The Effect Of The Ultrasound Activated Hematoporphyrin On The Mitochondrial Function Of EAT Cells In Vitro

Nowadays the most important task at home and abroad is the prevention of malignant tumors. The normal methods of the tumor treatment mainly includes resection, chemotherapy, radiotherapy, thermotherapy, hormonotherapy, photodynamic therapy(PDT) and so on. In 1978, American scholar Doughtery firstly put forward PDT, which was already applied to the clinical treatment of the tumor. Because of the undesirable penetration of the light, photodynamic therapy was used mostly to treat tumors located in human skins, but not tumors in the deep part of human bodies. In 1989, Japanese scholar Umemura raised the sonodynamic therapy(SDT) based on the PDT. SDT is a newly developed way in which the ultrasound is used to activate hematoporphyrin derivatives in order to treat the tumor, selectively killing tumors either in original parts or in other parts. Due to the special accumulation of hematoporphyrin derivatives in tumor cells and the selective irradiation of the ultrasound, SDT has no damage on healthy tissues and is suitable for many patients who cannot be cured with the operation or in need of chemotherapy through veins. With the continuous study on SDT, lots of internal and external scholars have already put forward many physical and chemical mechanisms, but they have been still exploring the specifically biological mechanisms. Early study in our laboratory suggested that the killing effect of the ultrasound on EAT cells cound be greatly increased by hematoporphyrin derivatives, which cound cause secondary damage on EAT cells. The sensitive killing site of hematoporphyrin derivatives in cells was demonstrated mainly in the membrane system, and mitochondria were probably the most vulnerable cellar organs.As a part work of "The Mechanism of Tumor Cell Apoptosis Induced by Ultrasound Activating Hematoporphyrin" supported by National Nature Science Foundation, the focused ultrasound sonicatioon at a frequency of 1.43MHz, an intensity level of 3W/cm~2 and the hematoporphyrin concentration of 0.05 mg/ml were applied to treat the EAT cells. The experimental conclusions are as follows:1. The relative survival rates of EAT cells were different in relate to the different ultrasound intensity by means of investigating the killing effect of the ultrasound with various intensity associated with hematoporphyrin on EAT cells. With the increasmentin the ultrasound intensity, the relative survival rates of EAT cells both in ultrasound groups and in ultrasound and hematoporphyrin groups decreased to some extent. With the same ultrasound intensity, the relative survival rates of EAT cells were much lower in ultrasound and hematoporphyrin groups than in ultrasound groups. Therefore, the parameter of the ultrasound intensity was determined to be 3W/cm2.2. The killing effect of the ultrasound combined with hematoporphyrin on EAT cells was examined at different time(0, 1, 2, 3, 4h) after the treatment. The results showed that the relative survival rates of EAT cells in hematoporphyrin groups had nothing to do with the treatment time, while the relative survival rates in ultrasound groups and ultrasound and hematoporphyrin groups dropped along with treatment time. With the same ultrasound intensity, the damage on EAT cells treated by ultrasound combined with hematoporphyrin was more serious than those treated by the ultrasound alone. Finally the time was determined to be 0,1,3hours in the following experiment.3. The expressions of pro-apoptosis proteins including Bax, Bid, cytochrome c and caspase-3 were examined by the method of the immunohistochemistry, indicating that the expressions remained the same in both control groups and hematoporphyrin groups. In ultrasound groups, four pro-apoptosis proteins started to express three hours after the sonication. However, the expressions in EAT cells started one hour after ultrasound activating hematoporphyrin, and more expressions were also detected three hours after ultrasound activating hematoporphyrin.4. The activities of mitochondrial cytochrome c oxidase and succinate dehydrogenase in respiratory chains of EAT cells were examined by means of enzyme histochemistry one hour after the treatment. The activities of both enzymes were decreased greatly in the ultrasound and hematoporphyrin groups, comparing to other three groups.5. The activities of both superoxide dismutase(SOD) in EAT cells and Mn-SOD in mitochondria were determined by xanthine oxidase method. The Mn-SOD in mitochondria almost does not exist. In the ultrasound and hematoporphyrin groups, the activity of superoxide dismutase(SOD) in EAT cells remarkably decreased immediately after the treatment and continued reducing one hour and three hours after the treatment, compared to three other groups.6. In the ultrasound and hematoporphyrin groups, the content of lipid peroxides in EAT cells examined by thiobarbituric acid reaction method notably increased immediately after the treatment and continued increasing one hour and three hours afterthe treatment, compared to three other groups.