Different Intensity Focused Ultrasound Combined With Protoporphyrin Injury Of S180 Cells In Vitro Culture

Sonodynamic therapy(SDT),a relatively new approach for cancer treatment,was firstly reported by Japanese Umemura in 1989.This therapy is based on preferential and long time retention of a sonosensitizer in tumor tissues and subsequent activation of the drug by ultrasound.Ultrasound, especially focused ultrasound,could penatrate deeply into organic tissues and be precisely focused on the target volume,which made it possible to effectively activate the cytotoxicity of sonosensitizers, while with minimal damage to peripheral healthy tissues.Comparison with photodynamic therapy (PDT),which is mainly used to cure surface tumor,SDT is a more potential and promising method for cancer treatment.The sonosensitizers most commonly used are hematoporphyrin(Hp) and their derivatives(HpD).ProtoporphyrinⅨ(PpⅨ) is as an effective compound of hematoporphyrin derivatives, which is found preferentially accumulated in rapid proliferating cancer cells.Previously,it has been reported that the PpⅨmight have more advantages to cause cytotoxicity than HpD in SDT in our experiment system.Concentration of PpⅨin tissue or cell could be increased not only by administration of PpⅨitself but also by administration its precursors such asδ-aminolevulinic acid (5-ALA) or 5-ALA derivatives.5-ALA mediated PDT at the cellular and tissue levels has been thoroughly investigated in the past decade,however,there are few reports about endogenous PpⅨby administration of 5-ALA in SDT and the cell killing effect by ultrasound activating exogenous PpⅨis still on the primary study.On the background of international research development and our previous results,this paper is supported by the National Natural Science Foundation of China(Grant No.39870240 and No. 30270383).Different intensities of ultrsound at a frequency of 1.065 MHz are used to activate protoporphyrinⅨin order to study the cell killing effect on S180 cells in vitro.The experimental conclusions are as follows:1.It was shown that the maximal excition wavelength was 407 nm and the emission spectrum of PpⅨgave a peak fluorescence value at 605 nm that were used for kinetic measurement. Treating S180 cells with exogenous PpⅨ,following by rising gradually,its intracellular content reached a maximum at 45 min and then remained constant after that.Exogenous PpⅨpharmacokinetics suggested that it displayed a dose dependent manner and exsited saturated situation.For endogenous PpⅨ,it also displayed a time-dependent manner,but no plateau was found.The porphyrin level in cells increased linearly at 0～12 h.High-performance liquid chromatographic analysis indicated that mainly PpⅨwas formed after administration of 5-ALA,not the other intermediates.Otherwise,it was clearly showed that the changes of PpⅨlevel in S180 cells coincided with the results of fluorence spectrophotometer.2.The dynamic changes of their subcellular localization patterns with the prolonged incubation time were evaluated by Laser scanning confocal microscope.Fluorescent images revealed that the exogenous PpⅨwas mainly accumulated in cell membrane in the early stage,whereas the ALA-derived PpⅨwas initially localized in the mitochondria.Apparently,the intracellular localizations of endogenous PpⅨand exogenous PpⅨwere different in the early stage after incubation.3.The fluence on the proliferation of S180 cells by the combination of endogenous PpⅨor exogenous PpⅨand ultrasound at the frenquency of 1.065 MHz and various intensities of 0,1.0, 3.0,5.0 W/cm~2 was investigated.Our study implied that when the content of exogenous PpⅨwas equal with endogenous PpⅨ,the cell viability of ultrasound plus ALA group and ultrasound plus PpⅨgroup had no remarkable difference with ultrasound alone group.While,when the PpⅨconcentration was 40μM,cell damage induced by ultrasound was increased significantly.4.Under the SEM and the TEM,changes in the cell ultrastructure observed in the combined treatment group were obvious,with severely broken cytoplasm membranes,many spaces appeared in cytoplasm,and so on.Thibabituric Acid(TBA) method according to the protocol of the MDA Detection Kit and enzymatic chemical methods were used to measure the lipid peroxidation levels and activities of extracellular LDH and intracellular ATPase after treatment.We found that the malondialdehyde content in S180 cells and activities of LDH in the supernate was remarkably enhanced,while,ATPase activities were obviously decreased at different level compared with the other three groups as the ultrasound intensity increased.The results indicated that S 180 cells exhibited damage by focused ultrasound in the presence of PpⅨ-leading to the destruction of cytoplasm membranes,improving the membrane lipid peroxidation,decreasing the activities of ATPase and ultimately inducing cell damage.The results indicated that cytoplasm membranes might be the main targets in the sonodynamic effect.