment and cryosectionsof the tumors were stained by the TUNEL method

12. apoptotic cells in vitro 4 fold all cells

13. in vivo theinhibition of blood vessel growth by arresten starves the tumor cellsand they undergo apoptosis

14. arresten (700 nM) or TNF-α 24h HMVEC endothelial cells Western blotting To understand the pathways involved in the pro-apoptotic activityof arresten is a pro-apoptotic molecule, whereas both Bcl-2 and Bcl-xL are anti-apoptotic Bax pro-apoptotic molecule Bcl-2 and Bcl-xL anti-apoptotic molecules

15. shift the balance and favor the pro-apoptoticsignaling

16. microvascular lung endothelial cells (MLEC) were isolated cells from both wild type and α1 integrin deficient mice

17. the T7peptide---corresponding to the active site oftumstatin---it inhibits angiogenesis via binding to integrin αvβ3 MTT Arresten inhibited cell survival of only the wild typecells, whereas T7 had an inhibitory effect on both wild type and α1integrin deficient cells

18. Matrigel plug assay----study the in vivo effect of arresten on the formation of newcapillaries in wild type and α1integrin deficient mice.

19. Arresten does not inhibit tumor growth in integrin α1deficient mice due to the lack of integrin α1 expression in thetumor vasculature arresten treatment ofthe α1 integrin deficient mice had no effect on thevascular density of tumors tumor size vascular density

20. Double staining of integrin α1 (green) and CD31 (red) of CT26 colon carcinoma tumors implanted on wild typeand integrin α1 deficient Balb/c mice

21. pro-apoptosis property antiangiogenicproperty mechanism Arresten

22. conclusion ♠significantly induces EC apoptosis by down-regulating the expressionof anti-apoptotic Bcl-family signaling molecules. ♠The antiangiogenicproperty of arresten is located within the last 113 aminoacids of this molecule. ♠arresten binds to α1β1 integrin, and that α1β1 integrin is afunctionally necessary receptor for the anti-angiogenic andantitumorigenicnature of arresten in the microvascular ECs in thetumor.