Studies On The Antitumor Mechanism Of Three Kinds Of Liriodenine Metal Complexes Inducing Cell Cycle Arrest And Apoptosis

The Liriodenine has significant antibacterial,antitumor,antiviral and other broad pharmacological activity.In this paper,with Liriodenine and its metal complexes as the research object from the three fields basic research in modern molecular oncology:cell cycle regulation,signal transduction and cell apoptosis,We deeply study its antitumor activity and mechanism in vitro.The main contents are as follows.1.Briefly introduce the Liriodenine and its metal compounds’ research progress of biological activity and the research progress of cell apoptosis signaling pathway and cell cycle regulation.And on this basis,this paper expounds the significance of the topic and the research content.2.By MTT assay,we conduct the preliminary screening of antitumor activity in vitro for Liriodenine and its metal compounds against 8 kinds of tumor cell lines(NCI-H460,T-24,MGC80-3,BEL-7404,Hep G2,HeLa229,MG-63,SK-OV-3)and human normal liver cell line HL-7702 and calculate the corresponding IC50 value.Liriodenine and its metal compounds are as follows:[Co2(LA)2Cl4(CH3OH)2](1-Co),Rh(LA)Cl3CH3OH](2-Rh),[Ir(LA)Cl3CH3OH](3-Ir),[Sm(LA)2(NO3)3](4-Sm),[Eu(LA)2(NO3)3](5-Eu),[Pr(LA)2(NO3)3](6-Pr).Through comparing the IC50,we find that 1-Co complex shows the higher inhibitory activity to NCI-H460,T-24 and MGC80-3 cells,However 4-Sm and 6-Pr complexes performance the higher inhibitory activity to T-24 and HeLa229 cells.In addition,most of the metal complexes in antitumor activity are the higher than the ligand.And we study the effect of these complexes on Cell morphology of T-24 cells by microscope observation.3.Using flow cytometry to detecte the effect of 1-Co and ligand on the cell cycle of NCIH460,T-24 and MGC80-3 and the effect of 4-Sm,6-Pr and ligand on the cell cycle of T-24 and HeLa229,The results show that 1-Co,4-Sm and 6-Pr make the corresponding tumor cells be arrested in S phase and have drug concentration dependence.However,the effect of ligand on the cycle arrest is not obvious.By confocal microscopy to detect Alexa Fluor? 488 and Hoechst 33342 staining.The results indicate that 1-Co,4-Sm，6-Pr can inhibit cell proliferation by affecting the DNA replication and cell cycle of the corresponding tumor cells.4.The 1-Co with better antitumor activity and less toxicity to normal cells were selected,and the NCI-H460 cells were choosed to screen differentially expressed genes in cell cycle regulation pathway and pick out candidate genes that change significantly by gene chip technology.Then we use fluorescence quantitative RT-PCR to verify the candidate genes,and detect its influence on cycle proteins and DNA damage by Western blot analysis.In the same way,we study the mechanism of cell cycle arrest in T-24 and MGC80-3 cells induced by 1-Co.The results show that the fluorescence quantitative RT-PCR results are basically consistent with the results of gene chip.The mechanism of 1-Co on three tumor cells is similar.1-Co can significantly up regulate the expression of p-ATM,p53,p21,p27 and yH2AX protein,and down regulate the expression of cMyc and CyclinA2 in the corresponding tumor cells.It can also damage DNA and activate ATM kinase.Activated ATM further modifies p53,and then promote the expression of p21,p27 which are CDK2 inhibiting factor.Thereby inhibiting CDK2 activity,and then hindering the formation of CyclinA2/CDK2 complexes.Ultimately,the ATM-p53-p21-CDK2 pathway is activated,which makes the corresponding tumor cells be arrested in S phase.5.By AnnexinV-FITC/P1 staining,mitochondrial membrane potential(JC-1)changes,intracellular reactive oxygen species(ROS)level changes,intracellular Ca2+level and Caspase 3/8/9 activity detection experiments,1-Co,4-Sm,6-Pr are detected for the effect on the corresponding tumor cell apoptosis.The results show that 1-Co,4-Sm,6-Pr can make corresponding tumor cells at the early stage of apoptosis.The level of ROS in the cells was increased,calcium ion release,resulting in the decrease of mitochondrial membrane potential,enhanced membrane permeability,and then induced apoptosis.Induction of apoptosis through non Caspase dependent pathway.1-Co activation Caspase-3/8/9 is not obvious in the corresponding tumor cells.4-Sm and 6-Pr can significantly activate Caspase-3/8/9 in T-24 cells.That 1-Co induces apoptosis pathway with mitochondria,But the endogenous pathway is not the main pathway of apoptosis.4-Sm and 6-Pr induce apoptosis through the endogenous pathway of mitochondria.1-Co activation of the corresponding tumor cells Caspase-3/8/9 is not obvious.4-Sm and 6Pr can significantly activate Caspase-3/8/9 in T-24 cells.That 1-Co induces apoptosis pathway with mitochondria,But the endogenous pathway is not the main pathway of apoptosis.4-Sm and 6-Pr induce apoptosis through the endogenous pathway of mitochondria;6.The mechanism of 1-Co on the apoptosis of the tumor cells is further studied from the protein level by means of Western Blot analysis.The experimental results show that 1-Co by down-regulating of Bcl-2/Bax ratio,mediated by the mitochondrial pathway induces cell apoptosis.By inhibiting Erk activation and reducing the expression of transcription factors c-Myc in the nucleus,1-Co mediates Ras/Erk/Myc signaling pathway to inhibit cell proliferation.In addition,it up regulate the expression of Caspase-12 protein and mediate endoplasmic reticulum pathway to make the cell apoptosis.In a word,this is a variety of apoptotic pathways which are interrelated and intertwined inducing apoptosis.