Sage Journals: Discover world-class research

Abstract

Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

Keywords

NK cell perforin NKG2D NCR granulysin

Introduction

Ganoderma lucidum (G. lucidum)—also called reishi, lingzhi or mannentake—is a lamellae Basidiomycete fungus that grows naturally on decaying logs of many broad leaf trees.¹ G. lucidum has been recognized as a potent medicinal mushroom for more than 2000 years in China owing to its beneficial effects on health, healing and longevity.¹

Previous studies have indicated that G. lucidum produces various beneficial effects on cultured cells and laboratory animals. For instance, G. lucidum exhibits antioxidant activity against acute ethanol-induced heart toxicity.² This fungus also protects mice against colitis induced by trinitrobenzene sulfonic acid,³ and prevents liver injury induced by Mycobacterium infection.⁴ Notably, G. lucidum produces a beneficial effect in an animal model of type 2 diabetes mellitus by lowering serum glucose levels through suppression of phosphoenolpyruvate carboxykinase expression in the liver.⁵

Several studies have shown that G. lucidum produces both stimulatory and inhibitory effects on immune cells. For instance, it enhances the activity of immune effector cells in immunosuppressed mice.⁶ However, this fungus also suppresses cell proliferation and LPS-stimulated inflammatory responses in macrophages,^7,8 and induces the phagocytic activity of human polymorphonuclear neutrophils⁹ and cytokine expression in mouse splenocytes¹⁰ by activating MAPK signaling pathways.

G. lucidum also inhibits the growth of leukemic cells and induces their differentiation into mature monocytes/macrophages.¹¹ It activates the proliferation and anti-tumor activity of cytokine-induced killer cells,¹² and it shows inhibitory effects on murine oral cancer,¹³ bladder cancer cells,¹⁴ hepatoma cells¹⁵ and breast cancer cells.¹⁶ G. lucidum produces anti-tumor effects by inducing cytokine release from activated macrophages and T lymphocytes.¹⁷ Furthermore, intraperitoneal administration of the closely-related Ganoderma tsugae increases splenic NK cytotoxic activity in a dose-dependent manner in C3H/HeN mice.¹⁸ Notably, preliminary evidence suggests that G. lucidum may provide beneficial effects in treating human cancers when used as an adjuvant treatment with conventional therapies.^19–21

Even though numerous studies have been performed to understand the effects of G. lucidum on cultured cells and animals, the molecular mechanisms underlying the effects of this fungus on NK cells have not been examined so far. NK cells are important cells of the innate immune system that are involved in early host defense against infections and tumors.^22–24 We, and others, have previously shown that the cytotoxicity of NK cells involves the secretion of cytolytic proteins, such as perforin, granzymes and granulysin towards the target cell.^25–28 These cytolytic proteins are exocytosed by activated NK cells, and they mediate the cell-killing activity by inducing apoptosis in target cells.

NK cell cytotoxic activity is modulated by signals that bind to activating and inhibitory receptors on the cell surface.²⁹ The major activating receptors are natural cytotoxicity receptors (NCRs), which include the constitutively expressed receptors NKp46³⁰ and NKp30³¹, as well as the induced receptor NKp44.³² Moreover, the natural-killer group 2D receptor (NKG2D), which is also found on human T cells,³³ also activates NK cell cytotoxicity.³⁴ The binding of ligands on the activating receptors leads to activation of a cascade of intracellular signals resulting in polarization and exocytosis of granules that lyse target cells.^29,35 In NK cells, ERK, JNK and p38 are critical intermediates of the MAPK signaling pathways that mediate the reorientation of the microtubule organizing center, and induce granule polarization and secretion.^36–38

In the present study, we examined the effects of a G. lucidum water extract on NK cell cytotoxicity. Our results indicate that G. lucidum activates NK cell cytotoxic activity by inducing NKG2D/NCR receptors, MAPK signaling pathways, and the release of perforin and granulysin.

