Cell Killing Effect And Its Potential Mechanism Induced By Ce6 Mediated Sonodynamic Action In Human Leukemia Cells

Leukemia is a malignant disease of the hematopoietic system.It is one of the top ten high-occurrence malignant tumors.Presently,chemotherapy and bone marrow transplantation are still the common strategies for leukemia treatment,but with many side effects such as drug resistance,higher recurrence,death by infection,etc.Therefore,in current biomedical research,the major issue is to explore new methods and strategies for clinical leukemia treatment.Sonodynamic therapy(SDT)involves the synergistic effect of ultrasound and sono-sensitizers.Ultrasound,especially focused ultrasound can be precisely focused on the target volume,which made it possible to effectively activate the cytotoxicity of some sono-sensitizers like porphyrins that preferentially accumulate in tumor tissues/cells,while with minimal damage to peripheral healthy tissues,this indicates that SDT has potential value for cancer therapy.Chlorin e6(Ce6)is a second-generation,porphyrin-related photo-sensitizer that has better selectivity and prolonged retention time in tumor cells.It has been demonstrated that Ce6 had great anti-tumor effect in photodynamic therapy as well as in sonodynamic therapy.Based on previous studies and the Natural Science Foundation support,this study was to investigate cell damage effect and its potential mechanisms of human leukemia K562 and U973 cells after Ce6 mediated SDT treatment,the obtained findings may provide valuable information for SDT therapy in leukemia clinical application.1.Cellular uptake and sub-cellular localization of Ce6 in human leukemia K562 and U973 cells:The intracellular concentration changes of Ce6 at different time points after addition to K562 and U937 cells were evaluated by mean fluorescence intensity as determined by flow cytometry.The results showed that,the cell uptake of Ce6 increased as Ce6 dose increased.At the same Ce6 concentration,immediately after administration,the intracellular Ce6 increased quickly for the first few hours,then slightly increased and peaked at 6 h,which almost sustained the same level after that.To assess the sub-cellular localization pattern of Ce6 in K562 and U937 cells,we co-loaded cells with the mitochondrial specific dye Mito Tracker Green(MTG)and Ce6.After 6 h of incubation,the fluorescence distributions of Ce6 and MTG were captured using laser scanning confocal microscopy,which indicated Ce6 mainly localized to the mitochondria of K562 and U937 cells.2.Cytotoxicity of Ce6-SDT on K562 and U937 cells:Cell killing effects of Ce6 dose and ultrasound intensity on K562 and U937 cells were evaluated by MTT assay.Results showed that,the relative cell survival decreased as Ce6 concentration increased.Ce6 dose at 20 pg/ml and 10 μg/ml might be the destruction threshold in K562 and U937 cells,respectively.Similarly,the relative cell survival decreased as ultrasound intensity increased,and the sonication threshold was approximately at 2 W/cm2 for the two cell lines.The killing effects of K562 and U937 cells by the combination of Ce6 and ultrasound showed some commonness and consistency,displaying significant synergistic anti-tumor effect.3.Damage effect of K562 cells induced by the combination of Ce6 and focused ultrasound:Ultrasound intensity at 2W/cm2 and Ce6 concentration at 20 μg/ml were chosen to study different cell death mode and the underlying mechanism in K562 cells,① SEM and TEM observation confirmed the ultra-structural changes of K562 cells after treatment.② Apoptosis of K562 cells after SDT treatment was analyzed using flow cytometer,laser scanning confocal microscope and western blot.The flow cytometry with Annexin V-PE/7-ADD staining indicated apoptotic cell death in K562 cells post SDT.The mitochondria membrane potential(MMP)of K562 cells was detected by flow cytometry with Rho123 staining,which showed significant MMP loss at 0.5 h post-SDT,and the MMP gradually restored as the incubation time prolonged.Obvious nuclear condensation was also found with DAPI staining in SDT treated cells.The apoptotic features such as Bax redistribution was examined by laser scanning confocal microscopy.Bax re-localized from cytoplasm to mitochondria as early as 0.5 h-2 h post SDT.The key apoptotic proteins such as Caspase-8,Caspase-9 and Caspase-3 were activated by SDT treatment,and the substrate of Caspase-3,poly ADP-ribose polymerase(PARP)was also cleaved at 2 h post SDT.These results implied a caspase-dependent pathway might be involved in Ce6-SDT induced apoptosis of K562 cells.