β 1 , 3-GLUCAN ANTICANCER EFFICACIES AND SYNERGIES : A REVIEW

β1,3-glucans from fungi, cereals, seaweeds and bacte ri have been shown to possess favourable biologica l and anti-carcinogenic activities including upregula tion of phagocytosis, cytokine production enhanceme nt, superoxide and nitrite production; antibody secreti on and stimulation of signalling pathways associate d with proto-oncogene expression. However, human dietary s upplements containing β1,3-glucans vary in efficacy due to glucan source, the lifecycle stage of the so urce at extraction, extraction methods, purity, concentration and combination with other immunomodu lators. A review of efficacy of some commercially available β1,3-glucan products is presented. Three apparently fficacious products in which β1,3-glucan was the only immunomodulator were identified: Gluca n #300®, Maitake Gold 404® (diluted Yukiguni Maitake MD Fraction®) and Betamune®. A trial of Mai take Gold 404® produced evidence of standardisation problems. It is recommended that Yu kiguni Maitake MD Fraction® (a more standardised alternative), Glucan #300® and Betamune® be compara tively trialled at optimal doses across immunological measures and tumor reduction. β1,3-glucans have been shown to be synergistic with conventional cancer therapies and monoclonal antibo dies, as well as immunomodulators including vitamin C, transresveratrol, humic acids and Ashwagandha ( Withania somnifera). Trialled commercially available products containing immunomodulator combinations ha ve been shown to be inefficacious, apart from RVB300®, a β1,3-glucan/transresveratrol/vitamin C combination. The efficacies of various combinations of β1,3-glucans with other immunomodulators and the det ails of specific β1,3-glucan/monoclonal antibody synergies in treating particular cancer cell lines, r quire systematic elucidation.


INTRODUCTION
The estimated number of Americans to receive a new cancer diagnosis in 2014 is 1,665,540, with almost 1,600 deaths per day predicted (ACS, 2014). Cancer is the cause of one in every four American deaths (ACS, 2014). Marked morbidities are associated with both cancer and chemotherapy (Nanba, 1997). The identification of novel pharmaceuticals containing effective treatment agents, is essential for evidence based pharmacy practice.Possible agents include βglucans: Complex, structurally diverse β-glucose homopolymers isolated from the cell walls of grains, fungi, seaweeds and bacteria (Jamois et al., 2005;Novak and Vetvicka, 2008;Vetvicka and Vetvickova, 2010;Vetvicka et al., 2002;Vetvicka and Yvin, 2004). Nomenclature designates glycosidic bond positions, position β(1,3) with side chains bound by 1,6 glycosidic linkages predominant in studies, due to their allegedly superior immunological and anti-carcinogenic activity (Novak and Vetvicka, 2009). Degree of branching, detailed structure and solubility and hence biological activity of β1,3-glucans differ according to source and isolation method (CMEC, Science Publications AJI 2006; Liu et al., 2009;Lowman et al., 2003;Novak and Vetvicka, 2008;Tada et al., 2007;Vetvicka et al., 2002;Zhang et al., 2007).

