| In order to investigate the effect of the group at site 1 of oxoaporphines alkaloid and its metal complexes on antitumor activity.Three kinds of oxoaporphines alkaloids with structure-activity relationship have been synthesized and charcterized.Utilization of this three kinds of oxoaporphines as ligands to react with Au,Co,Mn,Ru,Pt salts and other metal ions afforded seventeen metal complexes,and utilization of oxo-papaverine as a ligand to react with Ru([2,2’]Bipyridinyl)2Cl2 and Ru(4,7-Diphenyl-[1,10]phenanthroline)2Cl2 afforded two metal complexes.The main contents are as follows:1.The research progresses of the metal-base anticancer drugsand their clinical applications have been reviewed.The research advanceson the aporphine alkaloids with antitumour activity and action mechanism also have been summarized.Basing on these results,the study significance and basis have been provided.2.Three kinds of oxoaporphines alkaloids with structure-activity relationship have been synthesized.Seventeen metal complexes have been synthesized by reaction of these three kinds of oxoaporphines alkaloids with Au,Co,Mn,Pt,Ru salts The utilization of oxo-papaverine as a ligand to react with Ru afforded two metal complexes.All complexes as follows:[OG1:H][AuCl4]·2H2O(1)[Co(OG1)2(NO3)2](2)[Mn(OG1)2(CH3OH)2](ClO4)2(3)[Pt(OG1)(DMSO)Cl](4)[Ru(OG1)Cl2(DMSO)2](5)[Ru(OG1)(4,7-Diphenyl-[1,10]phenanthroline)2](PF6)2(6)[OG2:H][AuCl4]·2H2O(8)[Ru(OG1)([2,2’]Bipyridiny1)2](PF6)2(7)[Co(OG2)2(NO3)2](9)[Mn(OG2)2(CH3OH)2](ClO4)2(10)[Pt(OG2)(DMSO)Cl](11).[Ru(OG2)Cl2(DMSO)2](12)[Co(OG3)2(NO3)2](16)[OG3:H][AuCl4]-2H2O(15)[Ru(OD)([2,2’]Bipyridinyl)2](PF6)2(18).[Ru(OG2)(4,7-Diphenyl-[1,10]phenanthroline)2](PF6)2(13)[Mn(OG3)2(Cl)2](17)[Ru(OG2)([2,2’]Bipyridinyl)2](PF6)2(14)[Ru(OD)(4,7-Diphenyl-[1,10]phenanthroline)2](PF6)2(19)The structure of all the complexes were determined by 1H NMR,ESI-MS spectrometry and single crystal X-ray diffraction analysis.Beside,ICP-MS was used to detect the hydrophilic and lipophilic properties of the seventeen metal complexes.3.By MTT assay,we conduct the preliminary screening of antitumor activity in vitro for these three kinds of linds and their metal compounds against 5 kinds of tumor cell lines(MG-63,SK-OV-3,T-24,Hep-G2,A549),and human normal liver cell line HL-7702 and calculate the corresponding IC50 value.The antitumor activity in vitro assay results showed that the complex 6 on five kinds the tumor cell lines exhibited good activity;complex 10 on T-24 cell lines shows higher activity.Complex 16 on T-24,Hep-G2 and MG-63 cell lines exhibited good activity.In addition,compared with these three kinds of ligands,most of the metal complexes exhibited higher cytotoxicity.4.The antitumor mechanism of complexes 6 and 16 on MG-63 cell lines,complex 10 on T-24 cell lines,complexes 4 and 11 on Hep-G2 cell lines were investigated by confocal microscopy,flow cytometry,ICP-MS and Western Blot.The results are shown as follows:Ⅰ、Flow cytometry was used to detect the effects of complexes 4,6,10,11 and 16 on the cell cycle of corresponding tumor cell lines.The results show that complexes 6 and 16 caused MG-63 tumor cells arrest in G2 phase;complex 10 make the T-24 tumor cells be arrested in S phase;complex 4 make the Hep-G2 tumor cells be arrested in G2 phase while complex 11 make the Hep-G2 tumor cells be arrested in G1 phase.Ⅱ.By means of AnnexinV-FITC/PI staining,mitochondrial membrane potential(JC-1)changes,intracellular reactive oxygen species(ROS)level changes,intracellular Ca2+ level and caspase 3/8/9 activity assay experiments,complexes 4,6,10,11 and 16 on the corresponding tumor cell apoptosis were assayed.The results show that complexes 6,10,16 can induce the 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 pathways.Complexes 6 and 13 induce apoptosis through mitochondria-dependent and caspase-independent pathways.Complexes 10 and 16 can activate caspase 3/8/9 and induce apoptosis through mitochondria-dependent and caspase-dependent pathways.In addition,complexes 16 can induce autophagy on MG-63 tumor cells,while complex 10 causes both apoptosis and autophagic death of T-24 tumor cells.Ⅲ.Western Blotting was used to detect the expression of proteins related to cell cycle and apoptosis.The experimental results show that the complexes 6 and 16 can cause cell cycle protein Cyclin B1,CDK1 and Cdc25C decreased.In a word,complexes 6 and 16 that arrest cell cycle in G2 phase were closely related to CyclinBl-CDK1 and Cdc25C-CDK1.Compared with the control group,complex 10 can significantly up regulate the expression of p53,p21 and p27 protein,and down regulate the expression of Cyclin E1,Cyclin A2,CDK2 and Cdc25A in the corresponding tumor cells.Ultimately,the p53-p21-CDK2 pathway is activated,which makes the T-24 tumor cells be arrested in S phase.In addition,the complexes 6,10 and 16 also can cause the apoptosis protein Bcl-2 expression down regulated and the expression of apoptotic protein Bax increased on corresponding tumor cells.The results indicated that complexes 6,10 and 16 could mediate the mitochondrial pathway and induce the apoptosis by reducing the ratio of Bcl-2/Bax.At the same time,complexes 10 and 16 both caused the up regulation of LC3B expression,and the ratio of LC3B Ⅱ/LC3B Ⅰ increased,indicating that complexes 10 and 16 could induce autophagy in corresponding cells. |
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