ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the anti-angiogenic activity of peptide 21 obtained by modification of tumstatin, and its inhibitory effect on the growth and metastasis of human ovarian cancer transplanted in nude mice.</p><p><b>METHODS</b>The peptide 21 was purified by affinity chromatography. Human ovarian cancer SKOV3 cells were inoculated in nude mice and the transplanted tumor was treated with the peptide 21 to observe the tumor growth and metastasis. The microvessel density (MVD) and immunohistochemical staining index of PCNA, VEGF and MMP-2 and TIMP-2 were performed to assess the inhibitory effect of the peptide 21.</p><p><b>RESULTS</b>In the nude mice at 21 days after peptide 21 treatment, the inhibition rate of tumor growth was 53.17%, the tumor microvessel density was significantly reduced (P <0.05), the expression of PCNA, VEGF and MMP-2 were significantly lower (P <0.01), and TIMP-2 expression was significantly higher (P <0.01) in comparison with that of control group.</p><p><b>CONCLUSION</b>The peptide 21 generated in this study has a significant anti-angiogenetic activity, showing significant inhibitory effect on the growth of human ovarian cancer transplanted in nude mice. The mechanism of its inhibitory action on ovarian cancer growth may be mediated by reduction of neovascularization and reduction of expression of angiogenetic factors.</p>
Subject(s)
Animals , Female , Humans , Mice , Angiogenesis Inhibitors , Chemistry , Pharmacology , Antigens, CD34 , Metabolism , Antineoplastic Agents , Chemistry , Pharmacology , Autoantigens , Chemistry , Pharmacology , Cell Line, Tumor , Collagen Type IV , Chemistry , Pharmacology , Matrix Metalloproteinase 2 , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic , Pathology , Ovarian Neoplasms , Metabolism , Pathology , Peptides , Chemistry , Pharmacology , Proliferating Cell Nuclear Antigen , Metabolism , Recombinant Proteins , Chemistry , Pharmacology , Tissue Inhibitor of Metalloproteinase-2 , Metabolism , Tumor Burden , Vascular Endothelial Growth Factor A , Metabolism , Xenograft Model Antitumor AssaysABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>
Subject(s)
Animals , Humans , Mice , Angiogenesis Inhibitors , Pharmacology , Apoptosis , Autoantigens , Chemistry , Genetics , Cell Line , Cell Line, Tumor , Cell Proliferation , Collagen Type IV , Chemistry , Genetics , Dose-Response Relationship, Drug , Endothelial Cells , Cell Biology , Flow Cytometry , Mice, Inbred BALB C , Mice, Nude , Microscopy, Electron, Transmission , NIH 3T3 Cells , Neoplasms, Experimental , Pathology , Neovascularization, Pathologic , Pathology , Peptides , Chemistry , Genetics , Pharmacology , Recombinant Proteins , Chemistry , Pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.