Potential Sources of Variability in β1,3-Glucan Product Efficacy
The concentration of effective β-glucan in a product and the dose bear a strong relationship to immunological effects, as evidenced by (Deng et al., 2009), who measured the human oral dose response curves for Maitake Gold 404® (containing Yukiguni Maitake MD Fraction®) across a wide range of immunological parameters. Optimal dose for many parameters was around 5-7 mg/kg/day, with highest dose causing reductions in some immunological reactions. The effective dose of β-glucan within a product is affected by source, growth stage of the source, purity and extraction method.
Unfortunately, a significant part of research has been conducted with only partially purified β1,3-glucans, making assessment of efficacies of specific glucans problematic Xu et al., 2010). Individual fungal species incorporated in products may contain multiple ingredients which can contribute to immunological and biological activity , including many types of β-glucans: Numerous different β1,3-glucans have been identified in Maitake mushrooms and Saccharomyces cerevesiae or Candida albicans yeasts alone Ito et al., 2009;Lowman et al., 2003;Ohno et al., 1986;Tada et al., 2009;Vetvicka and Vetvickova, 2005;Zhang et al., 2007). There is significant growth condition-related variation in β1,3-glucan branching and molecular weight within individual sources and hence variable potentially anti-tumor properties (Jamois et al., 2005;Lowman et al., 2003;Novak and Vetvicka, 2009).
The increased heat and alkalic solvents commonly used in extraction may destroy triple helical structures underlying some mechanisms of action in highly branched β1,3-glucans (Novak and Vetvicka, 2008;Ohno et al., 1986;Vetvicka and Vetvickova, 2010;Zhang et al., 2007), such as Maitake MD fraction. Opinions vary as to whether, following hydrolytic removal of Maitake MD fraction side branches, the fraction lacks immunostimulatory activity Miyake et al., 2005). Therefore, hot water extraction is recommended above the less costly alcohol extraction, which removes side branches and may destroy some efficacy (Zhang et al., 2007).
The burgeoning of β1,3-glucan products commercially available as human dietary supplements (in 2014 over 400 on the U.S. market alone) raises the importance of identification of those with superior anticarcinogenic properties. Proprietary extracts from yeast alone vary in manufacturer, solubility and purity (Vetvicka and Vetvickova, 2010;Vetvicka et al., 2008). With the notable exception of the work of Vetvicka's group, direct comparisons of immunological and biological actions of β1,3-glucan products are almost non-existent (Vetvicka and Vetvickova, 2005;Vetvicka et al., 2008). From the perspective of cancer patients, evidencebased use of such products is critical.
Furthermore, β1,3-glucan products for human consumption containing additions of any number of other alleged immunomodulators, including Astralagus, Echinacea, Aloe vera or Hydrastis Canadensis (Goldenseal) are increasingly available (Vetvicka and Vetvickova, 2012c). The immunological effects and toxicities of such combinations are generally unknown. It is crucial to patient interests that combinations of commercially available β1,3-glucans with anti-cancer medications or other immunomodulators be evidence based.

COMPARATIVE STUDIES OF POTENTIALLY ANTI-CANCER IMMUNOMODULATION BY β1,3-GLUCAN PRODUCTS
In 2005 Vetvicka and Vetvickova compared the immunostimulating properties of lentinan and two different preparations of Maitake D or MD fraction: Maitake Gold 404® containing diluted patented MD fraction from Yukiguni biofarm, a β1,3-glucan-protein complex (Nanba and Kubo, 1997) and Grifron-Pro

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Maitake D Fraction®, a β1,3-glucan: Agents 1, 2 and 3 on Table 1 (Vetvicka and Vetvickova, 2005). Following oral administration to mice, all three preparations stimulated phagocytosis to the same extent. However, Maitake Gold 404® was the only product effective at low doses. It produced large between-sample phagocytosis stimulation variation specifically of neutrophils, suggesting a lack of standardisation of effective MD fraction concentration.
All glucans enhanced NK activity, but Maitake Gold 404® had the strongest effect. The researchers concluded that β1,3-glucans from Maitake possess similar types of immunomodulatory properties to those from yeasts and that in some ways they are more immunologically active.
The three agents stimulating the greatest migration of CD4 + and CD8 + T lymphocytes were Now Beta®, Glucan #300® and Maitake Gold 404®. Glucan #300® and Maitake Gold404® stimulated the highest IL-2 production. Glucagel T® and Glucan #300® stimulated the highest antibody response. Although it was possible for an agent to score highly on one measure of immunomodulation and negligibly on others (for example, Glucagel T ® stimulated well antibody formation, but on no other activity), clearly the most active agents across immunological measures were Glucan #300®, Now Beta® and Maitake Gold 404®. Senseiro®'s apparently low immunological activity was contrary to a study asserting the anti-tumour properties of Agaricus blazei fractions (Kawagishi et al., 1989). It might be possible that A. blazei contains components lacking in Senseiro® and responsible for its anti-tumor activity. Table 1. A partial list of β1,3-glucan products commercially available as human food supplements β1,3-glucan product Source 1) Lentinan Fruit body of Lentinula edodes (Shiitake) mushroom 2) Maitake Gold 404® Grifola frondosa (Maitake) mushroom a)

