Effects of cloned tumstatin-related and angiogenesis-inhibitory peptides on proliferation and apoptosis of endothelial cells.
BACKGROUND
Tumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide (peptide 21), to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity: specifically inhibiting tumor angiogenesis like tumstatin.


METHODS
Peptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy (TEM) and flow cytometry (FCM) were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density (MVD). To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors (VEGFs) by tumor tissue was semi-quantitatively analyzed.


RESULTS
The in vitro MTT test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner (P < 0.01); TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis (P < 0.01). Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group (P < 0.05), with a mean tumor inhibition rate of 67.86%; MVD of the tumor tissue in the peptide 21 group was significantly lower than in the control group (P < 0.05); the number of cells positive for VEGF in the peptide 21 group was significantly fewer than in the control group (P < 0.01).


CONCLUSIONS
Similar to the activity of tumstatin in specifically inhibiting tumor angiogenesis, peptide 21 may specifically inhibit tumor endothelial cell proliferation and induce their apoptosis, thereby suppressing tumor angiogenesis and indirectly inhibit the growth, infiltration and metastasis of tumors. Peptide 21 may exert its effect through down-regulating the VEGF expression of tumor cells and vascular endothelial cells.
Zhang
Guang-mei
GM
Department of Obstetrics & Gynaecology, First Clinical College, Harbin Medical University, Harbin, Heilongjiang 150001, China.
Zhang
Ying-mei
YM
Fu
Song-bin
SB
Liu
Xing-han
XH
Fu
Xue
X
Yu
Yan
Y
Zhang
Zhi-yi
ZY
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D024141:Collagen Type IV / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046529:Microscopy, Electron, Transmission; D041681:NIH 3T3 Cells; D009374:Neoplasms, Experimental / Q000098:blood supply / Q000473:pathology / Q000517:prevention & control; D009389:Neovascularization, Pathologic / Q000473:pathology / Q000517:prevention & control; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
D020533:Angiogenesis Inhibitors; D001324:Autoantigens; D024141:Collagen Type IV; D010455:Peptides; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain