**2. Results**

#### *2.1. FX Effect on RAW264.7 Cell Viability*

The cytotoxic effect of FX on RAW264.7 cells was determined using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay (Figure 1A). FX did not affect cell viability at a concentration of ≤5 μM. However, the number of viable cells after treatment with 10 μM of FX was significantly lower than the number of untreated cells was. Additionally, in contrast to the results of Das et al. [22], no cleavage of procaspase-3 and poly ADP ribose polymerase (PARP; Figure 1B) was detected in cells treated with ≤10 μM FX. Therefore, FX was used at a concentration of ≤5 μM in all subsequent experiments.

**Figure 1.** Effect of fucoxanthin (FX) on viability of RAW264.7 cells. ( **A**) Cells treated with different concentrations of FX, and cell viability was determined using 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay. Data are representative of six independent experiments and are expressed as mean ± standard error of mean (SEM); \* *p* < 0.05 versus FX-untreated cells (0 μM). (**B**) Procaspase-3 and poly ADP ribose polymerase (PARP) expression remained uncleaved upon treatment with ≤10 μM FX, unlike in pemetrexed-treated cells of lung-cancer cell line NCI-H3122.

#### *2.2. FX Inhibits Osteoclastogenesis*

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in differentiated osteoclasts, and is therefore used as a primary marker of osteoclastogenesis [28]. The treatment of soluble RANKL (sRANKL, 50 ng/mL) or costimulation with TNFα (50 ng/mL) and IL-6 (50 ng/mL) in RAW264.7 cells increased the number of TRAP-positive and multinucleated cells compared with that of untreated control cells (Figure 2A). Under these conditions, FX treatment decreased the number of TRAP-positive multinucleated cells in a dose-dependent manner (Figure 2B). In experiments using human CD14+ monocytes, 0 to 5 μM of FX dose-dependently suppressed RANKL- and TNF-α/IL-6-induced osteoclast differentiation from osteoclast precursors (Figure 2C). These results sugges<sup>t</sup> that FX inhibits the differentiation of RAW264.7 cells and human CD14+ monocytes to osteoclast-like cells.

**Figure 2.** Soluble receptor activator of nuclear factor-κB (NF-κB) ligand (sRANKL)- or tumor necrosis factor (TNF)-α/interleukin (IL)-6-induced differentiation into osteoclast-like cells. (**A**) Representative microscopic images of tartrate-resistant acid phosphatase (TRAP) stained RAW264.7 cells (red arrows; original magnification, 100×). Blue box in bottom corner is a magnified photograph of the smaller boxed area (original magnification, 400×). (**B,C**) Number of TRAP-positive multinucleated cells differentiated from (**B**) RAW264.7 cells and (**C**) human CD14+ monocytes decreased upon treatment with FX in a dose-dependent manner. Data are representative of three independent experiments and are expressed as mean ± SEM; \* *p* < 0.05 versus FX-untreated osteoclast-differentiated cells; † *p* < 0.05 by Jonckheere–Terpstra test. FX, fucoxanthin.

The resorption pit assay was performed to examine osteoclast activity. As shown in Figure 3, the resorption pit area was significantly decreased upon treatment with FX in a dose-dependent manner. These results collectively sugges<sup>t</sup> that FX exerts an inhibitory effect on both sRANKL-dependent and -independent bone-resorbing osteoclast activity.

**Figure 3.** Effect of FX on osteoclast activity. Statistical differences of resorption pit area and trends tests are presented in histograms. Data are representative of three independent experiments and are expressed as mean ± SEM. \* *p* < 0.05 versus FX untreated cells; † *p* < 0.05 by Jonckheere–Terpstra test.

#### *2.3. FX Downregulates Osteoclast-Specific Markers and Transcriptional Factors in RAW264.7 Cells*

NFATc1 is a master transcription factor for osteoclastogenesis [29]. The expression of NFATc1 in sRANKL- or TNF/IL-6-stimulated RAW264.7 cells decreased upon treatment with FX in a dose-dependent manner (Figure 4A). Dendritic-cell-specific transmembrane protein (DC-STAMP), another essential mediator for osteoclastogenesis, is upregulated upon osteoclastogenic stimulation. Increased DC-STAMP expression, in turn, upregulates the expression of osteoclast-specific markers such as *TRAP* [30]. As shown in Figure 4B, *DC-STAMP* mRNA expression was significantly decreased upon treatment with FX in a dose-dependent manner.

