As shown in Table 1, fucoxanthin (at the dose of 50 and 100 mg/kg) caused a significant decline of sarcoma weight in a dose-dependent manner compared with the model control group (p < 0.05 or p < 0.01). The tumor inhibitory rate of fucoxanthin was 41.0% and 72.2% at the dose of 50 and 100 mg/kg, respectively.

Our in vivo study provides information that fucoxanthin reduced the S180 sarcoma weight in a dose-dependent manner indicating its exact role in anticancer therapy, which appears consistent with previous in vitro researches [8,9,10,11,12,13,14,15,16,17,18,19]. Fucoxanthin at the high-dose showed better suppressive effect on the tumor growth of S180 sarcoma than that of cyclophosphamide (CTX). Moreover, during the experimental period, we found that some mice in CTX-treated group were in bad mental state, manifested as sluggishness and drowsiness, with thinning of hair (data not shown), which was not found in the fucoxanthin-treated groups.

Apoptosis in sarcoma was detected by in situ end-labeling of nuclear DNA fragments (TUNEL) staining. In the TUNEL assay (Figure 1), sarcoma sections from the model control group showed only slight background stained with a few TUNEL-positive cells (5.29 ± 0.28 apoptotic cells/×200 field), whereas in the CTX-treated group, the number of TUNEL-positive cells significantly increased (16.29 ± 0.68 apoptotic cells/×200 field, p < 0.01, vs. model control group). Following treatment with fucoxanthin (50 and 100 mg/kg), the number of TUNEL-positive cells also markedly elevated (9.03 ± 0.58 apoptotic cells/×200 field in 50 mg/kg fucoxanthin group; 17.53 ± 0.62 apoptotic cells/×200 field in 100 mg/kg fucoxanthin group, p < 0.05 or p < 0.01, vs. model control group).

We further investigated the expression levels of two key apoptosis-related genes—caspase-3 and bcl-2 in tumor tissue, which were determined by western blotting (Figure 2A,B). The pro-apoptotic cleaved caspase-3 protein level was obviously increased, while the anti-apoptosis gene bcl-2 protein level was decreased by CTX (p < 0.01, vs. model control group), fucoxanthin at the dose of 50 and 100 mg/kg (p < 0.05 or p < 0.01, vs. model control group).

To explore the anti-tumor mechanisms of fucoxanthin in S180 tumor, we examined the protein expressions of survivin and VEGF. Western blotting analysis was used to detect the effects of fucoxanthin on the expression of survivin in S180 tumor tissue. The result showed the expression of survivin was dramatically decreased by CTX and fucoxanthin at the dose of 50 or 100 mg/kg (p < 0.05 or p < 0.01, vs. model control group, Figure 3). Immunohistochemistry analysis showed that the expression of VEGF-positive cells were obviously down-regulated by CTX and fucoxanthin (Figure 4A). Western blotting analysis also confirmed that the expression of VEGF (Figure 4B) was decreased by CTX and fucoxanthin at the dose of 50 or 100 mg/kg compared with the control group (p < 0.05 or p < 0.01).

Survivin is the smallest and structurally unique member of the inhibitor of apoptosis (IAP) gene family preferentially expressed in a myriad of clinical cancers [30]. Absent in most adult tissues, surviving is selectively upregulated in many human tumors [31]. The over-expressed survivin protects against apoptosis by either directly or indirectly inhibiting the activation of effector caspases [32]. VEGF is another protein that is a potent stimulator of cancer angiogenesis, inducer of endothelial cell migration and vascular permeability. Notably, VEGF was reported in previous studies to contribute to high degree of vascularization in malignant tumor, where it is up-regulated by oncogenic expression and a variety of growth factors, and promote tumor progression, thus has become a central therapeutic target in treatment of malignancy [33,34]. In our study, western blot assay showed that both survivin and VEGF were overexpressed in model control group but displayed a significantly lower expression in fucoxanthin-treated groups at middle and high dose and CTX-treated group.

