Maitake beta-glucan MD-fraction enhances bone marrow colony formation and reduces doxorubicin toxicity in vitro

International Immunopharmacology 4 (2004) 91 – 99 www.elsevier.com/locate/intimp

Maitake beta-glucan MD-fraction enhances bone marrow colony $ formation and reduces doxorubicin toxicity in vitro Hong Lin a, Yu-Hong She b, Barrie R. Cassileth a, Frank Sirotnak b, Susanna Cunningham Rundles c,* a Integrative Medicine Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Department of Molecular and Experimental Therapeutics, Memorial Sloan-Kettering Institute, New York, NY, USA c Immunology Laboratory, Department of Pediatrics, Cornell University Weill Medical College, 1300 York Avenue, New York, NY 10021, USA b

Received 9 July 2003; received in revised form 6 August 2003; accepted 24 October 2003

Abstract Previous studies have indicated that MD-fraction (MDF), in which the active component is beta 1,6-glucan with beta 1,3branches, has anti-tumor activity as an oral agent and acts as an immune adjuvant. Since some other beta glucans appear to promote mobilization of hematopoietic stem cells, the effects of a beta glucan extract from the Maitake mushroom ‘‘MDfraction’’ on hematopoietic stem cells were examined in a colony forming assay. Here we report for the first time that MDF has a dose response effect on mouse bone marrow cells (BMC) hematopoiesis in vitro. Using the Colony Forming Unit (CFU) assay to detect formation of granulocyte-macrophage (CFU-GM) colonies, and the XTT cytotoxicitiy assay to measure BMC viability, the data showed that the addition of MDF significantly enhanced the development of CFU-GM in a dose range of 50 – 100 Ag/ml ( p < 0.004). The mechanism of action included significant increase of nonadherent BMC viability, which was observed at MDF doses of 12.5 – 100 Ag/ml ( p < 0.005). In the presence of Doxorubicin (DOX), MDF promoted BMC viability and protected CFU-GM from DOX induced toxicity. In addition, MDF treatment promoted the recovery of CFU-GM colony formation after BMC were pretreated with DOX. These studies provided the first evidence that MDF acts directly in a dose dependent manner on hematopoietic BMC and enhances BMC growth and differentiation into colony forming cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Beta-glucan; Doxorubicin; Hematopoiesis; CFU-GM

1. Introduction

$

Source of Support: the US Army Medical Research and Material Command under DAMD 17-01-1-0323, NIH NCI 29502, and the Children’s Blood Foundation. * Corresponding author. Tel.: +1-212-746-3414; fax: +1-212746 8512. E-mail address: [email protected] (S. Cunningham Rundles). 1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2003.10.012

Chemotherapeutic drugs, such as doxorubicin (DOX), exert a dose-dependent injury to bone marrow that suppress hematopoiesis [1 –4]. Patients with bone marrow suppression usually develop anemia, lymphopenia, thrombocytopenia and granulocytopenia, which markedly increase the risk of serious and potentially lethal infections. A range of different growth factors and cytokines, including interleukin-3 (IL-3), granulo-

92

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

cyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF), have been developed and shown capable of stimulating hematopoiesis and supporting peripheral recovery of granulocytes after chemotherapy [5– 9]. However, hematopoietic and immune recovery involves complex interactions among precursor cells, cytokines and growth factors which must recapitulate ontogeny in a fashion analogous to the reconstitution process following bone marrow transplantation [10,11]. Thus despite use of GM-CSF and G-CSF, myelotoxicity and delayed immune recovery continue to be major limiting toxicities of cancer chemotherapy. These complications have led to development of autologous bone marrow and stem cell rescue therapies [11] and a continuing search for alternative approaches. Beta glucans are among the emerging new agents that could directly support or enhance functional autologous hematopoietic stem cell recovery, as discussed below. Previous studies have indicated that beta glucans derived from bacteria and fungi may stimulate or enhance cytokine production and affect hematopoiesis or compartmentalization of blood cells. A beta-glucan derived from fungi has been shown to stimulate peripheral hematopoietic response in normal and myelosuppressed animals following intraperitoneal administration [12 – 15]. In normal mice glucans have been shown to increase the production of bone marrow pluripotent hematopoietic stem cells granulocyte-macrophage (CFU-GM), pure macrophage (CFU-M), and erythroid hematopoietic stem cells (CFU-e, BFU-E) when given intraperitoneally in pharmacological amounts [16]. However, for human use an orally active agent would be required, preferably from a plant source. Current work on a barley derived 1,3 glucan with 1,4 branched has shown promise. Using the immune deficient xenograft tumor models, Cheung et al. [17] showed that orally administered beta glucan enhanced cytotoxicity and synergized with a specific anti-tumor monoclonal antibody in killing tumor cells. In this study we have worked with a mushroom derived glucan and sought to evaluate the dose response effect of MDF on doxorubicin (DOX) induced bone marrow suppression in normal mice using colony forming unit (CFU) assay. This assay is a semisolid agar technique first developed by Bradley and Metcalf [18] and provides a clear means of quantitative assessment. The colony forming unit (CFU) assay also

