Study On HMME-PDT Induced Apoptosis Of Canine Breast Tumor Cells And The Caspase-3, Caspase-8 Activities

Canine breast tumor is common disease in veterinary clinics. As humanity's companion animal, dogs share many aspects which the humanity lives. In the pathogenesis,the tumor histology characteristic, the method of treatment and the prognosis extremely is all similar with human breast tumor, therefore studying the canine breast tumors is significance useful for studying the new treatment plan for humanbeings.The traditional treatments of cancer are surgery, chemotherapy and radiotherapy, but it is difficult to obtain ideal efficacy. With the development of new treatments of malignant tumors. Photodynamic therapy is one cytotoxicity treatment which can induce necrosis and apoptosis of cells, it is a product of integration of laser technique, photoconductive, optical information processing technique, biological photochemistry and modern medicine.It is a very promising treatment which can induce necrosis and apoptosis.Photodynamic effect of factors are include of light-sensitive material——photosensitizer,light,matrix and oxygen molecules.The Photosensitizer can be absorbed selectively by target pathological tissue,stimulated by the appropriate wave length light,it can produce active oxygen by the energy transformation,thereby causing damage to the target tissue.Hematoporphyrin monomethyl ether is one new phylloporphyrin photosensitizer, and has been proved that its highly effective in the experiments and the clinical trials.But,there are few researches about the function of HMME-PDT on canine breast tumor cell at home and abroad. Objective: Using the new photosensitizer——hematoporphyrin monomethyl ether,with a wavelength of 632.8nm He-Ne laser irradiation in vitro of canine breast tumor cells,To explore different doses of photosensitizer and laser irradiation of photodynamic effects on canine breast tumor cells.To detect apoptosis rate of canine breast tumor cells after HMME-PDT.To detect cell apoptosis with transmission electron microscope, fluorescence microscope and flow cytometry.To observe and detect the case of apoptosis, and detect the changes of the important participant(calcium,Caspase-3,Caspase-8 and telomerase) in apoptosis, Investigate the mechanism that HMME-PDT induce canine breast cells apoptosis.Methods: The breast tumors CHMm which were isolated and cultured from the metastatic breast tumor clinically,and subcultured to stable cell line.The inhibition rate of cells were detected by MTT method,determine the optimum doses of photosensitizer and laser irradiation of photodynamic effects.Cells were divided according to the illumination after 3h,6h,12h,24h,48h, at the same time set the blank control group.With the optimal dose treat cells, the morphology of apoptosis were observed at light microscope through Giemsa staining, fluorescence microscope through AO/EB and transmission electron microscope; The apoptosis were detceted with DNA LADDER;Apoptosis rate of cells were detected by Annexin V/PI with flow cytometry;The calcium ion within cells concentration([Ca2+]i) were detected with Fura-2/AM;Intracellular Caspase-3 and Caspase-8 changes were detected with spectrophotometric method;Telomerase activity of the cells were detected with Telomere Repeat Amplification Protocol (TRAP) assay.Results: 1.The MTT result indicated that HMME-PDT can significantly inhibit the growth of canine breast tumor cells CHMm. The optimum dose was 20ug/ml for phtosensitizer and 2.8J/cm2 for laser irradiation.2.Giemsa's staining,AO/EB double fluorescence staining and transmission electron microscope observed that HMME-PDT induced apoptosis of canine breast tumor cell line CHMm, the cells showed typical apoptotic morphology, and with the time expand after HMME-PDT, the level of apoptosis was gradually serious.3. Treated with HMME-PDT, cell electrophoresis showed typical DNA ladder bands and color depth with the cells after irradiation was increased. [0]It suggested that with the expand after HMME-PDT, the level of apoptosis was gradually serious.4. Annexin V/PI double fluorescence staining detected apoptosis with flow cytometry, the results showed that after irradiation with the time increasing their apoptosis rate increased gradually, there were significant differences compared with control group.5.In experimental group [Ca2+]i increased gradually,there were significant differences compared with control group.Indicated that Accompanied by deepening levels of apoptosis, [Ca2+]i increased gradually.6. Accompanied by deepening levels of apoptosism, in experimental group, activity of Caspase-3 and Caspase-8 increased gradually, activity of Caspase-8 compare with control group significant differences from 3h group.Caspase-3 compare with control group significant differences from 6h group.7.In experimental group, gel electrophoresis showed that on the ladder 6bp which can demonstrate telomerase activity, analyzed with Image VCD, the ladder average gray values steped down gradually, there were significant differences compared with control group. Indicated that Accompanied by deepening levels of apoptosis telomerase activity steped down gradually.HMME-PDT can induce canine breast cancer cell line CHMm apoptosis,and have time-dependent manner.The mechanisms of HMME-PDT induced cells were related with imbalance of calcium homeostasis mechanism, Caspase cascade mechanism(Caspase-8 in early apoptosis play"Initiator of apoptosis"role and Caspase-3 in late apoptosis play a"executor of apoptosis"role.) and inhibited the telomerase activity.