ABSTRACT
Background: Angiogenesis is important for the typical physiological activities such as cure from injury, menstrual cycle and embryo growth. It is also plays a crucial role in several pathological conditions in cancer. Antiangiogenesis, e.g., inhibition of blood vessel growth, is being investigated as a way to prevent the growth of tumors and other angiogenesis-dependent diseases. The chick embryo chorioallantoic membrane (CAM) is commonly used as an experimental in vivo assay to study both angiogenesis and antiangiogenesis in response to tissues, cells or soluble factors. Given the high occurrence of cancer worldwide and the major source of the discovery of new lead molecules are medicinal plants. The objective of the present research was to study the antiangiogenic property of “aqueous extract of Nigella sativa seeds” using chick chorioallantoic membrane (CAM) assayMethods: The chick chorioallantoic membrane (CAM) assay for screening the effect of Nigella sativa on anti-angiogenesis was performed according to the method given by Ribatti and co-workers.Results: The results of present study significantly increased the antiangiogenic effect on CAM by decreasing the proliferation of capillary networks in a dose (50 to 300 µg/egg) dependent manner which is probably related to the inhibition of neovascularization.Conclusions: It is concluded that aqueous extract of N. sativa seeds possesses significant antiangiogenic activity, and this is a possible rationale for its folkloric use as an anticancer agent.
ABSTRACT
Angiogenesis is controlled by number of growth factors, including vascular endothelial growth factor (VEGF). Plant derived anti-angiogenic molecules acting via VEGF are being investigated for curtailing angiogenesis dependent diseases. In this study, methanolic (CM), n-hexane (CH), ethylacetate (CE) and water (CW) extracts of the roots of Calotropis procera were tested for anti-angiogenic activity. In the chicken egg chorioallantoic membrane (CAM) assay, CM, CH and CE but not CW inhibited VEGFinduced neovascularization in a dose-dependent manner. Of all the tested extracts, CM at the dose of 10, 5 and 2.5 ng most effectively inhibited over 83, 71 and 64%, of neovascularization induced by 10ng of VEGF, respectively. Sponge implantation assay in mice further showed that at the dose of 100ng CM, CH and CE but not CW significantly inhibited neovascularization induced by VEGF (100 ng). Taken together, this study indicates that the root extracts of C.procera may possess anti-angiogenic activity.
ABSTRACT
Objective To optimize the conditions for enhancing the refolding of a novel recombinant human(rh)endostatin and test the biological activities of refolded endostatin.Methods The partial purified inclusion bodies of rh-endostatin were dissolved with 6 mol/L guanidine-HCl followed by combination of dilution and dialysis of the dissolved endostatin.The refolded endostatin was then purified by cation-exchange chromatography.The biological activities of purified rh-endostatin were assessed by endostatin-specific monoclonal antibody and chick embryo chorioallantoic membrane assay.Results A 46% refolding yield was achieved after optimizing the refolding conditions.The purified endostatin reacted with specific anti-endostatin monoclonal antibody and showed significant inhibition of angiogenesis in chick embryo ehorioallantoic membrane assay.Conclusion The method of highest refolding yield of human endostatin was developed.This optimized method significantly promotes the application of this novel human endostatin to preclinical and clinical studies.
ABSTRACT
The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.
Subject(s)
Animals , Cattle , Chick Embryo , Humans , Chorioallantoic Membrane/blood supply , Friend murine leukemia virus , Leukemia, Erythroblastic, Acute/pathology , Neovascularization, Pathologic/etiology , Umbilical Veins/cytology , Cell Proliferation , Endothelial Cells/pathology , Leukemia, Erythroblastic, Acute/metabolism , Tumor Cells, CulturedABSTRACT
Objective To express the recombinant human canstatin protein, and to examine its biological activity. Methods Canstatin cDNA was cut off from the plasmid pUCm-T/canstatin with restriction enzymes BamHⅠ and Hind Ⅲ. The cDNA fragment was then ligated into the correspondence sites of plasmid pET-22b(+) by T4 DNA ligation enzyme and transformed into E.coli BL21 which was induced to express proteins with isopropyl-1-thio-b-dgalactopyranoside (IPTG). The expressed proteins were analyzed by SDS-PAGE and purified through Ni-NTA column affinity chromatography. Chick chorioallantoic membrane (CAM) assay was performed to determine the activity of the recombinant protein. Results Canstatin cDNA from pUCm-T showed one clear objective DNA band with electrophoresis. Seven of positive colonies were selected and identified by restriction enzyme analysis with BamH Ⅰ and Hind Ⅲ. Electrophoresis revealed that all selected colonies had two specific bands,one near the location of primary plamid,the other near that of objective gene fragment. After IPTG induction, there was a new protein band about 24 000 on SDS-PAGE.The induced product over total bacterial proteins in 1,2, 3. and 4 hours after induction was 18. 2%, 18. 8%, 23.0% and 23.4%, respectively, by densitometry examination. CAM assay demonstrated that the recombinant canstatin protein significantly inhibited the embryonic neovascularization in a dose-dependent manner. Conclusion The prokaryotic expression vector of human canstatin gene has been successfully constructed, laying the foundation for further clinical study.
ABSTRACT
The MBT-2 mice bladder cancer tissues and the human bladder cancer tissues were implanted on the chorioallantoic membrance(CAM) of the immune deficient fertilized chicken eggs and the histopathologic changes of the CAM and gross morphologic changes of the implanted cancer tissues on CAM ere studied. The chemosensitivity tests using chicken CAM were performed for the 4 human bladder cancer tissues to mitomycin C, thiotepa and adriamycin. With this study, the following results were obtained: 1. The observation of the blood vessel on the chorioallantoic membrane was possible from the post-incubation 6th day group, but for the implantation of the cancer tissues, the blood vessels from the post-incubation 8th day group was appropriate. 2. The budding oif the host capillary vessel to the implanted cancer tissue were observed from the post-implantation second day. 3. The size of the post-implantation 7th day cancer tissues were varied from 2.3 to 9.2 folds to the size of the implantation day. 4. The total failure rate in experiment within post-operative 3rd day were 71.3 percent and the total failure rates in group who had the damage on the chorioallantoic membrance during operation was 82.5 percent. The failure rate of the experiment was declined acutely after post-operative 4th day. 5. The salvage of the eggs could be maintained until post-operative 7th day in 28.1 percent among chemosensitivity test group. 6. The 4 bladder cencer tissues which had the chemosensitivity test showed 1.6 to 7.1 fold growth to the inital implanted size and this meant resistance to the test drugs and these results were corresponded with clinical course.