| Photodynamic therapy (PDT) is a novel treatment for cancer and certain non-cancerous diseases, which can not only kill tumor cells directly, but also augment the host anti-tumor immune response. Methods for preparing tumor vaccine by photodynamic therapy have been established recently, but the method of preparing PDT-lung cancer vaccine has not been reported. Our present study used the murine lung adenocarcinoma cells (LA795) as a model to investigate the anti-tumor efficiency of vaccine generated by PDT, which may provide a new strategy for lung adenocarcinoma. Specific proteins separated and identified by proteomics which provide a powerful technology platform for the drug target and provide a new thought and method to find specific antigens in the vaccines.Objective1. To investigate the killing effects of DTPP-based photodynamic therapy on LA795cells in vitro, so as to search appropriate parameters of PDT for preparing vaccines.2. To study the mechanism of DTPP mediated photodynamic therapy.3. To analyze the protective effects of vaccine generated by PDT on LA795cells in vivo. To optimize the preparation conditions of PDT cell vaccine, and to study the effects of the vaccine on anti-tumor immune response in mice.4. Through compare of protein expression profiles of LA795cells before and after PDT treatment to search specific proteins associated to the killing effect and immunogenic of PDT-cells and to apply a new way for drugs targets finding and cancer vaccines preparation.Methods1. The uptake of DTPP in LA795cells was observed by fluorescent microscopy and determined by UV visible spectrophotometer. Sub-cellular localization of DTPP in LA795cells was observed by confocal microscopy. Detect of the effect of three elements on the cell killing effect of PDT including DTPP concentration, power density and energy density. Antioxidants Sodium Azide and Mannitol and so on were used to determine the reaction type of PDT. Detect the change of cell membrane and mitochondria after PDT.2. Using microscope observation of photodynamic therapy on the change in the cell morphology, the effect of PDT on apoptosis was observed by Hoechst fluorescent staining and TUNEL analysis, and apoptosis ratio was determined by Annexin V-FITC/PI flow cytometry technology. By studying of the cell cycle, ROS, GSH, NO, Ca2+, cell membrane potential, Caspase-9, Caspase-3, Cytochrome C, the Bc1-2and Bax and other factors to further clarify the mechanism of inhibition LA795cell proliferation.3. Prepare tumor vaccine/cell lysates by PDT, with concentration of photosensitizer, energy density and the number of PDT cells (immune dose) to optimize the PDT cell vaccine preparation conditions. Normal saline (NS) was used as control and vaccine prepared by freezing and thrawing (F/T) was used as positive control,7d after immunization, spleen lymphocyte subgroup was detected by the flow cytometry technology and the levels of IL-1and IFN-γ were detected by ELISA. The mouse was killed when tumor grew for20days and pathological changes of tumor were observed by HE staining.4. The protein of LA795cells before and2h after PDT treatment were extracted and separated by two-dimensional electrophoresis. MALDI-TOF-MS was used to compare protein expression profiles of cells before/after PDT to find specific proteins with3times difference expression. Meanwhile, the functions of these proteins and the interaction relationship were analysised by bioinformatics method to find specific proteins.Results1. The absorption plateau of DTPP was18-24h. Incubating time of24h and concentration of10μg/mL was selected as optimal parameters for PDT. DTPP located in the mitochondria. MTT test showed that the cell viability rate of cells increased with the increase of energy density and photosensitizer concentration. When using10μg/mL as DTPP concentrations, the cell survival rates decreased from63.61%for2.4J/cm2to10.79%for7.2J/cm2illumination dose. Reaction of Type I exists with but with the greater proportion than Type II after PDT. PDT damage cell membranes and mitochondria.2. Typical apoptosis morphological changes of LA795cells were observed24h after PDT by Hoechst fluorescent and TUNEL staining analysis. When the energy density is greater than2.4J/cm2, cells death was mainly in apoptosis pathway with the cell apoptosis rates increase from24.9%for2.4J/cm2to49.45%for7.2J/cm2illumination dose. However PDT promoted the cell from G1-phase into S-phase and so boosted cell proliferation. The mechanism of DTPP-PDT in lung cancer was that:mitochondrial cytochrome C is released firstly into the cytoplasm, then Caspases-9/Caspases-3was activated, Bcl-2decreased/Bax increases, thus initiated the cell apoptosis in mitochondrial pathway.3. Based on these results of tumor growth curves in PDT vaccine prophylactic treatments experiment for tumor-burdened, the optimal protocol for PDT vaccine preparation was found to be LA795cells exposed to10μg/mL DTPP for24h, then illuminated at7.2J/cm2and2×107PDT cell lysates injected per mouse. PDT vaccine can improve the immune function of mouse, reverse T cell subsets CD4+/CD8+ratio (1.72-1.96folds of control) in spleen lymphocyte, increase NK cell ratios (1.71-2.98folds of control), meanwhile enhance the levels of IL-1and IFN-γ in plasma. Histopathology showed tumor immunology as the PDT vaccine induced marked lymphocyte aggregation at the edge of tumor.4. Thirty one proteins were identified successfully, including six cytoskeleton proteins, five enzymes, three transfer/regulatory proteins, four apoptosis related proteins and functions of eleven proteins were unknown. Biology behavior of known proteins were divided into7categories, including oxidative stress, immune process, protein metabolism, signal transduction, cell movement, cell morphology and protein modification process.ConclusionDTPP with low toxicity and high effect located in the mitochondria, which mediated photodynamic therapy can kill LA795cells effectively. Proportion of Reaction Type I is greater than Type II and PDT could damage cell membranes and mitochondria. The apoptosis induces by DTPP based-PDT mainly in the mitochondria-involved pathway. Tumor cell vaccine generated by PDT in LA795cells can effectively enhance the anti-tumor immune response, retard the tumor growth in tumor-bear mice and has significant effects on prophylactic treatments for tumors. Proteomics study shows the most of different proteins treated by PDT are structural protein, enzyme apoptosis related proteins and functions of eleven unknown proteins need further study.Such differential proteins may be related to PDT regulation anti-tumor immune response in mice. |
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