It was reported that survivin and VEGF expressions are both upregulated primarily by activation of STAT3 signaling, which were known as STAT3-survivin signaling pathway [32] and STAT3-mediated VEGF pathway [23,35], respectively. STAT3 is an important member of the STAT family as DNA binding protein. The continuous activation of STAT3 can induce proliferation, differentiation, closely related genes high expression, thus promoting cell proliferation, malignant transformation, blocking cell apoptosis, displaying a strong effect that causes cancer [36]. At present, many STAT3 target genes have been identified including antiapoptotic gene bcl-xl, bcl-2, mcl-1, cyclin D, c-Myc, MMPs, survivin and VEGF [37]. Previous researches have proven that fucoxanthin down-regulated bcl-xl [13] and cyclin D [10,18]. In this research, we found fucoxanthin treatment groups inhibited the expressions of bcl-2, survivin and VEGF. Since these STAT3 target genes all are influenced by fucoxanthin, we raised up a question: could fucoxanthin regulate STAT3 expression in S180 tumor? To address this item, we focused our attention on STAT3 and its phosphorylation protein level. As expected, fucoxanthin decreased the expression of STAT3 and phosphorylated STAT3 (p-STAT3), which was consistent with our in vitro result [19]. At the presence of CTX and fucoxanthin at the dose of 50 or 100 mg/kg body weight, the protein expression of STAT3 (Figure 5A) and p-STAT3 (Figure 5B) were reduced than those of the model control group (p < 0.05 and p < 0.01, respectively), all these effects of fucoxanthin being presented in a dose-dependent manner.

STAT3 has been reported to be activated by interleukin-6 receptor (IL-6R) or EGFR in carcinomas, which may have important implications for responsiveness to therapeutics targeted at EGFR, IL-6R, or intermediary kinases [38]. EGFR pathway is one of the most dysregulated molecular pathways in human cancers. The activation of EGFR, an ubiquitously expressed transmembrane glycoprotein belonging to the ErbB/HER family of receptor tyrosine kinases, has been found to lead to the rapid phosphorylation of STAT3 on tyrosine 705 and the subsequent activation of STAT3-dependent gene expression [39]. On the other hand, activation of the EGFR pathway increases the production of tumor-derived VEGF that acts on endothelial cells in a paracrine manner to promote angiogenesis [40]. EGFR expression was determined by immunohistochemistry and western blot (Figure 6A,B). There were few positive immunostained cells in CTX positive control group, which was also evidenced by western blot. Treatment with fucoxanthin at the dose of 25, 50 and 100 mg/kg obviously reduced EGFR expression compared with the model control group (p < 0.05, Figure 6B). These data indicated that, consistent with those of STAT3 and VEGF, the expression of EGFR in the tumor tissue was remarkably declined by treatment with fucoxanthin, suggesting that down-regulation of STAT3/EGFR signaling appeared to involve in the in vivo anti-tumor effect and apoptosis induction of fucoxanthin.

Fucoxanthin was prepared as previous [19], and dissolved in soybean oil to a final concentration of 2.5, 5, 10 mg/mL and stored at 4 °C until used. CTX was purchased from Sigma, USA, and diluted with saline to a concentration of 2 mg/mL.

Male Kunming mice (6–8 weeks old, 20 ± 2 g) were obtained from the Experimental Animal Center of the Hubei Province (Grate II, Certificate No. SCXK-2008-0004, Hubei, China). S180 tumor cells used in experiment was given by Tongji Medical College of Huazhong University of Science and Technology. Mice were housed at a constant room temperature of 22 °C and maintained under controlled conditions of 12 h light/12 h dark photoperiod. All animals received human care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and the experiments were approved by the Animals Care and Use Committee of Wuhan University of Science and Technology Medicine College. After fed in our facility for 1 week, all 75 mice were randomly divided into 5 groups of 15 animals in each group: (1–3) fucoxanthin treatment groups: administered with fucoxanthin, at low (25 mg/kg), middle (50 mg/kg), and high (100 mg/kg) oral administration dose once per day respectively; (4) model control: orally administered with the same volume of soybean oil; (5) positive control group: CTX was injected intraperitoneally at a dosage of 20 mg/kg body weight once per day. After fucoxanthin pretreatment for 1 week, sarcoma 180 ascites tumor cells (2 × 10⁶ cell/500 μL) were implanted subcutaneously into the left hind groin of the mice in all groups. 24 h after inoculation, all animals were treated as planned. On day 14 after inoculation, all animals were sacrificed, and then the sarcoma tissues were isolated and weighted for TUNEL, immunohistochemistry and western blot analysis.

