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Fucoidan ingestion increases the expression of CXCR4 on human CD34+ cells

  • Mohammad R. Irhimeh
    Correspondence
    Offprint requests to: Mohammad R. Irhimeh, Discipline of Medicine, University of Tasmania, Hobart 7001, TAS Australia
    Affiliations
    Discipline of Medicine, Clinical School, University of Tasmania, Hobart, Australia

    Clinical Haematology and Medical Oncology Unit, Royal Hobart Hospital, Hobart, Australia
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  • J. Helen Fitton
    Affiliations
    Discipline of Medicine, Clinical School, University of Tasmania, Hobart, Australia

    Marinova Pty Ltd., Hobart, Australia
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  • Raymond M. Lowenthal
    Affiliations
    Discipline of Medicine, Clinical School, University of Tasmania, Hobart, Australia

    Clinical Haematology and Medical Oncology Unit, Royal Hobart Hospital, Hobart, Australia
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      Objective

      Transplantation of hematopoietic progenitor stem cells (HPC) is an important treatment modality for a variety of neoplastic diseases. HPC collection for transplantation with granulocyte colony-stimulating factor may be unsuccessful in patients who have received prior chemotherapy or for other reasons. Methods to improve mobilization of HPCs are required. Disruption of the interaction between the cell surface receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1) is a mechanism for HPC release from the bone marrow into the peripheral blood (PB).

      Methods

      We carried out a clinical trial to evaluate the effects of ingestion of a fucoidan, galactofucan sulfate (a putative HPC mobilizing agent) on circulating CD34+ cells, CXCR4 expression, and levels of SDF-1, interferon gamma (IFN-γ) and interleukin 12.

      Results

      Following ingestion of fucoidan, CD34+ cells increased significantly in the PB from 1.64 to 1.84 cells/μL after 4 days. The proportion of CD34+ cells that expressed CXCR4 increased from 45 to 90% after 12 days, the plasma level of SDF-1 increased from 1978 to 2010 pg/mL, and IFN-γ level increased from 9.04 to 9.89 pg/mL.