Materials and methods

Chemical reagents and fungal strain

Cell culture media, including alpha minimum essential medium (α-MEM) without ribonucleosides or deoxyribonucleosides, RPMI 1640 medium without phenol red, horse serum and FBS were purchased from Invitrogen (Carlsbad, CA, USA). Inositol, 2-mercaptoethanol, folic acid, glutamine, JNK inhibitor (SP600125), ERK1/2 inhibitor (PD98059) and p38 inhibitor (SB203580) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MAPK inhibitors were stored in DMSO at –20℃ until use. The G. lucidum strain initially selected and characterized at Chang Gung Biotechnology (Taipei, Taiwan) was validated by sequencing of rDNA and internal transcribed spacer DNA, which showed 99.39% identity with a database strain [European Molecular Biology Laboratory (EMBL) accession number AF506371; EMBL, Hinxton, UK]. The water extract was prepared by mixing 400 g of dried G. lucidum mycelium into 10 l of double-distilled water, followed by agitation at a speed of 150 rpm for 30 min at 12℃. The solution was cooled to room temperature (22℃) and centrifuged at 5894 g for 30 min at 4℃ using a Sorvall RC 3C Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was concentrated to a final volume of 2 l by using a Büchi R220 Rotavapor vacuum concentrator (Buchi, Flawil, Switzerland) at 65℃. The extract was sterilized at 121℃ for 20 min, and stored at 4℃ in dark glass bottles prior to use.

Cell isolation and culture

The human NK cell line NK92 (ATCC CRL-2407) was derived from a case of non-Hodgkin’s lymphoma, as described previously.³⁹ NK92 cells were maintained in culture with 100 IU/ml of IL-2 (Peprotech, Rocky Hill, CT, USA). Primary NK cells (pNK) were collected from healthy individuals following the method of Favier et al.⁴⁰ Briefly, pNK cells were isolated from peripheral blood using the EasySep NK cell depletion kit, as described by the manufacturer (Stem Cells, Grenoble, France). The purity of pNK cell preparations was verified by flow cytometry based on CD16, CD56 and CD3 expression. The pNK cell preparations used contained more than 80% CD16⁺CD56⁺ cells, but no CD3⁺ cells. pNK cells were activated by adding 200 IU/ml IL-2 (Peprotech) to the culture medium for 36–48 h. All cell lines were cultured in α-MEM containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 12.5% horse serum and 100 IU/ml IL-2. The K562 cell line was obtained from the ATCC (Manassas, VA, USA), and was maintained in RPMI 1640 containing 10% FBS.

Abs

Mouse anti-human Abs raised against perforin, granulysin, NKG2D, NKp30, NKp44 or NKp46 were obtained from R & D Systems (Minneapolis, MN, USA). Rabbit polyclonal Abs against ERK, JNK, p38, phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185) or phospho-p38 (Thr180/Tyr182) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-actin Ab was obtained from Chemicon International (Temecula, CA, USA).

MTT cell viability assay

pNK and NK92 cells (1 × 10⁵/well) were seeded in 96-well plates in which 90 µl of culture medium containing various concentrations of G. lucidum water extract were incubated for 24 h. Then, 10 µl of MTT reagent (5 mg/ml; MTT In Vitro Toxicology Assay kit; Sigma) was added to the wells and the plates were incubated for 3 h at 37℃. Then, 100 µl of solubilization solution was added prior to incubation overnight (18 h). Cell viability was calculated based on instructions from the manufacturer. All experiments were performed in triplicate.

Cytotoxicity assay

Cytotoxicity was evaluated using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) based on the colorimetric measurement of lactate dehydrogenase, a stable cytosolic enzyme released from target cells following lysis by NK cells. Briefly, NK effector cells (5 × 10⁴, 2.5 × 10⁴ and 5 × 10³ cells/well in U-bottom 96-well plates (Corning, Corning, NY, USA) were treated with different doses of G. lucidum water extract for 24 h and pretreated with MAPK inhibitors for 30 min or with MAPK small interfering RNA (siRNA) overnight, followed by treatment with 0.1–5% G. lucidum for 4 h. NK cells were then washed and re-suspended in RPMI 1640. A constant number of K562, A549, HepG2, MCF-7 and HT-29 target cells (5 × 10³/well) cultured in complete cell culture medium was then added at the E:T ratios of 1 : 1, 5 : 1 and 10 : 1, based on our previous experimental results.³⁸ The microplates were centrifuged for 4 min at 250 g to settle the cells before incubation for 4 h at 37℃ in 5% CO₂. After this co-incubation, supernatant (50 µl) was collected from each well, added to 50 µl of the provided Substrate Mix and incubated for 10 min at room temperature in the dark. The enzymatic reaction was stopped using the Stop Solution (50 µl), and absorbance was measured at 490 nm. Maximum release (TM) was determined by lysing target cells with 10 µl of lysis solution. Spontaneous release by either the target cells (TS) or effector cells (ES) was determined following incubation alone at the respective cell concentrations. Results were calculated and expressed as percentage of cytotoxicity using the following equation: [(experimental – ES – TS)/(TM – TS)] × 100.

