Studies On The Synthesis Of Metal Complexes And Antitumour Activity Of These Complexes Interaction With Human Serum Albumin

Two types of metal complexes have been synthesized in this dissertation. The first kinds of metal complexes are copper metal complexes derived from Schiff base thiosemicarbazones. The second types of metal complexes are derived from natural plumbaginas. The structures of these metal complexes were characterized by elemental analysis, IR, ESI-MS and single-crystal x-ray diffraction analysis. From the cellular level, antitumor activity of these metal complexes in vitro and tumor cells apoptosis induced by them have been examined by MTT assay, flow cytometry and fluorescence photographs. At the molecular level, we have used Western Blot analysis to detect the expression of proteins which were related to the cells apoptosis and cycle influenced by these metal complexes. The content of this dissertation mainly are as follows:1. Briefly introduces the tumor present situation and the general situation of the anticancer drugs. Summarizes the research progress of thiosemicarbazone Schiff base and its metal complexes of biological activity. The study of human serum albumin （HSA） as a drug carrier targeted therapy of cancer has made a brief introduction, and on this basis, a brief exposition of the topic of this paper and significance.2. Copper （Ⅱ） compounds are a promising candidate for next generation metal anticancer drugs and have been extensively studied. Therefore, four binuclear copper（Ⅱ） compounds derived from Schiff base thiosemicarbazones （L1-L4）, namely [CuCl（L1）]2 （Cl）, [CuNO₃（L2）]2 （C2）, [Cu（NCS） （L3）]2 （C3） and [Cu（CH₃COO） （L4）]2 （C4） were synthesized in this chapter. the four kinds of complexes were characterized by x-ray diffraction analysis. The antitumor activity in vitro and mechanism of complexes C1-C4 and C4-human serum albumin complex were determined by MTT assay, fluorescence microscope photograph, flow cytometry assay and Western Blot. The results are as follows: （1） The antitumor activity against the human cancer cells （BEL-7404, A549, NCI-H460） in vitro of Cu （Ⅱ） ion and ligand was evaluated by MTT method, but the effect is not good. However, the C1-C4 complexes have higher cytotoxicity to cancer cells than the free ligand and Cu （Ⅱ） ion. Among the C1-C4 complexes, C4 complex was found to have the highest cytotoxicity, and the C1 complex was the weakest cell toxicity. The cytotoxicity of C4 complex is two times of C2 and C3 complexes, as well as C2 and C3 complexes have twice toxicity comparing with Cl complex. The IC50 values of C4 complex in turn for the above three kinds of cancer cells is 0.486 ± 0.010,0.507 ± 0.021,0.235 ± 0.010 μM. Of course, once they interact with human serum albumin, the toxicity for the tumor cells will increase, but the IC50 values will be reduced. （2） Taking human hepato cellular carcinoma BEL-7404 as the representative, we studied the Cl-C4 complexes and C4-human serum albumin complex on the mechanism of tumor cells. Fluorescence staining assays （AO/EB） and FITC Annexin V/propidium iodide test showed that the complexes above could induce apoptosis. In order to further investigate their mechanism of antitumor, JC-1 dyeing experiment demonstrated that Cl-C4 complexes and C4-human serum albumin complex induced the decrease of mitochondrial membrane potential. After staining with JC-1, the cells gradually changed from red to green fluorescence, which showed that the cancer cells had apoptosis. ROS and GSH/GSSG experimental results showed that compared with the blank control group, C1-C4 complexes and C4-HSA complex raised the levels of reactive oxygen species in the tumor cells, however the GSH/GSSG ratios decreased. Among the C1-C4 complexes, C4 complex made the level of ROS become the highest and GSH/GSSG ratio the lowed, but Cl complex on the contrary, this may be related to the functional groups associated with the four complexes. （3） The levels of the Bcl-2 family protein, caspase-3, caspase-9 and cytochrome C of C1-C4 complexes and C4 interact with human serum albumin in BEL-7404 cells were measured by Western Blot. The results indicated that the expression of anti apoptotic protein Bcl-2 and Bcl-xl was down regulated, the expression of pro apoptotic protein Bax was up-regulated, resulting the ratio of Bax/Bcl-2 increased, which made tumor cell apoptosis be more likely to occur. In addition, the release of cytochrome C promoted the caspase cascade. The experimental results confirmed that the mechanism of antitumor activity induced by C1-C4 complexe and C4-HSA complex was mediated by intrinsic mitochondrial apoptotic pathways.3. Four copper （Ⅱ） metal complexes were synthesized with the natural plumbagin （PLN） as ligand:[Cu（PLN）2]·2H₂O （C1）, [Cu（PLN）（Bipy）（H₂O）]NO₃·2H₂O（C2）; [Cu（PLN）（Phen）]NO₃·0.25H₂O（C3）; [Cu（PLN）（Phen）（Br）]CH₃OH （C4）. The structures of these metal complexes were characterized by elemental analysis, IR, ESI-MS and single-crystal x-ray diffraction analysis. The results showed that the four copper metal complexes are foundation and the security for the subsequent research of antitumor biological activity and introduced by metal matrix drug. The folate （FA）-functionalized HSA carrier （FA-HSA） is promising for improving the target and efficiency of anticancer drugs. To develop FA-HSA carrier for metal anticancer drugs, we investigated anticancer properties and mechanism of FA-HSA carrier for Cu（Ⅱ） complexes derived from PLN. The fluorescence spectroscopy and molecular docking revealed that Cu（Ⅱ） complexes bind to IIA subdomain of HSA. The cytotoxicity introduced by the four copper complexes in the presence or absence of FA-HSA carrier were determined by the MTT assay against two human tumor cell lines （MCF-7 and HeLa） and normal cell HL-7702. The results showed that Compared with Cu（Ⅱ） complexes alone, FA-HSA-metallodrug complexes enhances cytotoxicity to FA-positive cancer cells （HeLa）, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent with separate copper（Ⅱ） metal complexes in tumor cells to some extent. The results of flow cytometry showed that FA-HSA-metallodrug complex has a stronger capacity for cell cycle arrest in the G2/M phase of HeLa cells, and down-regulating the expression of cyclin-dependent kinase 1 （CDK1） and cyclin B1. Moreover, FA-HSA-metallodrug complex promotes HeLa cells apoptosis through intrinsic reactive oxygen species （ROS） mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.In this paper,we mainly studied two kinds of metal complexes. Our studies may be helpful to provide the experimental and theoretical basis for the future development of novel anticancer metal complexes and develop targeting therapy of metal anticancer drugs through rational design HSA carrier...