      Conclusion

      Oral fucoidan significantly amplified the CXCR4+ HPC population. The ability to mobilize HPC using sulfated polysaccharides and mobilize more HPC with high levels of CXCR4 could be clinically valuable.
      Autologous transplants of hematopoietic progenitor stem cells (HPC) are used to treat a variety of neoplastic and other diseases. HPC can be mobilized from the bone marrow (BM) niche into the peripheral blood (PB) via the administration of granulocyte colony-stimulating factor (G-CSF) or other agents such as AMD3100 or fucoidan. The mechanisms for the mobilization may include modulation of serine proteases, metalloproteases, and of stromal derived factor-1 (SDF-1)-CXCR4 interactions [
      • Nervi B.
      • Link D.C.
      • DiPersio J.F.
      Cytokines and hematopoietic stem cell mobilization.
      ].
      The success of subsequent engraftment may be associated with the expression of the receptor CXCR4 on CD34+ HPC. This effect can be observed both clinically [
      • Spencer A.
      • Jackson J.
      • Baulch-Brown C.
      Enumeration of bone marrow “homing” haemopoietic stem cells from G-CSF-mobilised normal donors and influence on engraftment following allogeneic transplantation.
      ] and experimentally [
      • Brenner S.
      • Whiting-Theobald N.
      • Kawai T.
      • et al.
      CXCR4-transgene expression significantly improves marrow engraftment of cultured hematopoietic stem cells.
      ,
      • Lapidot T.
      • Dar A.
      • Kollet O.
      How do stem cells find their way home?.
      ]. Mobilization of adequate amounts of HPCs including CD34+ CXCR4+ is not always successful. Failure to achieve sufficient mobilization can occur in patients who have received multiple cycles of chemotherapy or for other, unknown reasons.
      Previous research has shown that intravenous (IV) fucoidan has a pronounced and extended mobilizing effect on HPC in mice and on nonhuman primates [
      • Sweeney E.A.
      • Lortat-Jacob H.
      • Priestley G.V.
      • Nakamoto B.
      • Papayannopoulou T.
      Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.
      ,
      • Sweeney E.A.
      • Priestly G.V.
      • Nakamoto B.
      • Collins R.
      • Beaudet A.
      • Papayannopoulou T.
      Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin engagement.
      ,
      • Frenette P.S.
      • Weiss L.
      Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms.
      ,
      • Sweeney E.A.
      • Papayannopoulou T.
      Increase in circulating SDF-1 after treatment with sulphated glycans, the role of SDF-1 in mobilization.
      ]. This effect is postulated to be the result of dissociation of the chemokine SDF-1 from the BM stroma, creating an attractive gradient into the peripheral circulation. Disruption of the interaction between the CXCR4 and its ligand SDF-1 is one of the mobilizing mechanisms also common to G-CSF [
      • Petit I.
      • Szyper-Kravitz M.
      • Nagler A.
      • et al.
      G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and upregulating CXCR4.
      ] and newer agents such as AMD3100 [
      • Broxmeyer H.E.
      • Orschell C.M.
      • Clapp D.W.
      • et al.
      Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.
      ,
      • Devine S.M.
      • Flomenberg N.
      • Vesole D.H.
      • et al.
      Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin's lymphoma.
      ].
      Fucoidan is a generic term for the sulfated, fucose-rich polysaccharides derived from brown macroalgae [
      • Berteau O.
      • Mulloy B.
      Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide.
      ] or echinoderms [
      • Mulloy B.
      • Ribeiro A.C.
      • Alves A.P.
      • Vieira R.P.
      • Mourao P.A.
      Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the 0-2 and 0-4 positions.
      ]. In animal models, ingestion of fucoidan has inhibitory effects on tumors, which appear to be associated with a rise in interferon-gamma (IFN-γ), interleukin-12 (IL-12), and stimulation of innate immunity [
      • Maruyama H.
      • Tamauchi H.
      • Hashimoto M.
      • Nakano T.
      Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida.
      ,
      • Irhimeh M.R.
      • Fitton J.H.
      • Lowenthal R.M.
      • Kongtawelert P.
      A quantitative method to detect fucoidan in human plasma using a novel antibody.
      ,
      • Mavier P.
      • Martin N.
      • Couchie D.
      • Pre´aux A.
      • Laperche Y.
      • Zafrani E.
      Expression of stromal cell-derived factor-1 and of its receptor CXCR4 in liver regeneration from oval cells in rat.
      ,
      • Funahashi H.
      • Imai T.
      • Mase T.
      • et al.
      Seaweed prevents breast cancer?.
      ]. In vitro treatment of BM mononuclear cells (MNCs) with IFN-γ can upregulate the expression of CXCR4 granulocyte precursors and monocytes [
      • Funahashi H.
      • Imai T.
      • Mase T.
      • et al.
      Seaweed prevents breast cancer?.
      ].
      Despite the available literature evidence that IV fucoidan had an effect in tumor animal models and mobilization of HPC, there are no reports to date about the clinical use of oral fucoidan to modulate or mobilize HPC. Although fucoidans synergize with G-CSF to increase mobilization 11 times over G-CSF alone in primates, to date no clinical trials in patients have been reported. In this study, we examined the effects of orally ingested Undaria pinnatifida fucoidan on the PB stem cells, the expression of CXCR4, and plasma levels of SDF-1, IL-12, and IFN-γ.

      Materials and methods

      Human volunteers

      In a single-blind, randomized, placebo-controlled clinical trial, 37 nonsmoker volunteers of either sex were divided into three groups after giving informed consent and after human ethics approval was obtained from the Southern Tasmania Health & Medical Human Research Ethics Committee. As placebos, six volunteers took 3 g of guar gum. Another six volunteers took 3 g of whole Undaria containing 10% w/w fucoidan, and another 25 volunteers took 3 g of 75% w/w fucoidan daily for 12 days. All volunteers took three capsules (0.33 g each) three times a day. During the study time, volunteers were asked not to eat any seafood, seaweed-derived products, drugs, or food supplements. Blood samples were collected as described later.

