Effects Of High Intensity Focused Ultrasound And Sonodynamic Effect On Ovarian Cancer Cells In Vitro

Being one of the most harmful genital malignant tumors, ovarian cancer has posed us many problems concerning diagnosis and treatment. Although many trials have been made to improve the low survival rate, the results are not satisfying. It is very important to look for new ways of treatment.Ultrasound medicine is used for diagnostic and therapeutic purposes. However, diagnostic ultrasound predominated by far. During the past several decades, therapeutic ultrasound has made some progress and many new terms have been introduced. Much attention has been given to high intensity focused ultrasound (HIFU) and sonodynamic therapy for their imposing effects of experiments. However, few reports have been found about their uses in treating ovarian caner.Based on these above, some experiments were done to identify the inhibitory effects of the two methods on ovarian cancer cells in vitro. Three parts were presented here. The first part investigated the effects of HIFU and the synergistic effects of Tween 80 and HIFU. Heat shock protein 70 expression was detected as well. The second part was about sonodynamic effect where hematoporphyrin was activated by HIFU. The last part studied the cytotoxic effect of methylene blue and the possible sonosensitizing character of methylene blue. It is demonstrated that both high intensity focused ultrasound and sonodynamic therapy may be the potential strategies to treat ovarian caner.PART IINHIBITORY EFFECTS OF HIGH INTENSITY FOCUSED ULTRASOUND ON OVARIANCANCER CELLS IN VITROObjectives: To study the effects of HIFU on ovarian cancer cells and the synergistic effects of Tween -80 and HIFU. To determine the expression level of heat shock protein 70 in ovarian cancer cells before and after HIFU treatment.Methods: Several different methods (including MTT assay, growth curve, and colony-forming efficiency) were used to determine the inhibitory effects of HIFU and Tween 80 on ovarian cancer cells. The morphological and ultra-structural changes were observed with scanning electron microscope and emission electron microscope. Flow cytometry (FCM) was used to determine the changes of apoptosis rate. Immunocytochemistry and western blotting were used to evaluate the expression of heat shock protein 70.Results: (1) HIFU could significantly suppress the growth and proliferation of ovarian cancer cells. With increasing ultrasound intensity and time, the significant decrease of cell survival rate was observed, K0.05. Furthermore, the growth curve and colony-forming efficiency were also inhibited. However, the survival rates were similar among three different cell lines. After HIFU treatment, cells became irregular and many pores were found on cell membranes. Apoptosis and necrosis were both found in cells. FCM results showed that apoptosis rate increased after HIFU treatment. (2) Tween 80 had slight killing effects on ovarian cancer cells. However, when it was given before HIFU, synergistic effects were found. Cell survival rate and colony-forming efficiency of the combining group both decreased compared with those of HIFU group and Tween 80 group. (3) Heat shock protein 70 expression increased after HIFU and it reached the highest level after 8h. Furthermore, its intracellular distribution changed with the differenttime after HIFU.Conclusions: HIFU could significantly suppress the growth and proliferation of human ovarian caner cells. HIFU and Tween-80 had synergistic effects. After HIFU heat shock protein 70 showed significant changes not only in the expression level but also in the intracellular distribution.PART IIEFFECTS OF HIGH INTENSITY FOCUSED ULTRASOUND AND HEMATOPORPHYRIN ON OVARIAN CANCER CELLS IN VITROObjectives: To study the effects of high intensity focused ultrasound and hematoporphyrin on human ovarian cancer cell line HRA in vitro and to clarify the possible mechanism of sonodynamic therapy (SDT).Methods: Hematoporphyrin (HP) was selected as a sonosensitizer and HIFU with certain intensity was used to activate it. Then MTT assay and colony-forming efficiency assay were applied to determine the growth inhibitory effects of ovarian cancer cells. Scanning electron microscope and emission electron microscope were applied to detect the morphological and structural changes of the cells. Flow cytometry was used to determine the changes of mitochondrial membrane potential and apoptosis rate of the cells. Reactive oxygen species (ROS) were also detected with fluorospectrophotometer.Results: HP had no significant inhibitory effects on the cells. However, when HP was used before HIFU, it could enhance the killing effects of ultrasound (P <0. 05). With the increasing concentration of HP and time of ultrasound treatment, the inhibitory effects increased (P<0. 05). After HP and ultrasound treatment, cells became irregular and many pores were.found on cell membranes. Apoptosis and necrosis were both found in cells. After HP plus HIFU treatment, cellular mitochondrial membrane potential decreased significantly compared with those of control and simple HIFU treatment group. Apoptosis rate and ROS were both found to increase after SDT and the increasing level was higher than that of simple ultrasound treatment group.Conclusions: Sonodynamic therapy could significantly suppress the growth and proliferation of ovarian cancer cells and it could induce apoptosis through mitochondrial way. ROS may be an important explanation.PART IIIEFFCTS OF METHYLENE BLUE AND HIGH INTENSITY FOCUSED ULTRASOUND ON OVARIANCANCER CELLS IN VITROObjectives: To investigate the effects of methylene blue (MB) and high intensity focused ultrasound on human ovarian cancer cell line HRA in vitro and to identify the possible mechanism of MB cytotoxicity and MB sonodynamic effect.Methods: HRA cells were incubated with MB for different periods of time. Then MB was also used before high intensity focused ultrasound. MTT assay was applied to determine the survival rate of HRA cells. Emission electron microscope was applied to detect the morphological and structural changes of the cells. Flow cytometry was used to determine the changes of mitochondrial membrane potential and apoptosis rate of the cells.Results: (1) MB had significant inhibitory effects on the cells and the inhibitory effects increased with the increasing concentration and treating time. (2) When used before HIFU, MB could enhance the cell-killing effects of ultrasound (KQ. 05). With the increasing concentration of MB and time of ultrasound treatment, the inhibitory effects increased (/><0. 05). After MB and HIFU treatment, apoptosis and necrosis were both found in cells. Furthermore, cellular mitochondrial membrane potential decreased significantly compared with those of control and simple HIFU treatment group. Apoptosis rate increased after treatment and the increasing level was higher than that of HIFU treatment.Conclusions: MB could significantly suppress the growth and proliferation of ovarian cancer cells and it showed the potential as a sonosensitizer in sonodynamic therapy.