1. Immunomodulatory effect

Host immune surveillance plays a crucial role in recognizing and destroying invading pathogens and host cells that become cancerous, so immunotherapy has become one of the important strategies for cancer prevention and treatment. Accumulating evidence implicates that G. lucidum polysaccharides(GLP) partially exert the anticancer action by stimulating the immune function. This is primarily attributed to the fact that GLP can activate T and B lymphocytes, macrophages, dendritic cells (DCs), and natural killer (NK) cells, which promotes proliferation of lymphocytes, and enhances phagocytosis, increases the production of cytokines, and augments NK cell-mediated cytotoxicity.

1.1. Effect of GLP on T lymphocytes

T lymphocytes (also known as T cells) are essential for cellular immunity. Several subtypes of T cells have distinct functions, such as T helper cells, cytotoxic T cells, memory T cells, suppressor T cells, natural killer T cells, and mucosal-associated invariant T cells. Numerous studies have suggested that GLP is an activator of T lymphocytes. It has been described that treatment with GLP significantly promotes concanavalin A (ConA)-induced mouse lymphocyte proliferation and IL-2 production. GLP can also enhance the DNA synthesis in mouse spleen cells in a mixed lymphocyte culture by inducing the expression of DNA polymerase α. GLP increases the expression of IFN-γ in the T-lymphocytes and IL-1, IL-2, and IFN-γ in mouse spleen cells. GLP increases the production of inositol triphosphate (IP3) and diacylglycerol (DAG) in resting T cells. However, it does not influence the production of IP3 and DAG in ConA-activated T cells, suggesting that both IP3/Ca2+ and DAG/ protein kinase C (PKC) pathways may be involved in the immunomodulatory effect of GLP on T cells. Further studies have shown that GLP can activate PKC and protein kinase A (PKA) in murine T cells.

B16F10 melanoma cells can secrete a large amount of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in the cell culture medium. It has been shown that treatment with the B16F10 cell culture supernatant prevents phytohemagglutinin (PHA) from stimulating the production of perforin and granzyme B, as well as the proliferation in lymphocytes. Interestingly, the addition of GLP can fully or partially antagonize the inhibitory effects of the B16F10 cell culture supernatant on lymphocytes. GLP also prevents B16F10 cells from reducing the expression of CD71 and Fas ligand (FasL) in lymphocytes. Furthermore, the plasma of lung cancer patients inhibits proliferation, CD69 expression, and perforin and granzyme B production in lymphocytes activated by PHA, which can also be fully or partially reversed by GLP treatment. GLP treatment of the co-culture of B16F10 melanoma cells and lymphocytes increases the production of CD69 (a co-stimulatory molecule for T cell activation and proliferation), FasL, and interferon (IFN)-γ in the medium.

1.2. Effect of GLP on B lymphocytes

B lymphocytes (also known as B cells) play an important role in humoral immunity. Unlike T or NK cells, B cells express B cell receptors on their cell membrane, allowing the B cell to bind a specific antigen and produce an antibody against the antigen. Besides, B cells also present antigens and secrete cytokines. GLP can activate B cells by increasing their proliferation and differentiation. It has been reported that GLP not only increases the percentage of B cells by 2.5–4 fold but also increases the size of B cells. Similarly, in vivo treatment with GLP activates spleen and bone marrow-derived B lymphocytes from sarcoma S180-bearing mice and induces proliferation of the B cells and production of large amounts of immunoglobulins in mice. Mechanistically, GLP can induce the expression of CD71 and CD25 on the B cell surface and increase the secretion of immunoglobulins by B cells by directly stimulating the expression of PKCα and PKCγ in B cells.

1.3. Effect of GLP on DCs

DCs are important professional antigen-presenting cells necessary for initiating the primary immune response of helper and cytotoxic T lymphocytes. GLP can stimulate the maturation of normal human monocyte-derived DCs and leukemic monocyte-derived DCs. GLP increases cell-surface expression of CD80, CD86, CD83, CD40, CD54, and human leukocyte antigen-DR (HLA-DR). It also enhances the production of IL-12 p70, IL-12 p40, and IL-10. The GLP-induced activation and maturation of human monocyte-derived DCs are mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways. GLP (0.8, 3.2, or 12.8 mg/ml) increases the co-expression of CD11c and I-A/I-E molecules on the surface of cultured bone marrow-derived DCs. It elevates mRNA expression of cytokine IL-12 p40 in DCs and increases protein production of IL-12 p40 in culture supernatants. GLP also enhances the lymphocyte proliferation of mixed lymphocyte cultures induced by mature DCs. Treatment of leukemic monocytic cell lines THP-1 and U937 with GLP (100 μg/mL) significantly increases the expression of HLA-DR, CD40, CD80, and CD86. GLP induces the differentiation of THP-1 leukemia cells to macrophage-like cells by enhancing cell adherence, superoxide production, cell cycle arrest, and expression of differentiation markers such as CD11b, CD14, CD68, matrix metalloproteinase-9 (MMP-9) and myeloperoxidase. Also, GLP-induced activation of caspases and p53 contributes to this differentiation.

