Trametes versicolor (Synn. Coriolus versicolor) Polysaccharides in Cancer Therapy: Targets and Efficacy

Coriolus versicolor (L.) Quél. is a higher fungi or mushroom which is now known by its accepted scientific name as Trametes versicolor (L.) Lloyd (family Polyporaceae). The polysaccharides, primarily two commercial products from China and Japan as PSP and PSK, respectively, have been claimed to serve as adjuvant therapy for cancer. In this paper, research advances in this field, including direct cytotoxicity in cancer cells and immunostimulatory effects, are scrutinised at three levels: in vitro, in vivo and clinical outcomes. The level of activity in the various cancers, key targets (both in cancer and immune cells) and pharmacological efficacies are discussed.


Introduction
According to the recent WHO figure [1], cancer is the second most leading cause of mortality in the world and accounts for an estimated 9.6 million deaths in 2018. Most of the cancer death (~70%) occur in developing or the so-called low-and middle-income countries where access to modern medicines are not widely available. The common cancer deaths are from the lung, colorectal, stomach, liver and breast cancers, respectively, while other common cancers include prostate and skin cancers [1]. The major control measure for cancer is chemotherapy by using a variety of small molecular weight compounds and biological agents. As always, nature has its fair share of abundance as a source of these agents and drugs like paclitaxel (Taxol ® ), podophyllotoxin derivatives and vinca alkaloids (vinblastine and vincristine) are our excellent examples for potential exploration of more novel anti-cancer agents from higher plants. On the other hand, doxorubicin, daunomycin, mitomycin C, and bleomycin are good representative examples of anti-cancer agents explored from fungal sources, particularly Streptomyces.
In addition to their nutritional value, medicinal mushrooms have emerged in recent years not only as a source of drugs but also as adjuvants to conventional chemo-or radiation-therapy to either enhance their potency or reduce their side effects ( see [2] and references therein). In this regard, one of by far the best investigated medicinal mushroom in recent years is Coriolus versicolor (L.) Quél. (Syn. Polyporus versicolor) which is now known by its accepted scientific name as Trametes versicolor (L.) Lloyd (family Polyporaceae). Its most common name in the Western world is Turkey Tail, and its distinct morphological features include the concentric multicoloured zones on the upper side of the cap (no stalk) and spore-bearing polypores underside (Figure 1). The fungus is common in temperate Asia, North America and Europe, including the UK, where it has been recorded in all regions [3]. Its medicinal value as part of the Chinese traditional medicine dates back for at least 2000 years and includes general health-promoting effects [4], including endurance and longevity. Both in China and Japan, preparations such as dried powdered tea of the fungus are employed in traditional medicine

Small Molecular Weight Compounds
Like all other mushrooms, the fruiting body of C. versicolor is harvested for its nutritional and medicinal values. The bracket or shelf mushroom body in the wild or the mycelial biomass collected from the submerged fermentation could all be used for this purpose. In addition to the major macromolecules (proteins, carbohydrates, and lipids) and minerals, the fungus is known to contain potential pharmacologically active secondary metabolites belonging to small molecular weight compounds. The study by Wang et al. [5] reported the isolation of four new spiroaxane sesquiterpenes (Figure 2), tramspiroins A-D (1-4), one new rosenonolactone 15,16-acetonide (5), and the known drimane sesquiterpenes isodrimenediol (6) and funatrol D (7) from the cultures. Readers should bear in mind that these compounds isolated from the ethyl acetate fraction are non-polar and are not expected to be available in the polysaccharide fractions of the fungus (see below). Janjušević

