Bystander Effect By γ-radiation And The Response Of Cells To The Change Of Antioxidases Induced By TBHP

The tumor radiotherapy is a "double blade sword", radioactive ray can wipe-out tumor cells. This is the main purpose of radiotherapy, but irradiated tumor cells can produce free radicals after accepting radiotherapy which severely injures the neighbour surrounding or somatic away from normal tissues and cells. Radiotherapy can not only kill tumor cell instantly, but also damage the nearby normal tissues and cells tardively. Therefore, "cocenric citcle effect" is proposed and defined as the dual roles of free radicals on tumor cell killing and the normal cell injury. Moreover,it is close-related to the mechanism of "bystander effect", and influences the whole progress and effect of radiotherapy.Radiation induced bystander effect(RIBE) refer to the responses detected in unirradiated cells when neighboring cells are irradiated, The responses include altering changes in gene expression, induction of genetic effects such as mutations, DNA damage, cell killing and malignant transformation which appear in cells irradiated. Bystander cell and irradiated-cell can be nearby or no-nearby. The radiation-induced bystander effects has been well established, the underlying mechanisms are still unknown. Some evidences showed that multiple signal transduction pathways were involved in this process, including soluble extracellular factors, oxidative metabolism and gap junction-mediated intercellular communication (GJIC), Oxidize the metabolism is a focal point of the research currently. Hydrogen peroxide (H₂O₂), a capital ROS, is an important effect or side-effect product of radiotherapy. The produced ROS level has a linear correlation with the radiation doses. In part one of the experiment, we mainly focused on the bystander effect caused by high doses of radiation, which caused injury to non-radiation cell. The study showed that high-level of ROS can induce cell apoptosis or death by oxidative damage. With the development of free radical biological research, the current study demonstrated that light level free radicals, especially H₂O₂, can affect a series of signal transduction pathways. Free radicals in cells can regulate the living or death of cells by adjusting its concentration. Apart from leading to cell apoptosis and necrotic, free radical has more vital roles on activing transcription factors and promoting cell proliferation and differentiation. Besides, second primary cancers in human arised from radiation-induced somatic genomic instability after radiotherapy in clinic. Investigators mainly concentrated on the relationships between the bystander effect and Epigenetic changes. However, few studies taked H₂O₂ as a signaling molecular and investigated its bystander effect-related diffusion effect on the organism's biological changes. We hypothesised that H₂O₂ paly an improtant role in causing second primary cancers in postoperation.Our study consisted of experiment one and experiment two. In part one of the experiment, we investigated the injury of the unirradiated cell, caused by RIBE induced by high-level irradiation. The study showed that middle-level and high-level ROS can induced cell apoptosis and death by oxidative stress. So we projected the part two of the study, and initially investigated the low-level H₂O₂ caused by low-level irradiation has the effect of cell proliferation and the relation with internal antioxidase.This study investigated the intervention function of antioxidant in RIBE, demonstrate that antioxidant can relieve RIBE. Moreover, low-level H₂O₂ can promote cell proliferative. This result showed that the mechanism might contribut to development of second cancer at postradiotherapy in clinic. The result has improtant meaning in demonstrating oxidation-metabolic mechanism and directing the guard of radiotherapy in clinic.1,The bystander effects ofγ- irradiated and the intervention of antioxidant The osteogenic sarcoma cells were irradiated by ⁶⁰Coγat the dose of 0,0.25,3,4Gy ⁶⁰Co-γ,then collected cell and cultivated it about 48h with 1×10⁴ density,MTT assay detect cell activity; DCFH-DA assay detect ROS content coused by irradiated. The result indicate that the irradiation at the dose of 0.25 Gy promote cell proliferation, cell proliferation have significant distinction with other groups and ROS content,especially H₂O₂ cantent, obviously increase,compared with negative group(P<0.05).Coculture of ⁶⁰Coγ- irradiated and nonirradiated osteogenic sarcoma U20S cells was observed to the bystander effects and the interfering effects of Trolox.Results: Compared with the control group, cells in the irradiated group exhibited decrease in survival rate (P<0.05), decrease in cell activity (P<0.05), decrease in colony formation efficiency (P<0.05), increase in micronucleus frequency (P<0.05) and increase in MDA content in the culture medium(P<0.05). With the intervention of Trolox, exhibitions of the above mentioned indexes were greatly improved. Radiation of ⁶⁰Coγinduces obvious cell damage and increase in oxidization. Radiation-hit cells have obvious bystander effects on the nonirradiated cells, which