Study On Mitochondrial Damage Of Canine Breast Cancer Cell Induced By HMME-PDT

Photodynamic therapy (PDT) was a new treatment for cancer patients, owing to combine the medical application of laser, light guiding and biological photochemical technologies, as well as the modern optical medicine. There a great advantage compared with conventional surgery, radiotherapy and chemotherapy treatment. PDT had been study in human health research and clinical trials on various antitumor. PDT was still in the basic theory research and exploratory stage on canine breast cancer. Canine breast cancer is a common neoplastic disease in clinical veterinary. Canine breast cancer has many likenesses with human breast cancer, as an excellent model for human breast cancer. CHMm cells apoptosis induced by HMME-PDT. Therefore, many issues would to be further study in the apoptosis induced by HMME-PDT. Mitochondrial pathway is one important way of apoptosis. Mitochondrial damage has not yet been report in CHMm cells apoptosis caused by HMME-PDT. Role of mitochondrial damage in CHMm cells apoptosis with HMME-PDT treatment was unclear.CHMm cell line collected and isolated from the clinical metastatic breast cancer, treated with HMME and a He-Ne laser at a wavelength of 632.8 nm in vitro. Changes of cell morphology determined following HMME-PDT treatment. Apoptosis was analysed using TUNEL. Cell total protein was detected by Coomassie Brilliant Blue G-250. Intracellular distribution of HMME was detected by fluorescence microscopy. Mitochondrial damage was detected by Regand staining. Mitochondrial DNA damage was determined by agarose gel electrophoresis. Changes of mitochondrial membrane potential were detected by flow cytometry. Changes of Ca²⁺ transport ability in cytoplasm and mitochondrial detected. Na⁺-K⁺-ATPase and Ca²⁺-ATPase detected by spectrophotometry.The results showed that CHMm cells vacuolated and died off at a time-dependent. Photosensitizer alone and separate laser inhibited cell proliferation at different levels, showing a slow proliferation, the smaller increase in density. We have examined the apoptosis using TUNEL assay, the nuclei of the experimental group cells at later time points showing high levels of (or bright) staining and pyknosis. The intensity and number of brightly stained cells increased as the toxicity or apoptosis progresses, significant different after 6 h (P<0.01). HMME was mainly distributed in the cytoplasm, displaying strong fluorescence around the nucleus. Mitochondria was stained blue cytoplasm granules, mitochondria stained gathered deeper. Cytoplasmic components stained shallow and particles decreased along with the time. The whole cell colouring shallow and reduce the number of cells. Mitochondrial DNA was fracture after HMME-PDT treatment, the fragment between 100²00bp bands in the experimental group, bands broadening and brightness after 3h. Mitochondrial membrane potential disappeared; Uptake ability of Ca²⁺ decreased within 4～12min significantly different (P<0.01). Activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase significantly reduced.Conclusions of all studies: CHMm cells vacuolated stop proliferating and died off after treated by HMME-PDT. Effects of separate photosensitizer and laser irradiation on cells were weak. HMME-PDT induced apoptosis and the killing effects increased prolong treatment time. HMME distributed in cytoplasm and mainly around the nucleus in CHMm cells. Cell mitochondrial damaged by HMME-PDT, mitochondrial density and membrane potential decreased. Mitochondrial Ca²⁺transport ability and Activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase decreased. Mitochondria's structure and function damaged by HMME-PDT.