Flow cytometry analysis

Flow cytometry analysis was performed using a FACS CaliburTM (Becton Dickinson, Franklin Lake, NJ, USA). Fluorophore bound Abs were used to sort cells: anti-NKG2D-allophycocyanin, anti-NKp46-fluorescein- isothiocyanate, anti-NKp30-phycoerythrin (R & D Systems) and anti-NKp44-phycoerythrin (Becton Dickinson). In brief, the non-specific antigens of 5 × 10⁵NK92 cells were blocked using cold 1% BSA–PBS at 4℃ for 30 min, prior to treatment with the Abs in the dark at 4℃ for 30 min. The cells were washed twice in cold PBS and re-suspended in 1 ml cold PBS before analysis.

RNA isolation and real-time PCR

mRNA was isolated from pNK and NK92 cells using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) based on the manufacturer’s instructions, and total RNA was converted to cDNA using the Taqman reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed with 1/50 of the cDNA preparation in an Mx3000P (Stratagene, La Jolla, CA, USA) in a final volume of 25 µl with Brilliant QPCR Master Mix (Stratagene). Real-time PCR protocols included an initial denaturation at 95℃ for 10 min, followed by 50 cycles of 95℃ for 30 s, 55℃ for 1 min, 72℃ for 1 min and, finally, 95℃ for 1 min, 55℃ for 30 s and 95℃ for 30 s.

Western blot analysis

NK92 cells (10⁶/well in six-well plates) were stabilized overnight in cultured medium, followed by addition of different doses of G. lucidum water extract. Washed cells were lysed in Mammalian Protein Extraction Reagent (Pierce Chemical, Rockford, MA, USA). Total lysate protein samples (40 µg per lane) were fractionated on a 10% SDS polyacrylamide gel and blotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk for 2 h at room temperature in TBS (Tris 10 mM, NaCl 150 mM, pH 7.6) containing 0.1% Tween 20, and probed with a primary Ab (1 : 1,000 for anti-perforin, anti-granulysin, anti-NKG2D, anti-ERK, anti-JNK, anti-p38, anti-phospho-ERK, anti-phospho-JNK and anti-phospho-p38; 1 : 10,000 for anti-actin) overnight at 4℃. Membranes were then incubated with the appropriate HRP-conjugated secondary Ab (1 : 10,000). Immunoreactive protein bands were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

siRNA knockdown and siRNA transfection

siRNA against human p38 (sc-29433), JNK-1 (sc-29380), ERK-2 (sc-35335), NKG2D (sc-42948), NKp44 (sc-72170), NKp46 (sc-63344), NKp30 (sc-42950) and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The siRNA was transfected into NK92 cells by electroporation using a commercial kit (Amaxa, Gaithersburg, MD, USA).

Data analysis

Statistical analysis was performed using ANOVA (SPSS 12.0 software; SPSS, Chicago, IL, USA) with a correction for multiple comparisons. A difference between the results of two assay conditions that gave a P-value < 0.05 was considered significant.

Results

Effect of G. lucidum water extract on NK cell viability and cytotoxic activity

We first verified whether the G. lucidum water extract may affect the viability of NK cells. The viability of pNK and the cultured NK92 cell line was not affected by treatment with 0.1% G. lucidum (Figure 1A, B). At 2 and 5%, we observed that G. lucidum instead increased the fraction of viable NK cells compared with untreated cells (Figure 1A, B). Next, we measured the cytotoxic activity of G. lucidum-treated NK cells against the human leukemia cell line, K562. NK cells were treated with G. lucidum at various concentrations (0.1, 2 and 5%), and the cytotoxic activity of the treated cells was measured against K562 cells at various effector:target (E:T) ratios (1 : 1, 5 : 1 and 10 : 1). We observed that treatment with G. lucidum stimulated the cytotoxicity of both pNK and NK92 cells in a dose-dependent manner (Figure 1C, D).

Figure 1.