3) Grifron-Pro Maitake®
Grifola frondosa (Maitake) mushroom a) 4) Glucan # 300® Yeast: Highly purified Whole Glucan Particle (WGP) 5) Now Beta® (also called "now Glucan"®) Yeast, mushrooms 6) Glucagel T® Barley 7) Immutol® Yeast 8) Immune Saccharomyces cerevisiae a) Described as β 1,6 glucans in Japanese studies (Kodama et al., 2002a;Nanba, 1997;Nanba and Kubo, 1997) Science Publications In an additional study , the same group examined murine cholesterol and blood glucose level changes, as well as immunological changes (phagocytosis by peripheral leukocytes, IL-2 production) following administration of Glucan #300®, Now Beta®, Krestin and ImmunoFiber® (agents 4, 5, 10 and 11 respectively, Table 1). Glucan #300® stimulated greatest phagocytosis and, along with Krestin, longest lasting increases in that measure. IL-2 production was most lastingly stimulated by Krestin, although the highest level of stimulation in the first two days after β1,3glucan administration was by Glucan #300®. Glucan #300® was again the most active sample. Vetvicka et al. (2008) examined changes in the release of seven cytokines and levels of phagocytosis in Science Publications AJI a murine and in vitro model following administration of different doses of β1,3-glucans WGP, Lentinan and Betamune® (agents 1, 12 and 13, Table 1). In vivo tumor inhibition and breast cancer related gene expression were also examined. WGP and Lentinan were only active at the highest dose. Betamune® was more active at lower doses than the otheragents, stimulated phagocytosis in a wider effector cell range, in general stimulated the strongest cytokine response and resulted in the greatest tumor weight reduction. It also stimulated increased expression of the widest range of protooncogenes, potentially associated with upregulation of the adhesion molecules and angiogenesis contributing to tumor metastasis and growth, a finding similar to that in other studies on glucans . This result should be considered in light of the complexities of the genetic landscapes underlying cancers (Novak and Vetvicka, 2009;Otake et al., 2007).

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Another study compared the β1,3-glucan-mediated recovery of phagocytosis, production of three cytokines, T and B cell proliferation and NK activity after mercuryinstigated immunosuppression (Vetvicka and Vetvickova, 2009). Following administration of β1,3-glucans 4-6 and 13 (Table 1), Glucan #300® was the only agent active on all measures and was consistently most active.
As in previous comparative study, many of the glucans tested (e.g., Swanson®), showed little immunological activity and some agents produced a high level of activity on some measures only. Glucan #300® showed the greatest activity across all measures ( Table 2). The authors have repeatedly hypothesised that this was partly because Glucan #300® had a high degree of purity compared to other agents: For example, Glucan #300® is 85% pure and Swanson® 70% (Vetvicka and Vetvickova, 2010).

β1,3-GLUCAN SYNERGIES WITH ANTI-CANCER MEDICATIONS AND IMMUNOMODULATORS
Clinical and experimental evidence has clearly established the anti-cancer synergy of a range of β1,3glucans with conventional cancer therapies including cyclophosphamide (Tada et al., 2007;, interferon-α (Louie et al., 2010), mitomycin C (Nanba, 1997), dendritic cell-based immunotherapy (Masuda et al., 2010) and radiotherapy (Gu et al., 2005). Kodama et al. (2002a) published a non-randomised clinical trial in which Yukiguni Maitake MD Fraction® plus whole Maitake powder, taken with various chemotherapies, enhanced immunocompetent activities by a factor of 1.2-1.4 compared with chemotherapy alone.A previous limited, non-controlled study published by Nanba found 12-28% improved efficacy of the whole Maitake MD fractionand chemotherapy above that of chemotherapy (Nanba, 1997). Both studies found tumor regression or complete remission in some patients. However, the MD fraction doses administered were significantly under the optimal ranges determined by (Deng et al., 2009) and the greater efficacy achieved with addition of whole Maitakepowder may have been the result of additional MD fraction related β-glucans in the powder rather than any true synergy of fraction and powder. A commercially available MD Fractionwhole Maitake powder product derived from Yukiguni Maitake and combined approximately in the ratio used in the Nanba study is Maitake QOL Extract® (Muir, 2012;Nanba, 1997).

