| Sonodynamic therapy (SDT) is a relatively new approach for cancer treatment, which was firstly reported by Japanese Umemura S in 1989. SDT is based on the antitumor effects by ultrasound combined with porphyrin drugs. Ultrasound can penetrate deep tissue and activate the sonosensitizer which can localize in tumor tissue specifically, therefore, SDT has a good application of the treatment prospects, which made it possible to effectively activate the cytotoxicity of sonosensitizers, while with minimal damage to peripheral healthy tissues. In recent years, our group in the SDT study found that low-intensity ultrasound activated hematoporphyrin, protoporphyrinâ…¨can induce apoptosis of tumor cells. As an active ingredient of hematoporphyrin derivative, Protoporphyrin IX combined with ultrasound induced apoptosis of tumor cells is more intense than hematoporphyrin. However, the apoptotic characteristics are diverse in different tumor cells treated with different ultrasound parameters and sonosensitizer. The specific mechanisms of tumor cell apoptosis induced by Ppâ…¨combined with ultrasound remains unclear.On the background of international research development and our previous results, this paper was supported by the National Natural Science Foundation of China (Grant No.30270383 and No. 10904087). Ultrsound at a frequency of 1.1 MHz was used to activate protoporphyrinâ…¨in order to study the apoptotic effects on L1210 murine leukemia cells in vitro. The molecular mechanisms of apoptosis induced by SDT were preliminarily discussed by detecting the changes of apoptosis-related protein expressions and physiological/biochemical changes of tumor cells.The experimental conclusions were as follows:1. MTT assay showed that the proliferation of L1210 murine leukemia cells in ultrasound combined with Ppâ…¨groups were significantly inhibited compared to Ppâ…¨groups and ultrasound groups. Cell viabilities dropped along with the increase of treatment time, and decreased to minimum (36.34%) at 8 hours after the treatment.2. Western blotting detected the expression of Caspase-3. It was found that SDT groups and ultrasound groups showed a visible Caspase-3 cleaved fragment, while control groups and Ppâ…¨groups not been found. And the expression of Caspase-3 cleaved fragment was significantly higher in SDTgroups than ultrasound groups. The percentages of apoptotic cells in different groups were detected by using Annexin V/7-AAD double staining and flow cytometry. It was found that the percentages of apoptotic cells in SDT groups were significantly higher than ultrasound groups. The results show that Ppâ…¨can not induce apoptosis in L1210 murine leukemia cells, while ultrasound and SDT can induce apoptosis in L1210 murine leukemia cells, and SDT induced apoptosis is more intense than the former.3. The subcellular localization of Ppâ…¨was evaluated by Laser scanning confocal microscope. Fluorescent images revealed co-localization of Ppâ…¨and MitoTracker Green, indicating that Ppâ…¨was mainly localized in the mitochondria. The result indicated that mitochondria might be one of the main targets in the sonodynamic effect.4. By using flow cytometry, it was found that SDT treatment could induce reactive oxygen species generation, mitochondrial membrane potential decrease, DNA fragments in L1210 murine leukemia cells. The results indicated that the above-mentioned might be the reasons for L1210 murine leukemia cells apoptosis caused by SDT.5. The expressions and subcellular localization of apoptosis related proteins in L1210 murine leukemia cells were detected by indirect immunofluorescence, western blotting and flow cytometry after SDT treatment. It was found that Bax transferred to the mitochondria, cytochrome C released to cytoplasm, Caspase-9 activated and cleaved. The expression of Caspase-9 cleaved fragment was increased, while the expression of Bcl-2 was significantly reduced.of Caspase-9 activation and expression of fragments have increased, suggesting that mitochondrial apoptotic pathway might involved in apoptosis of L1210 murine leukemia cells induced by ultrasound combined with Ppâ…¨. |
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