Small Molecular Weight Compounds
Like all other mushrooms, the fruiting body of C. versicolor is harvested for its nutritional and medicinal values. The bracket or shelf mushroom body in the wild or the mycelial biomass collected from the submerged fermentation could all be used for this purpose. In addition to the major macromolecules (proteins, carbohydrates, and lipids) and minerals, the fungus is known to contain potential pharmacologically active secondary metabolites belonging to small molecular weight compounds. The study by Wang et al. [5] reported the isolation of four new spiroaxane sesquiterpenes (Figure 2), tramspiroins A-D (1-4), one new rosenonolactone 15,16-acetonide (5), and the known drimane sesquiterpenes isodrimenediol (6) and funatrol D (7) from the cultures. Readers should bear in mind that these compounds isolated from the ethyl acetate fraction are non-polar and are not expected to be available in the polysaccharide fractions of the fungus (see below). Janjušević et al. [6] studied the phenolic composition of the fruiting body of C. versicolor of European origin.
In their HPLC-MS/MS-based study, they identified 38 phenolic compounds belonging to the flavonoid (flavones, flavonols, flavanone, flavanols, biflavonoids, isoflavonoids) and hydroxy cinnamic acids. Although the ethanol and methanol extracts are generally the richest sources of these phenolic compounds, the water extracts were also shown to contain (µg/g dry weight) considerable amount of baicalein (21.60), baicalin (10.7), quercetin (31.20), isorhamnetin (14.60), catechin (17.20), amentoflavone (17.20), p-hydroxybenzoic acid (141.00) and cyclohexanecarboxylic acid (80.40). The biological activities of the water extracts of C. versicolor, especially in the antioxidant area, must, therefore, account for the cumulative effects of the phenolic compounds. These compounds are, however, not established as the main components of the fungus, and further research is required to establish their potential contribution to the known biological activities of C. versicolor.  (17.20), p-hydroxybenzoic acid (141.00) and cyclohexanecarboxylic acid (80.40). The biological activities of the water extracts of C. versicolor, especially in the antioxidant area, must, therefore, account for the cumulative effects of the phenolic compounds. These compounds are, however, not established as the main components of the fungus, and further research is required to establish their potential contribution to the known biological activities of C. versicolor.

Polysaccharides
Like other edible mushrooms, the fruiting body of C. versicolor is composed of carbohydrates, proteins, amino acids, and minerals. The main bioactive components of C. versicolor are the polysaccharopeptides (PSPs), which are isolated from the mycelium as well as fermentation broth. As a commercial product, the main sources of these PSPs are China and Japan that produce them from the strains of "COV-1" (PSP in China) and "CM-101" (polysaccharide K (PSP Krestin or PSK, in Japan), respectively. Both products have been approved as medicines primarily as adjuvants in cancer therapy. Given that over 100 strains of the fungi are known to occur, one must recognise the diversity of these products coming from different genetic and environmental sources, including the in vitro culture conditions of their mycelial production. They are made from polysaccharides covalently bonded to peptides through O-or N-glycosidic bonds. Numerous studies have established that D-glucose is the principal monosaccharide of PSP and PSK, although other sugars such as arabinose and rhamnose are also found in small amounts (e.g., [6][7][8]). One noticeable difference in these products could be the composition of polysaccharide:peptide ratio, and their relative molecular weight. The (PSK and PSP) are both proteoglucans of about 100 kDa with variations in the individual sugar compositions such as glucose, fucose, galactose, mannose, and xylose.

Polysaccharides
Like other edible mushrooms, the fruiting body of C. versicolor is composed of carbohydrates, proteins, amino acids, and minerals. The main bioactive components of C. versicolor are the polysaccharopeptides (PSPs), which are isolated from the mycelium as well as fermentation broth. As a commercial product, the main sources of these PSPs are China and Japan that produce them from the strains of "COV-1" (PSP in China) and "CM-101" (polysaccharide K (PSP Krestin or PSK, in Japan), respectively. Both products have been approved as medicines primarily as adjuvants in cancer therapy. Given that over 100 strains of the fungi are known to occur, one must recognise the diversity of these products coming from different genetic and environmental sources, including the in vitro culture conditions of their mycelial production. They are made from polysaccharides covalently bonded to peptides through Oor N-glycosidic bonds. Numerous studies have established that D-glucose is the principal monosaccharide of PSP and PSK, although other sugars such as arabinose and rhamnose are also found in small amounts (e.g., [6][7][8]). One noticeable difference in these products could be the composition of polysaccharide:peptide ratio, and their relative molecular weight. The (PSK and PSP) are both proteoglucans of about 100 kDa with variations in the individual sugar compositions such as glucose, fucose, galactose, mannose, and xylose.
The distinction between the extracellular (EPS) and intracellular polysaccharides have also been made with respect to their backbone structure [7,8]. The EPS contains small amounts of galactose (Gal), mannose (Man), arabinose (Ara), xylose (Xyl) and predominantly glucose (Glc) and are composed of β-(1→3) and β-(1→6)-linked D-glucose molecules. On the other hand, PSP and PSK contain α-(1→4) and β-(1→3) glucosidic linkages in their polysaccharide moieties. D-glucose is the major monosaccharide present while fucose (Fru), Gal, Man, and Xyl are the other principal monosaccharides in PSK. Earlier studies [9] established the distinctive features of these two polysaccharides with the presence of fucose in the PSK and rhamnose and arabinose in PSP. Analysis of the polysaccharide moiety of PSP, showed the predominance of 1→4, 1→2 and 1→3 glucose linkages (molar ratio 3:1:2), together with small amounts of 1→3, 1→4 and 1→6 Gal, 1→3 and 1→6 Man, and 1→3 and 1→4 Ara linkages [9]. On the other hand, the peptide moiety of PSP contains 18 different amino acids, with aspartic and glutamic acid residues being most predominant [8]. More importantly, PSK and PSP polymers are soluble in water.