1.4. Effect of GLP on macrophages

Macrophages are the “big eaters” of the immune system, which engulf and digest apoptotic cells and pathogens (called phagocytosis) and produce immune effector molecules. In vivo treatment with GLP activates bone marrow-derived macrophages from sarcoma S180-bearing mice, producing immunomodulatory substances, such as IL-1β, TNF-α, and nitric oxide (NO). GLP increases phagocytosis of macrophages significantly and enhances macrophage-mediated tumor cytotoxicity. Also, GLP activates macrophages in vitro and increases the levels of various cytokines, including IL-1β, tumor necrosis factor (TNF)-α, IFN-γ, and IL-6 in the culture medium. GLP has been shown as an inducer of MAPKs- and Syk-dependent TNF-α and IL-6 secretion in murine resident peritoneal macrophages. GLP stimulates dectin-1, but toll-like receptor (TLR)-4 signaling is not involved in the biological activities of GLP. However, this is controversial since GLP has been found to directly bind to TLR-4, mIg of B cells, 7S ribosomal protein, and bZIP enhancer, and to transduce certain signalings via the TLR-4. Apart from the effects of GLP on the expression of the cytokines and chemokines mentioned above, GLP also induces the expression of inflammatory cytokine IL-1, which, in part, links to its anticancer activity. It has been described that GLP up-regulates the secretion of IL-1 and the expression of pro-IL-1 (precursor of IL-1) and IL-1-converting enzyme in human macrophages and murine macrophages (J774A.1). This is attributed to the activation of protein tyrosine kinase/protein kinase C/MEK1/extracellular signal-regulated kinase (ERK) and protein tyrosine kinase/Rac1/p21-activated kinase/p38 pathways.

1.5. Effect of GLP on NK cells

NK cells, unlike cytotoxic T-cells, can recognize stressed cells without antibodies and major histocompatibility complex (MHC), allowing for a much faster immune reaction. Hence, NK cells are critical to innate immunity. It has been shown that treatment with GLP increases the population of CD14+CD26+ monocyte/macrophage, CD83+CD1a+ DCs, and CD16+CD56+ NK cells by 2.9, 2.3, and 1.5 fold, respectively, in human umbilical cord blood mononuclear cells. Also, GLP enhances NK cell-mediated cytotoxicity by 31.7%. Oral administration of G. lucidum extract increases T-helper type 1 and macrophage cytokines (IL-6 and IFN-γ) and enhances the NK cell activities and phagocytosis in BALB/c mice. A GLP fraction binds to the TLR-4 receptor and activates ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK. GLP can stimulate the expression of IL-1, IL-6, IL-12, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) in mouse splenocytes. In addition, a GLP fraction increases the number of DCs and CD4, CD8, regulatory T, B, plasma, NK, and natural killer T (NKT) cells in the spleen of mice. In vivo treatment of mice with this fraction elevates the levels of 12 cytokines and chemokines, including KC (CXCL1), MCP-1 (CCL2), IL-6, MIP-1β (CCL3), IL-1 β, IL-12p40, IL-12p70, RANTES (CCL5), IL-1α, TNF-α, IL-10, and IL-13 in the serum of mice.

G.lucidum mitigates cyclophosphamide-induced decrease in body weight, NK activity, IFN-γ production, and cytotoxic T lymphocyte activity and inhibits the abnormal increase or decrease in IL-4 level due to cyclophosphamide administration. Chronic administration of 2.5 mg/kg GLP accelerates recovery of bone marrow cells, red blood cells, and white blood cells, as well as splenic NK cells and NKT cells. It enhances T and B cell proliferation responses compared to the treatment with vehicle. It also augments the phagocytosis and cytotoxicity of macrophages without any obvious side effects. Thus, the low-dose GLP treatment accelerates the recovery of immunosuppressed mice from leukopenia, myelosuppression, and immunosuppression, which should be beneficial to cancer chemotherapy.