G. lucidum enhances perforin and granulysin expression and NK cell cytotoxic activity. G. lucidum water extract produced no cytotoxic effect on NK cells. At 2% and 5%, G. lucidum enhanced the viability of pNK (A) and NK92 cells (B). The cytotoxic effect of pNK (C) and NK92 cells (D) against K562 cells was induced by G. lucidum in a dose-dependent manner at E:T ratios of 1 : 1, 5 : 1 and 10 : 1. Cytotoxicity was measured based on the release of lactate dehydrogenase, and data were expressed as percent of cytotoxicity. NK92 cells were treated with G. lucidum at the dose indicated. mRNA (E) and protein (F) levels of perforin and granulysin were measured. The data represent means of three independent experiments performed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001).

G. lucidum increases NK cell perforin and granulysin production

We next examined whether G. lucidum treatment may increase the production of the cytolytic proteins, perforin and granulysin in NK cells. We found that G. lucidum treatment increased the level of perforin and granulysin mRNA in a dose-dependent manner compared with untreated cells (Figure 1E). G. lucidum treatment also increased the production of perforin and granulysin protein in a dose-dependent manner (Figure 1F). These results suggest that G. lucidum treatment increases the cytotoxic activity of NK cells by inducing the expression and production of perforin and granulysin.

G. lucidum enhances expression of NKG2D and NCR cell surface receptors

NKG2D and NCRs represent cell surface receptors that activate NK cell cytotoxicity and induce the secretion of perforin and granulysin.^22–24,27 Therefore, we examined the effects of G. lucidum on NKG2D and NCR gene expression. As shown in Figure 2A, the mRNA level of NKG2D, NKp30, NKp44, and NKp46 increased in a dose-dependent manner following G. lucidum treatment. In agreement with the level of mRNA expression, production of the corresponding proteins also increased following G. lucidum treatment compared with control cells (Figure 2B). To further confirm the level of activating receptors on NK cells, we performed a flow cytometry analysis of G. lucidum-treated NK cells using fluorophore-bound Abs. We observed that the level of cell surface NKG2D and NCR proteins was enhanced by treatment with G. lucidum compared with control, untreated cells (Figure 2C). These results indicate that G. lucidum not only affects the production of perforin and granulysin, but also the expression and production of upstream signaling receptors.

Figure 2.

G. lucidum enhances NKG2D and NCR receptor expression. After treatment with G. lucidum (GL), total cellular mRNA from NK92 cells was prepared for quantification of NKG2D and NCR mRNA levels by real-time PCR (A) and receptor expression by Western blotting (B). Quantification of the receptors on NK92 cells was also monitored by flow cytometry (C). Data are representative of four independent experiments. APC: allophycocyanin; PE: phycoerythrin (*P < 0.05, **P < 0.01, ***P < 0.001).

G. lucidum induces intracellular MAPK signaling pathways

Previous studies have shown that MAPK signaling is activated in NK cells during tumor cell lysis.^37,41,42 In addition, inhibition of JNK and ERK-1/2 signaling has been shown to interfere with NKG2D-mediated NK cell cytotoxicity.³⁶ To identify the intracellular pathways modulated by G. lucidum in NK cells, we examined whether the mycelium extract regulated kinase signaling pathways. NK cells were treated with specific doses of G. lucidum prior to Western blotting analysis of MAPKs. As shown in Figure 3A, G. lucidum stimulated the phosphorylation of MAPKs in a dose-dependent manner. To confirm these results, we treated NK cells with specific MAPK inhibitors, and verified the cytotoxic activity of the treated cells. NK cells were treated or not with a MAPK inhibitor followed by treatment with G. lucidum, prior to challenge with target K562 cells at E:T ratios of 1 : 1, 5 : 1 and 10 : 1. Treatment with the MAPK inhibitors did, indeed, reduce the cytotoxicity of G. lucidum-treated NK cells (Figure 3B–D). Consistent with the reduced cytotoxicity, production of perforin and granulysin was reduced in NK cells treated with the MAPK inhibitors (Figure 3E –G).

Figure 3.

G. lucidum activates MAPK pathways in NK cells. (A) NK92 cells were treated with G. lucidum (GL) water extract for 30 min before measuring phosphorylation (P) and total (T) protein levels of ERK, JNK and p38. The effects of (B) MAPK inhibitors against JNK (SP, SP600125, 10 µM), (C) ERK1/2 (PD, PD98059, 10 µM), or (D) p38 (SB, SB203580, 10 µM) on the cytotoxic activity of NK cells treated or not with 5% G. lucidum water extract are shown. (E–G) The effects of MAP kinase inhibitors on perforin and granulysin expression in NK cells treated or not with 5% G. lucidum are shown. The results are representative of four independent experiments.