Anticancer Effect through Direct Toxicity to Cancer Cells
Studies during the 1990s established that C. versicolor polysaccharides such as PSK could inhibit hepatic carcinogenesis in rats induced by 3 -methyl-4-dimethylaminoazobenzene [13]. The direct effect of PSK on gene expression profile in cancer cells was also established back in the 1980s [14]. Studies on combination therapy with radiation further showed the increased survival rate of mice bearing MM46 tumours [15]. Corriolan as a β-(1→3) polysaccharide with some (1→6) and no (1→4) branched glucan from C. versicolor was shown to be effective (100 mg/kg for 30 days) in suppressing sarcoma 180 tumours in mice [16]. Since then, the direct anticancer effect of C. versicolor polysaccharides has been demonstrated in the various experimental models in vitro, in vivo and clinical trials (see below).

MCF-7 breast cancer cells
Inhibits cell proliferation through β-estradiol degradation and cell apoptosis; decreases in the mRNA levels of anti-apoptotic genes (BCL-2 and NF-kβ); increases the mRNA level of proapoptotic genes (p53).
Chauhan et al. 2019 [18] Polysaccharide-rich extracts Human colon carcinoma LoVo and HT-29 cells-proliferation; wound healing and invasion assays-10 or 100 µg/mL Inhibits human colon cell proliferation and induces cytotoxicity; inhibits oncogenic potential, cell migration and invasion in colon cancer cells; suppresses MMP-2 enzyme activity; increases the expression of the E-cadherin.
Luk et al. 2011 [29] Polysaccharopeptide (PSP)-Commercial source-Winsor Health Products Ltd, Hong Kong HL-60-25 µg/mL Reduces cell proliferation; inhibits cell progression through both S and G2 phase; reduces 3 H-thymidine uptake and prolonged DNA synthesis time; enhances the cytotoxicity of camptothecin; no effect on normal human peripheral blood mononuclear cells.
Wan et al. 2010 [30] Methanol extract of fruiting body of Serbian origin

B16 mouse melanoma cells-200 µg/mL
Induces cell cycle arrest in the G0/G1 phase, followed by both apoptotic and secondary necrotic cell death; see Table 2  Suppresses cell proliferation in a dose-dependent manner (IC 50 = 150.6 µg/mL); increases nucleosome production from apoptotic cells; increases Bax and downregulates Bcl-2 or increases Bax/Bcl-2 proteins ratio; increases the release of cytochrome-c from mitochondria to cytosol; other effects, see Table 2 Ho et al.

Preparation Experimental Model Key Findings References
Proteins and peptide bound polysaccharides (PSP) HL-60 cells-400 µg/mL Induces apoptosis; phosphorylated regulation of early transcription factors (AP-1, EGR1, IER2 and IER5) and downregulates NF-κB pathways; increases apoptotic or anti-proliferation genes (GADD45A/B and TUSC2) and the decrease of a batch of phosphatase and kinase genes; alters carcinogenesis-related gene transcripts (SAT, DCT, Melan-A, uPA and cyclin E1).
Zeng et al.
Lau et al.
As expected for apoptosis-inducing agents, genes and proteins that are associated with cancer cell survival (anti-apoptotic BCL-2, Bcl-xL, survivin) are shown to be suppressed while those markers of apoptosis (proapoptotic Bax) induction are upregulated by the C. versicolor polysaccharide preparations [18,32,35,36]. Induction of the intracellular level of reactive oxygen species (ROS) in cancer cells is a well-established mechanism of cell death by chemotherapeutic agents and this appears to be the case for C. versicolor in several cell lines [17,46]. The critical cell growth and death regulator mitogen-activated protein kinase (MAPK) is involved in the induction of cell death by C. versicolor polysaccharides, as shown by the enhancement of p38 MAPK phosphorylation [24][25][26]28]. Accordingly, the cytotoxicity of these polysaccharides in melanoma cells could be abolished in the presence of c-Jun N-terminal kinase (JNK) inhibitors [17] or the p38 MAPK inhibitor [24][25][26]. Key transcription factors that are involved in cancer development and metastasis could be inhibited by C. versicolor polysaccharides. This includes the well-defined cancer modulator, NF-κB, or its induced protein product, cyclooxygenase-2 (COX-2) [36,40]. The potential combination of C. versicolor polysaccharides with conventional chemotherapeutic agents has been demonstrated in vitro, as shown for camptothecin [30], doxorubicin and etoposide [32,38], and cisplatin [46]. Even at concentrations where direct toxicity was not observed, inhibition of cell migration and invasion was evident along with inhibition of key angiogenic enzymes such as matrix metalloprotease (MMP)-9 [23] or MMP-2 [19].