2. Anti-proliferative and pro-apoptotic effects on tumor cells

Increasing evidence has suggested that GLP functions as an anticancer agent by stimulating the immune response and displaying direct cytostatic and cytotoxic effects on tumor cells. It has been described that GLP inhibits the proliferation of mouse melanoma cells (B16F10), rat adrenal medulla pheochromocytoma cells (PC12), and human bladder cancer cells (HUC-PC and MTC-11). GLP down-regulation of cyclin D1 is associated with growth inhibition and cell cycle arrest in human ovarian OVCAR-3 cells. Studies have also revealed that phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and MAPK signaling pathways are involved in the anticancer effect of GLP. For instance, GLP reduces the expression of some signaling molecules in the PI3K/AKT/mTOR and MAPK pathways at both gene and protein levels. Treatment of inflammatory breast cancer cells (SUM-149) with GLP down-regulates the expression of mTOR downstream effectors at early treatment time points in vitro. Notably, GLP reduces the level of eukaryotic initiation factor (eIF) 4G (eIF4G), increasing the binding of eIF4E to eIF4E-binding protein 1 (4E-BP1), and decreases the formation of eIF4F complex, thereby inhibiting protein synthesis.

Also, GLP has been found to reduce cell viability in human colon cancer cells (HCT-116 and SW 480) in a concentration-dependent manner. GLP can induce apoptosis by enhancing the release of lactate dehydrogenase (LDH) and increasing the level of intracellular Ca2+, leading to activation of Fas-mediated caspase, mitochondrial, and JNK pathways in HCT-116 cells. A GLP fraction triggers apoptosis in THP-1 leukemia cells by up-regulating the expression of death receptor (DR3 and DR4/5) ligands (TNF-α and TRAIL). It results in death receptor oligomerization recruitment of specialized adaptor proteins and activating the caspase pathway.

Oral administration of G. lucidum extract (at a dose of 28 mg/kg/day) for 13 weeks does not affect the body weight of mice but inhibits breast SUM-149 xenograft volume and tumor weight by 58% and 45%, respectively. It is related to reduced expression of E-cadherin, mTOR, eIF4G, p70 S6 kinase (S6K), and activity of ERK1/2. Administration of GLP (100 mg/kg) 24 h after tumor implantation with Ehrlich’s ascites carcinoma cells shows 80.8 and 77.6% reduction in tumor volume and mass, respectively. In contrast, administration of GLP at the same dose before tumor inoculation exhibits 79.5 and 81.2% inhibition of tumor volume and mass, respectively. Different administration protocols of GLP can lead to up to 60 % inhibition of S180 xenografts in mice without apparent adverse effects on body weight. In Yoshida AH-130 ascites hepatoma cells implemented mice, administration of an aqueous extract of G. lucidum reduces the tumor weight in a dose-dependent manner compared to the control group, with inhibition rates of 25% and 47% at 200 and 400 mg/kg, respectively. GLP significantly suppresses tumor growth in hepatoma-bearing mice, associated with an increase in the ratio of regulatory T cell (Treg) and effector T cell (Teff). Moreover, GLP induces miR-125b expression and attenuates the inhibitory effect of Treg on Teff proliferation by increasing IL-2 secretion.

It has been of interest that mice immunized with an L-fucose enriched GLP fraction can produce IgM antibodies specific to tumor-associated glycans, which have cytotoxicity and reduce the production of tumor-associated inflammatory mediators in murine Lewis lung carcinoma cells. In addition, in vivo administration of GLP increases the Con A-induced proliferative response of splenocytes and induces anti-tumor activity against Lewis lung cancer in mice.

In addition, GLP can enhance the anticancer effects of chemotherapeutic agents. For instance, a combination of GLP and chemotherapeutic agents (cisplatin and arsenic trioxide) displays synergistic cytotoxicity in human urothelial carcinoma cells, including parental NTUB1; cisplatin-resistant, N/P(14); and arsenic-resistant, N/As (0.5). It has been proposed that GLP enhances these compounds’ cytotoxicity by activating p38 MAPK, down-regulation of AKT and XPA, induction of Fas, and activation of caspases 3/8, up-regulation of BAX and BAD, down-regulation of Bcl-2 and Bcl-xL, and increase of cytochrome c release. Besides, a combination of cyclophosphamide and G. lucidum has been found to inhibit tumor growth more potently than cyclophosphamide alone in MM 46-bearing mice. This is attributed to GLP’s alleviation of cyclophosphamide-induced decrease in body weight, NK activity, IFN-γ production, cytotoxic T lymphocyte activity, and inhibition of cyclophosphamide-induced abnormal expression of IL-4.

