The Effect Of The Ultrasound Activated Protoporphyrin On The Mitochondrial Function Of H22 Cells In Vitro

Malignant tumors has become one of the most fatal diseases in recent years. The mainly methods in tumor treatment include resection, chemotherapy, radiotherapy, ultrasound thermal therapy, photodynamic therapy (PDT), and tumor-immunity. In 1978, American scholar Doughtery firstly present PDT, which was already applied to the clinical treatment of the tumors. 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. The antitumor effect of SDT is based on that ultrasound could focalized in the deep part of human bodies and activated the photosensitive drugs there.With the domestic and overseas study on SDT until now, many physical and chemical mechanisms have been advanced, and we still need more biological mechanisms, recent study in our laboratory suggested that the killing effect of the ultrasound on H22 cells could be greatly increased by hematoporphyrin derivatives, which could cause secondary damage on H22 cells. The sensitive killing site was demonstrated mainly in the membrane system, and mitochondria were probably one of the most vulnerable celluar organs. And ultrosound activated protoporphyrin have cooperated effects on S180 tumor cells have been proved in our laboratory, whereas there was no study on the effect of ultrosound activated protoporphyrin on the H22 cells.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 1.8MHz, an intensity level of 1.0W/cm~2 and the protoporphyrin concentration of 0.02 mg/ml were applied to treat the H22 cells. The experimental conclusions are as follows:1. Holding intensity level at 1.0W/cm~2, the livability of tumor cells were examined after different treatment time (0s, 10s, 20s, 30s, 40s, 60s), the relative survival rates in ultrasound groups and ultrasound combined with protoporphyrin groups dropped along with the increase of treatment time while protoporphyrin groups were hardly influenced. And the mortality on H22 cells treated by ultrasound combined with protoporphyrin was more serious than those treated by the ultrasound alone. Finally the treatment time was determined to be 30s in order to reaserch its mechanism.2. The killing effect of the ultrasound combined with protoporphyrin on H22 cells was examined at different time after the treatment (0, 1, 2, 3, 4, 5h). The results showed that the relative survival rates of H22 cells in protoporphyrin groups had unnotably changes after the treatment, while the relative survival rates in ultrasound groups and ultrasound combined with protoporphyrin groups dropped after treatment delay. With the same ultrasound intensity, and the damage on H22 cells treated by ultrasound combined with protoporphyrin was more serious than those treated by the ultrasound alone within 5 hours. So the tests time was determined to be 0-5 hours in the following experiment.3. Morphology observation through sample preparation for different time that protoporphyrin can significant enhance the damage degree of H22 induced by low-intensity ultrasound and lingering damages, the sensitive killing sites of SDT are membrane systems and mitochondrion is one of the most sensitive site to be destroyed.4. Based on former tests, the permeability transition pore (PTP) of tumour cells was observed by UV-spectrometer, the function of PTP gradually decline after treatment in ultrasound group and ultrosond combined with protoporphyrin group.5. We detected the influence of protoporphyrin combined with ultrasound on mitochondrial membrane potential (labeled by rodamine123). Contrast with the control group, the mitochondrial membrane potential gradually descend after treatment in ultrasound group and ultrosond combined with protoporphyrin group.6. The expressions of pro-apoptosis proteins including Bax, Smac, cytochrome C and caspase-3 were examined by the method of the immunocytochemistry, which indicated that the expressions remained negtive in both control groups and protoporphyrin groups. In ultrasound groups, four pro-apoptosis proteins expression started to increase three hours after the sonication. However, the expressions started one hour after ultrasound activating protoporphyrin treatment, and more expressions were also detected three hours after ultrasound activating protoporphyrin.7. The ROS (reactive oxygen species) from mitochondrial and non-mitochondrial was labeled by DCFH-DA then the fluorescence were measured by laser scanning confocal microscope (LSCM). As a result, the cumulation of ROS of H22 tumor cells raised in ultrasound group and ultrasound combined with protoporphyrin group along with time delay, 90minutes later the ROS level visiblely droped in ulltrosound group.