Evidence of Efficacy through Animal Models
Many in vitro experiments that showed promising effects in direct cytotoxicity study were also extended to animal models of tumour-bearing mice. This was based on the injection of the cancer cells into mice and assess the size and spread (metastasis) of the tumour in the presence or absence of C. versicolor polysaccharides. Table 2 has a good summary of these data with the description of the polysaccharides, their route of administration and main outcomes [14,23,29,31,37,42,45,[50][51][52][53][54][55]. It appears that C. versicolor polysaccharides in the form of PSP, PSK, refined fractions, water or aqueous extracts exhibit anticancer effects in vivo when administered by either oral (p.o.), intraperitoneal (i.p.) or intravenous (i.v.) routs. In a combination approach, favourable responses were obtained with metronomic zoledronic acid [50] and docetaxel-taxane [51]. In addition to a reduction in the size and volume of the implanted tumours, the incidence of tumours [29,45] and angiogenesis via vascular endothelial cell growth factor (VEGF) expression have been shown to be inhibited. The combination increased more tumour suppression than either treatment alone-reduced tumor proliferation and enhanced apoptosis; other effects on immunomodulation (see Table  4).
Reduces tumour volume and anti-metastatic activity to the lungs; downregulates the expression of PLAU (uPA protein) and CXCR4 genes in breast tumors; no effect on genes associated with breast-to-lung cancer metastasis: ezrin (EZR), HRAS, S100A4, CDKN1A (protein p21) and HTATIP2 (protein TIP30).  Direct effect on the transcription and translation of genens (pPIC1, pPIC2 and pPDC1).

Evidence of Efficacy through Clinical Trials
Chay et al. [56] employed a human study on C. versicolor extract by recruiting fifteen eligible cases of hepatocellular carcinoma patients in Singapore who failed or were unfit for standard therapy. The randomised placebo-controlled trial using 2.4 g as a daily treatment for~5.9 weeks) showed a better quality of life without a significant difference in primary endpoint measure of the median time to progression. On the other hand, a pilot study of randomised, double-blind, and multidose study on dogs (not humans) revealed that treatment with PSP (e.g., 100 mg/kg capsules daily) could delay the progression of metastases of canine hemangiosarcoma [57]. The systematic review and meta-analysis studies by Eliza et al. [58] assessed the survival outcome in cancer patients from 13 clinical trials on C. versicolor. They reported an impressive result showing a significant survival advantage when compared with standard conventional anti-cancer agents alone. For example, a 9% absolute reduction in 5-year mortality was recorded with one additional patient alive for every 11 patients treated. They also reported a better 5-year survival rate in patients receiving combination treatment in cases of breast cancer, gastric cancer, or colorectal cancer. Database on ClinicalTrial.gov shows one terminated clinical trial on the potential benefit of C. versicolor for hepatocellular carcinoma and one currently recruiting for a vaginal gel based on C. versicolor medical device (PAPILOCARE) as a phase III trial. A further entry in this database is the USA (University of Minnesota), trial on C. versicolor extract in Stage I, Stage II, or Stage III breast cancer who have finished radiation therapy.

