Ling Zhi-8 Research Articles

Preclinical Safety Evaluation of Engineered Lactococcus lactis NZ3900 Biotherapeutics Using a 13-Week Repeated Dose Oral Toxicity Study in Rabbits

ABSTRACT Lactococcus lactis is one of the most important and safe microorganisms used in the dairy industry. Previously, we orally fed cholesterol-fed rabbits with recombinant Ling Zhi 8 (LZ8) protein from L. lactis NZ3900/pNZ8149 and showed anti-inflammatory effects during the development of fatty liver and atheroma. In order to generate a clinically effective oral immunotherapy for the treatment of allergy, L. lactis clones for Der p 2, Per a 2, and Cla c 14 were introduced into L. lactis NZ3900/pNZ8149 to obtain recombinant allergen-based vaccines. In this study, we developed a triple-antigen vaccine formulation, Lactis- IL-1Ra, Lactis-LZ8, Lactis-Triple, and evaluated their preclinical safety in rabbits by oral administration with repeated doses for 90 days. All of the rabbits survived the challenge without any adverse clinical signs or abnormal changes. A notable advantage of this vaccine is that it can be given orally, and thus self-administration at home would be feasible.

Ling Zhi-8, a fungal immunomodulatory protein in Ganoderma lucidum, alleviates CPT-11-induced intestinal injury via restoring claudin-1 expression.

CPT-11 (Irinotecan) remains an important chemotherapeutic agent against various solid tumors nowadays. Potential adverse effects, especially gastrointestinal toxicities, are the main limiting factor for its clinical utility. Ling Zhi-8 (LZ-8), a fungal immunomodulatory protein in Ganoderma lucidum mycelia, has potential for drug development due to its multiple bioactivities and functions. This study aimed to explore the influence of LZ-8 on CPT-11-treated IEC-6 cells in vitro and on mice with CPT-11-induced intestinal injury in vivo. The mechanism through which LZ-8 exerted its protective effects was also investigated. In the in vitro study, the viability and claudin-1 expression of IEC-6 cells decreased gradually with increasing concentrations of CPT-11, but LZ-8 treatment had no obvious influence on their viability, morphology, and claudin-1 expression. Pretreatment of LZ-8 significantly improved CPT-11-decreased cell viability and claudin-1 expression in IEC-6 cells. In mice with CPT-11-induced intestinal injury, LZ-8 treatment could ameliorate symptoms and mitigate intestinal damage. Meanwhile, LZ-8 restored claudin-1 expression in the intestinal membranes in CPT-11-treated mice. Collectively, our results demonstrated the protective effects of LZ-8 against CPT-11 damage in both IEC-6 cells and mice. LZ-8 can restore claudin-1 expression in intestinal cells following CPT-11 treatment, suggesting the role of claudin-1 in the scenario.

Prevention of dextran sulfate sodium-induced mouse colitis by the fungal protein Ling Zhi-8 via promoting the barrier function of intestinal epithelial cells.

The fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum (GL) regulates immune cells and inhibits tumor growth; however, the role of LZ-8 in protecting intestinal epithelial cells (IECs) is unknown. In this study, we aim to investigate the functional effect of LZ-8 on IECs. LZ-8 effectively rescued the pro-inflammatory cytokine-induced loss of tight junctions (TJs) by enhancing transepithelial electrical resistance (TEER), reducing permeability, and maintaining the distribution of TJ proteins, in Caco-2 cells. Mechanistically, LZ-8 blocked the upregulation of myosin light chain kinase (MLCK) and NF-kB activation by TLR2-mediated suppression of cytokine signaling (SOCS)-1 expression. Furthermore, LZ-8 pre-treatment reduced the pathological scores of dextran sulfate sodium (DSS)-induced colitis in mice. These results indicated that LZ-8 protected the barrier function of IECs against inflammation. Thus, LZ-8 may potentially be a novel candidate for treating inflammatory bowel disease (IBD).

Recombinant ling zhi-8 enhances Tregs function to restore glycemic control in streptozocin-induced diabetic rats.

To explore the effect of recombinant LZ-8 (rLZ-8) on streptozocin (STZ)-induced diabetic rats and further illustrate its underlying mechanism. Rats were intraperitoneally injected with single-dose STZ 50mg/kg for induction of type 1 diabetes (T1D), and then, the diabetic rats were treated with rLZ-8 for 3months. The clinical symptoms, fasting blood glucose, insulin, cytokines, histopathology, flow cytometry and immunofluorescence were used to evaluate the therapeutic effect and underlying mechanism of rLZ-8 on alleviating diabetes mellitus (DM). Treatment with rLZ-8 obviously alleviated the clinical symptoms of T1D and dose-dependently reduced the levels of blood glucose, blood lipid and haemoglobin A1c (HbA1c) in diabetic rat model. Meanwhile, rLZ-8 markedly increased insulin secretion and protected against STZ-induced pancreatic tissue injury. Additionally, rLZ-8 dramatically inhibited the levels of TNF-α and IL-1β, and obviously increased the level of IL-10 in serum and pancreas. Further investigation indicated that rLZ-8 treatment significantly increased the number of regulatory T cells (Tregs) and up-regulated the expression of Foxp3 to restore balance between anti-inflammatory and inflammatory cytokines. These data suggest that rLZ-8 can antagonize STZ-induced T1D, and its mechanism may be related to inhibit inflammation and enhance Tregs generation.

