Studies On Anticancer Mechanism Of Fivel Transition Metal Complexes With Schiff Bases Derived From Homopiperonylamine

In recent years,since Schiff base and its metal complexes were shown to have antibacterial,anticancer,and other biological activity,which have become the hot topic in the medicinal inorganic chemistry.This thesis focus on the studies on the anti-tumor activity and anti-tumor mechanisms of pepper amine Schiff base and its five transition metal complexes.The main contents are as follows.1.Firstly Introduces the Schiff base metal complexes and summarizes the research progress on apoptosis signaling pathways and cell cycle regulation.And the significance of the research wasdescribed.2.The in vitro antitumor activity of five transition metal complexes:[Cu2(La)2Cl2](1);[Pt(Lc)Cl(DMSO)](2);[Cu(BPA)Cl2](3);[Zn(BPA)Cl2](4);[C9H10AuCl2NO2](5)against T-24,DLD-1,NCI-H460,HCT116,BEL-7404,A549,SK-OV-3,Hep-G2,HeLa,MGC80-3 and HL-7702 cell lines were tested by MTT method.The results show:all the complexes on T-24 human bladder cancer cells and DLD-1 human colorectal adenocarcinoma epithelial cell lines exhibited higher inhibitory activity,and most metal complexes exhibited higher anti-tumor activity than the free ligand.Experimental results show that the T-24 cells and DLD-1 cells which were treated with complexes 1-5,respectively appeared early apoptotic phenomenon,and its rate of apoptosis was concentration-dependent manner.In DLD-1 cell,the complexes of 1,2 arrested the cell cycle in S phase and complexes 3,4,5 arrested the cell cycle in G1 phase.In T-24 cells in the cell cycle of testing,all the complexes arrested the cell cycle in S phase.3.Using confocal microscopy to detect Hoechst 33258 and DID staining,and TUNEL to detect early apoptotic cells.The results show that:the complexes can induce the T-24 cells apoptosis.4.Using flow cytometry and confocal microscopy to detect the decrease of mitochondrial membrane potential(JC-1),the release of Cytochrome C,ROS and Ca2+from apoptotic cells,the results show that complexes 1-5 can make Cytochrome C released from the mitochondria into the cytoplasm and cause the accumulation of ROS and the release of Ca2+within the T-24 cell.The experimental results suggest that complexes 1-5 can activate Caspase-3 Caspase-8,Caspase-9 by flow cytometry.The results show that:the complexes of inducing apoptosis is associated with mitochondrial pathway.5.Application of comet electrophoresis analysis,the experimental results show that complexes 2,5 can cause DNA damage in the T-24 cell.6.By RT-PCR method to detect effects of the compounds on the complexes 2,5 on apoptosis pathway and cell cycle pathways downstream signaling molecule mRNA expression levels,the results indicate that the complexes can inhibit caspase 12,Bcl-2 p33,CDK4,PCNA gene expression,and promote p53,Bax,Bid,Cyt-C,Apaf-1,Caspase-9,Caspase-8,Caspase-3,p21 gene expression.7.Application Western Blot analysis,the results revealed that Bcl-2 expression was down-regulated while p53,Bax,Cyt-C and Apaf-1 expression levels were strikingly increased,thereby activating the Caspase-9,further activate Caspase-3.Also found significantly increased death receptor pathway in Fas,Caspase-3,Caspase-8 protein expression.The Experiment proved complexes by mitochondrial apoptosis pathway and death receptor apoptotic pathway induced apoptosis in T-24 cell.The complexes caused cell cycle arrest in S phase by upregulating p53,p27,p21,p16 protein,and thus reduced c-myc,CDK2,CDK4,Cyclin A2,Cyclin E1,Cyclin D1,PCNA protein.The results indicate that the complexes regulate the cell cycle by CKI-Cyclin-CDK pathways.In summary,pepper amine Schiff bases metal complexes induce apoptosis of cells in T-24 cell is not a single underlying mechanisms,it may be combined effect multiple signaling pathways.(1)The potential mechanisms of the metal complexes induced T-24 cell line apoptosis are possibly by inducing DNA damage,activaing the p53 protein decreasing mitochondrial membrane potential,elevating levels of intracellular ROS and Ca2+,activating apoptotic factor cytochrome c release to activate Caspase-9,thus Bax protein upregulated,Bcl-2 protein down,further activate downstream execution type Caspase-3 induced apoptosis.(2)The potential mechanisms of the metal complexes induced T-24 cell line apoptosis are possibly leading to increased expression of Fas protein activates Caspase-8,further activation of downstream execution type Caspase-3 induced apoptosis.(3)The complexes exerts the inhibitory effects possibly by decreasing expression of c-myc protein,CyclinEl-CDK2 and CyclinA2-CDK2 protein,raised p21 protein expression and induce p27 protein increased regulation of p53 protein,and reduced PCNA protein,the cells were arrested in S phase.