provides a sensitive approach to study the potential hematopoietic toxicity of chemotherapeutic agents on bone marrow progenitor-derived clonogenic activity [1 –3,19]. The purposes of our current study were to (1) delineate the optimal dose range of MDF for putative enhancement of hematopoietic response of normal mouse bone marrow in vitro, (2) determine the feasibility of using MDF to protect bone marrow stem cells from DOX induced bone marrow suppression, (3) determine the feasibility of using MDF to enhance recovery from DOX-induced depletion of hematopoietic progenitor cells.

2. Materials and methods 2.1. Chemicals and reagents MDF is an extraction from fruit body of Maitake mushroom (Grifola frondosa), which was made and provided by Yuikiguni Maitake, under patented methods (Japan Pat. No. 2859843/US Pat. No. 5,854,404 ) and stored in a refrigerator at 0 –4 jC under dark conditions until use. The batch of MD-fraction used in this study was sent to NAMSA to test for endotoxin contamination using limulus amebocyte lysate (LAL) assay. The result showed that there is no detectable endotoxin in MDF. Adriamycin PFS (doxorubicin hydrochloride, DOX), Mr = 580; purity>98% (TLC) (Pharmacia, Columbus, OH). 2.2. Animals Mouse: B6D2F1 female mice, 12– 16 weeks old, were obtained from Jackson Laboratory. 2.3. Collection of mouse bone marrow cells Mouse bone marrow cells (BMC) were collected from femurs and tibiae of female mice B6D2F1 using a standard technique [20]. Briefly, fresh mouse bone marrow cells were collected by flushing femur and/or tibiae with 3 ml of cold RPMI-1640 containing 10% (v/v) FBS. The cell suspensions were passed up and down six times through an 18-gauge needle in RPMI1640-10% FBS to disperse cell clumps. Adherent bone marrow cells were removed after incubation at

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

37 jC, 5% CO2 for 24 h in RPMI-1640 containing 20% FBS, and non-adherent cells were collected and used as described below.

93

3. Results 3.1. Enhancement of BMC viability and CFU-GM response with MDF

2.4. In vitro colony-formation assay Colony-formation assay was carried out under defined conditions by standard techniques (StemCell Technologies Vancouver, Canada). Briefly, BMC were plated in a premixed methylcellulose culture medium (Methocut M3234, StemCell Technologies; Vancouver, Canada). Final adjusted concentrations were 1% methylcellulose, 15% FBS, 1% BSA, 10 Ag/ml insulin, 200 Ag/ml transferrin, 10 4 M 2-mercaptoethanol and 2 mM L-glutamine. Recombinant murine IL-3 (Intergen, Purchase, NY), and recombinant human G-CSF (Neupogen, AMGEN, Thousand Oaks, CA) were added at 10 ng/ml and 500 ng/ml, respectively. BMC suspension (2  105 cells/ml, 0.3 ml) were added to complete mixed culture medium (2.7 ml), were vortexed, and plated in Petri dishes (Falcon, Becton Dickinson), 1.1 ml/dish. Then all cultures were incubated in a water-saturated, 37jC, 5% CO2 atmosphere. After 7 days incubation, CFU-GM colonies consisting of 50 or more cells were scored using an inverted microscope.