Small pieces of sarcoma tissues taken from experimental animals were fixed in 10% formalin, dehydrated with alcohol, embedded in paraffin, and sectioned to a mean thickness of 4 μm. Then, the sections were used to perform TUNEL. Briefly, the tissue sections were hydrated with gradient alcohol (100, 95, 90, 80, 70%). The endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol for 3 min at 20 °C. After washing with PBS, the sections were permeabilized with Proteinase K solution (4 mg Proteinase K powder dissolved in 200 mL 10 mMTris/HCl to a final concentration of 20 μg/mL, pH = 7.4–8.0) for 1 min on the ice. After permeabilization, the sections were exposed to the mixture of TdT and dUTP at 37 °C for 60 min. A converter-POD solution was applied to the sections for 30 min at 37 °C in darkness. And then the sections were stained with DAB to generate a brown reaction product. Negative controls were performed at the same time. The number of positive cells was counted using the light microscope.

The paraffin-embedded sections were placed for 60 min at room temperature. Then the paraffin sections were dewaxed in xylene for 10 min, rehydrated with a series of gradient alcohol (100, 95, 90, 80, 70%), respectively. After that the sections were incubated in methanol containing 3% H₂O₂ for 10 min to inactivate endogenous peroxidase. Sections were incubated with Clean Vision™ blocking solution (ImmunoVision Technologies Co., CA, USA) for 1–2 h. Following washing three times for 5 min in PBS, the slides were incubated with the flowing antibody: rabbit anti-EGFR polyclonal antibody or mouse anti-VEGF monoclonal antibody (ab2430 abcam, ab1316 abcam, UK) at a dilution of 1:800 and 1:1000, respectively at room temperature for 2 h. And then a biotinylated goat anti-rabbit or goat anti-mouse antibody (Santa Cruz, CA, USA) was applied as secondary antibodies at room temperature for 2 h. Peroxidase activity was revealed by dipping the sections in a mixture containing 0.05% 3,3′-diaminobenzidine (DAB) and 0.03% H₂O₂ for 5 min. The sections were then counterstained with hematoxylin, coverslipped, and observed under a microscope.

The sarcoma tissues were removed from mice in each group and then washed twice with cold PBS before homogenized with a homogenizer in ProteoJET™ Mammalian Cell Lysis Reagent (MBI fermentas) followed by centrifugating at 4 °C, 14,000 rpm for 20 min. The protein concentration in the lysate was measured by the Lowry method [41]. The supernatants (50 µg protein/lane) were separated by 10% SDS-PAGE and then transferred to nitrocellulose membrane by electroblotting. The membrane was then blocked with TBS-T (20 mM Tris–HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature. The sources of primary antibodies used in this study were as follows: VEGF, EGFR (ab1316 abcam, ab2430 abcam, UK), survivin, bcl-2, caspase-3, STAT3, p-STAT3 (tyrosine 705), β-actin (sc-47750, sc-7382, sc-56055, sc-8019, sc-8059, sc-47778, Santa Cruz, CA, USA). The membrane was incubated with above antibodies for 2 h. After washing with 0.1% Tween-20 in TBS, the membranes were incubated with a secondary antibody horseradish peroxidase-conjugated anti-mouse IgG/anti-rabbit IgG (Santa Cruz, CA, USA) for 2 h at room temperature. Finally, the membrane was treated with the reagents in the enhanced chemiluminescence detection kit (ECL system, Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer’s instructions and exposed by using an X-ray film. The relative density of the immunoreactive bands was quantitated by Quantity One (Version 4.6.2, Bio-Rad Technical Service Department, USA). The relative amount of survivin, VEGF, caspase-3, bcl-2, EGFR, STAT3 and p-STAT3, in each lane was obtained after correcting with the amount of β-actin in the same sample.

All the grouped data were statistically evaluated with SPSS11 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test; p value of less than 0.05 were considered to indicate statistical significance. All the results were expressed as the mean ± SD for 15 animals in each group.