      Preparation of capsules

      Fucoidan is a highly sulfated, polyanionic soluble fiber derived from Tasmanian Undaria pinnatifida by Marinova Pty. Ltd. (Hobart, TAS Australia). We chose a neutral nonsulfated dietary fiber derived from guar gum as a placebo (Novartis Pty. Ltd., Mulgrave, VIC Australia). The capsules were prepared as described previously [
      • Irhimeh M.R.
      • Fitton J.H.
      • Lowenthal R.M.
      • Kongtawelert P.
      A quantitative method to detect fucoidan in human plasma using a novel antibody.
      ]. The structure and the therapeutic characteristics of these compounds have been described previously [
      • Berteau O.
      • Mulloy B.
      Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide.
      ,
      • Maruyama H.
      • Tamauchi H.
      • Hashimoto M.
      • Nakano T.
      Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida.
      ,
      • Irhimeh M.R.
      • Fitton J.H.
      • Lowenthal R.M.
      • Kongtawelert P.
      A quantitative method to detect fucoidan in human plasma using a novel antibody.
      ].

      Collection of blood samples

      Venous blood from the antecubital vein from the three groups of volunteers was collected using ethylenediamine-tetraacetic acid (EDTA) tubes. Plasma samples (platelet poor for SDF-1 assay) were collected and stored in aliquots at −80°C within 30 minutes of collection for later analysis. Complete blood counts were obtained using an automated cell counter (CELL-DYN-4000 System, Abbott Lab, IL, USA).

      Flow cytometry analysis

      The expression of different surface membrane markers on normal human PB HPCs was evaluated by fluorescein-activated cell sorting (FACS) direct immunofluorescence. Cells were Fc-blocked with 1 μg of human immunoglobulin G (IgG)/105 cells (Zymed Lab., San Francisco, CA, USA) for 15 minutes at room temperature, and then stained with the designated monoclonal antibody. FITC-CD34, Cy5-CD45 (Becton Dickinson, San Jose, CA, USA) and PE-CXCR-4 (R&D Systems Inc., Minneapolis, MN, USA) were used in the study. A negative control tube was prepared identically but contained isotype controls IgG1, or IgG2a antibodies (BD). FACS-fixed cells were analyzed using FACScan flow cytometer and the Cell Quest software package (BD).

      Preparation of MNCs and colony-forming unit granulocyte assays

      Human PB MNCs were isolated from healthy subjects using Histopaque-1077 kit and protocol from Sigma-Aldrich Co. (St. Louis, MO, USA). Briefly, 3 mL of EDTA blood was layered onto the Histopaque-1077 and centrifuged at 400g for 30 minutes. The opaque interface was mixed with 10 mL isotonic phosphate-buffered saline (PBS) then centrifuged at 250g for 10 minutes. The cell pellet was washed with 5 mL PBS twice and resuspended in 0.3 mL PBS. Peripheral blood mononuclear cells were plated at densities of 1 × 105 viable nucleated cells per 35-mm plate using growth medium (MethoCult GFH4534, StemCell Technologies, Vancouver, BC, Canada), then colony-forming unit granulocyte (CFU-GM) was performed. Petri dishes were incubated for 14 days at 37°C in a humidified atmosphere containing 5% CO2. CFU-GM colonies defined as clusters of ≥30 cells were then counted using an inverted microscope and recorded as the mean of quadruplicate counts. Plates were neglected if contaminated or >50 colonies were counted.

      Plasma cytokines and cytokine assays

      PB was collected from volunteers according to the designated schedule in tubes containing EDTA. Platelet-poor plasma was prepared within 30 minutes of blood collection and stored at −80°C for later analysis. Three different cytokines, SDF-1, IFN-γ, and IL-12 levels were analyzed directly after thawing plasma samples gradually on the day of test by enzyme-linked immunosorbent assay using kits and protocols from R&D Systems Inc.