Mouse bone marrow cells were collected as described in Materials and methods, and non-adherent

2.5. XTT cytotoxicity assay The direct effects of MDF on murine bone marrow cells and the effects on doxorubicin mediated bone marrow cytotoxicity were determined immediately after 24 hrs by microtiter 2,3-bis (2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay performed as directed (Sigma, St. Louis, MO). Briefly, after murine bone marrow cells were cultured with DOX in presence or absence of MDF, reconstituted XTT was added in an amount equal to 20% of the culture medium volume. XTT was reconstituted with serum-free medium, at concentration of 1 mg/ml, with 0.2% phenazine methosulfate (PMS). After adding XTT, cultures were returned to incubator for 2 –4 h. Spectrophotometrically we measured absorbance at a wavelength of 450 nm. 2.5.1. Data presentation Data presents as mean F SD. Statistical analysis was performed by paired Student’s t-test, using Sigma Plot 2001 for Windows version 7.0.

Fig. 1. (A) MDF enhancement of BMC CFU-GM. Non-adherent BMC were collected after incubating 24 h followed by two washes with serum free RPMI-1640 medium, then treated with MDF at different doses as indicated and then cultured in methylcellulose medium for 7 days, using cultures without MDF as controls. Data show mean colony counts of the combined results from three separate experiments, *p < 0.05 vs. controls, **p < 0.001 vs. controls. (B). Enhancement of BMC viability response with MDF. Non-adherent BMC were collected and seeded into 96 well plate with MDF at doses as indicated and incubated at 37 jC, 5% CO2 for 24 h. Cell viability was detected by XTT assay and compared with cultures in the absence of MDF. Data represent triplicates of combined BMC of seven mice. *p < 0.05, **p < 0.001 vs. controls.

94

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

MDF at dose of 12.5– 100 Ag/ml for 24 h. Neither mrIL-3 nor rhG-CSF was added. After incubation, cell viability was detected by XTT assay and compared between the cultures with and without MDF, using the cultures without MDF as control. As expected, freshly obtained non-adherent bone marrow cells were very vulnerable to cell death. A previous study showed that the viability of these cell cultures dropped to about 40% of controls during culture due to the absence of exogenous growth factors or cytokines [20]. In the presence of MDF, at doses lower than 100 Ag/ml, the BMC viability was significantly enhanced when compared with that of controls without MDF, as shown in Fig. 1B ( p < 0.001). Fig. 2. Dose response of BMC to MDF with DOX. Non-adherent BMC were harvested after incubation for 24 h followed by two washes with serum free RPMI-1640 medium and treatment with DOX (15 ng/ml) in the presence or absence of MDF at doses as indicated, and then cultured in methylcellulose medium for 7 days. Count the GM colony units. Data are given as mean colony counts for combined results of two separate experiments. *p < 0.05 vs. DOX alone.

bone marrow cells (BMC) containing presumptive monocyte/macrophage progenitor cells were collected for subsequent treatments. To detect the effects of MDF on non-adherent precursor cell hematopoietic activity, the collected cells were cultured in the presence of MDF in methylcellulose medium containing murine recombinant interleukin 3 (mrIL-3), and recombinant human G-CSF (rh G-CSF), (10 ng/ml and 500 ng/ml, respectively). As shown in Fig. 1A, MDF enhanced colony formation in a dose dependent fashion, at dose range from 50 –100 Ag/ml ( p < 0.05). MDF produced a modest increase in CFU-GM at the lowest dose of 25 Ag/ml, over control cultures without MDF. At a dose of 50 Ag/ml, addition of MDF increased the mean number of CFU-GM colonies and this difference was significantly different from the number of colonies found in control cultures ( p < 0.004). When MDF was added at a dose of 100 Ag/ml a very significant increase in colony formation compared with control cultures was observed ( p < 0.0003). But at a higher dose of 500 Ag/ml, addition of MDF led to a lower number of CFU-GM, about 70% of control. To investigate whether MDF exerted any direct effects on BMC viability, non-adherent BMC were incubated in RPMI-1640 containing 20% FBS and

