Mechanism And Cytotoxicity Of Anticancer Zn²⁺ Tetraazamacrocvcle Complex

Cancer is one of the leading diseases which cause death to human. Chemotherapy is used as a common method for the therapy of cancer. At present, metal-based anticancer agents mainly focus on some noble metals and heavy metal complexes. Although the curative efficacy of cancer has been improved, it is still urgent to find some new inexpensive anticancer agents with low toxicity, excellent curative efficacy and universality. Zn is abundant in mammalian cells, and thus the risk of cytotoxic effects for Zn is lower than other transition metal complexes. Therefore, it is significantly important to find new Zn(Ⅱ) complexes with low toxicity, high-efficiency and explore the anticancer mechanism. In this thesis, We chose five common cancer cells (Hela, MCF-7, NCI-H446, A549, HepG2) as model cells to study the cytotoxic properties of three different Zn2+ tetraazamacrocycle complexes by MTT method. And RT-PCR, Western blotting, immunefluorescence staining technologies were performed to study the apoptotic mechanism of Hela induced by Li-Zn which has the best cytotoxic property in the cells. Finally we further demonstrate that AIF palys an important role in cell apoptosis in our study. This thesis is mainly divided into the following two aspects:(1) Study of the anticancer activity of the Zn2+ tetraazamacrocycle complexesAll of the three different Zn2+ tetraazamacrocycle complexes have good anticancer activities in the five common cancer cells. When the five cells treated with the three different Zn2+ tetraazamacrocycle complexes after 48 h, all of the IC50 values are below 50μM with a lowest value of 3.73μM. A good linear relationship is observed between the inhibited activity and the doze of the Zn2+ tetraazamacrocycle complexes. The inhibited activities of L-Zn in the five cells increase with the elevated doze of Zn2+ tetraazamacrocycle complexes. Besides, the inhibited activity of cell proliferation is enhanced with the elevated incubation time of L-Zn in the cells. Comparing all the three different Zn2+ tetraazamacrocycle complexes, L2-Zn has the best anticancer activity in cancer cells. Its IC50 value is lower than cisplatin. It is worth further exploring the mechanism of the anticancer activity of L2-Zn.(2) Study of the anticancer mechanism of L2-ZnL2-Zn is selected for further study to explore the anticancer mechanism due to its best anticancer activity in Hela. RT-PCR results show that the expression of the Caspase inhibitor IN mRNA in Hela is concentration-dependent. The expression level increases with the elevated concentrations of L2-Zn. The expression of AIF mRNA only increases a little. No obvious difference is observed comparing with the control experiment. In the WB experiments, no activated form of caspase 3, caspase 6 and caspase 7 in Hela incubated with L2-Zn has been detected and the expression level of these Procaspase remains the same as in Hela without L2-Zn. These results demonstrate that the process of cell apoptosis induced by L2-Zn is Caspase-independent. We separated nucleus and cytoplasm from the cell and detected the expression level of AIF in whole cell, nucleus and cytoplasm, respectively. It is found that the expression level of AIF in whole cell only increases a little after addition of L2-Zn to Hela. It is consistent with the changes of nucleic acid levels. Besides, the expression of AIF in cytoplasm decreases and the expression of AIF in nucleus increases significantly. It is indicated that AIF transfers from the cytoplasm to nucleus during the incubated process with L2-Zn in Hela. The transfer of AIF leads to the cell apoptosis. We also used immunofluorescence staining and confocal microscopy to further confirm the position change of AIF from the cytoplasm to nucleus before and after addition of L2-Zn to Hela. The process of cell apoptosis induced by the zinc complex does not depend on the activation of the Caspase pathway, which is different from cisplatin. Therefore, this kind of complex may solve the tumor resistance and side effects caused by the classic platium drugs. It is promising to find some new inexpensive anticancer agents with low toxicity, excellent curative efficacy and universality.