To confirm the effect of astaxanthin on cell growth, proliferation of NPCs treated with different concentrations (1, 5, and 10 ng/mL) of astaxanthin for three days was evaluated by trypan blue exclusion. As shown in Figure 1A, this treatment significantly increased proliferation of NPCs in a dose-dependent and time-dependent manner. In particular, 10 ng/mL astaxanthin showed the highest proliferation of NPCs. Therefore, 10 ng/mL was determined to be the optimal treatment for the study of NPCs. A clonogenic assay was also performed to estimate the proliferation efficiency of astaxanthin-treated NPCs. Because colony-forming units (CFU) are single cell populations, increases in CFU values show that astaxanthin can actively stimulate proliferation of NPCs. Proliferation efficiency of CFU in astaxanthin-treated cells was assessed via visual colony counts. In the CFU assay, astaxanthin-treated NPCs showed increased colony formation compared with control NPCs (Figure 1B). In particular, 10 ng/mL astaxanthin-treated NPCs showed an approximately two-fold increase in colony formation (Figure 1B).

In both control NPCs and astaxanthin-treated cells, expression of molecular markers, including proliferation-related transcription factors and stemness genes, was assessed via RT-PCR. As shown in Figure 2A, 10 ng/mL astaxanthin applied for three days significantly induced upregulation of proliferation-related transcription factors (Rex1, CDK1, and CDK2), coupled with overexpression of stemness genes (OCT4, SOX2, Nanog, and KLF4) [25,26]. In particular, Rex1 expression was markedly increased in astaxanthin-treated cells. This result revealed that Rex1 expression is closely associated with proliferation of NPCs. In a recent study, we showed that Rex1 is a major gene, the expression of which is closely associated with proliferation of adipose tissue stromal cells [27]. Our present results are consistent with a recent report in which enhancement of Rex1 expression caused increased efficiency of cell proliferation. According to a recent report, four transcription factors (Oct4, Sox2, Klf4, and c-Myc) have been shown to reprogram primary mouse fibroblasts in culture [28]. Also, a balance between Klf4 and c-Myc is, in all likelihood, necessary for stable reprogramming in induced pluripotent stem cells [28]. In this study, astaxanthin-treated NPCs were shown to overexpress not only Oct4, Sox2, Nanog, and Rex1, but also Klf4 for the acquisition of active self-renewal activity (Figure 2A). Therefore, these results show that astaxanthin can induce active cell proliferation and that it improves stem cell potency in NPCs via stemness acting signals.

For identification of possible activated signaling molecules involved in active cell proliferation occurring after astaxanthin treatment, total protein levels of several proliferation-related proteins were assessed in NPCs by Western blot analysis. Figure 2B shows the results of Western blot in astaxanthin-treated NPCs for different lengths of time (0, 6, and 12 h). In Western blot analysis, astaxanthin induced significant activation of PI3K and its downstream mediators, p-Rac, p-c-Raf, p-MEK, p-ERK, p-Akt, p-mTOR, and p-Stat3 [29] in a time-dependent manner. This study then examined the relevance of the PI3K and MEK signaling pathways in cell growth in astaxanthin-treated NPCs. For these studies, inhibition assays were performed with specific inhibitors, LY294002 (a specific inhibitor of PI3K) and PD98059 (a specific inhibitor of MEK). Astaxanthin-treated NPCs were treated with LY294002 or PD98059, or were left untreated. After LY294002 and PD98059 treatment respectively, the relative cell proliferation rate of astaxanthin-treated NPCs was assessed by trypan blue exclusion: Results are shown in Figure 3A. PI3K and MEK inhibition have been shown to cause inhibition of cell growth in astaxanthin-treated NPCs in vitro. As shown in Figure 3B, the results of Western blot analysis indicated that LY294002 significantly induced downregulation of PI3K, p-Rac, p-c-Raf, p-MEK, p-ERK, p-Akt, p-mTOR, and p-Stat3 proteins.

In addition, PD98059 significantly induced reduction of p-MEK, p-ERK, and p-Stat3 proteins (Figure 3C). Results of RT-PCR also indicated that PD98059 caused downregulation of proliferation-related transcription factors (Rex1, CDK1, and CDK2) and stemness genes (OCT4, SOX2, Nanog, and KLF4) (Figure 3D). In this study, the activation of p-ERK in the astaxanthin-treated NPCs resulted in the induction of stemness transcription factor expression, particularly Rex1 expression (Figure 3). Conversely, inhibition of PI3K and MEK in NPCs induced a reduction in cell proliferation (Figure 3A) and stemness transcription factor expression (Figure 3D). Therefore, the upregulation of Rex1 production by astaxanthin is clearly required for cell proliferation. This result indicated that Rex1 is a major gene, the expression of which is closely associated with the proliferation of NPCs, and that astaxanthin increases NPCs proliferation efficiency via an enhancement of Rex1 expression. In conclusion, these results clearly showed that astaxanthin induces proliferation of NPCs via activation of the PI3K and MEK signaling pathways.

