Cytotoxicity Of Zn²⁺ Tetraazamacrocycle Complex And Arsenic In Cells

At present, metal-based anticancer agents mainly focus on some precious metals and heavy metal complexes, which has a wide range of applications, however, treatment gives great agony to patients at the same time., Platinum-based antitumor drugs have had a strong impact on cancer chemotherapy and constituted a cornerstone of the treatment for various solid tumors. Cisplatin, the typical representative of a class of platinum-containing antitumor drugs, has been used for more than three decades in standard chemotherapy regimens and still occupied broad market. However, it has been greatly hampered by drug resistance and severe side effects, like nephrotoxicity and neurotoxicity. Therefore, exploring new antitumor drugs, especially metal complexes with good biological compatibility, is one of effective methods.Acute promyelocytic leukemia (APL) is one of the most common malignancies in blood system. AS2O3used for clinical treatment of APL has made a success and become the most effective treatment measures, but the etiology of APL and the mechanism of AS2O3have not been entirely clear. Probing into the cause of the disease and developing effective drugs are of great significace to ease the the suffering of patients and improve the human health. The methylation of inorganic arsenic (iAs) is the main arsenic metabolism pathway in mammals, but the role played by the arsenic metabolietes in the treatment of malignant tumor has been ignored by many researchers. It is useful to establish transfected cells with stable expression of human Arsenic (+3oxidation state) methyltransferase(hAs3MT)to research the metabolism of AS2O3in recombinated leukemia cells, and control the methylation to generate only specific methylated arsenicals for deeply researching the effects on recombinated cells. Thus discussing the molecular mechanism of AS2O3in APL therapy will provide effective information for the comprehensive study of arsenic metabolism pathway and the clinical treatment for leukemia. In this thesis, we will focus on exploring the cytotoxic properties of trivalentarsenic and Zn2+tetraazamacrocycle complex from our laboratory.Chapter1reviews the history of antitumor drugs’ development, achievements and existied problems, especially the situation of metal complexes in this field.. The chapter also introduces the toxicity of arsenic in arsenic biological metabolism and treatment for leukemia. Besides, mechanism of action, and clinical resistance and experimental methods of cell apoptosis are also included in this chapter.Chapter2mainly studies the anti-tumor activity and mechanism of Zn2+tetraazamacrocycle complex, including cytotoxicity, cytomorphology, the cell apoptosis, etc. The complexes recognizes, binds and hydrolytically cleaves DNA in vitro, and cell experiments show that the complex also has good antitumor activity, especially considerable cytotoxicity against human Hela cells and MCF-7cells. Compared with cisplatin, the complex has a lower IC50in vitro, and stronger antitumor activity. In addition, from further research of cell apoptosis induction mechanisms of this complex, we find low concentration can induce cell death with the peculiar phenomenon, including pyknotic nuclei, crushing, PS evaginate and membranes damage. The process of cell apoptosis induced by the zinc complex does not depend on the activation of the caspase-3pathway, which appears to be different from cisplatin. Therefore this kind of complex may solve the problems of tumor resistance and other side effects caused by the classic platium drugs. A lot of metallic antitumor agents have been thoroughly studied in the anticancer drug design, such as platinum, gold, and ruthenium precious metals, but the function of zinc complexes have been seldom referred. Zn2+, which exists in biological systems, contributes widely to DNA synthesis, apoptosis, gene expression, protein stability and catalytic functions. Thus, exploring the effects of Zn2+tetraazamacrocycle complex on cells is of great significance, with the potential value of antitumor application, which may overcome some of the shortcomings of current antitumor drugs.Chapter3is a preliminary study of the molecular mechanism of arsenic metabolietes in the therapy of leukemia. In this chapter the necessary cell lines HepG2, HL60and NB4have been estabilshed. Based on HepG2cells, cytotoxicity of trivalent arsenic is studied and the metabolites of arsenic are analyzed by HPLC-ICP-MS. In addition, the eukaryotic expression vector of human Arsenic (+3oxidation state) methyltransferase (hAs3MT) pEGFP-N1-AS3MT is established and has been successfully transfected to HepG2cells. As-metabolites both in cell lysate and medium are also detected. We expect to establish a stable expression of hAs3MT in recombinated HL60and NB4cells in the next. We hope to clarify the role of arsenic metabolites in the apoptosis, differentiation and growth inhibition of the recombinated NB4and HL60cell lines, and finally determine the molecular mechanism of AS2O3in APL therpy. The difference arsenic tolerance between normal cells and transfected cells has been demonstrated, but time is limited, the arsenic metabolites has still not be determined, it is necessary to perform further study and optimize experimental methods.