3.2. Dose response of BMC to MDF after DOX treatment To determine whether MDF was capable of protecting the differentiation and growth of CFU-GM from BMC from the cytotoxic effects of DOX in a dose dependent manner, non-adherent BMC were cultured in methylcellulose medium containing mrIL-3 and rhG-CSF, with DOX (15 ng/ml) in the presence of a range of doses of MDF. As shown In

Fig. 3. MDF protects BMC viability from DOX cytotoxicity. Nonadherent BMC were harvested after incubation for 24 h. After two washes with RPMI-1640, cells were seeded into 96 well-plate with series doses of DOX alone or together with MDF at doses as indicated, and incubated at 37 jC, 5% CO2 for 24 h. Cell viability was detected using XTT assay. Data represent triplicates of combined BMC of seven mice. *p < 0.05 vs. DOX alone.

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

95

3.4. MDF protection of BMC colony formation from DOX toxicity The preceding experiments established the doses of MDF needed to protect BMC precursor cells from DOX induced toxicity. In the next experiment, we exposed BMC to a range of doses of DOX with or without optimal concentrations of MDF. As shown in Fig. 4, BMC colony forming ability decreased with increasing doses of DOX. Even though the actual number of CFU-GM obtained under optimal concentrations of MDF (50 and 100 Ag/ml) did decrease with increasing doses of DOX, we observed that this reduced number was significantly higher than with DOX alone ( p = 0.003, and p = 0.001, respectively). Fig. 4. MDF protects BMC colony formation from DOX cytotoxicity. Nonadherent BMC were harvested after incubation for 24 h, then were washed twice with serum free RPMI-1640 medium. BMC treated with serial doses of DOX in the presence or absence of MDF at the indicated doses, were cultured in methylcellulose medium for 7 days. Data are given as mean colony counts and represent the combined results of two separate experiments. *p < 0.05 vs. DOX alone.

3.5. Recovery of BMC CFU-GM from DOX induced cytotoxicity with MDF To further study the effects of MDF on cytotoxicity of DOX, we treated the non-adherent BMC with range of dilutions of DOX alone for 24 h. After removing the supernatant, the cell pellet was re-suspended in IMDM and placed into culture dishes with different

Fig. 2, MDF protected the development of BMC into CFU-GM colonies from the effects of DOX in a dose dependent manner. In the presence of MDF, the CFUGM forming ability of BMC exposed to DOX still remained around 80– 90%, whereas in the absence of MDF the level of colony formation dropped to 65% of control cultures without DOX or MDF ( p < 0.05). 3.3. MDF protects BMC viability from DOX toxicity To determine the sensitivity of fresh bone marrow cells to the short-term cytotoxic effects of DOX in the presence of MDF, non-adherent BMC were exposed to increasing concentrations of DOX with or without MDF. After 24 h, cell viability was measured by XTT assay. As shown in Fig. 3, in absence of MDF, DOX had a strongly cytotoxic effect on BMC, with an IC50 less than 1500 ng/ml after incubation for 24 h. In the presence of MDF, even at a low dose of 12.5 Ag/ml, BMC viability was significantly higher compared to viability with DOX alone. Thus the protective effect of MDF on viability was observed across the entire dose range from 12.5 Ag/ml to 100 Ag/ml ( p < 0.05).

Fig. 5. MDF promotes recovery BMC CFU-GM from DOX induced cytotoxicity. Nonadherent BMC were collected after incubation for 24 h, and were washed twice with RPMI-1640 medium, then were treated with serial dilutions of DOX for 24 h. BMC were then cultured with or without MDF at doses as indicated, in methylcellulose medium for 7 days. Data show mean colony counts representing the combined results of two separate experiments. **p < 0.001 vs. DOX alone.

96

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

concentrations of MDF. After incubation for 7 days, CFU-GM colonies were counted under the inverted microscope. As shown in Fig. 5, MDF (50 Ag/ml and 100 Ag/ml) enhanced recovery of the number of CFUGM colonies from the cytotoxic effects of entire dosage range of DOX being used. The average increase of 10% was significant ( p = 0.000066 and p = 0.0000086, respectively).

