| This research focused on the studies on the anti-tumor activity and anti-tumor mechanisms of the lysicamine(LY)and its four transition metal complexes.The main contents are as follows:1.Firstly,introduces liriodenine and lysicamine metal complexes and summarizes the research progress on apoptosis signaling pathways and cell cycle regulation.And the significance of the research was described.2.The antitumor activities of[Mn(LY)3](ClO4)2-3CH3Cl(1),[Zn(LY)2(C104)2](2),[Ru(LY)Cl2(DMSO)2]·3H2O(3)and[Rh(LY-OH)Cl3CH3OH](4)against a series of human tumor cell lines(T-24、Hep-G2、NCI-H460、BEL-7404)and human normal liver cell line HL-7702 were screeened by MTT method,and the cell apoptosis induction properties against Hep-G2 and NCI-H460 cell lines were examined by flow cytometry method(FCM).The results of MTT method indicated that complexes 1 and 4 exhibited higher antitumor activity against most of the tested tumor cell lines than that of the corresponding ligand LY.Intriguingly,their antiproliferative rates against Hep-G2,NCI-H460,T-24 cell lines were all higher than 50%,while did not show obvious growth inhibition against the normal liver cell line HL-7702(lower than 35%),suggesting that their potential cytotoxic selectivity for tumor cell lines.Among them,Hep-G2 cells showed the highest sensitivity to complex 4 with IC50 values of 7.56 ± 2.91 μm,which were nearly 2.5 times lower than that of cisplatin(IC50=18.51±0.78 μM),respectively.In addition,FCM results suggested that Hep-G2 and NCI-H460 cells were induced to early apoptosis under the treatment of complexes 1 and 4,with early apoptosis proportions of 13.0%and 17.2%,20.5%and 33.1%,respectively.3.Based on the results of cellular uptake,a panel of genes by RT-qPCR array,cell apoptosis,western blot of of cytochrome c,apaf-1,bcl-2 and bax proteins,measurement of ROS generation,Ca2+ fluctuation,caspase-3/9 activation assay,the cytotoxicity mechanism studies indicated that the apoptosis was likely caused by the disruption of mitochondrial function by treatments with complexes 1 and 4,which led to significant loss of mitochondrial membrane potential,increase of the reactive oxygen species,cytochrome c,bax,apaf-1,Ca2+ fluctuation and caspase-3/8/9 ratio and decrease of bcl-2,c-myc levels in the treated Hep-G2 and NCI-H460 cells,respectively.4.In molecular level,the cytotoxic activity of cell cycle arrest and cell cycle regulators between Hep-G2 or NCI-H460 cells and complexes 1 and 4 were investigated schemtically using flow cytometry method(FCM),a Panel of Genes by RT-qPCR Array and Western blot.Compared with the control cells,both complexes 1 and 4 induced Hep-G2 and NCI-H460 cell cycle arrest at the S phase.To investigate the effects of 1(14 μM)and 4(7μM)on the genes involved in cell cycle arrest and cell cycle regulators,we compared the mRNA expression profiles of untreated Hep-G2 cells and 1(14 μM)and 4(7 μM)treated cells using a human cell cycle PCR array(PAHS-020Z),which contains 84 well-known cell cycle and cell cycle regulators-related genes.Of the 84 genes,31 genes and 58 genes(i.e.,ATM、CDC25A、CDK2,etc.)were differentially expressed in mRNA levels by 1.5-fold or more after treated with 1(14μM)and 4(7 μM)for 24 h,respectively,which were in accordance with the results of cell cycle arrest by FCM.Furthermore,complexes 1 and 4 triggered the cell cycle blocked in S phase mainly via ATM-Chk2-Cdc25A-CDK2 and ATR-Chkl-Cdc25 A-CDK2 Chkl/Chk2-p53-p21-CDK2 pathways. |
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