To further confirm that G. lucidum activates NK cell activity through MAPK signaling, we used specific siRNA to deplete MAPKs, and examined whether this treatment affected perforin and granulysin production. Reduced levels of p38, JNK and ERK proteins confirmed that the MAPKs selected were efficiently silenced following siRNA transfection (Figure 4A –C). Notably, the amounts of perforin and granulysin protein were also decreased in G. lucidum-treated NK cells expressing MAPK siRNA. In agreement with these results, transfection of MAPK siRNA in NK92 cells markedly reduced the cytotoxic activity of these cells against the K562 cell line (Figure 4D –F). Based on these findings, we concluded that G. lucidum activates NK cell cytotoxic activity and the release of perforin and granulysin by inducing ERK1/2, JNK and p38 MAPK signaling pathways.

Figure 4.

G. lucidum stimulated MAPK activity leads to increased perforin and granulysin production and enhanced cytotoxic activity in NK cells. Effect of specific JNK (A), ERK (B) and p38 (C) siRNA on MAPK expression, perforin and granulysin production in NK cells treated or not with 5% G. lucidum (GL). NK cells were transfected with MAPK siRNA or scramble siRNA followed by stimulation with or without 5% G. lucidum water extract prior to cytotoxicity analysis (D–F). The results shown are representative of three independent experiments. (**P < 0.01, ***P < 0.001).

G. lucidum stimulates NK cell cytotoxicity against various cancer cell lines

To evaluate whether G. lucidum treatment increases NK cell cytotoxicity irrespective of the cell line used, we treated NK cells with various doses of G. lucidum, followed by co-culture with other cancer cell lines, including lung adenocarcinoma A549 cells, liver cancer HepG2 cells, breast cancer MCF-7 cells and colon cancer HT-29 cells. Our results showed that G. lucidum stimulated NK cell cytotoxic activity in a dose-dependent manner against all target cancer cell lines tested (Figure 5A –D, E:T ratios of 1 : 1, 5 : 1 and 10 : 1). G. lucidum thus induces NK cell cytotoxicity in a non-specific manner against a variety of tumor cell lines.

Figure 5.

Effect of G. lucidum on NK cell cytotoxicity against various target cells. (A–D) NK cells treated with 5% G. lucidum (GL) were incubated with various human cancer cell lines (A549, HepG2, MCF-7 and HT-29) at E:T ratios of 1 : 1, 5 : 1 or 10 : 1. The results shown are representative of three independent experiments.

Discussion

In this study, we reported that the underlying mechanism of activation of NK cells by G. lucidum occurs through activation of the NGK2D/NCRs, and p38, JNK and ERK, and MAPK signaling pathways. The enhanced NK cytotoxic activity induced by G. lucidum is effective not only against the human leukemia K562 cells but also against various cancer cell lines (A549, HepG2, MCF-7 and HT-29), indicating that G. lucidum activates NK cells in a non-specific manner. Our results thus indicate that G. lucidum may generally enhance tumor clearance through activation of NK cells. These findings provide a rational basis for the use of G. lucidum water extract as a food supplement for cancer prevention and treatment.

The results of the knockdown assay on the NKG2D and NCRs in this study suggest that the active compounds present in the G. lucidum water extract may directly bind to the activating receptors. Nonetheless, we cannot rule out the possibility of indirect immunomodulation effects occurring from the activation of other signaling pathways that may cross-talk with the NKG2D and NCR pathways. Accordingly, activation of MAPK signaling pathways can be achieved through other interconnected signaling pathways. In order to address this issue, a reporter assay (such as the IFN-γ promoter-based bioluminescence system) may be designed to monitor the activation or inhibition of the NKG2D and NCRs pathways in real time. This reporter assay may also help to identify the active compounds present in the G. lucidum water extract.

