Ultrasound Activated Hematoporphyrin Induced Ehrlich Ascites Tumor Cell Apoptosis

Malignant tumor is the first in the fatal diseases for human beings in recent years. People are afraid of talking cancer,so people do their best to find a way to overcome cancer. At present, there are many modalities being used for cancer therapy including radiotherapy, chemotherapy, surgical therapy and so on. Sonodynamic therapy (SDT) is a relatively new approach for cancer treatment, which involves the systemic administration of a sonosensitizer, then followed by local activation by ultrasound exposure to induce tissue or cell destruction.In 1989, Japanese scholar Umemura raised the sonodynamic therapy (SDT). 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 Ehrlich ascites tumor cells exposed to the combination of 50μg/ml hematoporphyrin and focused ultrasound at the frequency of 1.34MHZ, and the present results can be explained as follows:1. Combined with 50μg/ml hematoporphyrin, ultrasound sonication at a frequency of 1. 34MH_Z and an intensity level of 1.0W/cm² was delivered to EAT cells for 15S.Typan blue exclusion method was used to analyze the viability of EAT cells after ultrasonically activated hematoporphyrin;The effect of ultrasound combined with hematoporphyrin was significantly higher than treatment with ultrasound alone, and hematoporphyrin alone had little effect on EAT cells.2. Morphology observation through sample preparation for different time that hematoporphyrin can significant enhance the damage degree of EAT induced by low-intensity ultrasound and lingering damages, the sensitive sites of SDT are membrane systems which is the most sensitive site to be destroyed. Compared with other three groups, cell structure and microvilli over the surface of cells was significantly induced.Hoechst 33342 staining was used to detect the apoptosis of EAC cells after SDT treatment. The control cells were uniformly blue, and cells immediately after SDT had no visible nuclei changes. The apoptotic cells were blue and contain bright blue dots in their nuclei, representing the nuclear fragmentation. It was increased with time in SDT treatment groups.3. The expressions of apoptosis-relative proteins including Fas,FasL,Caspase-8 and Caspase-3 were examined by the method of the immunocytochemistry which indicated we found there were significant changes of apoptosis-relative proteins, including Caspase-3, Fas/FasL, and Caspase-8, suggested that the death receptor pathway might exist in both endogenous and exogenous apoptosis pathway.4. Combined with hematoporphyrin, high intensity focused ultrasound sonication at a frequency of 1.34MHz and an intensity level of 1 W/cm2 was delivered to EAT cells. Then the location of FADD was examined by immunofluorescence. Ultrasound activating hematoporphyrin could induce FADD translocation in EAT cells, which may indicate that apoptosis in EAT cells induced by ultrasound activating hematoporphyrin may occur through the death receptor pathway.5. The activaties of Ca²⁺-Mg²⁺-ATPase/Na⁺-K⁺-ATPase were detected by colorimetry. The activities of Ca²⁺-Mg²⁺-ATPase/Na⁺-K⁺-ATPase were obviously decreased after treated by ultrasound combined with hematoporphyrin.The decreased activities Ca²⁺-Mg²⁺-ATPase/Na⁺-K⁺-ATPase of in cells might be involved in mediating the killing effect of EAT cells in sonodynamic therapy.In this paper, we have investigated the ultrasonically induced cytotoxicity effect of hematoporphyrin on EAT cells, and preliminarily explored the biological mechanism about sonodynamic treatment. Our results imply hematoporphyrin has 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 hematoporphyrin is still in its primary stages, further investigations are needed to enrich and perfect this theory.