Based on our present data, we suggest a model, shown in Figure 4, for explanation of the astaxanthin-induced cell proliferation mechanism through activation of the PI3K and MEK signaling pathways.

Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) was purchased from Sigma Chemicals (St. Louis, MO, U.S.) and used in all of the cell culture experiments. A stock solution of astaxanthin was made with dimethyl sulfoxide (DMSO) and stored at 4 °C. The stock solution was diluted to working concentrations prior to use.

For preparation of NPCs, mice were anesthetized deeply using a pentobarbital in 0.9% sterile saline solution and sacrificed by decapitation. To obtain NPCs, the region of complete cervical enlargement (spinal cord level C3 through T1) was dissected out. The tissue was minced, washed in sterile Dulbecco’s phosphate buffered saline (DPBS), and digested in a solution of 0.125% of trypsin, DNase (0.01%, Sigma) in Hank’s balanced salt solution (HBSS) for 30 min at 37 °C. Cells were transferred to culture dishes containing serum free growth medium, which consists of neurobasal (NB) medium with B27 supplement, basic fibroblast growth factor (bFGF, 20 ng/mL), and epidermal growth factor (EGF, 20 ng/mL).

Cultured NPCs were seeded in 10 cm dishes at a density of 5 × 10⁵ and cultured in NB media at 37 °C in a CO₂ incubator. Cells were then treated with astaxanthin at various concentrations (1, 5, and 10 ng/mL) for 3 days. Cell viability was assessed by visual cell counts in conjunction with trypan blue exclusion. In all viability assays, triplicate wells were used for each condition, and each experiment was repeated at least three times.

Proliferation efficiency of colony-forming units (CFU) in astaxanthin-treated cells was assessed. Cultured NPCs were seeded in 10 cm dishes at a density of 5 × 10⁵ and cultured in NB media at 37 °C in a CO₂ incubator. Cells were then treated with astaxanthin (10 ng/mL) for 3 days. For the CFU assay, control NPCs and astaxanthin-treated NPCs were seeded in 10-cm dishes at a density of 2 × 10² and cultured in NB media at 37 °C in a CO₂ incubator. After 15 days, cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and stained with 0.1% toluidine blue in 1% PFA. Proliferation efficiency of the CFU was assessed via visual colony counts [30].

For confirmation of differentially expressed proteins after astaxanthin (10 ng/mL) treatment in cultured NPCs, NPCs and astaxanthin-treated cells were lysed in 500 μL of lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EGTA, 1 mM glycerophosphate, 1 mM Na₃VO₄, and 1 mM PMSF. Lysates were clarified by centrifugation at 15,000 × g for 10 min, and the total protein content was determined using a Bio-Rad (Millan, Italy) protein assay kit. For Western blotting, equal amounts (40 μg) of protein extracts in a lysis buffer were subjected to 10% SDS-PAGE analysis and transferred to a nitrocellulose membrane [31]; anti-phosphatidylinositol 3-kinase (PI3K; 1:1000), anti-p-Rac (1:1000), anti-p-c-Raf (1:1000), anti-p-mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK; 1:1000), anti-p-extracellular signal-regulated kinase (ERK; 1:1000), anti-p-Stat3 (1:1000), anti-p-Akt (1:1000), p-mTOR (Cell Signaling Technology, Danvers, MA, U.S.; 1:1000), and anti-β-Actin (Sigma) antibodies were then incubated with membranes. Relative band intensities were determined using Quality-one 1-D analysis software (Bio-Rad).

Total cellular RNA was extracted with Trizol (Life Technologies, Frederick, MD, U.S.). Concentration of RNA was determined spectrophotometrically, and concentration and integrity were further evaluated by agarose gel electrophoresis. Residual DNA, if any, was eliminated from the samples using DNase I (RNase-free, Takara Bio Inc, Kyoto, Japan). cDNA was synthesized from the RNA using an oligo-dT primer amplified by 35 cycles (94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min) of PCR using 20 pM of specific primers. PCR amplification was performed using the primer sets. Duplicate PCR reactions were amplified using primer designed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control for assessment of PCR efficiency and for subsequent analysis by 1.5% agarose gel electrophoresis. PCR products were stained with ethidium bromide [32].

To confirm the relevance of the PI3K and MEK signaling pathways in controlling the growth of astaxanthin-treated NPCs, cells were seeded in 10 cm dishes at a density of 5 × 10⁵ and cultured in NB media at 37 °C in a CO₂ incubator. Cells were then treated with astaxanthin (10 ng/mL) for 1 day. Astaxanthin-treated NPCs were treated with PI3K inhibitor LY294002 (10 μM; Promega, Madison, WI, U.S.), MEK inhibitor PD98059 (10 μM; Sigma), or were left untreated. Cells were analyzed by Western blot and RT-PCR [27].

All data were presented as mean ± S.E.M from five or more independent experiments. Statistical significance of the differences between groups was calculated by using the Student’s two tailed t-test.