Protease Inhibitors Screening And The Study Of Their Biological Activities

Both cysteine protease and MMP are important members of proteinase family. Studies have shown that the disorders of these proteinases activities are associated with many diseases, and the regulation of activities will contribute to the treatment of related diseases. The development of Protease inhibitor plays an important role for the effective regulation of protease activity in abnormal cells, and the regulation of proteinase activity disorders by proteinase inhibitor have become the treatment of related diseases. In another hand, the development of special inhibitors would contribute to the research of protease functionAutophagy is regarded as a cellular self-protective mechanism and keeps cells survival. during nutrient and growth factor deprivation and other stressful conditions. However, apoptosis is a programmed cell death mechanism that occurrence in physiological and pathological conditions. The current studies shown, in many cases, both apoptosis and autophagy were coexistence in several cell death processes notwithstanding the converse mechanism. Based mostly on the growing evidence that there must be a interconnection between autophagy an apoptosis, and this interconnection regulated survival or death of cells. So far, the regulatory mechanism of between autophagy and apoptosis was not clear.According to the reported, cysteine cathepsins (cathepsins) are involved in apoptosis and autophagic cell death. In the apoptosis pathway, cathepsins not only regulate apoptosis by the degradation of Bcl-2 family members, but also promote apoptosis through the activation of the procaspase. For the regulation of autophagy, lysosomal cathepsins play an important role in the ececution autophagy, and the inhibition of cathepsins activities can effectively control the occurrence of autophagy. At present, the interaction between apoptosis and autophagy is a matter of debate in different scientific communities, and the relationship need for more experiments to prove. Since cathepsins play important role in both Physiological and pathological processes, they may act as integrators of the two types of programmed cell death. the regulation of activities will control the development of autophagy and apoptosis and contribute to the treatment of related diseases.In this paper, cathepsin L is chosen as the target for inhibitors screening. We found that Sodium Tanshinone IIA Sulfonate (STS) can inhibit cathepsin L in vitro significantly. The inhibition activities of STS on cathepsin B, C and K are also detected, while the inhibitory effect is moderate. Furthermore, the inhibitory of STS on cathepsin L is time-dependent. Moreover, we also examined the inhibition of STS on caspase-3 and-8 which are cysteine proteinase, and play crucial role in apoptosis.Increasing evidence indicate that both cathepsins and caspases involved in the regulation machinery of cell death. In view of the inhibitory of STS on these cysteine proteinases, we wonder whether STS can regulate autophagy an apoptosis by the inhibition of these cysteine proteinases, especially in apoptosis accompany autophagy. Therefore, serum starvation was used to induce HT1080 cell death. In this model, Lysosomal Membrane Permeabilization (LMP), the enhancement of autophagic activity, the up-regulation of PARP degradation (which is a substrate of caspase-3) and.cell apoptosis were detected. These results proved serum deprivation induced autopahgy, and apoptosis. After treated this model with STS, LMP, the transition of LC3-â… to LC3-â…¡and the degradation of PARP were all reduced and apoptosis was inhibited. Surprisingly, the transition of LC3-â… to LC3-â…¡was reduced at 48 hours compared with 36 hours after serum deprivation. We presumed that cells exerted cytoprotective effect to keep alive at early serum deprivation, however, when excessive autophagy can not against long-time nutritional deficiency, cells executed apoptosis to replace autophagy. STS regulated autophagy to prevent occurrence of excessive autophagy, and delay apoptosis.Because both apoptosis and autophagy are involved in this model, in order to further clarify the function of STS in vivo, we have exmained pan-caspase inhibitor (zVAD-fmk) and broad-spectrum cysteine proteinase inhibitors (E-64 and E-64d) by using this model. The result that zVAD-fmk can not inhibit apoptosis effectively demonstrated the apoptosis induced by serum deprivation is predominate caspase independent apoptosis and can not be regulated effectively by the