Mechanisms by which natural polyphenols regulate the expression of cytoprotective genes
Epidemiological studies have shown that a diet high in fruit and vegetables has an inverse association with the occurrence of various degenerative diseases such as cancer, cardiovascular disease, neuro-degenerative disease and diabetes. Clinical and animal studies using fruit and vegetable extracts have shown that polyphenols abundant in plants may account for the beneficial effect of diets high in fruit and vegetables. Following these findings, laboratories worldwide are investigating the mechanisms underlying the health effects exerted by polyphenols. Although many studies have investigated the effect of various polyphenols on cancer cells, such as inhibition of cell growth, induction of apoptosis, tumorigenesis, few studies have been carried out to examine their effect on normal cells and whether they could prevent the initiation of cancer. DNA damage, one of the early steps in the tumorigenic process, may be caused by toxicants and oxidative stress either from the environment or from endogenous sources. Therefore, to prevent such damage, multiple mechanisms are deployed by cells to combat the toxic insult and reduce oxidative stress. The CNC-bZIP transcription factor, Nrf2, is known to regulate the expression of many of the genes involved in these processes. Under homeostatic conditions, the Nrf2 protein is targeted by its inhibitor Keap1 for ubiquitination in the cytosol. Upon redox stress, induced by low molecular weight electrophiles, Nrf2 can evade ubiquitination and translocate into the nucleus, where it heterodimerizes with members of the small Maf family of transcription factors and bind to the antioxidant response element (ARE) in the promoter region of many cellular defence genes, leading to the up-regulation of their transcription. In our study, we examined whether polyphenols could affect the Nrf2-ARE signalling pathway. Preliminary screening was first carried out using AREc32 cells to find flavonoids which have ARE-inducing ability. Flavonoids, which are present in relatively high concentration in fruits and vegetables and consumed most commonly, were chosen for screening. Quercetin and kaempferol showed the highest ARE-inducing ability and these two compounds were subsequently were used to examine their effect on Nrf2 and its target gene Nqo1 in rat liver RL-34 and mouse embryonic fibroblast MEF cells. By Nqo1 enzyme activity assay, Western blotting, and Taqman experiments, our study showed that these two flavonoids increased the enzyme activity, protein expression and mRNA level of Nqo1. By using Nrf2+/+ and Nrf2-/- MEFs, we found that such increases are Nrf2-dependent. The effect of quercetin and kaempferol on Nrf2 was therefore examined. Nrf2 protein, but not mRNA, was found to be elevated by quercetin and kaempferol. By cycloheximide-chase experiment, quercetin and kaempferol were shown to stabilize the Nrf2 protein by decreasing its turnover time. Furthermore, results from cellular fractionation and immunocytochemistry experiments showed that Nrf2 predominantly resides in nucleus under both normal and stressed conditions; the two flavonoids increased the accumulation of Nrf2 in both cytosol and nucleus, although the increase of Nrf2 protein in the nucleus was more pronounced. In addition, it was found that the flavonoids inhibited the ubiquitination of Nrf2. To address how the flavonoids inhibit the ubiquitination, we carried out mutagenesis experiments with quercetin and found Cys151 in Keap1 is required for stabilization of Nrf2, which indicates that quercetin may act as an electrophile and modify the Cys151 in Keap1, ultimately leading to the disruption of the association between Keap1 and Nrf2. The involvement of ARE in the regulation of Nqo1 by quercetin and kaempferol were examined by mutagenesis experiments, the results of which showed that the ARE was involved in both the basal expression of Nqo1 and its induction by quercetin and kaempferol. Besides the ARE, the promoter region of Nqo1 contains a xenobiotic responsive element (XRE) which can mediate gene regulation by AhR. With the evidence from previous research showing that Nqo1 can be up-regulated by the AhR agonist TCDD, we examined the involvement of XRE in the induction of Nqo1 by the flavonoids in mutagenesis experiments. The absence of XRE only affected the basal level but not the inducible level of Nqo1. However, by immunocytochemistry experiments, we showed that quercetin and kaempferol can act as AhR agonists. This was confirmed by Taqman experiments showing these two flavonoids can increase the mRNA level of Cyp1a1. Lastly, the effect of quercetin and kaempferol in vivo was also examined in animals by using C57BL/6 male mice. Only quercetin showed up-regulation of Cyp1a1 mRNA in the small intestine. An effect on Nqo1 and Nrf2 by the flavonoids observed in cells was not seen in the tissues. Taken together, the data presented in this thesis shows that the flavonols quercetin and kaempferol up-regulate the Nrf2-ARE signalling pathway by stabilizing the CNC-bZIP protein; such up-regulation also leads to the trans-activation of Nqo1. In addition, the two flavonoids are AhR agonists and increase the mRNA level of Cyp1a1 which is also observed in the mouse small intestine. Such increase of Cyp1a1 may indicate their implication in the prevention of gastrointestinal cancer
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