Study On The Effect Of Dianhydrogalactitol And It’s Anti-tumor Function Base On TopoⅡ In NCI-H460 Cells

Objective: To evaluate the antitumor activity of dianhydrogalactitol(DAG)in NCI-H460 cell line,and further clarify its underlying mechanisms.Methods:(1)Study on the effect of DAG on the proliferation of NCI-H460 cells.(1)The inhibitory effect of DAG in NCI-H460 cells was detected by CCK-8 assay and colony formation assay.The proliferation inhibition rates and half inhibitory concentration(IC50)of DAG were also determined.(2)The morphology of cells treated with DAG for 48 h was observed under optical microscope.(2)Study on the effect of DAG on the migration capabillity in NCI-H460 cells.(1)Wound healing assay was used to detect lateral-migration of NCI-H460 cells.(2)Transwell-migration assay was used to detect lateral-migration of NCI-H460 cells.(3)Study on the effect of DAG on DNA,Intracellular calcium level and mitochondrial membrane potential in NCI-H460 cells.(1)Nuclear morphology was captured by fluorescence microscopy after Hoechst 33342 staining.(2)DNA damage induced by DAG was detected by single cell gel electrophoresis.(3)Intracellular calcium level was measured by using Fluo-3 AM fluorescence dye staining.(4)Mitochondrial membrane potential was measured by using Rhodamine123 fluorescence dye staining.(4)Study on the effect of DAG on the expression level of topoisomerase Ⅱ(Topo Ⅱ).(1)Real-time PCR was used to analyze the expression level of topoisomerase Ⅱ(Topo Ⅱ)mRNA.(2)The protein expression level of Topo Ⅱ was detected by western blot.(3)Additionally,molecular docking approaches were used to predict the interaction between DAG and Topo Ⅱ.Results:(1)CCK8 assay and colony formation assay indicated that DAG exhibited potent antitumor activity in NCI-H460 cells,and inhibited cells proliferation persistently.The IC50 value of DAG treated for 48 h was 9.68±1.02μg/mL in NCI-H460 cells.(2)Wound healing assay and transwell-migration assay exhibited that DAG could inhibit the migration of NCI-H460 cells.(3)Mechanistic study indicated that DAG induced the apoptotic cell death in a dose-dependent manner.DAG obviously induced nuclear morphological changes of NCI-H460 cells,the adherent ability of cells weakened,and the nuclear condensed and chromatin gathered at edge or splintered.It was also observed that NCI-H460 cells treated with DAG showed increase of intracellular calcium level and depolarization of mitochondrial membrane potential.(4)Furthermore,DAG down-regulated the mRNA and protein expression levels of Topo Ⅱα,Topo Ⅱβ detected by Real-time PCR analysis and western blot,respectively.Molecular docking predicted that DAG can bind to Topo Ⅱ.Conclusion:(1)DAG significantly inhibited the proliferation of NCIH460 cells.(2)DAG significantly inhibited the migration capabillity of NCI-H460 cells.(3)DAG induced apoptosis of cells may be related to increase of intracellular calcium level,depolarization of mitochondrial membrane potential and DNA damage.(4)Its underlying mechanisms may be also relatedto down-regulation the mRNA and protein expression level of Topo Ⅱα,Topo Ⅱβ and directly bind to Topo Ⅱ,leading to cancer cell death.