Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2–ARE pathway

Abstract

Increased amounts of reactive oxygen species (ROS) have been implicated in many pathological conditions, including cancer. The major machinery that the cell employs to neutralize excess ROS is through the activation of the antioxidant-response element (ARE) that controls the activation of many phase II detoxification enzymes. The transcription factor that recognizes the ARE, Nrf2, can be activated by a variety of small molecules, most of which contain an α,β-unsaturated carbonyl system. In the pursuit of chemopreventive agents from marine organisms, we built, fractionated, and screened a library of 30 field-collected eukaryotic algae from Florida. An edible green alga, Ulva lactuca, yielded multiple active fractions by ARE–luciferase reporter assay. We isolated three monounsaturated fatty acid (MUFA) derivatives as active components, including a new keto-type C18 fatty acid (1), the corresponding shorter chain C16 acid (2), and an amide derivative (3) of the C18 acid. Their chemical structures were elucidated by NMR and mass spectrometry. All three contain the conjugated enone motif between C7 and C9, which is thought to be responsible for the ARE activity. Subsequent biological studies focused on 1, the most active and abundant ARE activator isolated. C18 acid 1 induced the expression of ARE-regulated cytoprotective genes, including NAD(P)H:quinone oxidoreductase 1, heme oxygenase 1, thioredoxin reductase 1, both subunits of the glutamate–cysteine ligase (catalytic subunit and modifier subunit), and the cystine/glutamate exchange transporter, in IMR-32 human neuroblastoma cells. Its cellular activity requires the presence of Nrf2 and PI3K function, based on RNA interference and pharmacological inhibitor studies, respectively. Treatment with 1 led only to Nrf2 activation, and not the increase in production of NRF2 mRNA. To test its ARE activity and cytoprotective potential in vivo, we treated mice with a single dose of a U. lactuca fraction that was enriched with 1, which showed ARE-activating effects similar to those observed in vitro. This could be owing to this fraction's ability to stabilize Nrf2 through inhibition of Keap1-mediated Nrf2 ubiquitination and the subsequent accumulation and nuclear translocation of Nrf2. The induction of many ARE-driven antioxidant genes in vivo and most prominently in the heart agreed with the commonly recognized cardioprotective properties of MUFAs. A significant increase in Nqo1 transcript levels was also found in other mouse tissues such as the brain, lung, and stomach. Collectively, this study provides new insight into why consumption of dietary seaweed may have health benefits, and the identified compounds add to the list of chemopreventive dietary unsaturated fatty acids.

Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis

“…To confirm activation of the Nrf2-ARE system in the present study, we measured the mRNA expression of Nrf2 gene and three antioxidant genes containing ARE at their promoter regions ( Nrf2 , contains two ARE sequences [33]; HO-1 , a well-characterized Nrf2 target gene [34]; NQO1 , reduces quinones to hydroquinones to protect against oxidative stress [35]; GCLC , combines Glu and Cys as the first step in GSH production [36]). Previous studies have been reported that the activation of Nrf2 induced increased expression of Nrf2, HO-1, NQO1 and GCLC [37,38]. Consistent with the results above, our founding showed an increase in mRNA expression of Nrf2 , HO-1 and GCLC during consecutive heat stress which may reversely promote the expression of oxidative-mediated antioxidant genes via activation of Nrf2 transcription factor (Figure 7).…”

Selecting Australian marine macroalgae based on the fatty acid composition and anti-inflammatory activity

“…Interestingly, n-3 PUFA have been reported as modulators of the activity of nitric oxide synthase (NOS) (Radosinska et al 2011), one of the primary enzymes involved in NO production. MUFA derivatives have also been isolated from Ulva lactuca which act as activators of the cytoprotective Nrf2 -ARE pathway (Wang et al 2013), linked to suppression of inflammatory responses (Chen and Kunsch 2004). Furthermore, the brown alga Eisenia bicyclis (order Laminariales) was evaluated for anti-inflammatory activity via inhibition against LPS-induced NO and other inflammatory mediators in RAW 264.7 cells as in this study.…”

Extracts of Fruits and Vegetables Activate the Antioxidant Response Element in IMR-32 Cells

“…Polyphenols and tertiary butylhydroquinone (tBHQ) activate endogenous antioxidant genes and also activate ARE reporter constructs in human IMR-32 neuroblastoma cells (11,(15)(16)(17). IMR-32 cells (ATCC) were grown and maintained in MEM (Life Technologies) containing 10% heat-inactivated FBS (hiFBS; Sigma), 13 nonessential amino acids (MEM NEAA; Life Technologies), 1 mM pyruvate (Hyclone), and 100 U/mL penicillin-streptomycin (Life Technologies).…”