      Statistical analysis

      Student's t-test and analysis of variance were used to analyze data. A p value of 0.05 was chosen as the limit of statistical significance. Triplicate readings for each sample were averaged. All other statistical parameters were calculated using Microsoft Office, Excel and SPSS, version 12.

      Results

      No side effects were reported, and none of the volunteers exhibited toxicity when 3 g of guar gum, 10% fucoidan, or 75% fucoidan extracts were taken orally three times a day for 12 days.
      Fucoidan ingestion caused mild leukopenia and lymphopenia but had no effect on neutrophils.
      We observed a nonsignificant decrease in the total number of leukocytes in the PB when 10% fucoidan was ingested, but when 75% fucoidan was ingested, the decrease was significant after 12 days (Table 1). There was a decrease in leukocytes from 5.74 cells/nL at baseline to 5.37 after 12 days (p = 0.05). Of the leukocyte fractions, lymphocytes were most affected. Ingestion of either 10% or 75% fucoidan decreased the lymphocyte count but the decrease was only significant with the 75% fraction. The absolute number of lymphocytes decreased from 2.18 cells/nL at baseline to 1.98 after 12 days (p = 0.03; Table 1). Neutrophil count was not affected after ingesting guar gum or 10% or 75% fucoidan. Furthermore, there was no effect on the expression of CD16 in the CD45+ population (results not shown).
      Table 1Average readings of all of the tests at four time points
      Treatment
      Volunteers ingested 3 g of each treatment three times daily.
      Test0 days
      The mean value at 0 day (baseline) was used in the t-test as first set of data to which other groups are compared.
      4 days8 days12 daysn
      Guar gumLeukocyte (cells/nL)7.37 ± 0.237.38 ± 0.617.03 ± 0.877.22 ± 0.716
      Lymphocyte (cells/nL)2.80 ± 0.132.82 ± 0.162.65 ± 0.152.83 ± 0.186
      Neutrophil (cells/nL)3.63 ± 0.443.52 ± 0.383.45 ± 0.323.43 ± 0.266
      SDF-1 (pg/mL)1977 ± 111978 ± 81981 ± 21974 ± 163
      IFN-γ (pg/mL)9.03 ± 0.189.14 ± 0.159.2 ± 0.159.17 ± 0.083
      10% fucoidanLeukocyte (cells/nL)7.48 ± 0.356.98 ± 0.29
      p < 0.05 using paired Student's t-test.
      7.00 ± 0.516.95 ± 0.596
      Lymphocyte (cells/nL)2.87 ± 0.122.73 ± 0.162.6 ± 0.302.62 ± 0.266
      Neutrophil (cells/nL)3.73 ± 0.283.42 ± 0.183.42 ± 0.243.35 ± 0.316
      SDF-1 (pg/mL)1973 ± 81977 ± 51980 ± 251979 ± 153
      IFN-γ (pg/mL)9.01 ± 0.139.01 ± 0.389.05 ± 0.198.97 ± 0.273
      75% fucoidanLeukocyte (cells/nL)5.74 ± 0.285.62 ± 0.335.48 ± 0.305.37 ± 0.37
      p < 0.05 using paired Student's t-test.
      25
      Lymphocyte (cells/nL)2.18 ± 0.142.06 ± 0.131.95 ± 0.14
      p < 0.01 using paired Student's t-test.
      1.98 ± 0.14
      p < 0.05 using paired Student's t-test.
      25
      Neutrophil (cells/nL)2.94 ± 0.192.97 ± 0.222.90 ± 0.192.82 ± 0.2225
      SDF-1 (pg/mL)1978 ± 261996 ± 312101 ± 33
      p < 0.01 using paired Student's t-test.
      2059 ± 4723
      IFN-γ (pg/mL)9.04 ± 0.429.41 ± 0.499.89 ± 0.39
      p < 0.01 using paired Student's t-test.
      9.82 ± 0.57
      p < 0.05 using paired Student's t-test.
      20
      Table shows the mean readings observed on baseline (0 day) and on 4th, 8th and 12th day after ingesting 3 g of each treatment. Guar gum is used as a placebo control. All values are average ± mean standard error. n = number of volunteers, different volunteers have been used for each treatment.
      § Volunteers ingested 3 g of each treatment three times daily.
      The mean value at 0 day (baseline) was used in the t-test as first set of data to which other groups are compared.
      p < 0.05 using paired Student's t-test.
      p < 0.01 using paired Student's t-test.