Monoclonal Antibodies
Combination of anti-tumor monoclonal antibodies (mAbs) with standard chemotherapy increases mAb efficacy, but adverse effect incidence and severity increase to an extent that limits utilisation (Liu et al., 2009). Enhanced effectiveness of immune responses to a range of cancers (including breast, lung and lymphoma), tumor shrinkage and prolongation of survival have repeatedly been demonstrated in murine and human xenograft models with co-administration of complement activating mAbs to tumor antigens, plus β1,3-glucans. The latterincludethe commercially available Glucan #300 and WGP: Agents 4 and 12 on Table 1, as well as a related yeast β1,3-glucan, PGG and a barley derived β1,3-glucan (Hong et al., 2004;Liu et al., 2009;Modak et al., 2013;Salvador et al., 2008;Yan et al., 2005).
PGG has the advantage of being the only tested β1,3glucan product which does not amplify inflammatory cytokine responses, a desirable attribute in mAb therapy (Liu et al., 2009). Unfortunately it is only available to patients via trials. Bevacizumab, an anti-vascular endothelial growth factor (VEGF) mAb, inhibits tumor microvasculature and triggers iC3b deposition on surface vasculature of a human ovarian carcinoma cell line, thus making such cells a target for PGG β-glucan primed neutrophil CR-3 related toxicity (Liu et al., 2009;Salvador et al., 2008). In one study, combination therapy prevented tumors from growing and led to long-term survival in 86% of murine subjects with tumors (Salvador et al., 2008). However, the combination provided no benefit above mAb alone in Colo38 tumors, since they do not express membrane bound VEGF (Salvador et al., 2008). Therefore the identification of cancer types possessing membrane-bound VEGF is crucial for the selection of patients to undergo combination PGG β-glucan bevacizumab therapy.
Similarly, cetuximab, a humanised anti-Epidermal Growth Factor Receptor (EGFR) mAb, when combined with PGG reduced tumor burden and greatly increased the rate of murine long-term survival (Liu et al., 2009). EGFR is highly expressed in very aggressive colorectal, lung, breast, pancreatic and gastrointestinal tumors and melanoma.
Anti-GD2 immunotherapy using mAb 3F8 has been shown to exert a small anti-tumor effect in chemoresistant neuroblastoma. The addition of a barley β1,3-glucan to 3F8 therapy in one study did not lead to increased CR3 mediated cytotoxicity (Modak et al., 2013). However, some patients developed anti-mouse antibody reaction. This was previously unobserved in 3F8 administration and associated with increased survival. Further investigation of the immunological effects of the combination is merited.
β-glucan binding of neutrophil CRs enhances specific humoral response targeting of tumor cells opsonised with complement iC3b and antibodies (Li et al., 2006;Novak and Vetvicka, 2009), constituting a mechanism of action for the synergy of mAbs and β-glucans in tumor shrinkage and enhanced host survival (Gelderman et al., 2004;Hong et al., 2004;Liu et al., 2009;Yan et al., 2005). Monoclonal antibodies with no inherent tumor regression activity may mediate regression which can be complete (Hong et al., 2004). For example, Herceptin and Erbitux are mAbs against Her2 and Her1 growth factors respectively which do not stimulate murine tumor regression unless administered with β1,3 glucans (Hong et al., 2004). Rituxan performs similarly (Hong et al., 2004). Vaccines which elicit anti-tumor antibodies may have increased efficacy if β1,3 glucan co-administration occurs (Hong et al., 2004;Liu et al., 2009;Yan et al., 2005), representing an area for future investigation.

