In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34 MHz for up to 60 s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80 lV PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80 lV PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2 h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4 h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30 s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm2 in the presence of 120 lM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (ie., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.

Sonodynamic therapy (SDT) is a novel tumor therapy method. We investigated membrane fluidity, activity of the enzymes and membrane morphology in vitro post hematoporphyrin-SDT treatment. Furthermore, the potential mechanisms behind the changes in membrane fluidity and enzymic activity were discussed. Tumor cells were exposed to ultrasound at 1.75 MHz for up to 3 min in the presence and absence of hematoporphyrin. Fluorescence polarization, contents of Malonaldehyde, and levels of free fatty acid were assessed. Activity of enzymes was checked by the plumbic nitrate detection method. For the morphologic study, a scanning electron microscope was used to observe the cellular surface. Ultrasonically induced cell damage increased in the presence of HPD (from 15% to 24%). Compared with ultrasound treatment alone, the fluidity decreased from 5.037 to 3.908, malonaldehyde content and free fatty acid level increased from 0.743 nmol/mL to 0.979 nmol/mL and from 237.180 lmol/L to 730.769 lmol/L, respectively, post ultrasound combined with HPD treatment. Inactivity of adenylate cyclase and guanylate cyclase and significant deformation of the cellular surface were also observed post SDT treatment. Our results suggested that alterations in membrane modality and lipid composition played important roles in SDT-mediated inhibition of tumor growth, even inducing tumor cell death, which might be attributed to a sono-chemical activation mechanism.

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. he results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm2. At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 lg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.

The ultrasonically induced cytotoxic effects of hematoporphyrin (Hp) on Hepatoma 22 (H22) cells in vitro and vivo were investigated. Tumor cells were suspended in saline and exposed to ultrasound at 1.43 MHz for up to 60 s in the presence and absence of Hp. The viability of cells was evaluated by trypan blue exclusion test. The ultra-structure changes of H22 cells induced by ultrasonic irradiation were evaluated by scanning electron microscope (SEM) and transmission electron microscope (TEM). Lipid peroxidation in cell was estimated by the thiobarbicturic acid (TBA) method. Our experiments indicated that the ultrasonic intensity of 2 W/cm2, the Hp concentration of 100 lg/ml and the ultrasound exposure time of 60 s were the best conditions for sonodynamic treatment in vitro. The tumor volume and weight after the combination of Hp with ultrasound were remarkably inhibited. SEM and TEM observation found the cell ultra-structure was significantly damaged, and lipid peroxidation level remarkably increased after sonodynamic treatment. This study suggested the ultra-structural changes may play a key role in cell destruction induced by sonodynamic treatment and the biological mechanism might be involved in mediating the killing effect on H22 cells in our experiment mode.