We have previously reported that resveratrol, a polyphenolic compound found in red wine, and 2,3-butanediol, a metabolite produced by various organisms, also stimulates the cytotoxic activity of NK cells.^38,43 An increase in the expression of NKG2D, MAPK activity and perforin production was observed when NK cells were treated with resveratrol, while NKG2D, NKp40, NKp46, MAPK activity and perforin production were activated by 2,3-butanediol. Compared with resveratrol and 2,3-butanediol, the G. lucidum water extract studied in the present study enhances NKG2D and NCR expression, and MAPK activity, as well as perforin and granulysin production. Thus, the mechanism of NK cell activation of G. lucidum, resveratrol and 2,3-butanediol may be different, an observation which is worthy of further studies.

The G. lucidum water extract may contain a variety of active components, and it may target different cellular components in NK cells. To clarify this issue, future experiments designed to isolate, identify and characterize single active compounds from the water extract will be necessary to obtain a detailed molecular and mechanistic view of G. lucidum compounds. Previous studies indicate that partially purified polysaccharides from G. lucidum enhance human CD56⁺ NK cells cytotoxicity in vitro⁴⁴ and in vivo⁶, including in advanced-stage cancer patients.⁴⁵ G. lucidum polysaccharides also inhibit tumor formation by activating host immune responses involving macrophages and T lymphocytes.^10,12,13,46 Other studies also indicate that the main activating compounds mediating these effects in G. lucidum water extracts may be polysaccharides.⁴⁷ However, we cannot rule out the possibility that other active compounds may also exist in the extract used here. For example, a previous study showed that an active glycoprotein isolated from a water extract of G. lucidum, designated fraction 3 (F3), stimulates mice spleen cell proliferation and cytokine expression.⁴⁸ Another study demonstrated that incubation with F3 alone not only increased the cytotoxicity of CD56⁺ NK-cells against the NK-sensitive tumor-cell line K562 but also altered the expression of cell surface markers.⁴⁴ Fractionation of G. lucidum water extracts using various chromatography techniques is currently under way in our laboratory to identify the active compounds responsible for these effects. In addition, we are also examining the effects of ethanol extracts of G. lucidum on NK cell function in order to identify the full range of bioactivities associated with this medicinal mycelium.

In conclusion, we found that a G. lucidum water extract activates NK cell cytotoxicity by activating NKG2D/NCRs and MAPK signaling pathways, and secretion of the cytolytic proteins perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts.^19–21 Experiments are currently in progress to isolate, purify and characterize the active components responsible for this activity.

Footnotes

Funding

This study was supported by Primordia Institute of New Sciences and Medicine, grants NSC101-2325-B-182-005 and NSC101-2320-B-030-011 from the National Science Council of Taiwan, and grant CMRPD1B0052 from Chang Gung Memorial Hospital (Linkou, Taiwan).

Conflict of interest

Y.-F.K. is President and an employee of Chang Gung Biotechnology Corporation. J.D.Y. is Chairman of the Board of Chang Gung Biotechnology Corporation. The other authors have no potential conflicts of interest to declare.

References

Sanodiya

Thakur

Baghel

Prasad

Bisen

. Ganoderma lucidum: a potent pharmacological macrofungus. Curr Pharm Biotechnol 2009; 10: 717–742.

Wong

Chao

Chan

Chang

Liu

. Antioxidant activity of Ganoderma lucidum in acute ethanol-induced heart toxicity. Phytother Res 2004; 18: 1024–1026.

Hanaoka

Ueno

Tanaka

Nagai

Onitake

Yoshioka

Chayama

. The water-soluble extract from cultured medium of Ganoderma lucidum (reishi) mycelia (designated as mak) ameliorates murine colitis induced by trinitrobenzene sulphonic acid. Scand J Immunol 2011; 74: 454–462.

Zhang

Wang

Teng

Lin

. Hepatoprotective role of Ganoderma lucidum polysaccharide against bcg-induced immune liver injury in mice. World J Gastroenterol 2002; 8: 728–733.

Seto

Lam

Tam

Chan

. Novel hypoglycemic effects of Ganoderma lucidum water-extract in obese/diabetic (+db/+db) mice. Phytomedicine 2009; 16: 426–436.

Zhu

Chen

Lin

. Ganoderma lucidum polysaccharides enhance the function of immunological effector cells in immunosuppressed mice. J Ethnopharmacol 2007; 111: 219–226.

Dudhgaonkar

Thyagarajan

Sliva

. Suppression of the inflammatory response by triterpenes isolated from the mushroom Ganoderma lucidum. Int Immunopharmacol 2009; 9: 1272–1280.

