| Reactive oxygen species (ROS) can function as signaling molecules that regulate numerous cellular processes such as proliferation, differentiation, and death, depending on their concentrations, pulse duration, and subcellular localization. During the past several decades, the role of antioxidants in counteracting ROS production has attracted much attention, but their action in promoting ROS formation has often been ignored. As a matter of fact, every antioxidant is a redox agent and thus might become a prooxidant to promote the formation of ROS under special conditions. Over the past few years, increasing experimental evidence has indicated that antioxidant-mediated production of ROS (prooxidant action) may be responsible for the induction of apoptosis of cancer cells and cancer chemoprevention (Nat. Rev. Drug Discov.2009,8,579). However, little is known about the structural basis and detailed prooxidative mechanism of polyphenolic antioxidants and the reason for their ability to differentiate between normal and abnormal tumor cells. Compared with normal cells, cancer cells exhibit increased intrinsic ROS stress and copper levels. Therefore, a further increase in ROS stress in cancer cells by using the prooxidant system constructed by polyphenolic antioxidants and Cu(Ⅱ) ions via Fenton-like reaction, is likely to cause elevation of ROS above the threshold level, thus leading to death of these cells. However, the same concentration of polyphenolic antioxidants in normal cells could not induce the ROS level to reach the toxic threshold, and thus the normal cells survive. To confirm the above speculations and solve these problems, we conducted a detailed investigation into the active mechanism of DNA damage and antiproliferation against cancer cells mediated by polyphenolic antioxidants-Cu(Ⅱ) ions from a physical organic chemistry point of view. More importantly, we demonstrated that the prooxidant system could selectively kill cancer cells while leaving normal cells survived. The thesis includes the following details: (1) Hydroxycinnamic acids were selected as the representative of phenolic antioxidants to probe their effectiveness, mechanism and biological implications to act as DNA-cleaving agents in the presence of Cu(Ⅱ) ions. Caffeic acid (CaA) bearing o-hydroxy groups on phenolic rings was the most active at causing DNA damage among the hydroxycinnamic acids investigated. The detailed prooxidative mechanism was well exploited by investigating the interaction between CaA and Cu(Ⅱ) ions and the influence of ethylenediaminetetraacetic acid (EDTA), argon gas, the solvent, and pH value on the interaction by using UV-Vis spectroscopic analysis. On the basis of these observations, it is proposed that it is the CaA phenolate anion, instead of the parent molecule, that chelates with the Cu(Ⅱ) ion as a bidentate ligand, hence facilitating the intramolecular electron transfer to form the corresponding CaA semiquinone radical intermediate. The latter undergoes a second electron transfer with oxygen to form the corresponding o-quinone and a superoxide followed by other ROS formation, which play a pivotal role in the DNA damage. The intermediacy of the semiquinone radical was supported by isolation of its dimer from the Cu(Ⅱ)-mediated oxidation products. Intriguingly, CaA was also the most cytotoxic compound among the hydroxycinnamic acids toward human promyelocytic leukemia (HL-60) cell proliferation. Addition of exogenous Cu(Ⅱ) ions resulted in an effect dichotomy on cell viability depending on the concentration of CaA, that is, low concentrations of CaA enhanced the cell viability and, conversely, high concentrations of CaA almost completely inhibited the cell proliferation. Moreover, when superoxide dismutase was added before, the two stimulation effects of exogenous Cu(Ⅱ) ions were significantly ameliorated, thus clearly indicating that the oxidative-stress level regulates cell proliferation and death. These findings provide important formation for prooxidant-mediated cancer chemoprevention and chemotherapy.(2) To verify that antioxidant-mediated production of ROS (prooxidant action) could exhibit its cytotoxicity selectively against cancer cells with minimal cytotoxicity against normal cells, we constructed the prooxidant system with resveratrol analogues and Cu(Ⅱ) ions, and investigated their antiproliferative and apoptosis-inducing activity as well as mechanism against HepG2 cells and their normal counterparts, L02 cells. It was found that resveratrol analogues bearing o-hydroxy groups on phenolic rings and exogenous Cu(Ⅱ) ions could induce synergistically cytotoxicity and apoptosis of HepG2 cells by promoting intracellular ROS production and the subsequent mitochondrial membrane potential collapse, whereas the system caused obviously decreased cytotoxicity on normal L02 cells probably due to its low and intrinsic ROS levels. Our results highlight the prooxidant system could selectively induce the death of HepG2 cells by the increase of intracellular ROS levels and provide direct evidence for prooxidant mediated cancer chemoprevention.Additionally, we investigated and evaluated the hepatoprotective potential of resveratrol in mice following two different routes (po and sc) of exposure to carbon tetrachloride (CCl4) in mice. Administration of CCl4 induced significant acute liver injury in mice and the treatment of resveratrol alleviated the CCl4 induced damage in liver. The results of the study suggest that resveratrol has potential to exert curative effects against liver injury. |
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