Ganoderma formosanum Exopolysaccharides Inhibit Tumor Growth via Immunomodulation.

Ganoderma formosanum (GF) is a medicinal mushroom endemic to Taiwan. Previous research established the optimal culture conditions to produce exopolysaccharide rich in ß-glucan (GF-EPS) from submerged fermentation of GF. The present study investigated the antitumor effects of GF-EPS in a Lewis lung carcinoma cell (LLC1) tumor-bearing mice model. In the preventive model, GF-EPS was orally administered to mice before LLC1 injection. In the therapeutic model, GF-EPS oral administration was initiated five days after tumor cell injection. The tumor size and body weight of the mice were recorded. After sacrifice, the lymphocyte subpopulation was analyzed using flow cytometry. Spleen tissues were used to analyze cytokine mRNA expression. The results showed that GF-EPS (80 mg/kg) effectively suppressed LLC1 tumor growth in both the preventive and therapeutic models. GF-EPS administration increased the proportion of natural killer cells in the spleen and activated gene expression of several cytokines. Our results provide evidence that GF-EPS promotes tumor inhibition through immunomodulation in tumor-bearing mice.

Structural characterization and amelioration of sulfated polysaccharides from Ganoderma applanatum residue against CCl4-induced hepatotoxicity.

Natural polysaccharides and their derivatives have attracted academic attention due to their extensive physiological activities. However, the hepatoprotective effects against carbon tetrachloride (CCl4) toxicity have not been well elucidated. The objectives of this study were to characterize the structural properties of sulfated Ganoderma applanatum residue polysaccharides (SGRP) and to evaluate their inhibitory effects on liver fibrosis caused by oxidative stress and inflammation. Our in vivo study showed that SGRP was hepatoprotective in CCl4-induced chronic liver injury mice. It reduced the histopathological damages, down-regulated CYP2E1 (cytochrome P450 2E1) expression, reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, improved the anti-oxidative and anti-inflammatory properties, inhibited TLR4/NF-κB signaling pathway, and reduced the release of inflammatory cytokines. The structural studies indicated that SGRP is a heteropolysaccharide with 7.8% sulfur content and α-linked residue. Our study projects SGRP as a potential candidate in anti-fibrosis treatment by using it as a food supplement or in medicines produced by pharmaceutical industries.

The Effect of Ganoderma Microsporum immunomodulatory proteins on alleviating PM2.5-induced inflammatory responses in pregnant rats and fine particulate matter-induced neurological damage in the offsprings.

Fine particulate matter 2.5 (PM2.5) induces free radicals and oxidative stress in animals, leading to a range of illnesses. In this study, Ganoderma Microsporum immunomodulatory (GMI) proteins were administered to alleviate PM2.5-induced inflammatory responses in mother rats, and PM2.5-induced inflammatory responses and neurological damage in their offspring. The results suggested that GMI administration decreased the risk of neurological disorders in mother rats and their offspring by reducing the white blood cell count, lessening inflammatory responses and PM2.5-induced memory impairment, and preventing dendritic branches in the hippocampi from declining and microRNAs from PM2.5-induced modulation.

Serological Reactivity and Identification of Immunoglobulin E-Binding Polypeptides of Ganoderma applanatum Crude Spore Cytoplasmic Extract in Puerto Rican Subjects.

BACKGROUND: The allergenic potential of Ganoderma applanatum basidiospores has been demonstrated previously in Puerto Rico. However, basidiomycete allergens are not available for inclusion in allergy diagnostic panels. Therefore, we sought to confirm allergic sensitization to G. applanatum crude spore cytoplasmic extract through reactivity in serological assays and detection of immunoglobulin E (IgE)-binding polypeptides. METHODS: Via an indirect ELISA, serological reactivity was compared between groups of individuals with different allergic profiles. Group 1 (n = 51) consisted of individuals with sIgE to the allergens included in the diagnostic panels; group 2 (n = 14) comprised individuals with no sIgE to the allergens tested; and group 3 (n = 22) included individuals with no allergic history. To visualize IgE-binding polypeptides, group 1 sera were examined via Western blotting (WB). Polypeptide bands with the highest reactivity were analyzed by mass spectrometry (MS) for putative identification. RESULTS: The serological reactivity of group 1 was significantly higher than that of group 3 in an indirect ELISA (p = 0.03). Sixty-five percent of group 1 individuals showed reactivity to polypeptide bands in WB. Bands of 81 and 56 kDa had the highest reactivity proportions among the reactive sera, followed by a 45-kDa band. MS analysis of these 3 polypeptides suggests that they are basidiomycete-derived enzymes with aconitate hydratase, catalase, and enolase functions. CONCLUSIONS: G. applanatum spores have allergenic components recognized by Puerto Rican individuals, which could eventually be considered as markers in cases of fungal allergy and be included in diagnostic allergen panels in Puerto Rico and tropical regions.

In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling.

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.

Skin test reactivity of allergic subjects to basidiomycetes' crude extracts in a tropical environment.