Vitamin C
Vitamin C and β1,3-glucans increase some of the same classes of immune responses: NK cell and macrophage activity amplification, specific antibody formation and cytokine levels (Schwager and Schulz, 1998;Vetvicka and Vetvickova, 2012a). When combined, vitamin C and β1,3-glucans produce synergistic increases in adaptive biological and immunological measures (Ditteova et al., 2003;Verlhac et al., 1998), most notably when the glucan is in liposomised form.

Transresveratrol (Trans-3,4',5-Trihydroxystilbene)
Transresveratrol is a nonflavonoid polyphenol with independent cancer chemopreventiveimmunomodulatory properties, typically isolated from grape skin or Japanese knotweed . It has been found to ameliorate the side effects of administration of doxorubicin and cisplatin chemotherapy (Do Amaral et al., 2007;Vetvicka and Vetvickova, 2012a).  combined transresveratrol with WGP and found synergistic potentiation of phagocytosis, proliferation of CD4 + T lymphocytes, production of potentially lymphocyte stimulating cytokines and splenic recovery from administration of 5-flourouracil. Synergistic upregulation of genes encoding for anti-apoptosis, cell growth and adhesion pathways (such as Cdc42 and NF-κB2) occurred.

Vitamin C Plus Transresveratrol
A recent study used a combination of vitamin C, transresveratrol and Glucan#300 against murine lung cancer and breast cancer in order to evaluate the treatment potential of the combination (Vetvicka and Vetvickova, 2012a). The triple combination was found to be the strongest enhancer of granulocyte numbers, phagocytosis and antibody formation, achieving the greatest reduction in metastasis (86%).
In a subsequent study, mercury or perfluorinated hydrocarbon poisoning were used to depress a range of immunological parameters in mice, including phagocytosis, NK cell activity, mitogen-induced proliferation and expression of CD markers, antibody formation and secretion of cytokines IL-6, IL-12 and IFN-γ (Vetvicka and Vetvickova, 2012b). A vitamin C/transresveratrol/Glucan #300 combination was shown to ameliorate immunosuppression.
In an additional study the same researchers compared five commercially available preparations, most of which combined β1,3-glucans with other immunomodulators (Table 3), with the commercially available vitamin C/transresveratrol/Glucan #300 combination "RVB 300®," in terms of levels of phagocytosis, respiratory burst (nitrite and superoxide production), IL-2 production, antibody response to ovalbumin and NK cell cytotoxicity (Vetvicka and Vetvickova, 2012c).
Only RVB 300® showed immunological activity across measures. Except for nitrite and superoxide anion formation, the other samples showed no such activity, despite their inclusion of β1,3-glucans with some evidence base. This may be due to low doses of active, poor isolation techniques or component immunomodulators negating each other's efficacy.

Humic Acids
Humic acids are high molecular weight aromatic polymers found mainly in decomposing peat, some of which have anti-viral and lymphocyte-proliferative effects .  used Glucan #300® and β1,3-glucan laminarin in combination with humic acids in testing. Two types of humic acids independently demonstrated synergy when combined in specific ratios with the β-glucans, in terms of production of specific cytokines (IL-2, IL-4, IL-5, IL-6 and TNF-α) and of MPC-1 and phagocytosis functions of neutrophils and macrophages. The most strongly aromatic humic acid in combination with glucan achieved the greatest reduction of mouse breast cancer tumor weight in all tested samples: 92% .

Ashwagandha (Withania Somnifera)
Ashwagandha is an herbal extract used for stress reduction in traditional Indian medicine. When administered with Yukiguni Maitake extract (MD fraction) a synergistic inhibition of stress-related corticosterone increase and cytokine reduction occurred (Vetvicka and Vetvickova, 2011b), with implications for maintenance of a healthy immune function during stress related to a cancer diagnosis for a human patient.

Whole Maitake (Grifola Frondosa) Powder
In a previously referred to non-randomised clinical trial (Kodama et al., 2002a) administered Yukiguni Maitake MD Fraction® plus Yukiguni whole Maitake powder to liver, lung and breast cancer patients. They found 58.3-68.8% cancer regression or rigorously defined symptomatic improvement. Nanba (1997) had previously reported similar findings. In each study the combination of products achieved superior results to those produced with MD Fraction alone, although it is unclear as to whether this may be due to β-glucan dose increase due to addition of glucan in the whole powder, or synergy of MD fraction with a component or components of the whole powder.

