*2.2. Fucoxanthin Induces Activation of Nrf2 and Enhances Binding of Nrf2 to the ARE in the Promoters of the GCLC and GSS Genes*

The genes encoding GCLC and GSS have an ARE sequence in their promoter regions. Nrf2 is an important transcription factor that regulates ARE-driven expression of these genes [22]. We examined whether fucoxanthin treatment activated Nrf2, resulting in the up-regulation of these enzymes. Fucoxanthin treatment increased protein levels of Nrf2 and phospho Nrf2 (active form) (Figure 2A), and resulted in the translocation of Nrf2 protein from the cytosol into the nucleus (Figure 2B). Moreover, chromatin immune-precipitation (ChIP) analysis revealed that binding of Nrf2 to the ARE in the promoters of the genes encoding GCLC and GSS was markedly increased in fucoxanthin-treated cells, as determined by comparison to binding of histone H3 as the internal control (Figure 2C). To verify the functional relevance of Nrf2 binding to the ARE sequence of these two genes, a construct was used that contained a promoter containing an ARE sequence (bearing the consensus Nrf2-binding site) linked to a luciferase reporter gene. Fucoxanthin treatment increased the transcriptional activity of Nrf2 (Figure 2D). These results suggest that Nrf2 mediates fucoxanthin-induced transcription of GCLC and GSS. 

**Figure 2.** Effects of fucoxanthin treatment on the expression, nuclear translocation, and antioxidant response element (ARE) sequence-binding activity of Nrf2. ( **A**) Nuclear extracts were prepared from HaCaT cells following treatment with 20 ΐM fucoxanthin for the indicated amount of time. Western blotting of the nuclear lysates was performed using Nrf2 and phospho Nrf2 antibodies. **\*** and **#** indicates significantly different from Nrf2 and phospho Nrf2 of control, respectively ( *p* < 0.05); ( **B**) An anti-Nrf2 antibody and a FITC-conjugated secondary antibody were used to detect Nrf2 localization (green) by using confocal microscopy. DAPI staining indicates the locations of nuclei (blue). The merged images show the nuclear localization of Nrf2 protein; ( **C**) Nuclear extracts were prepared from HaCaT cells treated with 20 ΐM fucoxanthin for 6 h. A ChIP assay was performed to assess binding of Nrf2 to the ARE in the promoters of the genes encoding GCLC and GSS; ( **D**) Transcriptional activity of Nrf2 in HaCaT cells following treatment with 20 ΐM fucoxanthin for 6 h was assessed by using luciferase reporter assay. **\*** Significantly different from control cells.

*2.3. Fucoxanthin Involves Nrf2-Driven GCLC and GSS via Phosphorylation of Akt* 

To further elucidate the up-stream signaling pathway involved in fucoxanthinmediated activation of Nrf2 and induction of GCLC and GSS, we examined activation of Akt, which is a signaling enzyme that is involved in the phosphorylation and nuclear translocation of Nrf2 [23]. Activation of Akt by fucoxanthin was assessed by performing Western blotting with an antibody against phosphorylated Akt. Fucoxanthin treatment increased phosphorylation of Akt (Figure 3A). A LY294002, phosphoinositide 3-kinase (PI3K)/Akt inhibitor, specifically represses the phosphorylation of Akt [24]. This inhibitor reduced the fucoxanthininduced phospho Akt expression (Figure 3B). Furthermore, this inhibitor suppressed the fucoxanthin-induced Nrf2, GCLC and GSS expression (Figure 3C,D). 

**Figure 3.** Effects of fucoxanthin treatment on Akt and its related protein. (**A**) Cells were incubated with 20 ΐM fucoxanthin for various amounts of time (0–12 h). Cell lysates were prepared and Western blotting was performed with anti-Akt and anti-phospho Akt antibodies. **\*** indicates significantly different from control cells (*p* < 0.05); After treatment with LY294002, cell lysates were subjected to electrophoresis (**B**) with anti-Akt, anti-phospho Akt. **\*** indicates significantly different from control cells (*p* < 0.05) and **#** significantly different from fucoxanthin-treated cells (*p* <sup>&</sup>lt; 0.05); (**C**) with anti-Nrf2 and anti-phospho Nrf2. **\*** and **\*\*** indicates significantly different from Nrf2 and phospho Nrf2 of control, respectively (*p* < 0.05), **#** and **##** indicates significantly different from Nrf2 and phospho Nrf2 of fucoxanthin-treated cells, respectively (*p* < 0.05); (**D**) with anti-GCLC and anti-GSS antibodies. **\*** and **\*\*** indicates significantly different from GCLC and GSS of control, respectively (*p* < 0.05), # and **##** indicates significantly different from GCLC and GSS of fucoxanthin-treated cells, respectively (*p* < 0.05). 