Anti-Cancer Effect Via Immunostimulation
Studies on the immunotherapeutic potential of the C. versicolor polysaccharides in cancer started in the late 1970s and accelerated in the 1980s and 1990s. In 1977, Kataoka et al. [59] reported that immuno-resistance in mice could be induced when protein-bound polysaccharides are administered together with L1210 murine leukemic cells. The suppression of TNF-α production in mice by cytotoxic antitumour agents (5-fluorouracil, cyclophosphamide and bleomycin) was shown to be ameliorated by PSK with an implication of immunotherapy potential [60]. The immunosuppressive effect of cyclophosphamide in rats could also be abolished by PSP [61]. Myelosuppressed mice due to chemotherapy could also be reversed by PSK, particularly when used in combination with granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3 [62]. These general immunostimulations or ameliorations of immunosuppression under cancer and depressed immune systems, either by cancer, splenectomy or other experimental agents, have been observed for C. versicolor polysaccharides [63][64][65][66][67][68][69].
Further studies in vitro showed the direct lymphocyte proliferative effect of PSP, while in mice, it reversed the inhibition of IL-2 production induced by cyclophosphamide along with restoration of the T-cell-mediated response [70]. The study by Kanoh et al. [71] also demonstrated that PSK could enhance the anti-tumour effects of IgG2a monoclonal antibody in the human colon cancer cell line, colo 205, both in vitro and in vivo via antibody-dependent macrophage-mediated cytotoxicity. Studies on PSK using mice bearing syngeneic plasmacytoma X5563 also showed that it enhances anti-tumour immunity by ameliorating the immunosuppressive activity of serum from tumour-bearing mice [72,73]. The tumour-induced immunosuppression could also be abolished by PSK in various cancer models in vivo [74]. Earlier in vitro studies further confirmed the direct effect of the polysaccharides on peritoneal macrophages [75], namely, interleukin-1 production by human peripheral blood mononuclear cells [76]. Further insight into the immunotherapeutic potential of C. versicolor polysaccharides is outlined below under the headings of in vitro, in vivo and human studies.

Evidence of Immunotherapy Potential through In Vitro Studies
Perhaps the best characterised pharmacological activity of C. versicolor relates to its immunostimulatory effects. Some of the key outcomes from in vitro studies with implications to cancer are shown in Table 3 [8,49,[77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. The proliferative effect of the polysaccharides on mononuclear cells, such as lymphocytes [78,79,86,92], monocytes [85] or macrophages [77] and others, including splenocytes [81], has been shown for the polysaccharides. The immunostimulatory effect also includes activation of immune cells, as shown in the LPS-induced cytokine (interleukin (IL)-1β and IL-6) expression by peripheral blood mononuclear cells (PBMCs) [77] or by blood lymphocytes) [78]. Enhancement of antibody production such as IgM and IgG1 by splenocytes was reported [82], while activation of dendritic cells was evident from the expression level of surface markers in mature cells [84]. Increased level of IgM production in B cells by PSK has also been reported. Similarly, cytokines expression, including upregulation of TNF-α expression, leads to enhanced breast cancer cell killing) [79]. The production of IL-10 in mouse B cells could be enhanced by up to 60-fold for some preparations [86], while the antibody-mediated cytotoxicity of natural killer (NK) cells against cancer cells could be enhanced through IL-12-dependent and independent mechanisms [87]. Selective induction of cytokines expression that promotes Th1 and Th2 lymphocytes have been shown [92]. Also, increased nitric oxide (NO) production in polymorphonuclear cells (PMNs) or mononuclear cells such as U937 and THP-1 have been reported for PSK and its fractions [49,94].
Considerable levels of research have been devoted to understanding how C. versicolor polysaccharides interact with the immune cells. One of the established recognition sites for the polysaccharides is the toll-like receptors (TLRs), of which effects via TLR4 are well-documented. In mouse peritoneal macrophages, the expression of cytokines and NF-κB activation by PSP was shown to be coupled with TLR4 activation [81]. Furthermore, the induction of TNF-α and IL-6 secretion in J774A.1 cells and primary splenocytes by PSP via TLR4 has also been well established and correlated with its effect on NF-κB p65 transcription and phosphorylation of c-Jun [88]. The expression of both TLR4 and TLR5 by PSP was shown in PBMCs of human origin, while TLR9 and TLR10 appear to be downregulated [89]. Fractionation of PSK further led to the identification of two motifs: a β-glucan recognised by the Dectin-1 receptor and lipid fraction with agonistic activity towards TLR2. Reduces cell growth; upregulates TNF-αexpression but not IL-1β and IL-6; enhances the proliferative response of blood lymphocytes associated with IL-6 and IL-1β mRNA upregulation.
Wang et al. 2015 [81] Polysaccharides-hot water extraction in house  Activates NK cells to produce IFN-γ and to lyse K562 target cells; enhances trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity ADCC against SKBR3 and MDA-MB-231 breast cancer cells; effect related to both direct and IL-12-dependent (indirect) mechanism.