DISCUSSION
Studies concerned with β1,3-glucan product efficacies and synergies are usually confounded by the issues of unknown optimal dose and unknown degree of product standardisation. Although (Deng et al., 2009) established the human oral dose response curves for Maitake Gold 404® across a wide range of immunological parameters,in general it is unknown as to whether murine and human studies used optimal doses of the range of β1,3-glucans tested, (and whether the optimal dose for one β1,3 glucan is the same as that for another). Nevertheless, β1,3-glucan products found to be efficacious as tumoricidal agents or carcinogenesis immunological mechanism inhibitors consistently include Betamune® , Glucan #300® and Maitake Gold 404® (Vetvicka and Vetvickova, 2005;Vetvicka and Vetvickova, 2009;Vetvicka and Vetvickova, 2010).
Maitake Gold 404® products are dilutions of Yukiguni Maitake MD fraction® and are produced by a range of US manufacturers (iHerb, 2014). Administration of a Maitake Gold 404® product in one study resulted in between sample variation in neutrophil stimulation and anomalous low IL-2 stimulation (Vetvicka and Vetvickova, 2005), suggesting product standardisation problems. Many additional marketed products contain non-patented substances claimed to be MD or D fraction, hence unknown doses of intact or authentic molecules of active (iHerb, 2014). Yukiguni Maitake MD Fraction® is extracted from bio farmed high β1,3-glucan content specimens, under uniform optimal growing conditions, with rigorous quality control, resulting in standardisation (Deng et al., 2009;Kodama et al., 2002a). Glucan extraction involves fractionation so as to obtain a relatively pure product, which is patented and proprietary (Nanba and Kubo, 1997). So that studies use a known dose of effective MD fraction, it is recommended that only MD fraction obtaineddirectly from Yukiguni be used in future trials, rather than manufacturer diluted or look-alike products. To date, apart from one known study (Vetvicka and Vetvickova, 2005), only Japanese studies have used the original product, generally with very efficacious results (Inoue et al., 2002;Kodama et al., 2002a;2002b;Masuda et al., 2012;Miyake et al., 2005). In addition, it is contrary to patient interests that numerous diluted products on the US market contain low levels of patented active compared with the original product and that recommended doses are subtherapeutic (Deng et al., 2009;iHerb, 2014).
Systematic identification of optimal dose for each β1,3-glucan product must be determined (preferably in human subjects), before meaningful comparisons of product efficacies may be made. After optimal doses are identified, it is suggested that Yukiguni MD fraction®, Glucan #300® and Betamune® effects be trialled across all immunological measures and tumor weight, so that conclusions on comparative immunological and anticancer effects may be derived.

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AJI tested which demonstrated favourable immunological effects (Vetvicka and Vetvickova, 2012c). Maitake QOL Extract® is composed of Yukiguni whole Maitake powder and Yukiguni Maitake MD Fraction®, combined in approximately the ratio shown by Nanbato produce cancer regression in human patients above that produced by MD Fraction alone (Kodama et al., 2002a). After the accurate determination of the MD fraction within the whole Maitake in QOL®, it is recommended that Maitake QOL Extract® and RVB300® be comparatively trialled at optimal dose across immunological measures and tumor weight. Systematic elucidation of synergies between β1,3-glucans and other immunomodulator combinationsis a promising area in product development.

CONCLUSION
Accumulated evidence shows that products containing β1,3-glucans can be anti-carcinogenic immunomodulators which possess utility as adjunctive therapy with chemotherapy, other cancer therapies and other immunomodulators. Unfortunately, many studies suffer from experimental design issues including nonrandomisation and incomplete control of dose of specific glucan. It is recommended that these issues be addressed in further research.

ACKNOWLEDGEMENT
The researchers wishes to thank the following advisorwithout whom the writing of this study would not have been possible: Mr Bruce Muir, Managing Director, QOL Nutraceuticals Incorporated.