| Flavones are a series of compounds that widely distributed in plants, and existed in our daily diet in a large amount. They rolled physiological, pharmacological and health activities into a one, nourished our body silently. They play an essential role from the daily regulation of the body to the prevention of major diseases such as tumors and cardiovascular disease. With further research, flavones have attracted more and more attention in the aspect of disease treatment due to their characteristics of natural, high efficiency and low toxicity.Macrothelypteris torresiana (Gaud.) Ching, widely distributed in the provinces in southwestern China, is a member of genus Mcrothelypteris in family Thelypteridaceae. The plant has the functions of clearing heat and removing toxicity and softening and resolving hard mass. It was often used in traditional Chinese medicine to stop the bleeding and the treatment of edema.In the recent years, several laboratories in the world have found that a particular flavonoid, protoapigenone, which detected in the Macrothelypteris torresiana (Gaud.) Ching have exhibited fine activities both in vivo and in vitro anti-tumor screen. What is more commendable to statement is that it has a wide spectrum of anti-tumor significant cytotoxicity to several tumor cells from different sources. Protoapigenone represent a series of flavonoids with a non aromatic B ring, which can exhibit a good anti-tumor activity in vivo and in vitro screening experiments are found through further researchs. Meanwhile the traditional flavones with similar structure have little anti-tumor effects.Based on the previous studies, used protoapigenone as a model, we adopted chemical synthesis methods to develop the new compounds RY10-4. It was specifically named as2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-pyran-4-one, including a core group:1-hydroxycyclohexa-2,5-dien-4-one. In preliminary in vitro screening, we adopted a liver tumor cells, lung tumor cells, breast tumor cells, prostate tumor cells as target tumor cells, among which breast were most sensitive to RY10-4. Besides the fine anti-tumor activities, no obvious side effects were observed. The results provided the basis for subsequent in-depth study and indicated the direction of further research. Through a follow-up study we found that the compounds can act on multiple pathways of tumor cells, such as the classic PI3K-AKT and MAPK pathways.In the task, taken the characteristics of small side effect of RY10-4into account, anti-angiogenic and anti-cell migration are two promising pathway to evaluate the specific anti-tumor mechanism. In anti-angiogenesis experimental, firstly human umbilical vein endothelial cells (HUVECs), which were essential cells in tube formation, were co-cultured with RY10-4, secondly tube formation inhibition and inhibition of cell motility also accomplished using HUVECs, finally transgenic zebrafish was used as a whole angiogenesis model to assess RY10-4. Through the above model, we evaluate the ability to anti-angiogenic of RY10-4by superficial test, which gave us a direction for further study. In order to explore the specific mechanism and behavior of RY10-4, we simulated hypoxic environment and established an AKT-mTOR-HIF-1-VEGF pathway evaluation system. We evaluated the role of RY10-4in the signal pathway in two human source tumor cells lines MDA-MB-231and MCF-7by immunoblotting. The results shows that the phosphorylation of key protein of the tumor cells in the pathways was inactivated after the tumor cells co-cultured with RY10-4, which may be the cause of inhibition of the pathway.As known that the invasion and migration of tumor cells are closely related to the behavior of angiogenesis around the tumor cells, so the human resource breast tumor cells MDA-MB-231, which were regarded to prone to migrate were used as cell models. Firstly, cell adhesion assay, tumor cell invasion and migration assay by in vitro transwell system were used to imitate the three processes of tumorigenesis: adhering to the adjacent tissue, degradation of extracellular matrix, migration.Then expression and distribution of the main structural support protein for cells: E-cadherin/β-catenin complex and extracellular matrix were detected by both immunofluorescence and immunoblotting. The results showed expression levels and distribution of E-cadherin and its corresponding β-catenin that constitute cytoskeleton in intracellular significantly changed after RY10-4treatment. The outcome exhibited as follows:β-catenin up-regulated in the cytoplasm, while down-regulated in the nucleus. E-cadherin increased in both expression levels and range. MMP-2/9acted as a major extracellular matrix degrading enzyme, their quantity was closely bound up of the amount of extracellular matrix. Therefore, we have adopted the Western blot method to evaluate MMP-2/9and its related proteins in the upstream pathway. The result shows the MAPK may be the other signal pathway RY10-4acts on the tumor cells. MAPK and PI3K-AKT signal pathway contribute to the expression of MMP-2/9, and the activation of the signal pathway can be blocked by reducing the phosphorylation levels of key proteins.As a compound with the prospects for the development, the subject established a HPLC-UV analysis method for RY10-4. Then we adopted SD rats to analysis the preliminary pharmacokinetic in vitro of RY10-4.The results show that whether it is in the blank or the animal tissues, RY10-4can be detected by HPLC-UV using a conventional mobile phase to get a high sensitivity. The tentative results show that there is a positive correlation between the tissue distribution and the amount of blood of RY10-4. The distribution of the compound exhibited only a little differences among the organs with plentiful bloods meanwhile it can sustain a relatively high concentration in the blood in a longer time. This is very beneficial for us to follow-up drug development work. |
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