Studies On Artificial Imitation Of Glutathione Peroxidases And Their Antitumor Activities

Selenium is an essential trace element in human body. It plays an important role in the human's metabolism. The deficiency and excessiveness of selenium will lead to the incidence of various diseases. The existent forms of selenium in human are selenoprotein, selenopolysaccharide and selenonucleic acid. Selenium displays its physiological function by means of selenoenzyme. Glutathione peroxidase is one of important selenoenzyme in human body, and plays an important role in the antioxidative defense system of organisms. It can catalyze the reduction of various peroxides by glutathione. It can block the ROS second order reactions induced by lipid peroxides and decrease the damage of body induced by peroxides. It can protect cell membrane and hold out the normal physiological function of cell. Selenopolysaccharide is a perfect selenium supplement and possesses the functions of antioxidation, improving the organism immunity and removal of harmful heavy metal, and so on. The contents of the two organic selenium compounds are very low in nature and affect their application in medical treatment and health protection. A large number of stable mimics could be obtained by chemical modification. Therefore, it becomes the study focus for researchers to mimic natural enzyme by chemical modification.GPX mimics have been studied for a long time, because the early studies only considered mimicking the active group of GPX and did not consider the effect of interaction between mimics and substrate, the activities of mimics were generally rather low. For example, the activity of Ebselen is only 0.99U/μmol. With the development of research, the importance of interaction between mimics and substrate is attracting more and more attention from researchers. A series of GPX mimics with GSH binding site were produced now. Because the poor conformation blocks the approach of GSH to the active site and and reduces the specifically binding ability, the mimics have low activities. Therefore, it become an ideal strategy to obtain the mimics which has the similar activities with natural GPX using chemical mutation of the Ser to Sec in the active site of protein scaffold which has GSH binding site. By this way, we have successful obtained a new mimic which activities are same order of magnitude as natural GPX.The early research of selenopolysaccharide focused on that it is a perfect selenium supplement, but with the development of research, it was found that selenopolysaccharide has synergism between selenium and polysaccharide. Specially significant is its antitumor activity. Due to the lower content in nature, people often use the method enrichment of selenium in plant to obtain a large of selenopolysaccharide. However, the method has long cycle and output instability, a number of resources were wasted. A large of mimics can be obtained by chemical methods. Therefore, the chemical modification becomes an ideal method for obtaining more selenopolysaccharide. After -OH of the polysaccharide were mutated to -SeH, a large of selenopolysaccharide will be harvested.In studying bioactivities of the two organic selenium compounds, we found that they all have antitumor functions. So we studied the antitumor activity of 2-SeCD and selenopolysaccharide in our lab, and further explore their mechanism in the course of antitumor. The data will provide valuable information for its application in clinical medicine.1. Chemical modification of glutathione transferase to imitate GPXThe key to construct mimics with high GPX activities is that the mimic must have GSH binding site and the Sec in the active site must be close to GSH. This means the microenvironment of the active center of mimic should be similar with that of the natural enzymes. According to this principle, we chose hGST Z1-1 as protein scaffold of chemical modification. The GSH binding site of hGST Z1-1 is similar with that of the natural GPX. The active site of hGSTZ1-1 is located in a deep crevice between the N- and C- terminal domains, nestling into a hydrophobic pocket. The characteristic motif of hGSTZ1-1, SSC (Ser14, Ser15, Cys16), locates in the N-terminal region and lines the port of the hydrophobic pocket. The location of both Ser is benefited to change to Sec by chemical mutation and is easy to approach GSH. Without the steric hindrance effect of structure, both the mutated Sec can easy to approach and bind GSH. We have successfully harvested Se-hGSTZ1-1 with high GPX activity by chemical modification, its activity is 8602.3U/μmol and is higher than that of some native GPX. And its activity is also higher than (1.5 fold) that of rabbit liver GPX. Catalytic kinetic analysis of Se-hGSTZ1-1 shows that the double reciprocal plots of the initial velocity versus the concentration of substrates were a family of parallel lines, consistent with a Ping-Pong mechanism similar with that of native GPX.2. Study of the antitumor activity of 2-SeCD and its action mechanism2-selenium-bridgedβ-cyclodextrin (2-SeCD) is a glutathione peroxidase mimic synthesized in our lab previously. It possesses potent antioxidant ability by catalyzing the reduction of hydroperoxides with glutathione and its GPX activity is 7.4 U/μmol that is 7.6 folds of that of Ebselen. In the study of its biological activities, we found low dose of 2-SeCD could inhibit the UV-B induced cell injury and inhibit cell apoptosis, but the effects of high dose of 2-SeCD to cell has not been reported. Here, we have studied the effects of high and low dose 2-SeCD on human cervical cancer cells (HeLa cells) death and proliferation. We found low dose of 2-SeCD could inhibit HeLa cell apoptosis. The antiapoptotic mechanism may be its antioxidant ability and it can protect cells against oxidative damage. High dose of 2-SeCD can induce HeLa cells apoptosis. The apoptotic mechanism may be that it can largely and rapidly deplete GSH, activate caspase-3 and induce the HeLa cell apoptosis, directly. To investigate the reason for decrease of the intracellular GSH, we studied the other biological activity of 2-SeCD, we found it has potential dehydroascorbic acid reductase- and thioltransferase-like activities. In the course of catalysis, 2-SeCD is reduced by GSH, generated CD-SeH and GSSG in the preincubation process and CD-SeH is involved in main catalytic reaction. In a whole catalytic cycle, 1 mole of 2-SeCD can deplete 4 moles of GSH. When the dose of 2-SeCD is very high, the consumption of GSH increases, the content of GSH cannot meet the needs for tumor cell to grow, leading to the change of redox state in tumor cell, activating caspase-3 and inducing the HeLa cell apoptosis. The results show the dual effect of 2-SeCD on HeLa cells growth, which has great potential in the tumor treatment.3. Preparation of selenoproteoglycan and its antitumor activitySelenopolysaccharide has the biological activity of both selenium and polysaccharide, simultaneously. The biological activities of selenopolysaccharides are better than polysaccharides. For example, it can enhance the immunity and antitumor. In nature, the content of selenopolysaccharide is very low and is far from meeting the demands in human medical treatment and health protection. Therefore, it becomes the focus for researchers to artificially synthesize selenopolysaccharide mimic. We gained a selenoproteoglycan (Se-GTP) with GPX activity by converting the–OH of Ganoderma tsugae. proteoglycan (GTP) to–SeH. The chemical modification method was used to study the GPX mimics in our lab. We also studied the antitumor activities of GTP and Se-GTP. Antitumor experiment in vivo showed that the average tumor inhibition percentage is 74.83%. The study of immunocompetence showed the antitumor activity of GTP is correlative with its immunocompetence and related with many factors. Antitumor experiment of GTP and Se-GTP in vivo showed the ability for GTP to kill tumor is very low, but the ability for Se-GTP to kill tumor is better than GTP. The cell death percentages are 86.68% and 15.79%, respectively, when the concentration of Se-GTP and GTP is at 500μg/ml.Se-GTP can induce the increase of Bax expression and decrease of Bcl-2 expression, the balance of Bcl-2/Bax is destroyed and induced cell death. Data show that the mechanism of Se-GTP killing tumor cell relates the direct reduction of cell apoptosis. The immunologic activity research indicated that Se-GTP retained the GTP original immunity function, enhanced the NK cell activity and promoted spleen lymphocyte multiplication. These data show that Se-GTP both retain the GTP biology function and increase the new activity, it has excellent potential as an antitumor medicine.