*2.4. Fucoxanthin Promotes the Synthesis of GSH Catalyzed by GCLC and GSS* 

GSH is a tri-peptide formed via GCLC and GSS, and has powerful antioxidant effects against free radicals. GSH was detected by confocal microscopy using 7- amino-4-chloromethylcoumarin (CMAC), a dye that specifically labels GSH. The fluorescence intensity of CMAC, indicative of the level of GSH, was notably higher in fucoxanthin-treated cells than in control cells (Figure 4A). Consistently, fucoxanthin increased the concentration of GSH, as determined by a GSH detection kit (Figure 4B). To evaluate whether fucoxanthin induced GSH production to protect cells against ultraviolet B (UVB)-induced oxidative stress, cells were pretreated with fucoxanthin and then exposed to UVB irradiation. The level of GSH was reduced by UVB exposure, and this decrease was significantly restored in cells pretreated with fucoxanthin (Figure 4C,D). These data suggest that fucoxanthin partially recovers the reduction of GSH induced by UVB. 

**Figure 4.** Effect of fucoxanthin on the level of reduced glutathione (GSH). The level of GSH was assessed in cells treated with 20 ΐM fucoxanthin for 12 h by ( **A**) performing confocal microscopy after CMAC staining and ( **B**) using a GSH detection kit. **\*** indicates significantly different from control (*p* < 0.05). The level of GSH in UVB-treated HaCaT cells incubated for 12 h, with or without pretreatment with 20 ΐM fucoxanthin, was detected by (**C**) performing confocal microscopy after CMAC staining and ( **D**) using a GSH detection kit. **\*** indicates significantly different from control ( *p* < 0.05) and **#** significantly different from UVB-irradiated cells ( *p* < 0.05). 

(**C**) 

## **3. Materials and Methods**

## *3.1. Materials*

Anti-TATA-binding protein (TBP) and anti-phospho Nrf2 antibodies were purchased from Abcam, Inc. (Cambridge, MA, USA). Anti-Nrf2, anti-Akt, and antiphospho Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Fucoxanthin, anti-GCLC and anti-GSS antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). [3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium] bromide (MTT) and an anti-Ά-actin antibody were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Cell Tracker™ Blue CMAC was purchased from Molecular Probes (Eugene, OR, USA). LY294002 was provided by Calbiochem (San Diego, CA, USA). All other chemicals and reagents were of analytical grade. 

## *3.2. Cell Culture*

The human keratinocyte cell line HaCaT was supplied by Amore Pacific Company (Gyeonggi-do, Korea) and maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO2 and 95% air. Cells were grown in RPMI 1640 medium containing 10% fetal calf serum, streptomycin (100 ΐg/mL), and penicillin (100 units/mL). 

## *3.3. Reverse Transcription-PCR (RT-PCR)*

Total RNA was isolated from cells using the easy-BLUE™ total RNA extraction kit (iNtRON Biotechnology Inc., Seongnamsi, Korea). cDNA was amplified using 1 ΐL of reverse transcription reaction buffer, primers, dNTPs, and 0.5 U of Taq DNA polymerase in a final volume of 20 ΐL. The PCR conditions were initial denaturation at 94 °C for 5 min, followed by 26 cycles of 94 °C for 30 s, 63 °C for 45 s, and 72 °C for  1 min, and a final elongation step at 72 °C for 7 min. The following primers were used: human GCLC, forward (5ȝ-AACCAAGCGCCATGCCGACC-3ȝ) and reverse (5<sup>ȝ</sup>-CCTCCTTCCGGCGTTTTCGC-3<sup>ȝ</sup>); human GSS, forward (5<sup>ȝ</sup>-GCCCCATTCACGCTCTTCCCC-3ȝ) and reverse (5<sup>ȝ</sup>-ATGCCCGGCCTGCTTAGCTC-3ȝ); human GAPDH, forward (5<sup>ȝ</sup>-TCAAGTGGGGCGATGCTGGC-3ȝ) and reverse (5<sup>ȝ</sup>-TGCCAGCCCCAGCGTCAAAG-3ȝ). The amplified products were mixed with blue/orange 6 × loading dye, resolved by electrophoresis on a 1% agarose gel, stained with RedSafe™ nucleic acid staining solution (iNtRON Biotechnology Inc., Seongnamsi, Korea), and photographed under UV light using Image Quant™ TL analysis software (Amersham Biosciences, Uppsala, Sweden). 