**Figure 4.** Effect of FX treatment on expression of osteoclast-specific markers. (**A**) Nuclear factor of activated T cell 1 (NFATc1) protein expression and (**B**) dendritic-cell-specific transmembrane protein (*DC-STAMP*) mRNA expression in sRANKL- and TNF/IL-6-stimulated RAW264.7 cells decreased upon treatment with FX in a dose-dependent manner. Data are representative of three independent experiments and expressed as mean ± SEM; \* *p* < 0.05 versus FX untreated cells; † *p* < 0.05 by Jonckheere–Terpstra test.

Matrix metallopeptidase (MMP)-9 is a well-established proteolytic effector of osteoclastmediated bone resorption. To examine whether FX affects MMP-9 production in differentiated osteoclast-like cells, MMP-9 levels in culture supernatant were measured by

ELISA. As shown in Figure 5, cells treated with 5 μM FX showed a significant decrease in MMP-9 levels. Moreover, MMP-9 production decreased following FX treatment in a dose-dependent manner.

**Figure 5.** Effect of FX treatment on MMP-9 levels in culture supernatant of osteoclast differentiated RAW264.7 cells. MMP-9 concentration significantly decreased upon treatment with 5 μM FX. Data are representative of three independent experiments and expressed as mean ± SEM; \* *p* < 0.05 versus FX untreated cells; † *p* < 0.05 by Jonckheere–Terpstra test.

To better understand the mechanism of the FX-induced inhibition of osteoclast differentiation, we performed immunoblot analysis of RAW264.7 cells to measure the expression of molecules known to be critically involved in the osteoclast signaling pathway, including MAPKs (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38), NF-κB, and phosphoinositide 3-kinase (PI3K) [31]. The treatment of RAW264.7 cells with FX significantly reduced the phosphorylation of ERK and p38 in a concentrationdependent manner. However, JNK, NF-κB, and PI3K phosphorylation was not altered following FX treatment (Figure 6).

**Figure 6.** Effect of FX treatment on signaling pathways during osteoclastogenesis. FX inhibited extracellular signal-regulated kinase (ERK) and p38 activation in both RANKL- and TNF-α/IL-6- stimulated conditions. However, c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase (PI3K), and NF-κB levels were not significantly altered. Data are representative of five independent experiments and expressed as mean ± SEM; \* *p* < 0.05 versus FX untreated cells; † *p* < 0.05 by Jonckheere–Terpstra test.

Previous studies demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) is a negative regulator of osteoclastogenesis [32–34]. Nrf2 deficiency augments the RANKLinduced activation of ERK and p38 MAP kinases in mouse bone-marrow-derived osteoclast precursor cells [32]. Moreover, FX activates Nrf2 in nonbone and RAW264.7 cells [27,35–37]. We assessed the levels of phosphorylated Nrf2 and Nrf2 proteins in total cell extracts, and the nuclear and cytosolic fractions of cell lysates by Western blotting (Figure 7A). Nrf2 levels in total cell lysates were not affected by FX treatment. Expression of nuclear Nrf2 significantly increased, while cytosolic Nrf2 expression dose-dependently decreased. The proportions of phospho-Nrf2 expression in the nucleus compared to in the cytoplasm were significantly augmented by FX. This effect increased with concentration (Figure 7B). These results sugges<sup>t</sup> that FX induces the dose-dependent phosphorylation and nuclear translocation of Nrf2 in RAW264.7 cells.

**Figure 7.** Effect of FX treatment on phosphorylated nuclear factor erythroid 2-related factor 2 (p-Nrf2) expression and nuclear localization of Nrf2 during osteoclast differentiation. Total cellular proteins were extracted from RAW264.7 cells and p-Nrf2 and Nrf2 expression were assessed by Western blotting. Cell lysates were fractionated into nuclear and cytosolic extracts, and identical experiments were performed. (**A**) Representative immunoblots and graphs for Nrf2 in total cell lysate, nucleus, and cytosol, and (**B**) nuclear/cytoplasmic p-Nrf2 from three independent experiments are shown. Data expressed as mean ± SEM; \* *p* < 0.05 versus FX untreated cells; † *p* < 0.05 by Jonckheere–Terpstra test.
