Ultrasound Activated Protoporphyrin Ix Disodium Salt Damage Effects In L1210 Murine Leukemia Cells

Sonodynamic therapy(SDT) is a relatively new approach for cancer treatment,which was firstly reported by Japanese researchers in 1989.This therapy is based on preferential and long time retention of sonosensitizers in tumor tissues and subsequent activation of the drugs by ultrasound. Ultrasound could penetrate 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.As a new method,SDT is a more potential and promising method for cancer treatment and is paid lots of attendtion by domestic and overseas researchers until now.So many foundation works had been done,especial on the choosing of sonosensitizers and the parameters of ultrasound,and some physical and chemical mechanisms had been advanced just like singlet oxygen and free radicals,but concrete biological mechanisms are explored by the researchers.On the background of international research developments and our previous results,this paper is supported by the National Natural Science Foundation of China(Grant No.39870240 and No.30270383).The L1210,mouse leukemic cells,were found to be poor immunogenicity cells and were more like to the leukaemia of human.It is beneficial to research the damage effects of L1210 cells induced by combination of protoporphyrin IX disodium salt and ultrasound and the possible mechanisms.This may be some useful information for the clinic therapy to treat the leukaemia. Different intensities of ultrsound at a frequency of 1.065 MHz are used to activate protoporphyrinⅨdisodium salt in order to study the cell killing effects of L1210 cells in vitro.The experimental conclusions are as follows:1.Treating L1210 cells with protoporphyrinⅨdisodium salt,it displayed a time-dependent manner and its intracellular content reached a maximum at 60 min with initial concentrations 2,10 and 20μM,then decreased with the prolonged incubation times and then remained constant on 120 min.With differents initial concentrations of protoporphyrinⅨdisodium salt,the tendency was the same way which contents of rotoporphyrinⅨdisodium salt in L1210 was changed as incubation times changed.2.The dynamic changes of protoporphyrinⅨdisodium salt with red fluorescence and Mito Tracker Green as the probe of mitochondrion subcellular localization patterns with 0,30,45,60,90, 120 min were evaluated by Laser scanning confocal microscope.Fluorescent images revealed that the subcellular localization of protoporphyrinⅨdisodium salt was time-dependent manner, accumulated in cell membrane in the early stage,then localized in intracellar coincidence with the green fluorescence of Mito Tracker Green,and then appeared extracellular.After the treatment of ultrasound,the locating of protoporphyrinⅨdisodium salt in L1210 cells had not been impaceted clearly.3.When the protoporphyrinⅨdisodium salt concentration was 40μM,there was little influence on the viabilities of L1210 cells,but the viabilities induced by the combination of various concentrations protoporphyrinⅨdisodium salt and ultrasound at the frenquency of 1.065 MHz appeared distinct decreasing,cell damage induced by ultrasound was increased significantly,with the concentration 40μM of protoporphyrinⅨdisodium salt which was choosed as the parameters of ultrasound for the future studies.4.The killing effects of L1210 cells by the combination of protoporphyrinⅨdisodium salt and ultrasound at the frenquency of 1.065 MHz and various intensities of 1.0,3.0 and 5.0 W/cm~2 were investigated.The study implied that cell viabilities of ultrasound plus protoporphyrinⅨdisodium salt group had remarkable difference with ultrasound alone group especially when the ultrasound was 5.0 W/cm~2.5.The changes of cell membrane of L1210 cells treated by the combination of protoporphyrinⅨdisodium salt and ultrasound were evaluated by means of scanning electron microscope(SEM) and the damage of cell membrane was evaluated by measurement of LDH release.The effects were more significant induced by ultrasound plus protoporphyrinⅨdisodium salt than ultrasound alone, and the difference was even more severity when the ultrasound was 5.0 W/cm~2.6.The changes of L1210 cells treated by the combination of protoporphyrinⅨdisodium salt and ultrasound were studied by light microscope and transmission electron microscope(TEM).The damage effects were more severity induced by the combination of protoporphyrinⅨdisodium salt and ultrasound than ultrasound alone,such as nuclei,cytoplasm and organelles.7.The activities of intracellular ATPase decreased after treatment in a way of depended on the increasing intensity of ultrasound.The difference between the group of ultrasound plus protoporphyrinⅨdisodium salt and ultrasound alone was significant,when the ultrasound was 5.0 W/cm~2.8.The levels of reduced glutathione hormone(GSH) and total antioxidant capacity were significant changed and increased with the enhanced intensity of ultrasound,the differences of the groups of ultrasound plus protoporphyrinⅨdisodium salt and ultrasound alone compared with control cells were obvious,but there was no conspicuous difference between the previous two groups.The difference was especially striking,when the intensity of the ultrasound was 5.0 W/cm~2.9.The levels of the lipid peroxidation were found to be increased at different levels after treatment,when the ultrasound was 5.0 W/cm~2,the change was obviously more significant.