Superoxide Anions Confers Therapeutic Activity To Dendritic Cells In A Mouse Model Of Breast Cancer

AIM: Dendritic cell (DC) is known as the most powerful antigen presenting cell which specializes in inducing the proliferation of native T cells and initiating the body's immune activities. It has been paid more attention since finding their significant roles in inducing anti-tumor immune activities. DCs functions are closely associated with their differentiation stage. The mature DCs can presents antigens to antigen-native T cells and induce powerful immune activities, so it is the initiating factor of body immune reactions and plays important roles in inducing anti-infective and anti-tumor immune activities. So far, many factors such as TNFα, compound of CD40-CD40L and LPS have been proved to be able to stimulate DC maturation, and other new agents which can stimulate DC maturation are being seeked.Recent research findings have showed that reactive oxygen species (ROS) and free radicals (FR) are effective in stimulating antigen-presenting cells and have powerful abilities to stimulate body immune activities. As the important member of free radical family NO has been proved to be able to stimulate DC maturation and strengthen DCs immune functions, we put forward a new hypothesis that the other important member of free radical family superoxide anion (O₂) maybe have the same effects on DC maturation and immune functions. In the present studies of O₂-, its effects have been mainly focused on DCs phenotype maturation, and there is no report on whether O₂- can affect DCs immune activities. So, in this study, we investigated the effects of O₂- on phenotype changes and immune activities of DC and the therapeutic activity of antigen sensitive DC to a mouse breast cancer model. We purpose providing experimental and theoreticalfoundations for using O₂' as a new immune enhancement agent in cancer biotherapy and study the effect of O₂- on DCs maturation stimulation and therapeutic activity to the mouse breast cancer model and examine whether antioxidant can be used in tumor treatment with DC at the same time. METHODS: In this study we used the system of Xanthine Oxidase and Xanthine (XaXO) to produce O₂- administrated in the culture medium for stimulating DC maturation. The expression of phenotype molecules closely related to DC maturation and antigen presentation and the change of DCs internalization capacity were detected by fluorescence-activated cell sorting (FACS). The effect of O₂ on DCs capacity of inducing the proliferation of congelleric T cells and its effective dosage were determined by CCK-8 method. DC was pulsed with freezing and thawing lysates of EMT6 tumor cell in vitro, then the antigen specific cytotoxic T lymphocyte (CTL) was induced by the tumor antigen sensetive DC. The tumor antigen specific CTL's performance of killing target cells was detected by MTT assay. To establish a mouse model of breast cancer, the cultured EMT6 cells was injected into animals subcutaneously. The cancer-bearing animals were given deferent therapeutic strategies, then the size of tumor and the animals' life span were examined.RESULTS: (1) We have successfully gained dendritic cells derived from murine bone marrow monocytes in vitro, and the cultured DC has been identified by inverted microscope and FACS observation. (2) The results from FACS observation showed that O2- produced by the XaXO system could promote DCs phenotype and function maturation, with the expression of MHCâ…¡ molecule Iab and costimulate molecule CD40 and CD86 increasing, on the other hand, the capacity of DC of internalizating Dextran-FITC decreased. The effect of O₂- on DC phenotype and function is similar to that of LPS. (3) FACS results showed that O₂- could change the cell cycle of DC, while single cell gel electrophoresis (SCGE) showed that high dose of O₂-caused DC DNA damage indicating harmful to DC by high dose administration of O2*. (4) Results from CCK-8 experiment showed more powerful stimulation on congelleric T cells proliferation by DC compared inducement by O₂- to untreatment. (5) MTT experiment showed that O₂-hastened the kill ability of CTL induced by antigen sensitive DC. (6) We have successfully established the mouse breast cancer model by using the...