Original Contribution
Structure activity relationship of carotenoid derivatives in activation of the electrophile/antioxidant response element transcription system

https://doi.org/10.1016/j.freeradbiomed.2009.06.008Get rights and content

Abstract

Induction of phase II detoxifying enzymes is a major cellular strategy for reducing the risk of cancer. We previously reported that carotenoids activate the electrophile/antioxidant response element (EpRE/ARE) transcription system and induced the expression of phase II enzymes. Various electrophilic phytonutrients have been shown to induce the EpRE/ARE system by disrupting the inhibitory activity of Keap1 on Nrf2, the major EpRE/ARE activating transcription factor. However, hydrophobic carotenoids such as lycopene lack any electrophilic group and, thus, are unlikely to directly activate Nrf2 and the EpRE/ARE system. Here we demonstrate that carotenoid oxidation products are the active mediators in the stimulation of the EpRE/ARE system by carotenoids. Two lines of evidence support this conclusion. (A) The oxidized derivatives, extracted by ethanol from partially oxidized lycopene, transactivated EpRE/ARE with a potency similar to that of the unextracted lycopene mixture, whereas the intact carotenoid showed a nonsignificant effect. (B) Using a series of characterized mono- and diapocarotenoids that potentially can be derived from in vivo metabolism of carotenoids we defined the following structure–activity rules for activation of EpRE/ARE: (I) aldehydes and not acids are the active molecules; (II) the activity depends on the relative position of the methyl group to the terminal aldehyde which determines the reactivity of the conjugated double bond; (III) the optimal length of a dialdehyde derivative is 12 carbons in the main chain of the molecule. The apocarotenals inhibited breast and prostate cancer cell growth with a similar order of potency to the activation of EpRE/ARE. These results may provide a mechanistic explanation for the cancer preventive activity of carotenoids.

Introduction

While there is considerable epidemiological evidence suggesting an association between consumption of fruits and vegetables and reduced cancer incidence [1], some recent studies have provided conflicting results. For example, no association was found between fruit and vegetable consumption and a reduction in the risk of bladder cancer [2]. In addition, Lee et al. reported reduced risk of renal cell cancer in men but no clear association in women [3]. Carotenoids, a major phytonutrient component of diets rich in fruits and vegetables, have been suggested to have a cancer preventive effect [4], [5]. For example, in a comprehensive analysis of the epidemiological literature on the relation of tomato consumption and cancer prevention by Giovannucci [6] it was found that most of the reviewed studies reported an association between tomato intake or blood lycopene level and the risk of various types of cancer. Other, more recent studies generally support this view [7], [8], [9].

Direct inhibition of specific cellular processes such as growth factor signaling [10] and cell cycle progression [10], [11] has been suggested as a mechanism for the cancer preventing activity of carotenoids. In addition, we and others have demonstrated that carotenoids regulate various transcription systems [12], [13], [14], [15]. Particularly, we recently reported that carotenoids stimulate the electrophile/antioxidant response element (EpRE/ARE) transcription system and its major activating transcription factor, Nrf2 [15]. This resulted in the induction of phase II detoxifying enzymes, which is a major cellular strategy for reducing the risk of cancer, inflammation, and other chronic degenerative diseases. Under unstimulated conditions, Nrf2 (nuclear factor E2-related factor 2) is bound to its cysteine-rich partner, Keap1, which is known to repress Nrf2 activity. Various phytonutrients, such as polyphenols and isothiocyanates [16], interact with Keap1 and activate Nrf2 and EpRE/ARE. Inducers of this system are diversified in their chemical structure but all are chemically reactive and nearly all are electrophiles which react with Keap1 to disrupt its inhibitory activity on Nrf2. However, hydrophobic carotenoids such as lycopene lack any electrophilic group and thus are unlikely to interact directly with Keap1. We hypothesized that oxidation products are the active mediators in the stimulation of the EpRE/ARE transcription system by carotenoids based on the observation that ethanolic extract of partially oxidized lycopene containing unidentified hydrophilic derivatives activated EpRE/ARE with a similar potency to lycopene [15]. To identify the putative activators, we designed a series of pure synthetic and fully characterized compounds that potentially can be derived from in vivo metabolism of lycopene and other carotenoids or during their spontaneous oxidation. The activity of these compounds to stimulate the EpRE/ARE transcription system was determined in order to elucidate the structure–activity relationship of these derivatives and their mechanism of action at the molecular level.

Section snippets

Materials

Crystalline lycopene preparations, purified from tomato extract (>97%), were a gift of LycoRed Natural Products Industries (Beer Sheva, Israel). β-Carotene was a gift of DSM Nutritional Products (Bazel, Switzerland). Synthetic carotenoid derivatives, shown in Table 1 (>99% purity), were synthesized and provided by BASF (Ludwigshafen, Germany). The compounds were characterized using UV/VIS spectroscopy, HPLC, and 1H and 13C NMR and proved to be in all-E-configuration. Tetrahydrofuran (THF),

Carotenoid derivatives and not intact carotenoids transactivate EpRE/ARE in prostate cancer cells

We examined whether different carotenoid preparations induce the EpRE/ARE system in LNCaP prostate cancer cells. We found that crystalline preparations of tomato lycopene and synthetic β-carotene as well as tBHQ, the classical activator of Nrf2, induced EpRE/ARE reporter gene activity in the prostate cancer cells (Fig. 1A). In addition, both lycopene and tBHQ elevated the protein levels of the phase II enzyme, NAD(P)H:quinone oxidoreductase (NQO1) (Fig. 1B). However, whereas lycopene was a

Discussion

The main finding of the current study is that hydrophilic oxidation products of carotenoids, and not the intact molecules, are the active mediators in the stimulation of the EpRE/ARE transcription system. Two lines of evidence support this conclusion. (A) The oxidized derivatives extracted by ethanol from partially oxidized lycopene transactivated EpRE/ARE reporter activity with a potency similar to that of the unextracted lycopene mixture, whereas the intact carotenoid molecule showed a

Acknowledgments

We thank Dr. Zohar Nir and Mrs. Tanya Svedlov, LycoRed Natural Products Industries, Beer Sheva, Israel, for donating purified lycopene and for the extraction of lycopene. We thank Dr. Regina Goralczyk, DSM Nutritional Products, Human Nutrition and Health, Basel, Switzerland, for donating β-carotene. We also thank Dr. J.J. Gipp, University of Wisconsin Medical School, Madison, WI, USA, for providing the ARE reporter constructs, NQO1hARE-tk-luc.

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