4. Discussion Maitake is the Japanese name for the G. frondosa mushroom that has been used for centuries as a dietary supplement. Recent reports have provided possible evidence for effects of various extracts of maitake on tumors, diabetes, obesity, hyperlipemia, and AIDS [21 – 25]. Interest in this mushroom derived beta glucan centers on possible oral activity. Yet few, if any of these reported studies, including animal models where maitake has been administered i.v. or i.p., showed dose response relationships or provided clear evidence for specific mechanism of action. Most of the research on maitake has focused on the use of a fairly defined extraction procedure. However the starting material has been taken from variably grown sources of mushroom, which is the probable reason for general lack of a defined dose response relationship. In contrast, the maitake extract MD-fraction (MDF) used in the studies described here is derived from a highly standardized source grown under defined conditions. The active component of MDF is an isolated beta-glucan, a protein-bound polysaccharide compound. MDF has very unique structure that is beta-1,6 glucopyranoside main chain with branches of beta-1, 3-linked glucose, with a molecular weight of about 1,000,000 that has been shown to have oral activity in a mouse model [26,27]. Glucans in general comprise a wide and varied class of compounds of which only a few have been studied in detail. Current studies using mushroom glucans have also not shown dose response relationships which is a potential limitation to specific application of this approach [30]. Most investigators have used MDF as a single agent, given either prophylactically or concurrently to suppress growth of transplanted tumor cells [31,32]. ‘‘MD-fraction’’ is described as a potent immunostimulant capable of: enhancing the cytotox-

icity of natural killer cells, activating macrophages, and increasing antibody responses when given i.p. in murine models. Some reports suggest that MDF may act as an adjuvant in the mouse by activating various effector cells to attack tumor cells, possibly through potentiation of the innate immune cytokine response including TNF-a and IL-1 [33]. In some studies, MDF appeared to protect the host by stimulating immune response toward the tumor, indicating that MDF potentially activates the host tumor immune response, perhaps through moblization of cells or an adjuvant [33]. Recent studies indicate that when orally administered MDF was associated with 60 – 70% tumor inhibitory rate in mice compared with 30% using a standard chemotherapeutic drug, mitomycin-c, suggesting adjuvant-like activity [26]. MD-fraction (MDF) is one of the fractions of extract from the fruit body of G. frondosa, which has shown the strongest anti-tumor effect [26,29,33]. The preparation of MDF used in these studies is a standardized form of beta-glucan with a molecular weight of 1,000,000, which has a unique and complex structure. According to the 13C-NMR spectrum, the beta glucan found in MD-fraction contains both a 1,6 main chain having a greater degree of 1,3 branches, and 1,3 main chain having 1,6 branches [26,27]. Most other mushroom derived beta-glucan have a 1,3 main chain with 1,6 branches only [29]. The unique polysaccharide structure and the degree of branching have been suggested as the likely basis for reported greater immune stimulatory properties of MDF compared with other beta-glucans [26,29,33]. Previous studies have shown that a gel-forming (1->6)-branched (1,3)-h-D-glucan, GRN, prepared from liquid-cultured mycelium of G. frondosa appeared to cause increased production of interleukin-6, interleukin-1a, and tumor necrosis factor-a from the mouse macrophage cell line RAW264.7 in vitro. However, dose –response relationships were not clear [34]. Adachi et al. [33] have also suggested that MDF might activate the immune system through a range of adjuvant effects including enhancement of natural killer (NK) cells, cytotoxic T cells activity and through increasing interleukin-1 (IL-1) and superoxide anion (SOA) production in mice responding to tumor. In this study, we examined the effect of MDF on the hematopoiesis of non-adherent murine bone marrow cells. To our knowledge this is the first time that MDF has been demonstrated to cause direct enhance-

