**Jian Zheng, Mei Jing Piao, Ki Cheon Kim, Cheng Wen Yao, Ji Won Cha and Jin Won Hyun \***

School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju 690-756, Korea; E-Mails: zhengjian0317@hotmail.com (J.Z.); mjpiao@hanmail.net (M.J.P.); svv771@hotmail.com (K.C.K.); vane1989923@hotmail.com (C.W.Y.); cjw102700@hanmail.net (J.W.C.) 

**\*** Author to whom correspondence should be addressed; E-Mail: jinwonh@jejunu.ac.kr; Tel.: +82-2-64-754-3838; Fax: +82-2-64-702-2687. 

*Received: 4 June 2014; in revised form: 1 July 2014 / Accepted: 4 July 2014 / Published: 10 July 2014* 

**Abstract:** Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by 

glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3- kinase (PI3K)/Akt inhibitor, suppressed the 

fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased  GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling. 

**Keywords:** fucoxanthin; NF-E2-related factor 2; oxidative stress; cytoprotection; PI3K/Akt; GCLC; GSS; GSH 

## **1. Introduction**

Oxidative stress is the most common cause of skin aging and can be effectively eliminated by the organism itself, pharmacological agents, and natural antioxidants. There are two theoretical methods to deal with these harmful stimuli, namely, early and delayed responses. Early responses rapidly remove reactive oxygen species (ROS) and free radicals via chemical reactions soon after their generation [1]. By contrast, delayed responses involve the expression of genes encoding antioxidant enzymes and proteins to reduce the generation of noxious substances [2]. Nuclear factor-erythroid 

2-related factor (Nrf2) is often the central signaling switch that modulates the activation of phase II bio-transferase/antioxidant enzymes, which include glutamatecysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS) [3,4]. As an extremely important antioxidant, GSH, which is synthesized by GCLC and GSS [5,6], not only scavenges free radicals [7], but also maintains the redox-sensitive active sites of many enzymes from an oxidized form to a reduced form [8]. Therefore, the correct balance between reduced GSH and oxidized GSH is required for cellular homeostasis [9]. 

Genes that encode antioxidant enzymes, such as GCLC and GSS, contain an antioxidant responsive element (ARE) in their promoter region [3]. Transduction of the ARE sequence-containing genes encoding GCLC and GSS mainly occurs via activation of Nrf2 protein [10]. Nrf2 is a transcription factor that detects variation in oxidative stress within cells [11] and induces the transcription of its target genes by binding to the ARE in their promoters. The target genes of Nrf2 include many antioxidant and phase II detoxifying genes [12], including those encoding GCLC and GSS. The synthesis of GSH catalyzed by GCLC and GSS via up-regulation of Nrf2 is associated with protection of cells against oxidative stress [13]. 

Fucoxanthin is a major carotenoid found in edible brown seaweeds [14] and contains several functional groups, including an unusual allenic bond, a conjugated carbonyl group, and an acetyl 

group [15]. Fucoxanthin has antioxidant, anti-cancer, anti-obesity, anti-diabetic, and anti-photoaging activities [16–20]. We previously reported that fucoxanthin reduced levels of ROS, inhibited DNA damage, restored mitochondrial membrane potential, and suppressed apoptosis [21]. In the present study, we examined whether fucoxanthin could increase the level of GSH by inducing GCLC and GSS expression via the Akt/Nrf2 pathway. 
