Studies On The Cell Cycle Arrest And Cell Apotosis Mechanism Of Oxoaporphine Derivative And Its Seven Transition Metal Complexes

The whole course of the cell cycle regulation is complicated and careful.Cell cycle checkpoints utilizing p53 can enhance cell survival and limit mutagenic events following DNA damage,concomitant interference with the ATM-Chk2-Cdc25A-CDK2 and ATR-Chkl-Cdc25A-CDK2 pathways.Mitochondria are important organelles in cells,which take part in the course of the cell apoptosis.Mitochondria can release a variety of apoptotic factors:Apaf-1,cytochrome c,caspases and so on into the cytoplasm,which cause apoptosis.According to lots of studies,mitochondrial pathway（intrinsic pathway）is the primary signal control and transduction of apoptotic way.This thesis investigated antitumor activity and antitumor mechanism of 9-hydroxyl-2-isopropyloxy-110-dimethoxy-7-oxoaporphine and its seven transition metal complexes.The mainly contents are as follows:1.This thesis firstly introduces the discovery of the cell cycle arrest and the mitochondrial apoptosis pathway and their relevant research progresses.Basing on these findings,the significance of the thesis has been proposed.2.The antitumor activity in vitro,cell cycle arrest and cell apoptosis of 9-hydroxyl-2-isopropyloxy-1,10-dimethoxy-7-oxoaporphine and its seven transition metal complexes（[Ru（OD）Cl₂（DMSO）₂]·CHCl₃（1）;[Rh（OD）Cl₃（CH₃CH₂OH）]（2）;[Co（OD）₂（H₂O）₂]（ClO₄）₂（3）;[Ni（OD）₂（CH₃OH）₂]（ClO₄）（4）;[Zn（OD）₂（H₂O）₂]（C10₄）₂（5）;[Ni（OD）₂（NO₃）₂]（6）;[Zn（OD）₂（NO₃）₂]（7））had been investigated.The IC50 value of complexes 3,4,5 in Hep-G2 cell lines is 0.66±0.11,0.20±0.09,0.39q0.03μM,while in T-24 cell lines is 0.45±0.17,0.23±0.13,0.51 ±0.17μM.According to flow cytometric analysis,Hep-G2 cell cycle were arrested at S phase after treatment with complexes 3,4,5,T-24 cell cycle were arrested at S phase by complexs 3 when complexe 4 and 5 disturb T-24 cell cycle arrest in G2 phase.3.The antitumor mechanism studied results from comet assay,RT-PCR,Western Blot analysis indicated complexes 3,4,5 triggered DNA damage in Hep-G2 cells,and initiated S phase arrest,as shown by the down-regulation of Cdc25 A,PCNA,Cyclin A2,and CDK2 and up-regulation of Chkl,Chk2,p53,p27 and p21.Finally,we found that complexes 3,4,5 exerted the cell cycle blocked in S phase via ATM-Chk2-Cdc25A-CDK2,ATR-Chkl-Cdc25A-CDK2 and Chkl/Chk2-p53-p21-CDK2 pathways.4.The antitumor mechanism studied results from nuclear staining（Hoechst 33258）,terminal deoxynucleotidyl transferase-mediated nick end labeling（TUNEL）analysis,acridine orange/ethidium bromide（AO/EB）staining and the mitochondrial membrane potential（JC-1）indicated that complexes 3,4,5 can induce Hep-G2 and T-24 cells apoptosis.Using a laser confocal microscopy to observe the reactive oxygen species（ROS）level and calcium ions increased,cytochrome c released into the cytoplasm after incubation with the.complexes 3,4,5,which is likely that the apoptosis is caused via a mitochondrial dysfunction pathway.Moreover,caspase-3/8/9 activated,decrease of Bcl-2,Bcl-xl,c-Myc expression,increase of Apaf-1,PARP expression in Hep-G2 cells after treatments with the complexes 3,4,5 by caspase-3/8/9 ratio,RT-PCR and Western Blot experiments.In short,apoptosis was caused by the disruption of mitochondrial function.Taken together,complexes 3,4,5 with 9-hydroxyl-2-isopropyloxy-1,10-dimethoxy-7-oxoaporphine alkaloid（OD）trigger the cell cycle blocked in S phase via ATM-Chk2-Cdc25A-CDK2 and ATR-Chk1-Cdc25A-CDK2 Chk1/Chk2-p53-p21-CDK2 pathways and the cell apoptosis through a mitochondrial dysfunction pathway.