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

ment of the CFU-GM response of BMC progenitors and to enhance recovery of the CFU-GM response after DOX induced hematopoietic suppression. The effects of MDF on hematopoiesis were demonstrated in non-adherent bone marrow cells by assay of colony forming unit potential. Normal mouse bone marrow CFU-GM progenitor cell activity was significantly increased in the presence of MDF in the dose range of 50 – 100 Ag/ml. Using different endpoints involving expression of immune response, few previous studies have identified any dose effects with MDF when given i.v. or i.p. or when added in vitro. Using bone marrow precursor cells we were able to see a clear titrational effect, perhaps suggesting that these cells are central to the effect of MDF. Supportive recent studies have shown that a ion-exchange chromatography purified h-glucan extracted from fruit bodies of Sparasis crispa, SCG, showing enhancement of hematopoietic response as indicated by increased numbers of white blood cells in the peripheral in cyclophosphamide (CY) induced leukopenia over a wide range of concentrations. The fact that these studies did not define a dose-dependent response perhaps reflects the role of other factors required in the transit of hematopoietic cells into different blood cell compartments or that effects may occur at more than one level [28]. While there are numerous reports indicating that various glucans stimulate reticuloendothelial, immunologic or hematopoietic functions, these effects have been hard to define as well as variable perhaps due to the presence of other active components in the various extracts. As reported by Shen Q. et al, different genotypes and nutrient supplements of Maitake (G. frondosa) have different biological characteristics and qualities [35,36]. A beta-glucan derived from zymosan, which is a glucopyranose polysaccharide, after i.p. injection into mice, produced a relative and absolute increase in the number of pure macrophage colonies from bone marrow and spleen. Serum from glucaninjected mice in the study had high colony stimulating activity levels, suggesting that the augmentation of macrophage colony might have been due to the increased of colony stimulating factor production in vivo [37]. However, a beta-glucan, which was isolated from the inner cell wall of the yeast Saccharomyces cerevisiae given by intravenous injection into normal mice, did not affect on the number of CFU-GM of murine

97

bone marrow cells, and in fact led to a significant decrease in comparison to control [13]. By contrast, the CFU-GM found in peripheral blood and spleen was increased [13,38]. Recently, Turbull et al. [39] have shown in vitro that submaximal concentrations of rhGM-CSF with the polysaccharide PGG-glucan purified from the cell walls of the yeast S. cerevisiae, caused a significant increase in human bone marrow mononuclear cells and CD34+ myeloid colony formation comparable to the increase observed with either rhIL-3 or stem cell factor (rhSCF). These in vitro data also support our study suggesting that another beta glucan directly stimulates granulocyte-monocyte committed myeloid precursors and enhances their proliferative capacity. In general, however, glucans that could stimulate immune response have appeared to act in a relatively nonspecific fashion and the effects may have been attributable to the activation state of the system, usually in response to transplanted tumor. Doxorubicin based chemotherapy represents the main treatment for cancer patients, but myelosuppression is a major limiting factor for the use of these anticancer agents [19,40]. Our study showed that toxicity of DOX on bone marrow is attenuated by MDF. Even without DOX, bone marrow cell viability is dramatically decreased within 24 h without the support of growth factor in vitro [20]. Our current study showed that in the presence of MDF, bone marrow cell viability was significantly higher despite lack of added growth factor. Moreover, we observed that MDF protected the bone marrow progenitor cells from irreversible damage associated with prior DOX induced toxicity, indicating that MDF supports bone marrow cells progenitor maintenance or viability in the face of DOX toxicity. Further, MDF treatment promoted recovery of hematopoiesis from bone marrow following pretreatment with DOX. These data indicate that MDF directly affects bone marrow cell survival from DOX. These studies suggest that MDF has the potential to reduce hematopoietic suppression induced by chemotherapy. In our study, essentially all accessory cells and mature cells were removed by overnight incubation to remove adherent cells. The non-adherent bone marrow cells were thereby enriched for precursor cells, primarily immature myeloid stem cells. These immature myeloid stem cells were incubated with MDF directly in the presence of mrIL-3 and rhG-CSF. The

98

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

enhancement of the number of CFU-GM by MDF may have been due to a direct effect of MDF on myeloid stem cells. A previous study using a different beta glucan also suggested that MDF might partially replace growth factors required for colony formation. Turnbull et al. [39] reported that PGG-glucan has a stimulatory effect in vitro on granulocyte – monocyte committed myeloid precursors in human peripheral blood. The data presented here suggest that MDF enhancement of CFU-GM is mediated directly through inducing both proliferation and differentiation of progenitor myeloid stem cells.

