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
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.