Sdt Induced Signal Transduction Pathway And Mechanism Of S180 Ascites Tumor Cell Apoptosis

In 2008, World Health Organization reported that cancer will become the number one killer of human health in 2010. Even in 2030,27 million people may have cancer all over the world, and 17 million people will die for this disease. Therefore, scientists are exploring new anticancer therapy day after night in order to find out more effective ways. At present, clinical therapies in common use include surgical, chemotherapy and physiotherapy. These anticancer strategies can cure some kind of cancer, but their own shortages and side-effects restrict the application especially for cancer in deep.With the research carrying on, scholars find multifactor treatments can kill cancer cells in effect. In 1989, Yumita and Umemura discovered that ultrasound could activate hematoporphyrin to anticancer, and named this treatment as "Sonodynamic Therapy, SDT". Ultrasound can penetrate deep tissue and focus on certain area. This is a unique advantage when compared to electromagnetic modalities such as laser beams in the noninvasive treatment of nonsuperficial tumors in clinical treatment. Hematoporphyrin is a intermediate product in haemachrome synthesize in normal human body. It can selectively accumulate in tumor tissue and be reserved for a long time. SDT is a promising anticancer therapy with more pertinency and efficiency. Although the synergistic anticancer effect of ultrasound and sonosensitizers has been proved, and the clinical experiment has been carried out, there is little report about the SDT inducing apoptosis. The exact chemical and mechanism of ultrasonic cytotoxic effect is still unclear.In this research,1.75 MHz continuous-wave ultrasound was used to investigate that(1)whether death receptor-and mitochondria apoptosis signaling pathway are activated; (2) the regulation effect of Bcl-2 family members (Bax, Bid and Bcl-2) in SDT induced S180 cells apoptosis; (3) activation of tumor suppressor protein p53 and the relation between p53 and sonosensitivity of tumor cells;(4) the mechanism of ultrasound-and SDT-induced apoptosis. To analyze the protein and mRNA, flow cytometer, laser scanning confocal microscope, single cell gel electrophoresis, western blotting, RT-PCR and DNA sequencing were used. The results are as follow:(1) The ultrasound alone and SDT can induce S180 cells apoptosis at same condition, whereas 20μg/ml hematoporphyrin significantly enhances the cytotoxic effect of ultrasound. Ultrasound and hematoporphyrin have a synergistic antitumor effect.(2) The death receptor and mitochondria apoptosis signaling pathway are activated in SDT-induced S180 cells apoptosis. In this process, FADD and caspase-8 translocated from cytoplasm to plasma membrane, and caspase-8 was activated. At the same time, mitochondrial depolarization and permeabilization of the inner mitochondrial membrane were found. Cytochrome-c was released to cytoplasm to promote cleavage of caspase-9. Activated caspase-8 and-9 in turn cleave caspase-3, and PARP.The activation of death receptor signaling pathway was a direct consequence after SDT treatment but not a signaling magnification of mitochondria signaling pathway. The Bid which can transmit apoptosis signaling between death receptor and mitochondria signaling pathway did not be activated.(3)The SDT can also initiate caspase-independent pathway to mediate apoptosis. AIF was released and translocated to nuclear to promote apoptosis.(4) The SDT can regulate apoptosis-relative protein expression. After SDT treatment, the protein and mRNA level of Fas and Bax increased to promote apoptosis, whereas the level of apoptosis-inhibitor Bcl-2 declined.(5) In the SDT-induced S180 and H-22 cells apoptosis, tumor suppressor protein p53 expressed and translocated to mitochondria to accelerate apoptosis by transcription-dependent and transcription-independent way. The downstream protein PUMA,Bax and Fas were also activated. P53 was not involved in EAC cells apoptosis after SDT treatment.(6) There is a relationship between p53 protein activation and sonosensitivity of tumor cells. The S180 cells are the most sensitivity to SDT treatment. The level of p53 increased and p53 translocated to mitochondria. The sonosensitivity of H-22 cells was in the middle.After SDT treatment, p53 expressed, and translocated after a period of time. The p53 was not activated in the EAC cells, and the EAC cells were resistant to SDT treatment.(7) The mechanism of SDT-inducing apoptosis included multiform apoptosis pathway and death mode. After SDT, the death receptor and mitochondria apoptosis signaling pathway are activated, and initiate caspase cascade to mediate apoptosis. The nuclear factor NF-κB may play pivotal role in regulating ultrasound-induced apoptosis. When S180 cells underwent sonication,NF-κB expressed immediately and translocated to nuclear.In the present research, for the first time, we systemicly studied the apoptotic signaling pathway in SDT-induced tumor cell apoptosis and discussed the mechanism underline ultrasound-and SDT-induced apoptosis to provide a basis for explaining the synergistic effect of ultrasound and Hematoporphyrin and facilitate SDT application in clinical anticancer treatment.