Chemistry And Biology Mechanisms Of HepG2 Cell Apoptosis Induced By Cu(Ⅱ) Reactors And Electrophiles

Currently, prooxidant-mediated cancer chemoprevention and chemotherapy strategy has attracted much attention. This strategy is based on a main biochemical property difference between cancer and normal cells:the formers, compared to the latters, need increased generation of ROS to maintain their malignant phenotypes, making them more vulnerable to further production of ROS. This thesis focused on this theme, and studied on chemistry and biology mechanisms of HepG2 cell apoptosis induced by hydroxychalcones and resveratrol analogs as Cu(II) reactors and curcumin as electrophiles. The main contents are as follows:(1) Low concentrations of 3,4,3’,4’-tetrahydroxychalcone and Cu(II) ions could effectively induce DNA damage and apoptosis of human hepatoma HepG2 cells by promoting ROS production. Chemical mechanism studies indicate that sequential proton loss electron transfer (SPLET) steers the oxidation process. Additionally, ortho-dihydroxy groups on A (the aromatic ring directly attached to a carbonyl group) and B (the aromatic ring connected by a double bond) rings harbor different sensitivity and role for the oxidation; the former provides acidic hydrogen, the latter results in ortho-quinone.(2) A short-term cooperation of Cu(Ⅱ) ions and resveratrol analogs bearing ortho-dihydroxy groups could preferentially and synergistically kill HepG2 cells. Chemical and biological mechanism studies suggest that under aerobic conditions, the prooxidant system produce extracellularly ROS through a SPLET mechanism followed by their diffusion into cells; ROS then act as second messengers to complete the downstream events including disruption of cellular redox homeostssis by inducing down-regulation of the ratios of GSH/GSSG and Bcl-2/Bax proteins, dissipation of mitochondrial membrane potential, cytosolic release of cytochrome c and death caspase activation, which eventually lead to occurrence of cell apoptosis. The selectivity comes from the fact that compared to L02 cells, HepG2 cells exhibit increased intrinsic ROS levels, and therefore are more vulnerable to further ROS stress reaching the toxic threshold. This work not only confirms the feasibility of the preferential cancer cell killing by the prooxidant strategy, but also provides evidence that polyphenols fuction as cancer chemopreventive agents by their prooxidant properities.(3) Through researching on biological mechanism of HepG2 cell apoptosis induced by curcumin and its analogs (2a and 3a) with the central methylene group omitted by Knoevenagel condensation and insertion of a benzene ring, we found that the increased activity, compared to that of the parent molecule, is directly related to their electrophilic ability to promote intracellular ROS production. The driving force of ROS production comes from their irreversible inhibition of thioredoxin reductase (TrxR), and the NADPH oxidase-like activity induced by their covalently modified TrxR. Intracellular ROS production is associated with disruption of redox homeostasis, collapse of mitochondrial membrane potential, cytosolic relase of cytochrome c and activation of caspase-9 and 3, ultimately resulting in cell apoptosis. This work confirms the feasibility in designing curcumin-inspired cancer chemopreventive agents by the prooxidant strategy, and gives us useful information on how to design them including the relation between apoptosis-inducing activity and the Michael addition acceptor moiety-depended ROS-generating ability which depends not only on the TrxR-inhibitory activity, but on NADPH oxidase-like activity induced by the modified TrxR.