Studies On Anticancer Mechanism Of Oxoaporphine Alkaloid Of Sugar Base Class And Its Transition Metal Complexs

Primary liver cancer is one of the most common malignant tumors in clinical,ranking sixth in the global cancer incidence and third in the mortality rate.Especially,liver cancer shows higher incidence in China,ranking the second in the mortality rate in all the malignant tumors.Therefore,it is urgent to search for anticancer drugs towards liver cancer with high efficiency and multi-targeting characters.The oxoaporphine alkaloid is a kind of natural products yielded from many herbal plants,most of which showing significant antibacterial,antitumor,antioxidant activities as well as other bioactive mechanism.The prospect of oxoaporphine alkaloids is attractive for their medicinal applications.Our research group has primarily developed the research field on the syntheses and antitumor activity studies of oxoaporphine alkaloids in their metal complexations for more than ten years.However,the detailed antitumor mechanism of these metal complexes of oxoaporphine is still not fully understood.So it is necessary to make more efforts to futher explore and discuss the exact antitumor mechanism of the metal complexes of oxoaporphine alkaloids.In this dissertation,we studied the in vitro anticancer activity and anticancer mechanism of 9-hydroxyl(acetyl glucose ester)-2-isopropyloxy-1,10-dimethoxy-7-oxoaporphine alkaloid and its five transition metal complexes.This thesis mainly includes the following contents:Firstly,the research discovery and progress of the S phase cell cycle arrest and apoptosis inducing by death receptor pathway was introduced,as well as the relationship between cell cycle,cell apoptosis and autophagy.On this basis,the elaboration of selection significance and the strategy of this research were contained in this thesis.The in vitro antitumor activity of four transition metal complexs of 9-hydroxyl-2-isopropyloxy-1,10-dimethoxy-7-oxoaporphine alkaloid(OD),which were OD-Ru(Ⅱ)(complex 1),OD-Rh(Ⅲ)(complex 2),OD-Ni(Ⅱ)(complex 3),OD-Zn(Ⅱ)(complex 4)and one transition metal complex of 9-acetyl glucose ester-2-isopropyloxy-1,10-dimethoxy-7-oxoaporphine alkaloid(OD-G),which was OD-G-Zn(II)(complex 5)were tested by MTT and CCK8 method,towards a series of human tumor cell lines,including MGC80-3 human gastric cancer cells,BEL-7404 human liver cancer cells.NCI-H460 human lung cancer cells,Hep-G2 human liver cancer cells,MG-63 people osteogenesis cells and HL-7702 human normal liver cells.The results showed that complexes 1,2,3,4 and 5 exhibited higher inhibitory activity on Hep-G2 human liver cancer cells line.In addition,flow cytometry(FCM)was used to analyze the cell cycle and apoptosis on HepG2 cells.The results indicated that complexes 3,4,and 5 could arrest cell-cycle on S phase,and cell apoptosis was induced on HepG-2 cells after treatment with complex 5,and the apoptotic rate increased in a dose-dependent manner,but complexes 3 and 4 could not induced obvious cell apoptosis.This result was confirmed by morphological analysis experiment with Hoechst 33258 and DID as cell staining indicator.The intracellular ROS level and the concentration of Ca2+ were further detected by flow cytometry.The results showed that ROS level and the concentration of calcium increased with the increasing concentrations of complexes 3,4 and 5,comparing with the OD ligand,the primary metal salts,which demonstrated the similar variation trend in ROS level and the concentration of Ca2+.But ROS level and the concentration of Ca2+ were significant diverse,when the cells were treated with complex 4 and 5,both of which were coordinated with the same central metal,Zn(II).But the ROS level and the concentration of calcium fluctuated with increase of concentration of complexes 3 and 4.The expression level of LC3B which was the marker protein of autophagy,was detected with confocal laser scanning microscope,indicating that the treatment of Hep-G2 cells with complexes 3,4 and 5 for 24 h could induce autophagy,and 5 could caused significant autophagy and enhances apoptotic cell death.According to the mentioned results,we can infer that complexes 3 and 4 cannot trigger cell apoptosis due to activating cellular self-protection autophagy.On the contrast,complex 5 could induce autophagic apoptosis on Hep-G2 cells.The activation of Caspase3,Caspase8,Caspase9 in Hep-G2 cells treated with complexes 3,4 and 5 for 24 h was further studied.Compared with the control,Caspase3,8 and 9 could be significantly activated to induce apoptosis by complex 5,and the mechanism might be involved in the death receptor.However.only Caspase3 could be activated after treating cells with complexes 3 and 4.The distribution of the metal complexes in the treated HepG-2 cells were measured by ICP-MS.The results have shown that a large amount of metal accumulated in nucleus after treatment with complexes 3 and 4,and complex 5 was mainly located on cell membrane and nucleus.RT-PCR assay and Western Blotting were used to detect the expression levels of RNA and protein related to cell cycle and apoptosis.It was found that epidermal growth factors receptor(EGFR)in Hep-G2 cells could be activated,following transducing signals to a series of downstream factors,ultimatedly arresting cell cycle when processing with complexes 3 and 4 for 24 h.The expression levels of cell cycle protein in S phase,such as Akt was unchanging and p21,p27were increased and PCNA,CDK2,Cdc25A,c-myc,CyclinA2,CyclinE1 were decreased.In a word,complexes 3 and 4 that arrest cell cycle in S phase were closely related to Akt-p27-CDK2,Akt-CyclinD-CDK2 and c-myc-Cdc25A-CDK2,c-myc-p21-CyclinE1-CDK2 signal pathways.Furthermore,complex 5 could up-regulate the expression leves of apoptosic related protein:Fas,Caspase 8,Caspase 3,PARP and autophagic related protein:LC3B and Beclinl.All together,complex 5 could trigger HepG-2 cell apoptosis via death receptor signal pathway,meanwhile induced cell autophagy.In conclusion,due to structure-activity relationship,complexes 3,4 and 5 possessed different antitumor mechanism:1.The antitumor mechanism of complexes 3 and 4 was various in Hep-G2 cells.It is likely to be caused by both autophagy and cell cycle arrest in S phase.2.The antitumor mechanism in Hep-G2 cells of complex 5 was versatile.For instance,(1)cell cycle arrest on s phase,and(2)the cause of autophagy enhances ROS-dependent apoptotic cell death,via death receptor signal pathway.