Recombinant Ganoderma lucidum Immunomodulatory Protein Improves the Treatment for Chemotherapy-Induced Neutropenia.

Ganoderma lucidum, also known as LINGZHI, has a long tradition of use in folk medicine of the Far East, which is documented in the oldest Chinese pharmacopoeia, declaring it a superior medicine. LINGZHI-8 (LZ-8) is an immunoregulatory fungal protein isolated from the fruiting body of Ganoderma lucidum. Neutropenia is a condition with an abnormally low levels of neutrophils in the blood, which is caused by numerous medical conditions or medications, such as chemotherapy. The current study demonstrated that recombinant LZ-8 (rLZ-8) from Pichia promoted the differentiation of bone marrow hematopoietic stem cells (HSCs) into granulocytes in a neutropenia mouse model induced by cyclophosphamide. Also, it regulated the CXCR4-SDF1 axis to promote the mobilization of HSCs and the release of neutrophils from the bone marrow to peripheral blood. Binding of rLZ-8 to the colony stimulating factor 1 receptor (CSF1R) promotes the differentiation of HSCs into primitive CFU colonies. These results suggested that rLZ-8 has a potential effect in the treatment of chemotherapy-induced neutropenia.

Ganoderma lucidum Immune Modulator Protein rLZ-8 Could Prevent and Reverse Bone Loss in Glucocorticoids-Induced Osteoporosis Rat Model.

Immune modulation has been recognized as an effective anti-osteoporosis strategy since the pivotal role of the RANK/RANKL/OPG signaling in bone metabolism and remodeling was discovered. To investigate the potential preventive and/or therapeutic effects of immune modulator protein Ling Zhi-8 (LZ-8) on osteoporosis, the osteoporosis animal model was established in Wistar rats by intramuscular injection of dexamethasone (DEX), namely glucocorticoids-induced osteoporosis (GIOP) rat model. To investigate the potential preventive effect of rLZ-8 on GIOP, we co-treated the rats with DEX and rLZ-8 intraperitoneally during the GIOP modeling stage and analyze the bone mass measured by bone mineral content (BMC) and bone mineral density (BMD), as well as levels of phosphorus (Pi), calcium (Ca2+) and hydroxyproline (HOP) in femur of GIOP rats. Consistently, all results suggested that rLZ-8 could prevent bone loss in the femurs of GIOP rats. Through analyzing the trabeculae morphology and the trabeculae amount by H&E staining, we found rLZ-8 could also improve the structural deterioration in femurs of GIOP rats. In order to further verify the results and its mechanism obtained from bone analysis, multiple biomarkers, including minerals metabolism (Pi and Ca2+), bone formation markers (osteocalcin, ALP and IGF-1), bone resorption markers (TRACP5b, CTX-1 and HOP), cytokines (IL-1β, IL-6 and TNF-α), oxidative stress indicators (GSH-px, SOD and MDA) and hormone molecules (testosterone, estradiol, calcitonin and parathyroid hormone) have been detected in serum or urine of rats. Results of these biomarkers in serum or urine confirmed rLZ-8’s protective effect in GIOP. Through analyzing the relative expression level of OPG and RANKL in femurs via western blot, we foundrLZ-8 could increase OPG/RANKL ratio which could impede osteoclastogenesis process. To test the potential therapeutic effect of rLZ-8 on successfully generated GIOP rats, we administrated rLZ-8 to rats for three weeks starting from the ending day of 7 weeks treatment of DEX. We found rLZ-8 could also reverse the bone loss in GIOP rats. Through the BWs and organ coefficient analysis, we found rLZ-8 has little toxicity to the rats. Our results suggested that rLZ-8 may be developed into promising anti-osteoporosis drug with both preventive and therapeutic properties.

