Effect Of Cell Cycle Phase On The Sensitivity Of SAS Cells To Sonodynamic Therapy Using Low-intensity Ultrasound Combined With 5-aminolevulinic Acid In Vitro And Vivo

Objective: Sonodynamic therapy(SDT)with 5-aminolevulinic acid(5-ALA)can effectively inhibit various tumors in vitro and in vivo.However,the relationship between the efficacy of SDT and cell cycle phase remains unknown.5-ALA might generate different amounts of sonosensitizer protoporphyrin IX(Pp IX)in different phases of the cell cycle,which may result in differences in sensitivity to 5-ALA-induced SDT.This study aimed to investigate the effect of the cell cycle on susceptibility of SAS cells to SDT after synchronization to different cell cycle phases.Methods:To synchronize cells to the G0/G1 phase,S phase and G2/M phase,SAS cells were arrested by serum starvation,thymidine and HU,and nocodazole.After SDT with the same parameter,Spectra Max 5 microplate reader to detect 5-ALA-induced Pp IX,CCK-8 to determine cell viability,flow cytometry to detecte apoptosis and Western Blot to detecte protein(cyclin A,caspase-3 and Bcl-2).Xenograft in vivo experiments using BALB/CA nude mice by subcutaneous injecting SAS cells,Mice were randomly divided into three groups: control group,SDT group,HU group,and SDT+HU group.Portraying the growth of tumor and detecting the apoptosis by tunnel.Results: Our results indicate that the cell mortality rate and apoptosis rate of SAS cells in the S and G2/M phases were significantly higher after SDT than in G1-phase cells and unsynchronized cells,with a corresponding increase in Pp IX in S and G2/M cells.Different Pp IX levels produced from 5-ALA may elicit differing sensitivities to SDT in certain phases of the cell cycle.Moreover,caspase-3 increased while Bcl-2 decreased greatly in S and G2/M cells after SDT.Further,Cyclin A was more highly expressed in S and G2/M cells than in G1-phase cells.SDT also caused a significant upregulation of cyclin A in all cell cycle phases,but this was most marked in S and G2/M cells.After 5-ALA injected mice abdominal cavity 4h,the Pp IX levels in HU group is higher than in the control group.Combination of hydroxyurea followed by SDT enhanced the reduce of tumor growth and cell apoptosis introduced by SDT.Conclusions:Taken together,these data demonstrate that cells in S and G2/M phases generate more intracellular Pp IX,have high levels of cyclin A,and are therefore more sensitive to SDT-induced cytotoxicity.These findings provide a novel prospect of preventing cancer onset by combining cell-cycle regulators with SDT.This sequential combination therapy could be a simple and cost-effective way of enhancing the effects of SDT in clinical settings.