is related to the free radicals released from cells caused by radiation-induced oxidative stress. Trolox can restrain the bystander effect as an antioxidant.The result show that U2OS take place obvious oxidative damage after acceptingγ- irradiated. Radiation-hit cells have obvious bystander effects on the nonirradiated cells by coculture of irradiated cells and nonirradiated cell, which is related to the free radicals released from cells caused by radiation-induced oxidative stress. Trolox can restrain the bystander effect as an antioxidant. First, The result further demonstrate that ROS was main active medium contributing to RIBE and antioxidan can restrain the bystander effect. Micronuclear rates are main index responding DNA mutational, the little restrain function of Trolox to Micronuclear rates show that single antioxidan has limit effect.2,low-level H₂O₂ promoted tumor cell proliferation and its relationship with antioxidase.We survey that H₂O₂ influence cell proliferation by adding exogenous H₂O₂ in cultured U2OS and radiating U2OS resuling into secondum endogenous H₂O₂. The result of study shows:1) Exogenous H₂O₂ represented dose-dependent in promoting cell proliferation and apoptosis. Different concentration tBHP were added into the cultured U2OS cell. IC50 was calcuated by MTT assays in order to get a series of proper concentrations that can promote cell proliferation. Then the cells were devided into 4 groups according to different treatment: negative, 1.00μmol·L^-1 tBHP, 10.00μmol·L^-1 tBHP, 40.00μmol·L^-1 tBHP group, 100.00μmol·L^-1 tBHP group. Cell proliferations and apoptosis were observed.Our results showed : H₂O₂ could promote U2OS cell proliferation and apoptosis growth but not apoptosis at the concentrations of 1.00 and 10.00μmol·L^-1 compared to the negative group(P<0.05). And the most obvious proliferation effects were obseved by MTT assay at 4h after the treatment. 40.00 and 100.00μmol·L^-1 tBHP caused cytotoxicity and inhibited cell proliferation. Besides, in 40μmol·L^-1 tBHP group, the rate of non-viable apoptotic cell and the expression of Caspase-3 increased compared with other groups(P<0.05). 100μmol·L^-1 tBHP induced cells death, dead cell ratio increased obviously.2) Exogenous H₂O₂ switched on irritability change of antioxidase. We determined content of MDA and bioactivity of antioxidase. Observe the mRNA and protein-level of Nrf2 and antioxidase,consist of CAT,SOD,GPX with RT-PCR,Western- Blot assay. The result: â‘ Antioxidase activites of cells treated with 1.00 and 10.00μmol·L^-1 tBHP decreased compared with negative group. We hypothesised that the antioxidase activites decreased to cooperate the signal role of H₂O₂ rather than responsively increased to scavenging H₂O₂ as an oxidant, and the function of signal factor insteads of oxidization. MDA content lower than negative group, which demonstrate that low-level H₂O₂ don't cause oxidative stress in cell. The increase of MDA content in 40μmol·L^-1 group indicated that 40μmol?L^-1 H₂O₂ can cause oxidative stress,H₂O₂ might swith on a regulatory mechanism of antioxidase in cell with the change of H₂O₂ cantent.â‘¡The change of antioxidase mRNA and protein-level: the distinction between Cu/Zn-SOD mRNA and GPX1 mRNA is obviously ,moreover, the distinction in protein level is similar with it in mRNA, the protein level is up-regulation in 40,100μmol·L^-1 groups compared with 1.00,10.00μmol·L^-1group(P<0.05).â‘¢Nrf2 was known as a regulatory factory, the result of Western-blot show that the Nrf2 protein level is a little down regulation in 1.00,10.00μmol·L^-1group compared with negative group, and is up-regulation in 40,100μmol·L^-1 groups, protein level is highest in 100μmol·L^-1 group.In conclusion,low-level H₂O₂(1.00,10.00μmol·L^-1)can promote tumor cell proliferation by palying a role of signal factor and can be clean up by antioxygen system. The mRNA and protein level expression of Cu/Zn-SOD and GPX1 were up-regulation and Nrf2 protein level expression was up-regulation that showed a certain consentration H₂O₂ can regulate the expression of these genes by regulating antioxidant enzymes mediated through Nrf2.This study based on cell model and investigate that low-level H₂O₂ have the function of promoting the cell proliferation form two aspect about exogenous and endogenous H₂O₂ by the funcation of signaling molecule,moreover, low-level H₂O₂ don't cause oxidative damage because of being clean-up by antioxidase. Exogenous H₂O₂ may cause oxidative damage to cell when H₂O₂ increase to certain level, and regulate the expression of antioxidase by regulating the increaesing of Nrf2 expression, resulting to activing antioxidase system.The produce of H₂O₂ is inevitable during the process of radiotherapy. Endogenic H₂O₂ can cause a series of biological effect which lead to oxidative damage of tissue and cell,or promote cell proliferation. Which function the H₂O₂ perform that depend on H₂O₂ level. Meanwhile H₂O₂ regulate the expression of antioxidase by the increasing of Nrf2 expression.The result of the research offers a new thinking about studying the occurrence and development of second cancer during postradiotherapy in clinic.