Imitation Of Synergy Between GPX And SOD And Anti-tumor Effect Of The Novel Selenium-containing Peptide

Reactive oxygen species (ROS), mainly superoxide anions (O₂^·-), hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH), are produced as by-products of mitochondrial respiration in living organisms. Under normal conditions, there concentrations are determined by the balance between their rates of production and their rates of clearance by various antioxidant compounds and enzymes. In certain pathogenic states, the production of ROS is enhanced and excess ROS oxidize lipids, protein, DNA, and carbohydrates, and therefore results in the breakdown of normal cellular function and induces various ROS-mediated diseases. ROS-related diseases include reperfusion injury, Keshan disease, Parkinson's disease, Alzheimer's disease, cataract and cancer. The enzymatic antioxidant system consists of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT).SOD, a kind of the important antioxidant enzyme in living organisms, dismutates superoxide anions to H₂O₂, and therefore prevents normal cells from injuries by superoxide anions. SOD has strong antioxidant ability and it is important for preventing inflammation and cancer, and age-related diseases. GPX is the well-known antioxidant selenoenzyme in organisms, which scavenges harmful peroxide and H₂O₂ and maintain the metabolic balance of ROS, thus protecting the biomembrances and other cellular components from oxidative damage.However, antioxidant enzymes must cooperate with each other to effectively scavenge reactive oxygen species because anyone of them can not scavenge all forms of ROS alone. Moreover, excessive H₂O₂ produced by SOD in the absence of GPX or CAT produces more toxic hydroxyl radical through Fenton reaction and destroys living organisms. Thereby, it is necessary that SOD cooperates with GPX. The available information from molecular biology and genetics indicates that many diseases relate to ROS and activities of antioxidant enzymes. Due to their pivotal role in scavenging ROS, antioxidant enzymes could act as promising antioxidant drug. However, native enzymes have some drawbacks such as solution instability, limited cellular accessibility and cost of production. These factors limit the pharmacological use of the naturally occurring enzymes. So many scientists have made a great deal of efforts to study small antioxidant enzymes mimics to scavenge ROS.On the other hand, selenium component not only composes GPX, but also is a kind of effective anticancer agent. Selenium compounds, such as selenite, Se-methylselenocysteine and ebselen, have been shown their antitumor activities, and ebselen is also a GPX mimic. Thereby, it is significant to study the effect of GPX mimics in tumor cells and the relationship between their antitumor and antioxidant activities.1. Selenium-containing peptide, Sec-RGD, mimics GPXIn selenium compounds with antitumor activities, due to poor solution instability and great cytotoxicity, selenite, Se-methylselenocysteine and ebselen are hard to apply for antitumor therapy directly. Selenocysteine (Sec), in possession of less toxicity as the major biological form of selenium in human, but poor solubility and short half-lives similarly limit its utilization. Due to theα_vβ₃ integrin is highly expressed on many tumor cells, but not strongly expressed on normal cells, and peptides with an Arg-Gly-Asp (RGD) sequence have shown to have a high affinity forα_vβ₃, we synthesize Sec-RGD by Fmoc chemistry, and DGR-Sec-Sec-RGD (Sec-RGD peptide oxide form) is the final product, which is named as Se8P. In the steady state kinetics of the Se8P, a family of parallel lines is given, consistent with a ping-pong mechanism of the native GPX. Our results show that Se8P is a GPX mimics. Moreover, we show that this novel peptide is able to prevent injury induced by H₂O₂ in African green monkey kidney cell line Vero cells.2. Study of the antitumor activity of Se8P and its action mechanismIn our study, Se8P can decrease cell viabilities and increase leakages of LDH, therefore kill human melanoma A375 cells. The morphological changes and DNA fragmentation suggested that the death of the A375 cells was resulted from the cells apoptosis. Most of antitumor drugs can produce ROS and induce tumor cells apoptosis. In order to clarify the cellular mechanism of Se8P-induced apoptosis, we examined the intracellular ROS levels in A375 cells by fluorescence microscope and flow cytometry. The change of intracellular ROS levels was as follows: rapid increase at the beginning and gradual decrease later. Moreover, mitochondrial membrane is destroyed due to gradual decrease of the potential (△Ψm), and would release cytochrome C and deliver other signals of apoptosis. In addition, intracellular GSH, as a direct antioxidant and GPX substrate protects against Se8P-induced ROS in A375 cells. Our results suggest that Se8P could be a candidate of cancer-treating medicine. 3. Construction of selenopeptide with GPX/SOD activitiesIn living organism, it is important that SOD, GPX and CAT cooperate with each other to scavenge reactive oxygen species. Previously, Prof Luo'group constructed a GPX mimic-15SeP and a SOD mimic-17CuP. On the basis of their sequences, we synthesize a bifunctional precursor 32P, and then obtain the target product by the methods of selenation and chelation with copper (named as Se-Cu-32P). The analysis of selenium and copper content shows the existence of a diselenium bridge and only one copper atom in per peptide molecule. The GPX activity of Se-Cu-32P is found to be 146 units/μmol, and the SOD activity to be 164 units/mmol. These results indicated that Se-Cu-32P is a novel bifunctional enzyme. But the GPX and SOD activities of Se-Cu-32P have some decrease compared to their original peptides duo to their disturbance each other in structure.We construct two damage systems (H₂O₂ and XOD/Xanthine/ Fe²⁺) to evaluate the biological effects of Se-Cu-32P in cell level. We show that this novel bifunctional selenopeptide is able to prevent injury induced by H₂O₂ and XOD/Xanthine/ Fe²⁺ in African green monkey kidney cell line Vero cells. Moreover, in XOD/Xanthine/ Fe²⁺ system, native SOD not only has not protective effect but also damages vero cells even more because of the accumulation of H₂O₂. These prove the importance of cooperation of antioxidant enzymes. Therefore, this bifunctional enzyme is a kind of promising antioxidant drug.