      Increase in CD34+ cell count in PB after fucoidan ingestion

      A slight increase in the circulating CD34+ was observed after ingesting fucoidan. When 10% fucoidan (3 g/d) was ingested, a nonsignificant increase in CD34+ in PB, from 1.07 to 1.29 cells/μL (p = 0.06, n = 6) after 12 days, was observed. However, when 75% fucoidan was ingested, the CD34+ count increased from 1.64 cells/μL to 1.84, 1.80, and 1.79 cells/μL at 4, 8, and 12 days, respectively. This increase was significant at day 4 (p = 0.04). Some volunteers presented a large increase in the CD34+ on days 8 and 12 but were considered by the statistical program as outliers and were not included in the calculated median.

      Increase in the expression of CXCR4 on CD34+ cells after fucoidan ingestion

      When 3 g/d of 10% fucoidan was ingested, a nonsignificant increase in the CD34+CXCR4+ was observed. However, when 3 g/d of the 75% fucoidan was ingested, the CD34+CXCR4+ count increased significantly (p < 0.0002) from 0.75 cells/μL at baseline to 1.65 cells/μL after 12 days (Fig. 1). The proportion of CD34+CXCR4+ increased from 45 to 90% after 12 days of treatment (Table 2). A few volunteers showed a large increase in the CD34+ cell count but were considered by the statistical program as outliers and were not included in the calculated median.
      Figure thumbnail gr1
      Figure 1Total number of PB CD34+/CXCR4+ cells at baseline and after 4, 8, and 12 days of taking 75% fucoidan. Black lines represent medians for 23 duplicate experiments representing 23 volunteers. The boxes represent the median (50% of the population), and the error bars represent the mean standard error. +, outliers or high responders.
      Table 2Average percentage of cells that are CXCR4+ out of the total CD34+ cells at four time points
      Treatment
      Volunteers ingested 3 g of each treatment three times daily.
      0 days
      The mean value at 0 days (baseline) was used in the t-test as first set of data.
      4 days8 days12 daysn
      Guar gum62.33 ± 4.6464.15 ± 2.8166.43 ± 3.1863.66 ± 3.096
      10% fucoidan67.27 ± 8.3365.49 ± 9.1371.78 ± 9.0171.96 ± 13.096
      75% fucoidan49.75 ± 7.1984.57 ± 4.74
      p < 0.01 using paired Student's t-test.
      84.4 ± 5.57
      p < 0.01 using paired Student's t-test.
      91.15 ± 3.63
      p < 0.01 using paired Student's t-test.
      23
      The table shows the average readings observed on baseline (0 day) and on 4, 8, and 12 days after ingesting 3 g of each treatment. Guar gum is used as a placebo control. Values shown are average % of CD34+/CXCR4+ ± mean standard error. n = number of volunteers; different volunteers have been used for each treatment.
      Volunteers ingested 3 g of each treatment three times daily.
      The mean value at 0 days (baseline) was used in the t-test as first set of data.
      p < 0.01 using paired Student's t-test.

      Fucoidan ingestion has no effect on PB MNCs in CFU-GM

      Generally, there was a nonsignificant decrease in the number of CFU-GM per microliter of l blood after ingesting the 75% fucoidan. The mean CFU-GM count at baseline was 1.87/μL (±mean standard error = 0.29) and decreased to 1.71 (±0.26), 1.4 (±0.29), and 1.31 (±0.35) after 4, 8, and 12 days (p = 0.50, 0.19, 0.14; n = 13) of ingesting 75% fucoidan, respectively.

