**4. Conclusions**

In this study, we demonstrated that fucoxanthin induced activation of nucleus Nrf2 via PI3K/Akt, which in turn activated the transcription of ARE-driven GCLC and GSS genes, leading the synthesis of antioxidant GSH. We recently reported that fucoxanthin did not show toxic effect on HaCaT cells at 20 ΐM and this concentration prevented cells against oxidative damage [21]. Therefore, we chose the same concentration to examine the effect of fucoxanthin on GSH induction in the present study. 

GSH is an important biological antioxidant and has diverse functions in nutrient metabolism [25], gene expression, and DNA/protein synthesis [26], and particularly in eliminating oxidants [27]. GSH is formed from glutamate, cysteine, and glycine in a reaction that is catalyzed by two cytosolic enzymes, namely, GCLC and GSS [6]. The GSH is associated with the inhibition of tumor cell growth [28], prevention of apoptosis [29], and reduced inflammation [30]. In this study, fucoxanthin increased the mRNA and protein expression of GCLC and GSS (Figure 1). Nrf2, a major transcription factor of antioxidant enzymes, is tightly controlled by a master regulator Keap-1. Keap-1 has a high affinity for Nrf2 owing to its cysteine residues that form a covalent bond with Nrf2 [31]. Subsequently, Nrf2 signaling is switched off by Keap-1-mediated ubiquitination and degradation of Nrf2 [32]. However, the phospho form of Nrf2 can translocate into nucleus [33]. 

The nuclear Nrf2 recognizes the ARE sequence within the promoters of its target genes [34], and binding of Nrf2 to ARE sequences stimulates the transcription of genes that are involved in cellular defense. As shown in data, the phosphorylated Nrf2 expression was induced by fucoxanthin treatment (Figure 2A,B). The Nrf2 then binds to ARE sequence in GCLC and GSH promoters, which was assessed by ChIP assays (Figure 2C). The increased Nrf2 binding ability to ARE sequence in fucoxanthin-treated cells led to the increased transcriptional activity of Nrf2 (Figure 2D). These results indicated that fucoxanthin promotes release of Nrf2 from Keap-1 and subsequent translocation to nucleus, which induced the synthesis of GSH by enhancing expression of GSS and GCLC through interaction between Nrf2 and ARE sequence. It recently reported that fucoxanthin enhanced heme oxygenase-1 and  NAD(P)H:quinone oxidoreductase 1 expression via activation of Nrf2/ARE system [35]. The phosphorylation of Nrf2 plays a pivotal role in its nuclear accumulation, and this phosphorylation can occur via Akt pathways [36]. Akt is a classic signaltransducing protein which can activate the primary cellular defense mechanism Nrf2/ARE in skin cells [37]. In our study, fucoxanthin treatment increased the level of phosphorylated Akt, which is the active form of this kinase (Figure 3A) and could elevate the nuclear level of Nrf2 (Figure 2A). Furthermore, LY294002, a specific inhibitor of PI3K/Akt, significantly suppressed the active form of Akt (Figure 3A) which resulted in reduction of Nrf2 accumulation, following decreased protein expression of GSS and GCLC (Figure 3B–D). In addition, working in concert with the effects of fucoxanthin on GSS and GCLC expression, the amount of GSH was increased in fucoxanthin-treated cells, as indicated by the increased fluorescence intensity of CMAC (Figure 4A) and the increased concentration of GSH (Figure 4B). In our system, UVB irradiation, an inducer of oxidative stress, suppressed the GSH level. However, treatment with fucoxanthin prior to UVB damage partially mitigated the reduction in GSH levels (Figure 4C,D). 

In conclusion, fucoxanthin substantially increased the mRNA and proteins levels of GCLC and GSS in human keratinocytes, and these effects were dependent on the nuclear translocation of Nrf2 following its phosphorylation by the protein kinase Akt. In addition, this study demonstrates that the Akt/Nrf2 pathway plays an essential role in the mechanism underlying the effects of fucoxanthin. Taken together, one of the major ways by which fucoxanthin treatment prevents or eliminates oxidative damage is to enhance the Akt/Nrf2/GSH-dependent antioxidant response. 

## **Acknowledgments**

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-C1ABA001-2011-0021037). 

## **Author Contributions**

Jian Zheng designed the research, performed the experiments, and wrote the manuscript. Mei Jing Piao and Ki Cheon Kim analyzed some data. Cheng Wen Yao and Ji Won Cha gave the advice on some experiments. Jin Won Hyun designed the research, analyzed and interpreted data, drafted the manuscript.

## **Conflicts of Interest**

The authors declare no conflict of interest. 