Rios

. Effects of triterpenes on the immune system. J Ethnopharmacol 2010; 128: 1–14.

Hsu

Chern

Chen

Yang

. Extract of sporoderm-broken germinating spores of Ganoderma lucidum activates human polymorphonuclear neutrophils via the p38 mitogen-activated protein kinase pathway. Chang Gung Med J 2012; 35: 140–147.

10.

Chen

Tsai

Lin

Khoo

Lin

Wong

. Studies on the immuno-modulating and anti-tumor activities of Ganoderma lucidum (reishi) polysaccharides. Bioorg Med Chem 2004; 12: 5595–5601.

11.

Lieu

Lee

Wang

. The effect of Ganoderma lucidum on induction of differentiation in leukemic u937 cells. Anticancer Res 1992; 12: 1211–1215.

12.

Zhu

Lin

. Effects of Ganoderma lucidum polysaccharides on proliferation and cytotoxicity of cytokine-induced killer cells. Acta Pharmacol Sin 2005; 26: 1130–1137.

13.

Gao

Zhang

Gao

Zhang

. Study of the extraction process and in vivo inhibitory effect of Ganoderma triterpenes in oral mucosa cancer. Molecules 2011; 16: 5315–5332.

14.

Jin

Zhang

Heber

. Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro. Cancer Lett 2004; 216: 9–20.

15.

Weng

Chau

Chen

Yen

. The anti-invasive effect of lucidenic acids isolated from a new Ganoderma lucidum strain. Mol Nutr Food Res 2007; 51: 1472–1477.

16.

Jiang

Slivova

Harvey

Valachovicova

Sliva

. Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of akt/nf-kappab signaling. Nutr Cancer 2004; 49: 209–216.

17.

Wang

Hsu

Tzeng

Lee

Shiao

. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and t lymphocytes. Int J Cancer 1997; 70: 699–705.

18.

Won

Lin

. Ganoderma tsugae mycelium enhances splenic natural killer cell activity and serum interferon production in mice. Jpn J Pharmacol 1992; 59: 171–176.

19.

Sliva

. Ganoderma lucidum (reishi) in cancer treatment. Integr Cancer Ther 2003; 2: 358–364.

20.

Yuen

Gohel

. Anticancer effects of Ganoderma lucidum: A review of scientific evidence. Nutr Cancer 2005; 53: 11–17.

21.

Jin

Ruiz Beguerie

Sze

Chan

. Ganoderma lucidum (reishi mushroom) for cancer treatment. Cochrane Database Syst Rev 2012; 6: CD007731–CD007731.

22.

Elhaik-Goldman

Kafka

Yossef

Hadad

Elkabets

Vallon-Eberhard

. The natural cytotoxicity receptor 1 contribution to early clearance of Streptococcus pneumoniae and to natural killer-macrophage cross talk. PLoS One 2011; 6: e23472–e23472.

23.

Yokoyama

Kim

French

. The dynamic life of natural killer cells. Annu Rev Immunol 2004; 22: 405–429.

24.

Brill

Larkin

Canaday

Kaplan

Boom

Silver

. Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis h37rv via granule-independent mechanisms. Infect Immun 2001; 69: 1755–1765.

25.

Podack

Young

Cohn

. Isolation and biochemical and functional characterization of perforin 1 from cytolytic t-cell granules. Proc Natl Acad Sci U S A 1985; 82: 8629–8633.

26.

Trapani

Smyth

. Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol 2002; 2: 735–747.

27.

Law

Lukoyanova

Voskoboinik

Caradoc-Davies

Baran

Dunstone

. The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 2010; 468: 447–451.

28.

Liu

Young

. Lymphocyte-mediated cytolysis and disease. N Engl J Med 1996; 335: 1651–1659.

29.

de Saint Basile

Menasche

Fischer

. Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat Rev Immunol 2010; 10: 568–579.

30.

Elboim

Gazit

Gur

Ghadially

Betser-Cohen

Mandelboim

. Tumor immunoediting by nkp46. J Immunol 2010; 184: 5637–5644.

31.

Wang

Mariuzza

. Structure of the human activating natural cytotoxicity receptor nkp30 bound to its tumor cell ligand b7-h6. J Exp Med 2011; 208: 703–714.

32.