      Increase in plasma levels of SDF-1 and IFN-γ with fucoidan ingestion

      Ingestion of guar gum and 10% fucoidan did not affect the levels of SDF-1 and IFN- γ in plasma (Table 1). Volunteers who ingested 3 g of the 75% fucoidan had an elevation in the plasma level of SDF-1 after 8 days from 1978 to 2101 pg/mL (p = 0.00005).
      Further, the plasma level of IFN-γ was assessed because this cytokine has been associated with the upregulation of CXCR4. Volunteers who ingested 3 g of the 75% fucoidan showed a significant elevation (p = 0.04) in the plasma IFN-γ, from 9.04 to 9.82 pg/mL (Table 1). However, there was no detectable change in the IL-12 plasma (results not shown).

      Discussion

      We carried out this clinical study to determine the effects of ingested Undaria-derived fucoidan on PB. We found that there was a small increase in CD34+ cells and a profound increase, from 40 to 90%, in the proportion of CD34+/CXCR4+ when 75% fucoidan was ingested. A smaller increase was noted when 10% fucoidan was ingested. We also observed a significant increase in IFN-γ and SDF-1 in the 75% fucoidan group but not in the control or 10% groups. We did not observe an increase, but rather a nonsignificant decrease in CFU-GM, despite the increase in the CD34+ cell count. This slight increase after oral intake contrasts with the large and sustained response elicited by IV fucoidan [
      • Sweeney E.A.
      • Lortat-Jacob H.
      • Priestley G.V.
      • Nakamoto B.
      • Papayannopoulou T.
      Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.
      ,
      • Sweeney E.A.
      • Priestly G.V.
      • Nakamoto B.
      • Collins R.
      • Beaudet A.
      • Papayannopoulou T.
      Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin engagement.
      ,
      • Frenette P.S.
      • Weiss L.
      Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms.
      ,
      • Sweeney E.A.
      • Papayannopoulou T.
      Increase in circulating SDF-1 after treatment with sulphated glycans, the role of SDF-1 in mobilization.
      ], AMD3100 [
      • Broxmeyer H.E.
      • Orschell C.M.
      • Clapp D.W.
      • et al.
      Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.
      ], or G-CSF [
      • Spencer A.
      • Jackson J.
      • Baulch-Brown C.
      Enumeration of bone marrow “homing” haemopoietic stem cells from G-CSF-mobilised normal donors and influence on engraftment following allogeneic transplantation.
      ].
      Previous studies have shown that IV fucoidan produces rapid mobilization of murine HPCs with long-term BM repopulating potential in mice and nonhuman primates [
      • Sweeney E.A.
      • Priestly G.V.
      • Nakamoto B.
      • Collins R.
      • Beaudet A.
      • Papayannopoulou T.
      Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin engagement.
      ,
      • Frenette P.S.
      • Weiss L.
      Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms.
      ,
      • Sweeney E.A.
      • Papayannopoulou T.
      Increase in circulating SDF-1 after treatment with sulphated glycans, the role of SDF-1 in mobilization.
      ,
      • Petit I.
      • Szyper-Kravitz M.
      • Nagler A.
      • et al.
      G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and upregulating CXCR4.
      ]. CXCR4 was not assessed in those studies. Fucoidan has also been used as a tool to examine the effects of binding SDF-1. Mavier and colleagues [
      • Mavier P.
      • Martin N.
      • Couchie D.
      • Pre´aux A.
      • Laperche Y.
      • Zafrani E.
      Expression of stromal cell-derived factor-1 and of its receptor CXCR4 in liver regeneration from oval cells in rat.
      ] demonstrated that after experimental hepatic destruction, IV fucoidan blocked the SDF-1 expression of liver stem cells and markedly decreased their accumulation. Ingestion of fucoidan has been shown to inhibit tumors of various kinds [
      • Funahashi H.
      • Imai T.
      • Mase T.
      • et al.
      Seaweed prevents breast cancer?.
      ], an effect that may be attributable to a stimulation of the nonspecific immune system [
      • Maruyama H.
      • Tamauchi H.
      • Hashimoto M.
      • Nakano T.
      Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida.
      ,
      • Shimizu J.
      • Wada-Funada U.
      • Mano H.