Preparation Experimental Model Key Findings References
PSP Human T lymphocyte proliferation-100 or 500 µg/mL Exhibits similar and additive inhibitory effects to ciclosporin to suppress activated T-cell proliferation, Th1 cytokines; reduces CD3 + /CD25 + cell expression but not Th2 cytokine expression.

Evidence of Immunotherapy Potential through In Vivo Studies
The animal studies on C. versicolor polysaccharides also support the general immunostimulatory effect (Table 4) [23,31,48,78,81,84,87,[95][96][97][98][99][100][101][102][103][104]. Increased cytokine and ROS production and NF-κB activation have been reported in rats [95]. Through IL-10-dependent mechanism, an enhancement of cytokine production that was associated with T helper (Th2 and Th17 cells) (e.g., IL-2, -4, -6, -10, -17A and IFN-α and -γ) were observed for a glucan product of C. versicolor in cancer-bearing mice [96]. By increasing the level of IL-6, PSP could also increase the duration of endotoxin fever in rats [98]. The proinflammatory effect of C. versicolor is also evident from in vivo effect of PSP in inducing a writhing response in animals, which was associated with induction of the release of prostaglandin-E2 (PGE2), TNF-α, IL-1β, and histamine from macrophages and mast cells [101]. In agreement with the in vitro experiments, combination with acacia gum resulted in a selective increase in IgG level in mice treated by PSP while the IgA or IgE levels were not affected [97]. Small peptide fractions of the polysaccharides have also shown to increase IgG level in vivo as well as white blood cell (WBC) count in tumour-bearing nude mice [48]. The animal studies in rats also suggest that high temperature exposure (hyperthermia) could suppress the cytokine production by C. versicolor polysaccharides [78]. Furthermore, PSP has been shown to rapidly lower temperature in rats by elevating the level of TNFα [99].
The correlation between TLR4 activation by PSP and anti-tumour potential was studied in mice. This was substantiated from the fact that its anti-tumour effect and increased thymus index and spleen index were evident in tumour-bearing C57BL/10J (TLR4 +/+ ) mice but not in C57BL/10ScCr (TLR4 − ) mice [81]. PSK could also enlarge lymph nodes, activate dendritic cells, and stimulate T-cells to produce cytokines, including IFN-γ, IL-2, and TNF-α [84]. The methanol extract of C. versicolor also induced a higher level of tumouricidal potential of peritoneal macrophage, as revealed by the study in mice subjected to melanoma cancer [31].
The beneficial effect of PSK in combination treatment with docetaxel in prostate-carrying mice was shown to be associated with immunostimulatory effects. In this case, the number of WBCs count under the combination treatment was much more favourable than docetaxel alone [51]. The potentiation effect of PSK in anti-HER2/neu mAb therapy of Neu transgenic mice with cancer was reported [87]. The potential application of PSP in potentiating radiation therapy has also been investigated where increased lymphocyte and granulocyte counts in the blood and spleen tissues were observed [102]. When combined with acacia gum, increased total IgG titre levels (day 4) while decreasing IgM titre had no effect on IgA or IgE titre levels.
Combination treatment of (FRH + PBP) decrease IL-1β, IL-6 and TNF-α mRNA expression in peripheral blood mononuclear cells; see Table 3  Increases IL-2, 6, 12, TNF-α and IFN-γ productions from the spleen lymphocytes; see Tables 1 and 2 for  other effects Luo et al. 2014 [23] PSK As an adjuvant to OVAp323-339 vaccine in vivo-DC activation 1000 µg-one injection by intradermal route Enlarges draining lymph nodes with higher number of activated DC; stimulates the proliferation of OVA-specific T-cells, and induces T-cells that produce multiple cytokines (IFN-γ, IL-2, and TNF-α; see Table 3 for in vitro effect. Engel et al. 2013 [84]  Decreased the number of acetic acid-induced writhing by 92.9%; PSP itself induces a dose-dependent writhing response; increased the release of PGE2, TNF-α, IL-1β, and histamine in mouse peritoneal macrophages and mast cells both in vivo and in vitro (1-100 µM).

