Mechanistic Study On The Inhibition Of Matrix Metalloproteinases By Scutellarein

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells in mammalian tissues. The ECM is composed of 3 major classes of biomolecules. There are structural proteins, specialized proteins and proteoglycans. The ECM can be degraded by some proteolytic enzymes. These enzymes are important factors in cancer invasion and metastasis. And they also play important roles in cancer cell proliferation, cancer escaping from the immune system and tumor angiogenesis. So, ECM degradation enzymes such as matrix metalloproteinases (MMPs) become good targets for anticancer metastasic therapy.MMPs are a family of over 20 zinc-containing endopeptidases which can degrade various components of the ECM. All MMPs are produced as latent, pro-enzymes, which must be proteolytically processed to be activated. Activity of MMPs can be inhibited by the tissue inhibitors of metalloproteinases (TIMP). Excessive avtivity of MMPs is believed to be linked with many pathological processes including metastasis and invasion of tumor. So, MMPs inhibitors are therefore potentially useful for blocking cancer progression. The goal of this project is to find good MMPs inhibitors. To get active MMPs as targets for screening inhibitors, catalytic domains of MMPs were expressed in E.coli as inclusion bodies. The recombinant protein was isolated by affinity chromatography in a nickel column under denature conditions with 8 moler urea. Then purified and refolded MMPs were used in the enzyme activity assays. We also performed the kinetic assays to determine the kinetic paremeters to understand the efficiency of MMP-16 to convert substrate into product.Scutellarein is a compound which screened from the library for tranditional Chinese medicine. There are three hydroxyl groups linked with benzol in scutellarein structure. We detected the inhibitory ability of scutellarein to some MMPs and some members of cathepsin family. We found that scutellarein can inhibit activities of MMPs with IC50 less than 10μM. Next we analyzed the interaction between MMPs and scutellarein by using Autodock system. According to computer mimic result, we know that scutellarein is bind to S1 pocket of MMPs catalytic domain. This is different with others MMPs inhibitors which bind top zinc of MMPs catalytic domain.To understand the effection of scutellarein on MMPs at cell level. We choose HT1080 cells as tumor cell line. Acording to RT-PCR result, scutellarein can inhibit MMP-2, 3, 7, 9, 14, 15 mRNA expressions in dose-dependent and time-dependent manner. And according to Western Blot result, scutellarein can inhibit MMP-14 protein expression in dose-dependent and time-dependent manner. In addition, Zymography result indicates that scutellarein also can inhibit MMP-2, -9 activities in dose-dependent and time-dependent manner. The influence of scutellarein on mobility of HT1080 cells was measured by the closure of a wound in confluent monolayer. 24 hours after wounding, scutellarein induced less closure of the wound compared with control. We used 3D cell culture models to eximine the effect of scutellarein on organization of HT1080 cells. After 48h, HT1080 cells formed the structure shaped of stellats and migrated into the collagen matrix. However, scutellarein treatment greatly reduced the number and the lengh of the sprouts. We seeded the same amount HT1080 cells into six invasion chambers, treated them with increasing concentrations of scutellarein. After 48h, the amount of invaded cells was dramatically decreased.Last, we detected changes of some signal pathway components by different concentrations of scutellarein. We found that with increasing doses of scutellarein, the beta-catenin decreased and E-cadherin level increased. This is very interesting. It means the effect of scutallarein on MMPs expression is involved in Wnt signaling pathway.