      • Matahira Y.
      • Mitsuaki K.
      • Wada M.
      Proportion of murine cytotoxic T cells is increased by high molecular weight fucoidan extracted from Okinawa mozuku (Cladosiphon okamuranus).
      ]. Fucoidans are well-known experimental selectin blockers. In vitro, the binding of L-selectin on lymphocytes by fucoidan enhanced the expression of CXCR4 in lymphocytes [
      • Ding Z.
      • Issekutz T.B.
      • Downey G.P.
      • Waddell T.K.
      L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration.
      ]. Clinical use of a fucoidan preparation was made in the 1960 s. Claudio and Stendardo [
      • Claudio F.
      • Stendardo B.
      Contributo clinico sperimentale sull'uso un fitocolloide in oncologia.
      ] reported favorable results from patients with leukopenia and leukocytosis with increases in the general condition of patients.
      In a prior work, two preparations of fucoidan to mobilize HPCs have been used; 100 mg/kg IV of sulfated linear fucan from the sea urchin Lytechinus variegatus [
      • Sweeney E.A.
      • Priestly G.V.
      • Nakamoto B.
      • Collins R.
      • Beaudet A.
      • Papayannopoulou T.
      Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin engagement.
      ,
      • Sweeney E.A.
      • Papayannopoulou T.
      Increase in circulating SDF-1 after treatment with sulphated glycans, the role of SDF-1 in mobilization.
      ] and 25 mg/kg intraperitoneally of branched fucoidan fraction from Ascophyllum nodosum (from Fluka) [
      • Sweeney E.A.
      • Lortat-Jacob H.
      • Priestley G.V.
      • Nakamoto B.
      • Papayannopoulou T.
      Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.
      ]. Despite the differences in fucoidans, similar mobilizations were seen and attributed to the creation of SDF-1 gradient into the PB.
      The fucoidan used in our studies is derived from Undaria pinnatifida. Our previous work using an antibody-based detection method indicated that when 3 g of 75% fucoidan was ingested daily, plasma concentration was elevated up to 4 mg/L after 4 days, and then 13 mg/L after 12 days despite the fact that fucoidan is a large-molecular-weight material [
      • Irhimeh M.R.
      • Fitton J.H.
      • Lowenthal R.M.
      • Kongtawelert P.
      A quantitative method to detect fucoidan in human plasma using a novel antibody.
      ]. Acidic conditions in the stomach may cause a limited hydrolysis of the fucoidan. Humans do not produce enzymes capable of breaking down fucoidans, and the latter also appear to be unaffected by human fecal flora [
      • Michel C.
      • Lahaye M.
      • Bonnet C.
      • Mabeau S.
      • Barry J.L.
      In vitro fermentation by human faecal bacteria of total and purified dietary fibers from brown seaweeds.
      ]. We hypothesized that small quantities of fucoidan may cross the intestinal wall as whole molecules probably by endocytosis. The slight but significant changes in HPCs shown here do not indicate stand-alone utility of this substance as a clinical entity but instead indicate a need for further investigation of its potential. Further work is needed to investigate the repopulating potential of the cells, and the timing of peak values for CXCR4 and total numbers of cells.
      AMD3100, which is a reversible inhibitor of the binding of SDF-1α to its cognate receptor CXCR4, is currently in clinical trials as a mobilizing agent. It significantly improves the mobilization capacity of G-CSF when used in combination with it in mice [
      • Cottler-Fox M.A.
      • Lapidot T.
      • Petit I.
      • Kollet O.
      • DiPersio J.F.
      • Link D.
      • Devine S.
      Stem cell mobilization.
      ]. We postulate that fucoidan may have a similar mechanism of action on SDF-1/CXCR4 that could play a role in the mobilization of CD34+ cells from BM to PB especially if fucoidan is used IV.
      The increase in CD34+CXCR4+ cells was marked in this study, although the increase in the CD34+ cells was small. Although we observed no significant change in SDF-1 levels at 4 days, when CD34 levels increased, SDF-1 levels were increased by day 12. Either the rise in SDF-1 is not correlated with the enhanced CD34+ cell numbers or CXCR4 expression, or the effect is only at the BM level at the 4 day stage.