Evidence of Immunotherapy Potential through Human Studies
Perhaps the most promising immunostimulatory effect of C. versicolor polysaccharides resides on the reported promise in human cancer patients. In breast cancer patients, for example, PSP has been shown to upregulate cytokine genes for L-12, IL-6 and TNF-α in PBMCs [104]. A comprehensive study with 349 gastric cancer patients receiving PSK (3 g/day) as adjuvant immunotherapy also revealed a greatly improved 3-year recurrence-free survival (RFS) rates when patients were MHC class I-negative [105]. A freeze-dried mycelial powder preparation of the fungus was also reported to show the trend of increased lymphocyte counts when applied at 6 and 9 g/day doses [106]. Although only 9 women were involved in this experiment, dose-related increases in CD8 + T-cells and CD19 + B-cells (not CD4 + T-cells or CD16 + 56 + NK cells) were reported. The application of PSK in gastric cancer -(Stage II/III) studied using large group (138 patients)-further revealed a relapse-free survival rate after post-operation or when compared to oral fluorinated pyrimidine anti-metabolites alone or in combination [107]. The double-blind placebo-controlled randomised trial study by Tsang et al. [108] employed 34 patients who had completed conventional treatment for advanced non-small cell lung cancer. They showed that PSP capsules of 340 mg each, 3× daily for 4 weeks, could lead to an improvement in blood leukocyte and neutrophil counts, serum IgG and IgM. Finally, Zhong et al. [109] undertook a meta-analysis study on randomised controlled trials of C. versicolor along with others. They reported that the treatment had a favorable effect on elevated levels of CD3 and CD4. Earlier studies in human cancer patients also substantiate this argument [110][111][112][113].

Other Benefits of C. versicolor Polysaccharides
Given oxidative stress is a prominent feature in cancer patients and experimental animals transplanted with tumours, the benefits of C. versicolor polysaccharides have also been tested as antioxidants. In both rats bearing with Walker 256 fibrosarcoma and human cancer patients, oral administration of PSK (daily dose of 3.0 g in humans and 50 mg/kg in rats) could normalise the disease-associated oxidative stress [114]. The immunostimulatory effect of C. versicolor polysaccharides has also been shown to be associated with increased superoxide dismutase (SOD) activities of lymphocytes and the thymus [115]. It is also worth noting that C. versicolor polysaccharides have been shown to ameliorate obesity [116] or experimental diabetes in rodents [117]. As anti-inflammatory agents, they further showed their benefit in experimental animal models of osteoarthritis [118], inflammatory bowel disease [119] or induction of analgesia [120]. Their organ protective effect was also proven through experimental models of alcoholic liver injury [10] and diabetic cardiomyopathy [121]. Their immunomodulatory effect in cancer is also extended in defenses against bacteria, including against intracellular parasites such as Neisseria gonorrhoeae [122].
While C. versicolor is regarded as an edible and medicinal mushroom, there is no report on sever toxicity induced by the fungus in humans. Experiments in rats using the standardised water extract has shown no mortality and signs of toxicity in acute and sub-chronic toxicity (up to 28 days) studies for doses up to 5000 mg/kg (p.o.) [123]. Monoclonal antibody against PSK has been developed [124], and, in principle, such antibodies could reduce the long-term use of the peptide-bound polysaccharides. For the doses indicated in the various animal experiments and human studies indicated herein, the toxicity of C. versicolor polysaccharides is not suggested as a concern.