inhibition of caspase activities. So, we ascertained STS carried out the inhibition of apoptosis by targeting other pathway. Meanwhile, E-64d inhibits autophagy and promotes apoptosis. This means autophagy is a cytoprotective mechanism, and the inhibition of autophagy promotes the development of apoptosis. However, E-64 can not impact the development of apoptosis. We conjectured that may be due to the poor membrane permeability of E-64. These results demonstrate STS regulate other unknown target rather then caspase to inhibit apoptosis induced by serum deprivation. In order to determine whether inhibition of STS on caspase can regulate apoptosis, cisplatin-induced caspase-dependent apoptosis was tested. The results showed that STS could not inhibit caspase-dependent apoptosis, but zVAD-fmk as a positive control effectively inhibited caspase-dependent apoptosis. These results documented that the inhibition of caspase by STS is insufficient to inhibit caspase-dependent apoptosis.It was reported lysosomal membrane permeability (LMP) was shown to be involved in cell autophagy and apoptosis. Therefore, our experiment also examined changes of LMP. After serum deprivation, the increase of LMP was detected, and STS can decrease the level of increased LMP. However, neither cysteine proteinase inhibitors (E-64 and E-64d) nor caspase inhibitor (zVAD-fmk) can inhibit increased LMP. So we can determine the change of LMP is not related to cysteine proteinase activities. Meanwhile, the inhibition of autophagy by E-64d could not decrease LMP, this mean LMP is the upstream of autophagy or there was not relationship between LMP and autophagy. Increasing evidences indicate LMP can regulate caspase-dependent and caspase-independent apoptosis, we presumed STS inhibited the increase of LMP to inhibit caspase-independent apoptosis.In addition, in our experiment, the degradation of poly ADP-ribose polymerase (PARP) was detected to determent the activity of caspase-3. However, we surprisedly detected there are considerable degradation of PARP notwithstanding the apoptosis induced by serum deprivation is a caspase independent apoptosis, and STS could inhibit effectively the degradation of PARP. According to recent researches, many cysteine proteases degraded PARP to different molecular weight. So, we presumed STS inhibit the degradation of PARP by the inhibition of cysteine protease.We also detected the ability of asparagines which inhibit autophagic-lysosomal delivery serum deprivation induced apoptosis and autophagy on autophagy and apoptosis induced by serum deprivation. Asparagine can inhibit LMP and apoptosis, but this result contrary to the effect of E-64d on serum deprivation induced apoptosis. Autophagy is generally considered to be a cellular protective mechanism, so we presumed that large concentration of STS (10mM) could target to other unknown pathway to regulate apoptosis.In summary, the serum deprivation-induced apoptosis is a caspase independent apoptosis with autophagy, and the inhibition of autophagy promotes apoptosis. The inhibition of autophagy by STS is through the regulation of cysteine proteases activities. Meanwhile, we presumed the inhibition of apoptosis is through the regulation of LMP.In addition to the regulation of apoptosis and autophagy, cathepsin is also involved in the development of tumor cells. It is reported that cathepsins promote tumor cell migration and invasion through the degradation of extracellular matrix components and adhesion-related proteins. Yoko Hashimoto and his colleagues demonstrate that approximately 10-fold higher mature cathepsin L activity was secreted in a medium of human fibrosarcoma (HT 1080) cells, compared with their intracellular activity and speculate that the cathepsin L found in HT1080 cell medium is involved in cancer invasion and metastasis. Therefore, we have investigated the inhibitory effect of STS and E-64 on HT1080 cell migration and invasion. The results showed that STS and E-64 could inhibit HT1080 cell migration and invasion effectively.Further more, matrix metalloproteinase (MMP) is chosen as target for inhibitor screening. Traditional Chinese medicine (TCM) has a significant effect in the treatment of cancer, but the mechanism is not clear. We wonder to know whether TCM can inhibit MMP activity to treat cancer. So we screened MMP inhibitor from TCM. Stir-baked Fructus Gardeniae, Polygoni Multiflori Gaulis, and Ramulus Cinnamomi can inhibit MMP activity in vitro and regulate the cell behavior in vivo. Our findings will help to understand the mechanism of these TCM.