      Previously, CXCR4 expression on HPCs has been shown to increase after administration of G-CSF within both human and murine BM, reaching peak levels at the time of mobilization [
      • Petit I.
      • Szyper-Kravitz M.
      • Nagler A.
      • et al.
      G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and upregulating CXCR4.
      ], although SDF-1 levels did not rise. Interestingly, we observed a small decrease in CFU-GM over 12 days in the 75% fucoidan group. This effect may perhaps be attributed to the rise in IFN-γ. Constitutive expression of low levels of IFN-γ by stromal cells has been noted to have a profound inhibitory effect on hematopoiesis [
      • Selleri C.
      • Maciejewski J.P.
      • Sato T.
      • Young N.S.
      Interferon-gamma constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition.
      ].
      CXCR4 plays an important role in regulating the trafficking of HPCs and their homing/retention in BM, and it modulates several biologic processes in more differentiated cells [
      • Majka M.
      • Ratajczak M.Z.
      Biological role of the CXCR4-SDF-1 axis in normal human hematopoietic cells.
      ].
      In this study, a small, significant decrease in leukocytes and lymphocytes was observed after 12 days of ingesting the 75% fucoidan, although this decrease was within normal clinical range. In previous studies, there was a fall in circulating leukocytes immediately after G-CSF was given [
      • Wright D.E.
      • Wagers A.J.
      • Gulati A.P.
      • Johnson F.L.
      • Weissman I.L.
      Physiological migration of hematopoietic stem and progenitor cells.
      ,
      • Abkowitz J.L.
      • Robinson A.E.
      • Kale S.
      • Long M.W.
      • Chen J.
      Mobilization of hematopoietic stem cells during homeostasis and after cytokine exposure.
      ,
      • Bradford G.B.
      • Williams B.
      • Rossi R.
      • Bertoncello I.
      Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment.
      ].
      The presence of normal number of functional neutrophils is important for mobilization of HPCs [
      • Liu F.
      • Poursine-Laurent J.
      • Link D.C.
      Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF.
      ,
      • Winkler I.G.
      • Levesque J.P.
      Mechanisms of hematopoietic stem cell mobilization: when innate immunity assails the cells that make blood and bone.
      ]. In this study, we have demonstrated that 3 g of oral fucoidan has no effect on neutrophil count and does not cause neutropenia.
      We observed that there was an increase in the plasma level of IFN-γ, consistent with a previous study that showed an increase in HPCs accompanied by an increase in the level of SDF-1, IFN-γ, and IL-12 [
      • Sweeney E.A.
      • Papayannopoulou T.
      Increase in circulating SDF-1 after treatment with sulphated glycans, the role of SDF-1 in mobilization.
      ]. The level of IL-12 in our study did not change (results not shown). It was shown previously that in vitro treatment of BM MNCs with IFN-γ can upregulate the expression of CXCR4 on granulocyte precursors and monocytes [
      • Lee B.
      • Ratajczak J.
      • Doms R.W.
      • Gewirtz A.M.
      • Ratajczak M.Z.
      Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for human immunodeficiency virus–type 1 infection.
      ]. This may, in part, reflect our observation of increased expression of CXCR4 on CD34+ cells. It could be more relevant to look at the BM cytokine levels as well and compare the changes between PB and BM levels especially as PB SDF-1 may originate from the BM pool.
      In summary, oral administration of fucoidan significantly amplified the CXCR4+ HPC population. The ability to mobilize HPCs with high levels of CXCR4 expression could be clinically valuable. However, the effect of IV fucoidan in humans remains to be determined.

      Acknowledgments

      We would like to thank Marinova Pty. Ltd., Hobart, TAS Australia, for kindly providing the Undaria fucoidan extracts as a gift. Dr. Scott Ragg advised on the methodology, and Dr. Greg Woods kindly assisted in proofreading the manuscript. M.R.I. is a Ph.D. candidate at University of Tasmania, and this work will be submitted in partial fulfillment of the requirement for the Ph.D.

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