Fungal allergies can be detected by the skin prick test with extracts of the organisms, but not all fungi, including the basidiomycetes, are being examined. We determined the level of sensitization to basidiomycetes in allergic subjects and compared their reactivity to commercial extracts commonly used to detect allergies. Crude spore extracts of the basidiomycetes Ganoderma applanatum, Chlorophyllum molybdites, and Pleurotus ostreatus, which are known to release numerous spores, were examined along with commercial extracts on 33 subjects with asthma, allergic or non-allergic rhinitis. Overall, affected subjects showed the highest reactivity to mites (36%), followed by Ganoderma applanatum (30%), grass (27%) Chlorophyllum molybdites (12%) and Pleurotus ostreatus (12%). Allergic rhinitis patients were most reactive to mites (58%), grass (42%), Ganoderma applanatum (25%), Penicillium spp. (25%), and cat (17%). Those with asthma primarily responded to mites (44%), Ganoderma applanatum (44%), grass (33%), and Pleurotus ostreatus (22%). IgE levels correlated with positive basidiomycetes extracts. This finding, coupled with higher reactivity to basidiospores as compared to mitospores, and the similar sensitivities of patients to G. applanatum and mites, suggest that basidiomycetes are important allergen sources in the tropics.

GMI, a Ganoderma immunomodulatory protein, down-regulates tumor necrosis factor α-induced expression of matrix metalloproteinase 9 via NF-κB pathway in human alveolar epithelial A549 cells.

Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.

Comparative studies on the immunomodulatory and antitumor activities of the different parts of fruiting body of Ganoderma lucidum and Ganoderma spores.

Ganoderma lucidum (GL, Lingzhi) has been suggested as a candidate for immunomodulation and cancer treatment. The present study aimed at comparing the different parts of the fruiting body (whole fruiting body, pileus and stipe) of GL as well as Ganoderma spores (sporoderm-broken and -unbroken), with regard to their antitumor and immunomodulatory activities in S-180 sarcoma-bearing mice. The hot water extracts of different parts of GL or the Ganoderma spores were orally administered to the sarcoma-bearing mice. The results showed that GL whole fruiting body, stipe and sporoderm-broken spore possessed stronger inhibitory activities on sarcoma growth when compared with the pileus extract. Higher immunomodulatory activities in terms of enhancing the proliferative responses and the cytokines (IFN-gamma, IL-4 and IL-6) production of spleen lymphocytes were also found in GL stipe and sporoderm-broken spore treatment groups. The sporoderm-broken spores had higher stimulatory effects on mitogen-activated spleen lymphocytes of healthy mice than those of sarcoma-bearing mice. In addition, the immunostimulatory activities of GL hot water extracts and Ganoderma spores were shown to be comparable; hence the latter did not show superiority in efficacy. This is the first comparative study on the immunomodulatory activities of Ganoderma spores and the fruiting body extracts.

Human nonmetastatic clone 23 type 1 gene suppresses migration of cervical cancer cells and enhances the migration inhibition of fungal immunomodulatory protein from Ganoderma tsugae.

The authors investigate the effects of human nonmetastatic clone 23 type 1 (nm23-H1 ) gene and fungal immunomodulatory protein-Ganoderma tsugae (FIP-gts) on the metastatic potential of cervical cancer cells and assess whether nm23-H1 can influence the action of FIP-gts using cell migration and invasion assays and gelatin zymography. The nm23-H1 gene was stably transfected into Caski cells, which lacked nm23-H1 expression. The results show that nm23-H1 stably transfected Caski cells exhibit reduced cell migration but no change of cell invasion and matrix metalloproteinase (MMP)-2 and -9 activities. FIP-gts reduced cell migration in SiHa and nm23-H1 transfected Caski cells more significantly compared with Caski cells and reduced invasion in Caski and nm23-H1-transfected Caski cells, but it exerted no influence on MMP-2 and MMP-9 activities in them. Conclusively, the nm23-H1 gene suppresses cervical cancer cell migration but not invasion and activities of MMP-2 and MMP-9 and enhances the inhibition of FIP-gts upon migration.

[A comparison study on the physical/chemical properties and immunomodulatory activities of mycelial polysaccharide extracts from five Ganoderma species].

Five Ganoderma species, including G. lucidum, G. tsugae, G. oerstedii, G. resinaceum and G. subamboinens, were parallel studied under an identical condition. These species were cultivated using liquid fermentation and their mycelia polysaccharides were extracted and compared on the physical/chemical properties and in vitro immunomodulatory activities. These results showed that the polysaccharide yields varied markedly among different species, and G. oerstedii was the highest among the five. However, HPLC analysis showed all the polysaccharide extracts had similar molecular weight distributions and monosaccharide compositions. They all contained glucose, galactose, mannose, glucosamine hydrochloride and fucose. In vitro assays, these polysaccharide extracts significantly stimulated phagocytosis and nitric oxide production by RAW 264.7 macrophage cell line, and G. subamboinens exerted the strongest potency. When Con A was not or presented, they all showed an up-or-down immunomodulatory effect on mouse splenocyte proliferation. The results illustrate that in addition to G. lucidum and G. tsugae, which are the two mostly studied and applied species, other Ganoderma species can also produce polysaccharides with similar physical/chemical properties and with similar immunomodulatory activities.

Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae in Sf21 insect cells.

The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.