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

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 thedevelopment of tumor metastasis★ shown to bind to α1β1 integrin and heparan sulphateproteoglycans(HSPG)★ many of the anti-angiogenic properties of arresten are mediated through α1β1 integrin

Methods and ResultsCalf pulmonary aortic endothelial (C-PAE) cellsHT1080 fibrosarcomacells renal cell carcinoma cells (786-0)Human prostate adenocarcinoma cellsHuman tongue squamous cell carcinoma cells (HSC-3)Human umbilical vein endothelial cells HUVEC (ATCC CRL-1730)human primary microvascular endothelial (HMVEC,Lonza) cellsCT26 colon carcinoma cells

Production of recombinant arresten and arrestendeletion mutants

arresten

Arr-1 (the first 115amino acids) Arr-2 (the last 115 amino acids)

Annexin V-FITC assay

C-PAE cells

arre

sten

Arr-1 Arr-2 TNF-α

PB

S

Annexin V-FITC assay

apoptosis

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

whether the apoptosis-inducing property of arresten is endothelial cell specific

HMVEC endothelial cells, HSC-3 oral carcinomacells, PC-3 prostate adenocarcinoma cells, 789-0 renal cellcarcinoma cells, HT1080 fibrosarcoma cells and primary gingival fibroblasts

various concentrations of arresten (350, 700and 1400 nM)

TNF-α

Caspase-3 assay

the pro-apoptotic effect of arresten is endothelial cell specific in vitro.

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

TUNEL assay to study apoptotic activity of arresten in vitro and in vivo

in vitro

arresten or TNF-α

in vivo

C-PAE cells

tumor-bearing mice were sacrificed after 16 days of arresten or PBS treatment and cryosections of the tumors were stained by the TUNEL method

4 fold

apoptotic cells

all cells

in vitro

the inhibition of blood vessel growth by arresten starves the tumor cells and they undergo apoptosis

in vivo

arresten (700 nM) or TNF-α

24h

HMVEC endothelial cells

Western blotting

To understand the pathways involved in the pro-apoptotic activity of arresten

pro-apoptotic molecule

is a pro-apoptotic molecule, whereas bothBcl-2 and Bcl-xL are anti-apoptotic

Bax

Bcl-2 and Bcl-xL anti-apoptotic molecules

shift the balance and favor the pro-apoptotic signaling

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

the T7 peptide---corresponding to the active site of tumstatin---it inhibits angiogenesis via binding to integrin αvβ3

MTT

Arresten inhibited cell survival of only the wild type cells, whereas T7 had an inhibitory effect on both wild type and α1integrin deficient cells

Matrigel plug assay----study the in vivo effect of arresten on the formation of newcapillaries in wild type and α1 integrin deficient mice.

Arresten does not inhibit tumor growth in integrin α1 deficient mice due to the lack of integrin α1 expression in the tumor vasculature

tumor size vascular density

arresten treatment of the α1 integrin

deficient mice had no effect on thevascular

density of tumors

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

antiangiogenic property

pro-apoptosis property

mechanism

Arresten

conclusion

♠significantly induces EC apoptosis by down-regulating the expression of anti-apoptotic Bcl-family signaling molecules.

♠The antiangiogenic property of arresten is located within the last 113 amino acids of this molecule.

♠ arresten binds to α1β1 integrin, and that α1β1 integrin is a functionally necessary receptor for the anti-angiogenic and antitumorigenic nature of arresten in the microvascular ECs in the tumor.