| Malignant tumor is one of the most deadly diseases for human beings. At present, there are many modalities being used for cancer therapy including radiotherapy, chemotherapy, surgical therapy and so on, but which all can not cure cancer patient effectively due to the unclear mechanism about tumor occurrence. Sonodynamic therapy (SDT) is a relatively new approach for cancer treatment basing on photodynamic therapy (PDT), which involves the systemic administration of a sonosensitizer, then followed by local activation by ultrasound exposure to induce tissue or cell destruction. Ultrasound, especially focused ultrasound, could be precisely focused on the target volume, which made it possible to effectively activate the cytotoxicity of sonosensitizers that preferentially accumulate in tumor sites while with minimal damage to peripheral healthy tissues, this indicates that SDT has potential value for cancer therapy. Since SDT was proposed, it has been widely studied by many researchers by using different sono-sensitizers, different ultrasound devices, and different kinds of tumor cells, and they also proposed many cell killing mechanism including sono-cavitation, free radicals, lipid peroxidation, and so on. But until now, we still do not have a unanimous conclusion about SDT, it seems that the sono-sensitizer used, the ultrasound exposure parameters and the type of biological system being irradiated are co-determinant factors for the specific mechanism of sono-sensitization.Basing on the international development and earlier experiment results, this paper has done some work about the National Natural Science Foundation of China (Grant No.39870240 and No. 30270383). The sonodynamical effect was investigated on EAT cells exposed to the combination of 80μmol/L protoporphyrinⅨ(PPⅨ) and focused ultrasound at the frequency of 2.2 MHz, and the present results can be explained as follows:1. The cellular uptakes of PPⅨare estimated by fluorescence photometer. The results showed that there exsited saturated situation, that was, the concentrations in EAT cells was not always raised as incubation time increased. And EAT cells absorbed PPⅨat relative saturation point after 20 minutes, what was the best incubation time interval before ultrasound treatment.2. The cytotoxic effect of PPⅨon isolated EAT cells induced by ultrasound was investigated, and the best acoustic parameters were chosen. Our study implied that the ultrasonically induced cell damage increased as the ultrasound intensity, PPⅨdose and exposure time increased. The intensity threshold for ultrasonically induced cell damage was observed to be around 3 W/cm~2. Cell damage increased rapidly with ultrasound intensity above the threshold. The concentration of PPⅨhad a relatively low threshold for ultrasonically induced cell damage, since cell damage was increased significantly when the PPⅨconcentration was greater than 80μM. The ultrasonically induced cell damage was enhanced as the exposure time increased, which became more significant when the exposure time was above 45 s.3. EAT cells were exposed to ultrasound for 45 seconds' duration, at the frequency of 2.2 MHz and an acoustical power of 3 W/cm~2 in the presence of 80μM PPⅨ. Treatment with ultrasound and PPⅨtogether made cell damage rate high to 50.22%, while treatment with ultrasound alone made cell damage rate to 29.24%, and PPⅨalone kept this rate no changed. Under the SEM and the TEM, changes in the cell ultra-structure observed in the combined 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 in cytoplasm and nucleoplasm were partly lost, and so on. The results indicated some cytoplasm membranes, nuclear membranes, and mitochondria might be the main targets in the sonodynamic effect.4. The expressions of pro-apoptosis proteins including caspase-3, cytochrome c and Smac/DAIBLO were examined by the method of the immunocytochemistry, which indicated that the expressions remained negtive in both control groups and PPⅨgroups. In ultrasound groups, three pro-apoptosis proteins expression started to increase 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. It suggested that endogenous pathway might also exist in the death process.5. The Subcellular localization of PPⅨwas estimated by laser scanning confocal microscopy (LSCM). We found that PPⅨand rhodamine 123 are colocalized. It revealed that PPⅨlocasized mainly in the mitochondria. And mitochondria may be the target organelle in the SDT.6. The mitochondrial membrane potential (MMP) was determined. Results showed that loss of mitochondria membrane potential was the most noticeable in ultrasound plus PPⅨgroups. It revealed that the function of mitochondria were severely lost in EAT cells after ultrasound activating PPⅨ.7. The generation of oxygen free radicals in cell suspensions was immediately detected after treatment using 2',7'- dichlorodihydrofluorescin diacetate (DCFH-DA). Lipid peroxidation induced by the ultrasonic action was measured using the Thibabituric Acid (TBA) method according to the protocol of the MDA Detection Kit. Results suggested that the content of ROS and the cell lipid peroxidation increased significantly immediately after treatment (P<0.01) compared with ultrasound alone and PPⅨalone treatment groups. It indicated that oxygen free radicals may play an important role in improving the membrane lipid peroxidation and ultimately leading to cell damage.In this paper, we have investigated the ultrasonically induced cytotoxicity effect of PPⅨon EAT cells, and preliminarily explored the biological mechanism about sonodynamic treatment. Our results imply PPⅨhas great potential as a sonosentizer for sonodynamic tumor treatment and provide useful information about the application of SDT. However, the research on the effects of ultrasound activating PPⅨis still in its primary stages, further investigations are needed to enrich and perfect this theory. |
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