Photodynamic Therapy To Ovarian Cancer Caused By Hemporphin

Background and Objective:Ovarian cancer is one of the three most common cancer in women' reproductive system neoplasia. And most of the patients are at advanced stage when diagnosed, owning to the lack of unique symptoms and sensible screening methods. Although the treatment of ovarian cancer improved a lot recently , most patients had recurrent cancer and at last died of it. To improve life quality and to prolong life span are the main theraputic purpose for patients with advanced and recurrent ovarian cancer. But the effect of operation or chemotherapy is still on debate. Phtodynamic therapy(PDT) is newly developed treatment for tumor since the 19th century. It is a treatment combining photosensitiser (Ps) and light illumination to produce photocytoxicity to cells. It has been popularly used in clinical therapy for benign vascular neoplasia. For patients with advanced or recurrent ovarian cancer, photosensitiser can be given firstly, then laser light was used locally through laparoscope or laparotomy, which is called intraperitonneal photodynamic therapy(IPPDT) .Hematoporphyrin monomethyl ether(HMME) is the second generation of photosensitiser in china. It has relatively pure component and higher orientation. In our previous research, we found that HMME has effective phototoxicity to ovarian cancer in vivo or invitro. The main death mode is necrosis. In this study, we intend to induce apoptosis by lowering the concentration of photosensitiser.And we investigated whether the death mode induced by HMME-PDT is dose-depended by using the control of high dose HMME-based PDT. We also explore the the phototoxicity and mechanism of different dose of HMME to the ovarian cancer cell line 3AO. Methods:After co-incubation with subcellular organelle probe(Rh123 or Lucifer Yellow) and different levels of photosensitizers (3ug/mLHMME or 30ug/mL HMME ) ,the fluorescence microscopy was applied to collect the fluorescence images in cells at different time point. The cytotoxicity of PDT was determined using a Cell Viability Analyzer. Mechanisms of cell death during PDT was determinded by Annexin V/PI double staining technique and analyzed by flow cytometer. Fluorecencein Active Caspase Staining Kit was used to explore the pathway of apoptosis duing PDT. DAPI staining was also used to show the morphological changes of cancer cells after HMME-based photodynamic therapy. To measure the change of MMP, RH123 was introduced to label the mitochondria. The diffusion and accumulation of RH123 is proportional to the degree of MMP. JD801 Image analysis system (JieDa, JiangSu, China) was applied to measure the fluorescence intensity of RH123 (excitation 488 nm and emission 530 nm) in the cells. Results:HMME can located both in the mitochondrion and in the lysosome after different incubation time combined with their fluorescence probe. Low dose of HMME intend to located in mitochondria as early as 1h after co-incubation with Rh123,while located in lysosome 3h after co-incubation with Lucifer yellow.And high dose of HMME intend to located in mitochondria 30m after co-incubation while loacated in lysosome 1h after co-incubation. The cytotoxicity and death mode were dose-dependent for HMME-based PDT.And apoptosis was induced instead of necrosis when the concentration of photosensitiser decreased. Mitochondria play an important role in the procedure of apoptosis, caspase activity was determined in the pathway.Neither Laser irradiation nor phtosensitiser alone has any influcence on the survival rate of cells. Obvious mitochondria damages were observed both after low and high dose of HMME-based PDT through the MMP investigation. Conclusions:HMME had obvious phototoxicity to ovarian cancer cells in vitro and is hopely to be used as a photosensitizer in photodynamic therapy to ovarian cancer. Necrosis was the predominant mode of cell death during PDT in higher concentration, while apoptosis take a major part in the lower concentration of HMME-based PDT. We determined that the effect of HMME-based PDT on ovarian cancer cell line 3AO is dose dependent. The pathway of apoptosis induced by low dose of photodynamic therapy is mitochondria involved.