3. Anti-metastatic effect on tumor cells

Cancer metastasis, one of the characteristics of malignant tumors, is the primary cause of death in most cancer patients. Tumor cell migration is a prerequisite for metastasis. Thus, targeting tumor cell motility has received significant attention for cancer therapy. Studies have shown that GLP can inhibit tumor cell motility and invasion in vitro and tumor metastasis in vivo. For example, GLP inhibits cell adhesion and motility in HCT-116, human lung carcinoma PG cells, and MT-1 human breast carcinoma cells. GLP inhibits cell adhesion in MT-1 breast cancer cells by reducing β1-integrin expression. The water extract of G. lucidum inhibits cell motility in breast MDA-MB-231 and prostate PC-3 cancer cells in a concentration-dependent manner. This is associated with inhibition of constitutive activation of NF-κB and AP-1, reducing the expression of the urokinase-type plasminogen activator uPA and the uPA receptor (uPAR) in the cells. Besides, GLP inhibits oxidative stress-induced migration of MCF-7 cells by suppressing ERK1/2 signaling, resulting in down-regulation of c-fos expression and inhibition of NF-κB and AP-1. GLP inhibits 4-aminobiphenyl-induced migration by inducing actin polymerization and focal adhesion complex formation in human bladder cancer cells (HUC-PC and MTC-11). Reishi extract inhibits cell invasion by downregulation of MMP-9. In a Lewis lung carcinoma cell mouse xenograft model, administration of cyclophosphamide increases metastasis of the tumor cells to the lung in C57BL/6 mice, which can be effectively suppressed by pre-feeding with the G. lucidum-containing diet.

4. Anti-angiogenic effect

Angiogenesis refers to forming new blood vessels from pre-existing vessels, which is crucial in promoting tumor growth and metastasis. Thus, targeting angiogenesis has become an attractive intervention for cancer therapy. Many studies have demonstrated that GLP can inhibit angiogenesis. It has been shown that GLP suppresses VEGF overexpression and tumor angiogenesis in metastatic mouse melanoma B16F10 cells in vitro and Yoshida AH-130 ascites hepatoma xenografts in vivo. GLP peptide markedly reduces the microvessel formation as detected by chorioallantoic membrane assay. A G. lucidum extract (standardized to 13.5% polysaccharides and 6% triterpenes) suppresses prostate-cancer-dependent angiogenesis by inhibiting the secretion of VEGF and TGF-β1. It is by inhibiting AKT/ERK-mediated AP-1 activity.

GLP peptide inhibits angiogenesis by directly inhibiting cell proliferation of human umbilical cord vascular endothelial cells (HUVEC). GLP peptide can also directly induce cell death of HUVEC by reducing Bcl-2 anti-apoptotic protein expression and increasing Bax pro-apoptotic protein expression. Furthermore, GLP peptide treatment decreases the secretion of VEGF in human lung carcinoma cells under hypoxia conditions. The above results suggest that GLP peptide inhibits angiogenesis by directly inhibiting cell proliferation, inducing cell death in vascular endothelial cells, and indirectly suppressing VEGF production in tumor cells.

5. Anti-inflammatory effect

Chronic inflammation triggers cellular events that can promote the malignant transformation of cells and carcinogenesis. Several inflammatory mediators, such as TNF-α, IL-6, TGF-β, and IL-10, have been shown to participate in cancer’s initiation and progression. GLP possesses an anti-inflammatory effect in a dose-dependent manner. Administration of GLP (100 mg/kg) results in 58% inhibition of inflammation, as evaluated by carrageenan-induced (acute) and formalin-induced (chronic) inflammation assays.

6. Antioxidant effect

GLP possesses potent scavenging activities against O2−, SO, HO˙, H2O2, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro and in vivo. It has been shown that 5 mg/ml of GLP extract can reduce DPPH radical strikingly within 2 minutes and completely scavenge the radical in 15 minutes. GLP extract induces the superoxide dismutase (SOD), catalase, phase II detoxification enzyme NAD(P)H: quinone oxidoreductase 1 (NQO1), and glutathione S-transferase P1 (GSTP1) via the Nrf2-mediated signaling pathway in colorectal cancers in vitro. GLP also enhances the activities of SOD and glutathione peroxidase activities and reduces the malonaldehyde level in vivo in a dose-dependent manner either in mice exposed to γ-irradiation or in rats with cervical carcinoma.

7. Other activities related to cancer

GLP protects mouse bone marrow from 60Co γ-irradiation and diminishes the occurrence of micronuclei as a sign of γ-irradiation-induced DNA damage in the bone marrow. It has been observed that a GLP fraction possesses radio-protective activity and facilitates DNA repair, which may be attributed to its antioxidant effect. Furthermore, G. lucidum extract decreases cisplatin-induced kaolin intake dose-dependently as a reflection of cisplatin’s nausea and vomiting action. It also alleviates cisplatin-induced food intake reduction in rats in a dose-dependent manner.