References [1] Gibaud S, Andreux JP, Weingarten C, Renard M, Couvreur P. Increased bone marrow toxicity of doxorubicin bound to nanoparticles. Eur J Cancer 1994;30A:820 – 6. [2] Pessina A, Piccirillo M, Mineo E, Catalani P, Gribaldo L, et al. Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM). Life Sci 1999;65:513 – 23. [3] Masse A, Ramirez LH, Bindoula G, Grillon C, WdzieczakBakala J, et al. The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Goralatide) protects from doxorubicin-induced toxicity: improvement in mice survival and protection of bone marrow stem cells and progenitors. Blood 1998;91:441 – 9. [4] Minderman H, Linssen P, van der LN, Wessels J, Boezeman J, et al. Toxicity of idarubicin and doxorubicin towards normal and leukemic human bone marrow progenitors in relation to their proliferative state. Leukemia 1994;8:382 – 7. [5] Beveridge RA, Miller JA, Kales AN, Binder RA, Robert NJ, et al. Randomized trial comparing the tolerability of sargramostim (yeast-derived RhuGM-CSF) and filgrastim (bacteria-derived RhuG-CSF) in cancer patients receiving myelosuppressive chemotherapy. Support Care Cancer 1997;5:289 – 98. [6] Kushner BH, Heller G, Kramer K, Cheung NK. Granulocytecolony stimulating factor and multiple cycles of strongly myelosuppressive alkylator-based combination chemotherapy in children with neuroblastoma. Cancer 2000;89:2122 – 30. [7] Mangi MH, Newland AC. Interleukin-3 in hematology and oncology: current state of knowledge and future directions. Cytokines Cell Mol Ther 1999;5:87 – 95. [8] Wang CH, Wang HM, Chen JS, Chang WJ, Lai GM. Intensive chemotherapy plus recombinant human granulocyte-colony stimulating factor support for distant metastatic nasopharyngeal carcinoma. A preliminary report. Oncology 1997;54:34 – 7. [9] Weaver CH, Schulman KA, Buckner CD. Mobilization of peripheral blood stem cells following myelosuppressive chemotherapy: a randomized comparison of filgrastim, sargramostim, or sequential sargramostim and filgrastim. Bone Marrow Transplant 2001;27(Suppl. 2):S23 – 9.

[10] Janowska-Wieczorek A, Marquez LA, Nabholtz JM, Cabuhat J, Montano J, et al. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood 1999;93:3379 – 90. [11] Locatelli F, Maccario R, Comoli P, Bertolini F, Giorgiani G, et al. Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children. Bone Marrow Transplant 1996;18:1095 – 101. [12] Hashimoto K, Suzuki I, Ohsawa M, Oikawa S, Yadomae T. Enhancement of hematopoietic response of mice by intraperitoneal administration of a beta-glucan, SSG, obtained from Sclerotinia sclerotiorum. J Pharmacobio-dyn 1990;13:512 – 7. [13] Patchen ML, Lotzova E. Modulation of murine hemopoiesis by glucan. Exp Hematol 1980;8:409 – 22. [14] Patchen ML, Liang J, Vaudrain T, Martin T, Melican D, et al. Mobilization of peripheral blood progenitor cells by Betafectin PGG-Glucan alone and in combination with granulocyte colony-stimulating factor. Stem Cells 1998;16:208 – 17. [15] Patchen ML, Vaudrain T, Correira H, Martin T, Reese D. In vitro and in vivo hematopoietic activities of Betafectin PGGglucan. Exp Hematol 1998;26:1247 – 54. [16] Patchen ML, MacVittie TJ. Temporal response of murine pluripotent stem cells and myeloid and erythroid progenitor cells to low-dose glucan treatment. Acta Haematol 1983;70:281 – 8. [17] Cheung NK, Modak S, Vickers A, Knuckles B. Orally administered beta-glucans enhance anti-tumor effects of monoclonal antibodies. Cancer Immunol Immunother 2002;51:557 – 64. [18] Bradley TR, Metcalf D. The growth of mouse bone marrow cells in vitro. Aust J Exp Biol Med Sci 1966;44:287 – 99. [19] Minderman H, Linssen PC, Wessels JM, Haanen C. Doxorubicin toxicity in relation to the proliferative state of human hematopoietic cells. Exp Hematol 1991;19:110 – 4. [20] Lin H, Chen C, Chen BD. Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells. Biochem J 2001;353:299 – 306. [21] Adachi K, Nanba H, Otsuka M, Kuroda H. Blood pressurelowering activity present in the fruit body of Grifola frondosa (maitake). I. Chem Pharm Bull (Tokyo) 1988;36:1000 – 6. [22] Kubo K, Aoki H, Nanba H. Anti-diabetic activity present in the fruit body of Grifola frondosa (Maitake). I. Biol Pharm Bull 1994;17:1106 – 10. [23] Kubo K, Nanba H. The effect of maitake mushrooms on liver and serum lipids. Altern Ther Health Med 1996;2:62 – 6. [24] Kubo K, Nanba H. Anti-hyperliposis effect of maitake fruit body (Grifola frondosa). I. Biol Pharm Bull 1997;20:781 – 5. [25] Mayell M. Maitake extracts and their therapeutic potential. Altern Med Rev 2001;6:48 – 60. [26] Hishida I, Nanba H, Kuroda H. Antitumor activity exhibited by orally administered extract from fruit body of Grifola frondosa (maitake). Chem Pharm Bull (Tokyo) 1988;36:1819 – 27. [27] Nanba H, Hamaguchi A, Kuroda H. The chemical structure of an antitumor polysaccharide in fruit bodies of Grifola frondosa (maitake). Chem Pharm Bull (Tokyo) 1987;35:1162 – 8. [28] Harada T, Miura N, Adachi Y, Nakajima M, Yadomae T, et al. Effect of SCG, 1,3-beta-D-glucan from Sparassis crispa on the

