Crypotanshinone Induced The Apoptosis Of Endothelial Cells

Angiogenesis is a critical event participated in tumor growth and metastasis. As the basic unit of angiogenesis, vascular endothelial cells are persistent proliferation and movement in the continuous expansion of tumor blood vessels. Nowadays, vascular endothelial cells are the target cells in variety of treatment strategies. Cryptotanshinone (CPT) is one of fat-soluble active ingredients of Salvia Miltiorrhiza Bunge. We found that CPT had the activity of anti-angiogenesis in tumor. This result has been confirmed by several research groups. At present, there are few reports about it. So, we selected CPT to affect on the proliferating HUVEC and the underlying mechanisms.Firstly, we utilized the method of tube formation, angiogenesis of rat aortic ring and transplanted B16F10in C57BL/6J mice. Then, we confirmed that CPT had a high potency in terms of anti-angiogenesis. Next, Cryptotanshinone was the search term in PubMed and the texts data was mined by Cytoscape. It suggested that CPT induced ROS overloading, inhibited the MAPK pathway, and regulated the Bcl-2protein family to inhibited angiogenesis. MTT assay, flow cytometry and Western blot were used to detected the effects of CPT on HUVEC. The results showed that CPT inhibited the proliferation of HUVECs in dose-and time-dependent manner. It mainly related to induce the apoptosis of HUVEC. Furthermore, CPT dcreased the level of mitochondrial cyt-c in dose-dependent manner, on the contrary, the level of cytoplasmic cyt-c is increasing. Then the cytoplasmic cyt-c activated the intrinsic mitochondrial apoptotic pathway. We check the release of Cyt-C caused by CPT at different time point. CPT induced the cyt-c release in time-dependent. It could be detected in6hours with the treatment of CPT. Cyclosporin A, specific inhibitor of mitochondrial penneability transition pore, could not reverse the release of cyt-c by CPT. It indicated that the release of Cyt-C by CPT during6hours was not caused by the opening of mitochondrial permeability transition pore. Elevations in intracellular concentrations of free calcium ions and ROS levels were usually considered as the mechanical damage of mitochondrial outer membrane. During6hours, the release of cyt-c from the mitochondria was not regulated by the mechanical damage of mitochondrial outer membrane. With the treatment of CPT, the levels of Bcl-2and Bcl-xl were lower. Conversely, the levels of Bim and Bax were higher. But more important, Bim expression was significantly changed than others at6-hrs. Then we employed the Bim siRNA to knock down the expression of Bim. It inhibited the release of Cyt-c which was caused by CPT. What’s more, the salience of Bim did not affect the proliferation and tube formation of HUVEC. The Results showed that the release of Cyt-C caused by CPT was via the Bcl-2family-mediated mitochondrial outer membrane permeability. It mainly concerned with the pro-apoptotic protein Bim. Furthermore, Bim physically interacts with Bax at the mitochondrial outer membrane, and then combined with VDAC to form pores which allow the release of cyt-c.In summary, our study demonstrated that CPT up-regulated the expression of Bim and enhanced the interaction between Bim and Bax. Then they can form the pores which triggering the cyt-c release from mitochondria into the cytosol. The cytoplasmic cyt-c activated intrinsic mitochondrial apoptotic pathway. It induced the apoptosis of HUVEC. Finally, CPT inhibited angiogenesis.In adult, most of vascular endothelial cells are in resting. Whether did the CPT affect them? As excluding, we constructed the model of resting endothelial cells to test the effects of CPT on it. We found that CPT was insensitive to it.