General Summary and Conclusions
A great deal of attention has been given to medicinal mushrooms in recent years, with emphasis to their polysaccharide-active components. Most of these fungi are highly exploited as commercial products in far eastern countries such as Japan and China. In this regard, the edible mushroom Dictyophora indusiata (Vent. Ex. Pers.) Fischer (Syn. Phallus indusiatus) as a source of polysaccharides with main components as β-(→3)-D-glucan with side branches of β-(1→6)-glucosyl units have been established. Their chemistry, along with potential applications in cancer and immunotherapy, inflammatory and CNS diseases, among others, have been reviewed [2]. Another excellent example of the potential application of fungal polysaccharides in cancer therapy was demonstrated for Ganoderma species, which is reviewed by Cao et al. [125]. Similarly, PSP, PSK as well as other polysaccharides from C. versicolor have now been established to induce direct cytotoxicity to cancer/tumour cells. They also increase the release of cytokines such as TNF-α with direct implication to tumour cell killing. The overall, anti-cancer pharmacology of these polysaccharides through a direct effect on cancer cells and an indirect effect via immunostimulation is depicted in Figure 3.
Dictyophora indusiata (Vent. Ex. Pers.) Fischer (Syn. Phallus indusiatus) as a source of polysaccharides with main components as β-(→ 3)-D-glucan with side branches of β-(1→ 6)-glucosyl units have been established. Their chemistry, along with potential applications in cancer and immunotherapy, inflammatory and CNS diseases, among others, have been reviewed [2]. Another excellent example of the potential application of fungal polysaccharides in cancer therapy was demonstrated for Ganoderma species, which is reviewed by Cao et al. [125]. Similarly, PSP, PSK as well as other polysaccharides from C. versicolor have now been established to induce direct cytotoxicity to cancer/tumour cells. They also increase the release of cytokines such as TNF-α with direct implication to tumour cell killing. The overall, anti-cancer pharmacology of these polysaccharides through a direct effect on cancer cells and an indirect effect via immunostimulation is depicted in Figure 3. Overall, the polysaccharides of C. versicolor have been shown to induce direct cell growth inhibitory effect and apoptosis in cancer cells. Cell cycle arrest, even in some cases at concentrations lower than 100 μg/mL in vitro, has been observed. This moderate level of activity should be considered significant since the active components are large molecular weight compounds or mixtures. Given that carbohydrates taken through the oral route are subjected to hydrolysis by intestinal enzymes, there is always a question of whether they could maintain their therapeutic value in vivo. Interestingly, C. versicolor polysaccharides, including PSP and PSK, have been shown to demonstrate anti-cancer effect in vivo following oral administration. The other well-established Overall, the polysaccharides of C. versicolor have been shown to induce direct cell growth inhibitory effect and apoptosis in cancer cells. Cell cycle arrest, even in some cases at concentrations lower than 100 µg/mL in vitro, has been observed. This moderate level of activity should be considered significant since the active components are large molecular weight compounds or mixtures. Given that carbohydrates taken through the oral route are subjected to hydrolysis by intestinal enzymes, there is always a question of whether they could maintain their therapeutic value in vivo. Interestingly, C. versicolor polysaccharides, including PSP and PSK, have been shown to demonstrate anti-cancer effect in vivo following oral administration. The other well-established mechanism of the anti-cancer effect by C. versicolor is via immunostimulant action, as evidenced by their ability to increase the production of cytokines such as IL-12, which is Th1 related. Th-lymphocyte subsets, including Th1, Th2, Th17 or Treg, mainly through the production of key cytokines and lymphocyte subsets (B-cells, CD4 + and CD8 + T-cells, NK cells, and different stages of differentiated T-cells), have been extensively studied for their response to C. versicolor polysaccharides. The further induction of cytokines such as IFN-γ in T cells was evident, which, together with TNF-α, induce cancer/tumour killing. Other cytokines, including IL-1 and IL-6, have been shown to be augmented by the polysaccharides. All these events appear to enhance antibody production in T-cells while enhancing the activity of other mononuclear cells, including monocytes/macrophages. By interacting via the membrane Ig (B-cell antigen receptor) and TLR4, C. versicolor polysaccharides have been shown to activate B-cells via the phosphorylation of ERK 1/2 and p38 MAPK [82]. In human PBMCs, for example, PSP activates cells through TLRs family (e.g., TLR4, TLR5, TLR6 and TLR7) and their adaptor proteins (e.g., TICAM2, HRAS, HSPA4, HSPA6, and PELI2) leading to genes activation for key cytokines (including IFN-γ, G-CSF, GM-CSF, IL-1α, IL-6) and NF-κB and TRAF6 [89]. In the latter case, it appears that PSP appears to involve the TRAM-TRIF-TRAF6 pathway of immunomodulation. With TRAM acting as a bridge between TLRs (e.g., TLR4) and TRIF6, activation of mononuclear cells to orchestrate an inflammatory response has been well-established [126]. Other important cell surface receptors for the polysaccharides include the TLR2, and Dectin-1, which are shown to be linked to the immunogenic activity of PSK [80]. Dendritic cells being an important component of the immune system, they appear to be the target for C. versicolor polysaccharides. For example, PSK as an adjuvant to vaccines has been demonstrated to induce the production of cytokines (e.g., IL-12, TNF-α, and IL6) in these cells both in vitro and in vivo [84]. Hence, the immunostimulatory effect coupled with direct toxicity to cancer cells by C. versicolor polysaccharides implies application even more than an adjuvant therapy. The evidence for signal transduction pathways, including that for TLR4 as well as other cell surface recognition markers of the polysaccharides (e.g., Dectin-1 as a β-glucan receptor), are evolving current research. The structural moieties of the polysaccharides that attribute to the various pharmacological effects also need further research.