H. Lin et al. / International Immunopharmacology 4 (2004) 91–99

[29]

[30]

[31] [32] [33]

[34]

hematopoietic response in cyclophosphamide induced leukopenic mice. Biol Pharm Bull 2002;25:931 – 9. Ohno N, Saito K, Nemoto J, Kaneko S, Adachi Y, et al. Immunopharmacological characterization of a highly branched fungal (1 – >3)-beta-D-glucan, OL-2, isolated from Omphalia lapidescens. Biol Pharm Bull 1993;16:414 – 9. Ohno N, Asada N, Adachi Y, Yadomae T. Enhancement of LPS triggered TNF-alpha (tumor necrosis factor-alpha) production by (1->3)-beta-D-glucans in mice. Biol Pharm Bull 1995;18:126 – 33. Nanba H. Activity of maitake D-fraction to inhibit carcinogenesis and metastasis. Ann NY Acad Sci 1995;768:243 – 5. Nanba H, Kubo K. Effect of Maitake D-fraction on cancer prevention. Ann NY Acad Sci 1997;833:204 – 7. Adachi K, Nanba H, Kuroda H. Potentiation of host-mediated antitumor activity in mice by beta-glucan obtained from Grifola frondosa (maitake). Chem Pharm Bull (Tokyo) 1987;35:262 – 70. Adachi Y, Okazaki M, Ohno N, Yadomae T. Enhancement of cytokine production by macrophages stimulated with (1->3)-

[35]

[36]

[37] [38]

[39]

[40]

99

beta-D-glucan, grifolan (GRN), isolated from Grifola frondosa. Biol Pharm Bull 1994;17:1554 – 60. Shen Q, Royse DJ. Effects of genotypes of maitake (Grifola frondosa) on biological efficiency, quality and crop cycle time. Appl Microbiol Biotechnol 2002;58:178 – 82. Shen Q, Royse DJ. Effects of nutrient supplements on biological efficiency, quality and crop cycle time of maitake (Griifola frondosa). Appl Microbiol Biotechnol 2001;57:74 – 8. Burgaleta C, Golde DW. Effect of glucan on granulopoiesis and macrophage genesis in mice. Cancer Res 1977;37:1739 – 42. Patchen ML, MacVittie TJ. Dose-dependent responses of murine pluripotent stem cells and myeloid and erythroid progenitor cells following administration of the immunomodulating agent glucan. Immunopharmacology 1983;5:303 – 13. Turnbull JL, Patchen ML, Scadden DT. The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines. Acta Haematol 1999;102:66 – 71. Lohrmann HP. The problem of permanent bone marrow damage after cytotoxic drug treatment. Oncology 1984;41:180 – 4.