③ Autophagy of SDT treated cells was examined by using western blotting,transmission electron microscopy,gene transfection and laser scanning confocal microscope.The results indicated that LC3 conversation form LC3-Ⅰ to LC3-Ⅱ occurred obviously as early as 0.5 h post SDT,and the autophagic vacuoles(AVOs)were also observed under TEM.After SDT,eGFP-LC3 transfected K562 cells appeared more green fluorescent aggregation or deletion region,suggesting LC3 protein redistribution occurred in autophagy,and the mitochondria damage might be involved in initiating the process.④ We monitored MAPK pathway related protein changes in K562 cell by western blotting.The results suggested that p38 MAPK was phosphorylated as early as 0.5 h post SDT and this phosphorylation rapidly returned to the level of control at 2 h after treatment.While,the total p38 protein expression level almost sustained the same level with or without SDT.In addition,at 0.5 h post SDT,JNK signaling pathway were also activated,JNK phosphorylation level significantly increased compared with control and the upstream factor MKK4,downstream substrates such as c-Jun,ATF-2,were also phosphorylated by SDT treatment.In ERK signaling pathway,the MEK1/2 phosphorylation level was at a relatively higher level form 0.5 h to 2 h post SDT and resumed to normal level at 4 h post-treatment.Total MEK1/2 protein expression level was not changed before and after SDT treatment.ERK 1/2 was also slightly phosphorylated,and the total ERK was significantly decreased at 4 h post treatment,suggesting that SDT may inhibit K562 cell proliferation through inhibition of ERK pathway.4.Damage effect of U937 cells by Ce6 mediated SDT therapy:This study mainly investigated cell apoptosis of U937 cells and the potential mechanisms of the action.SDT parameter was 2W/cm2 ultrasound combination with 10 μg/ml Ce6.① Obvious ultra-structure damage was observed with scanning electron microscopy and transmission electron microscopy.② Apoptosis of U937 cells was evaluated using flow cytometer,laser scanning confocal microscope and western blot.The ViaCount assay showed 50.1%cell survival after SDT treatment.About 41.3%apoptosis rate was detected by using Annexin V-PE/7-AAD apoptosis detection kit.The MMP as detected by flow cytometry with Rhl23 staining significantly decreased in Ce6-SDT treated cells as early as 0.5 h post SDT,which was further enhanced at 1 h post treatment,then gradually restored with prolonged incubation time.Obvious nuclear condensation was found with DAPI staining in SDT treated cells.PARP cleavage was also detected at 2 h post SDT,and this phenomenon sustained until 24 h after SDT.③Some key MAPK related protein changes in U937 cells were detected by western blot.Results indicated that p38 phosphorylation were significantly increased in U937 cell at 0.5 h post SDT,peaked at 2 h and then sustained at a higher level with 8 h,after that,the phosphorylation phenomenon gradually weakened.JNK was also significantly phosphorylated at 0.5 h post SDT,and its upstream factor MKK4,downstream substrates such as c-Jun and ATF-2 were all differently phosphorylated by SDT treatment.In ERK pathway,the phosphorylation of MEK and ERK1/2 was also detected at the early stage of SDT,which quickly declined with increased incubation time.The phosphorylation of ERK1 was slightly higher than that of ERK2.The total MEK and ERK 1/2 didn’t change any more after treatment.④ The inhibitory study showed that,the p38 MAPK pathway inhibitor SB203580 and the JNK pathway inhibitor SP600125 could somewhat alleviate Ce6-SDT induced U937 cell death.While,the ERK signal pathway inhibitors PD98059 and U0126 could enhance SDT induced cytotoxicity of U937 cells,and the later was more obvious than the former.⑤Some further studies were performed to explain the enhanced cytotoxicity of SDT treated cells with U0126 pre-incubation.The results suggested more intracellular ROS generation and more severe mitochondria damage may contribute to the increased cell damage in SDT plus U0126 treated cells.