Design Of Curcumin-inspired Cancer Chemopreventive Agents Based On The Prooxidant Strategy

Reactive oxygen species (ROS) are a family of oxygen-containing molecules produced in the mitochondrial electron-transport chain and oxygen-metabolizing enzymatic reactions as a consequence of aerobic life. Compared to normal cells, cancer cells exhibit increased levels of ROS and an altered redox status, due to many factors such as oncogenic stimulation, increased metabolic activity and mitochondrial malfunction, which makes it more vulnerable to further ROS stress. This provides an opportunity for the development of potential cancer treatment strategies, namely, using an exogenous ROS-generating agent (prooxidant) or redox intervention further escalates cellular ROS production to kill cancer cells. Redox intervention can be achieved using small molecule electrophiles containing a Michael acceptor (?, ?-unsaturated carbonyl) that form adducts with nucleophilic anionic thiolates on redox-active proteins.Curcumin, a natural polyphenol with two ?, ?-unsaturated carbonyl moieties, features prominently in dietary cancer chemoprevention agents and has attracted much attention. We synthesized a series of mono-carbonyl curcumin analogs with different substituents at different positions on the aromatic rings, and evaluated systematically their activity, chemical and biological mechanisms against cancer cells. It was found that the ortho-disubstituted compounds (Al, Bl and Cl) exhibited remarkably increased antiproliferative, apoptosis-inducing and cell cycle arrest activities against HepG2 and A549 cells compared with curcumin. Interestingly, The activities were directly related to their ability to promote intracellular ROS-generation by Michael acceptor. It was confirmed that they irreversibly modified thioredoxin reductase (TrxR) in vitro and in A549 cells, and the modified TrxR showed a strongly induced NADPH oxidase activity that resulted in an increased generation of ROS. Most importantly, ROS-generation ability of curcumin and its active analogs depends not only on their TrxR-inhibitory activity, but on NADPH oxidase activity induced by their modified TrxR which is even more important than the former for the ability. ROS-generation was associated with disruption of redox homeostasis, induced subsequently lipid peroxidation, increase of Ca2+ influx, collapse of mitochondrial membrane potential, activation of caspase-9 and 3 and ultimately resulted in the cell apoptosis. This work confirms the feasibility and gives us useful information in designing curcumin-inspired cancer chemopreventive agents by the prooxidant strategy. The main findings are as follows:(1) This work provides an excellent example in development of curcumin-inspired cancer chemopreventive agents based on the prooxidant strategy. A 2,2'-trifluoromethyl curcumin analog, Al, has surfaced as an important lead compound, displaying remarkably increased antiproliferative, apoptosis-inducing and cell cycle arrest activities against HepG2 and A549 cells compared with this parent molecules.(2) The electron and position effects of substituents on the aromatic rings play an important role in antiproliferative activity of the mono-carbonyl curcumin analogs against HepG2 and A549 cells. Introduction of ortho-trifluoromethyl on the aromatic rings are the most efficient way to improve the activity.(3) This work demonstrates a strong correlation between Michael acceptor-dependent ROS generation and apoptosis induction by curcumin and its active analogs in A549 cells(4) The apoptosis-inhibition offered by GSH or NAC is mainly due to Michael addition reactivity between GSH or NAC and cucumin analogs.(5) The mechanism by which ROS production occurs is that the modified TrxR by curcumin or its active analogs shows an increased NADPH oxidase activity.(6) ROS-generation ability of curcumin and its active analogs depends not only on their TrxR-inhibitory activity, but on NADPH oxidase activity induced by their modified TrxR which is even more important than the former for the ability.(7) The connection events between ROS generation and cell apoptosis induced by A1 include disruption of redox homeostasis, lipid peroxidation, increase of Ca2+ influx, collapse of mitochondrial membrane potential, activation of caspase-9 and 3.Our previous work showed that inserting a benzene ring to omit the active methylene group of curcumin resulted in the bischalcone (BCl) that exhibited higher apoptosis-inducing activity against HepG2 cells than this parent molecule. Inspired by the first part of this dissertation and the important role of trifluoromethyl group in improving cellular membrane permeability and increasing metabolism stability of drugs, we further synthesized a novel 2,2'-trifluoromethyl-bischalcone (BC2) and found that it was more active against cancer cells than curcumin, BCl and Al, due to its highest ROS-generation ability among the compounds investigated.