Campbell

Yusa

Kikuchi-Maki

Catina

. Nkp44 triggers nk cell activation through dap12 association that is not influenced by a putative cytoplasmic inhibitory sequence. J Immunol 2004; 172: 899–906.

33.

Barber

Sentman

. Nkg2d receptor regulates human effector t-cell cytokine production. Blood 2011; 117: 6571–6581.

34.

Rao

Groh

Spies

Gattuso

Kaufman

. Major histocompatibility complex class i-related chain a/b (mica/b) expression in tumor tissue and serum of pancreatic cancer: Role of uric acid accumulation in gemcitabine-induced mica/b expression. BMC Cancer 2011; 11: 194–194.

35.

Maghazachi

. Insights into seven and single transmembrane-spanning domain receptors and their signaling pathways in human natural killer cells. Pharmacol Rev 2005; 57: 339–357.

36.

Nicotra

Stern

Kopcow

Chen

Strominger

. Jnk map kinase activation is required for mtoc and granule polarization in nkg2d-mediated nk cell cytotoxicity. Proc Natl Acad Sci U S A 2008; 105: 3017–3022.

37.

Chini

Boos

Dick

Schoon

Leibson

. Regulation of p38 mitogen-activated protein kinase during nk cell activation. Eur J Immunol 2000; 30: 2791–2798.

38.

Chen

. Resveratrol enhances perforin expression and nk cell cytotoxicity through nkg2d-dependent pathways. J Cell Physiol 2010; 223: 343–351.

39.

Gong

Maki

Klingemann

. Characterization of a human cell line (nk-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8: 652–658.

40.

Favier

Lemaoult

Lesport

Carosella

. Ilt2/hla-g interaction impairs nk-cell functions through the inhibition of the late but not the early events of the nk-cell activating synapse. FASEB J 2010; 24: 689–699.

41.

Jiang

Zhong

Gilvary

Corliss

Hong-Geller

Wei

Djeu

. Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat Immunol 2000; 1: 419–425.

42.

Wei

Gamero

Liu

Daulton

Valkov

Trapani

. Control of lytic function by mitogen-activated protein kinase/extracellular regulatory kinase 2 (erk2) in a human natural killer cell line: Identification of perforin and granzyme b mobilization by functional erk2. J Exp Med 1998; 187: 1753–1765.

43.

Lai

Chang

Yang

Hsu

Chen

Lin

. Activation of nk cell cytotoxicity by the natural compound 2,3-butanediol. J Leukoc Biol 2012; 92: 807–814.

44.

Chien

Cheng

Chang

Tien

Tsao

Chang

. Polysaccharides of Ganoderma lucidum alter cell immunophenotypic expression and enhance cd56+ nk-cell cytotoxicity in cord blood. Bioorg Med Chem 2004; 12: 5603–5609.

45.

Gao

Zhou

Jiang

Huang

Dai

. Effects of ganopoly (a Ganoderma lucidum polysaccharide extract) on the immune functions in advanced-stage cancer patients. Immunol Invest 2003; 32: 201–215.

46.

Wang

Kuo

Zhuang

Khoo

Liu

Chen

. Polysaccharides purified from the submerged culture of ganoderma formosanum stimulate macrophage activation and protect mice against Listeria monocytogenes infection. Biotechnol Lett 2011; 33: 2271–2278.

47.

Lin

Liang

Lee

Chiang

. Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the nf-kappab and p38 mitogen-activated protein kinase pathways. J Leukoc Biol 2005; 78: 533–543.

48.

Wang

Khoo

Chen

Lin

Wong

Lin

. Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (reishi) polysaccharides: Functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorg Med Chem 2002; 10: 1057–1062.

Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin

Abstract

Keywords

Introduction

Materials and methods

Chemical reagents and fungal strain

Cell isolation and culture

Abs

MTT cell viability assay

Cytotoxicity assay

Flow cytometry analysis

RNA isolation and real-time PCR

Western blot analysis

siRNA knockdown and siRNA transfection

Data analysis

Results

Effect of G. lucidum water extract on NK cell viability and cytotoxic activity

G. lucidum increases NK cell perforin and granulysin production

G. lucidum enhances expression of NKG2D and NCR cell surface receptors

G. lucidum induces intracellular MAPK signaling pathways

G. lucidum stimulates NK cell cytotoxicity against various cancer cell lines

Discussion

Footnotes

Funding

Conflict of interest

References