| Malignant tumor is one of the most deadly diseases for human beings.At present, there are many modalities being used for cancer therapy includingradiotherapy, chemotherapy, surgical therapy and so on, but which all can not curecancer patient effectively due to the unclear mechanism about tumor occurrence.Sonodynamic therapy (SDT) is a relatively new approach for cancer treatment basing onphotodynamic therapy (PDT), which involves the systemic administration of asonosensitizer, then followed by local activation by ultrasound exposure to induce tissueor cell destruction. Ultrasound, especially focused ultrasound, could be preciselyfocused on the target volume, which made it possible to effectively activate thecytotoxicity of sonosensitizers that preferentially accumulate in tumor sites while withminimal damage to peripheral healthy tissues, this indicates that SDT has potentialvalue for cancer therapy. Since SDT was proposed, it has been widely studied by manyresearchers by using different sono-sensitizers, different ultrasound devices, anddifferent kinds of tumor cells, and they also proposed many cell killing mechanismincluding sono-cavitation, free radicals, lipid peroxidation, and so on. But until now, westill do not have a unanimous conclusion about SDT, it seems that the sono-sensitizerused, the ultrasound exposure parameters and the type of biological system beingirradiated are co-determinant factors for the specific mechanism of sono-sensitization.Basing on the international development and earlier experiment results, this paperhas done some work about the National Natural Science Foundation of China (GrantNo.39870240 and No. 30270383). The sonodynamical effect was investigated onsarcoma 180 cells exposed to the combination of 120μmol/L protoporphyrinⅨ(PPIX)and focused ultrasound at the frequency of 2.2 MHZ, and the present results can beexplained as follows:1. With the fluorescence photometer, we firstly detected the maximal excitionspectrum (402 nm) and emission spectrum (596 nm) of protoporphyrinⅨ. In order todetermine the optimum time for ultrasonic irradiation of the tumor, we measured thetime courses of PPIX concentrations in plasma, tumor, muscle and skin by thefluorescence intensity of tissues extractions with a fluorescence photometer based on the standard curve. The tumor was exposed to ultrasound at the time when the PPIXconcentration ratios in the tumor/tissues were relatively higher, which were veryimportant in order to maximize treatment effect while minimizing side effect. Ourexperiments suggested that a time of 24 h after the administration of PPIX was chosenas the best time for ultrasound exposure.2. The anti-tumor effect of ultrasound intensity and PPIX dose on S180 solidtumors was shown respectively that: Ultrasound alone at the intensity of 5 W/cm~2showed a threshold for anti-tumor effect, and which was enhanced by PPIX in a dosedependent manner, the synergistic inhibitory effect was significant when PPIX dose wasabout 5 mg/kg or higher.3. In this study, the in vivo effect of the combination of PPIX and ultrasoundexposure on a subcutaneously implanted S180 solid tumor was investigated, withultrasound at a frequency of 2.2 MHz and PPIX at a dose of 5 mg/kg. Anti-tumor effectswere estimated by measuring tumor size and calculating the average surviving time oftumor bearing mice after SDT treatment, additionally, the morphological changes ofS180 cells were evaluated by transmission electron microscope (TEM) observation.Results indicated that, 5 mg/kg PPIX alone had almost no inhibitory effect, whileultrasound alone showed a slight anti-tumor effect. The synergistic effect of PPIX andultrasound on inducing anti-tumor effect was more and more remarkable as timedelayed after treatment, and the tumor size was constantly about a half of the untreatedgroup 3 days after treatment. The average surviving time of tumor bearing mice afterdifferent treatments showed that ultrasound plus PPIX group mice showed significantlylonger average surviving time than any other three groups (p＜0.01). TEM observationindicated the synergistic effect was obviously stronger than ultrasound alone treatment,and cell morphological changes with severely broken cytoplasm membranes might playan important role in resulting in cell damages with SDT.4. The cytotoxic effect of PPIX on isolated sarcoma 180 cells induced byultrasound was investigated, and the best acoustic parameters were chosen. Our studyimplied that the ultrasonically induced cell damage increased as the ultrasound intensity,PPIX dose and exposure time increased. The intensity threshold for ultrasonicallyinduced cell damage was observed to be around 3 W/cm~2. Cell damage increasedrapidly with ultrasound intensity above the threshold. The concentration of PPIX had arelatively low threshold for ultrasonically induced cell damage, since cell damage was increased significantly when the PPIX concentration was greater than 120μM. Theultrasonically induced cell damage was enhanced as the exposure time increased, whichbecame more significant when the exposure time was above 30s.5. S180 cells were exposed to ultrasound for 30 seconds' duration, at the frequencyof 2.2 MHz and an acoustical power of 3 W/cm~2 in the presence of 120μM PPIX.Treatment with ultrasound and PPIX together made cell damage rate high to 50.91%,while treatment with ultrasound alone made cell damage rate to 24.24%, and PPIXalone kept this rate no changed.Under the SEM and the TEM, changes in the cell ultra-structure observed in thecombined treatment group were obvious, with severely broken cytoplasm membranes,many spaces appeared in cytoplasm, mitochondria were swelling with crista decreased,cytoplasm became less dense, chromatin condensation appeared in nuclear, materials incytoplasm and nucleoplasm were partly lost, and so on. The results indicated somecytoplasm membranes, nuclear membranes, and mitochondria might be the main targetsin the sonodynamic effect.6. The effect of active oxygen scavengers on the ultrasonically induced celldamage in vitro in the presence and absence of 120μM PPIX was studied. Thegeneration of oxygen free radicals in cell suspensions was immediately detected aftertreatment using the reactive oxygen detection kit. Lipid peroxidation induced by theultrasonic action was measured using the Thibabituric Acid (TBA) method according tothe protocol of the MDA Detection Kit. Copper reagent colorimetry method was used tomeasure the level of FFAs released into cell suspensions by the process of cell damageinduced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuringthe activities of key antioxidant enzymes (ie, SOD, CAT, GSH-PX) in S180 tumor cells.Results suggested that: The enhancement of ultrasonically induced cell damage by PPIXwas significantly reduced in the presence of 100μg/ml histidine, but not significantly inthe presence of either 100μg/ml SOD or 100μg/ml mannitol; Cell lipid peroxidationincreased significantly immediately after treatment (p＜0.01) compared with ultrasoundalone and PPIX alone treatment groups; Colorimetry and enzymatic chemical methodsshowed that the level of FFAs in cell suspensions significantly increased post thetreatment, while the activities of all the enzymes above in tumor cells decreased atdifferent levels, which indicated that oxygen free radicals may play an important role inimproving the membrane lipid peroxidation and ultimately leading to the destruction of oxidation resistant system and cell damage.In this paper, we have investigated the ultrasonically induced cytotoxicity effect ofPPIX on S180 cells, and preliminarily explored the biological mechanism aboutsonodynamic treatment. Our results imply PPIX has great potential as a sonosentizer forsonodynamic tumor treatment and provide useful information about the application ofSDT. However, the research on the effects of ultrasound activating PPIX is still in itsprimary stages, further investigations are needed to enrich and perfect this theory.
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