RecombinantLactococcus lactisExpressing Ling Zhi 8 Protein Ameliorates Nonalcoholic Fatty Liver and Early Atherogenesis in Cholesterol-Fed Rabbits

Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis, including dyslipidemia, type 2 diabetes mellitus, and metabolic syndrome, all of which lead to lipid deposition in the liver causing inflammation and fibrosis. Several clinical trials have shown that certain Chinese herbal medicines with anti-inflammatory effects can be used as adjuvant therapy to prevent the development of cardiovascular events and liver disease. Ling Zhi 8 (LZ8) is an immunomodulatory protein isolated from a medicinal mushroom and has been well documented to possess a broad range of pharmacological properties. This study aimed to evaluate the protective effects of recombinant Lactococcus lactis expressing LZ8 protein on NAFLD and atherogenesis in a cholesterol-fed rabbit model. Twelve rabbits were divided into three groups and fed with syrup only, L. lactis vehicle, or recombinant L. lactis-LZ8 once a day on weekdays for five weeks, respectively. The gene expression of IL-1β in the aorta was significantly suppressed after oral administration of L. lactis-LZ8. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the subendothelial space. LZ8 also inhibited the expression of IL-1β in the liver, decreased fat droplet deposits and infiltration of inflammatory cells, and improved liver function by decreasing liver enzymes in an animal model. Our results suggest that the Lactococcus-expressing LZ8 appears to be a promising medicine for improving both NAFLD and early atherogenesis owing to its anti-inflammatory effect. Furthermore, it is available as a low-cost food-grade product.

P6204Oral vaccination of Lactococcus lactis expressing Ling Zhi 8 protein prevents nonalcoholic fatty liver and early atherogenesis in cholesterol-fed rabbits

Abstract Background Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis. Epidemiology studies have indicated that NAFLD may be an independent risk factor for atherosclerosis. Anti-inflammation therapy by inhibiting IL-1β led to a significantly lower cardiovascular event rate in recent clinical trial (CANTOS trial). Ling Zhi 8 (LZ8) is an immunomodulatory protein that possesses a broad range of pharmacological properties, including anti-inflammatory activities. Methods and findings In this study, we developed an oral vaccination of Lactococcus lactis expressing LZ8 protein in a nisin-controlled gene expression system and investigated its anti-inflammation properties. Experimental rabbits received commercial rabbit chow supplemented with 2% cholesterol for 5 weeks and recombinant LZ8 L. lactis vaccine once a day on weekdays. The expression of IL-1β in the aorta (Figure A) was significantly suppressed after LZ8 vaccination. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the sub-endothelial space (Figure C). LZ8 also inhibited the expression of IL-1β in the liver (Figure B), decreased fat droplet deposits and infiltration of inflammatory cells (Figure D), and improved liver function by decreasing liver enzymes. Figure 1 Conclusions Our results suggest that LZ8 could be used as a therapeutic tool to improve both atherosclerosis and NAFLD due to its anti-inflammatory effect. Acknowledgement/Funding TCVGH-1067317C, TCVGH-1063108C

Involvement of N-glycan in Multiple Receptor Tyrosine Kinases Targeted by Ling-Zhi-8 for Suppressing HCC413 Tumor Progression.

The poor prognosis of hepatocellular carcinoma (HCC) is resulted from tumor metastasis. Signaling pathways triggered by deregulated receptor tyrosine kinases (RTKs) were the promising therapeutic targets for prevention of HCC progression. However, RTK-based target therapy using conventional kinase-based inhibitors was often hampered by resistances due to compensatory RTKs signaling. Herein, we report that Ling-Zhi-8 (LZ-8), a medicinal peptide from Ganoderma lucidium, was effective in suppressing cell migration of HCC413, by decreasing the amount and activity of various RTKs. These led to the suppression of downstream signaling including phosphorylated JNK, ERK involved in HCC progression. The capability of LZ-8 in targeting multiple RTKs was ascribed to its simultaneous binding to these RTKs. LZ-8 may bind on the N-linked glycan motif of RTKs that is required for their maturation and function. Notably, pretreatment of the N-glycan trimming enzyme PNGase or inhibitors of the mannosidase (N-glycosylation processing enzyme), kifunensine (KIF) and swainsonine (SWN), prevented LZ-8 binding on the aforementioned RTKs and rescued the downstream signaling and cell migration suppressed by LZ-8. Moreover, pretreatment of KIF prevented LZ-8 triggered suppression of tumor growth of HCC413. Our study suggested that a specific type of N-glycan is the potential target for LZ-8 to bind on multiple RTKs for suppressing HCC progression.

Computational Insights into the Molecular Mechanism of the High Immunomodulatory Activity of LZ-8 Protein Isolated from the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes).

Ling zhi-8 (LZ-8) is the first fungal immunomodulatory protein (FIP) isolated from the lingzhi or reishi medicinal mushroom, Ganoderma lucidum. LZ-8 effectively induces interleukin 2 expression and secretion by forming a stable homodimer, and it is regarded as a good candidate to become a new therapeutic agent and/or functional food supplement. However, the molecular mechanism by which LZ-8 dimerization influences the regulation of interleukin 2 is not clear. In this study we performed structure-based multiple alignment of LZ-8 and an FIP from Volvariella volvacea, compared the electrostatic potential of their protein surfaces, and developed a model summarizing the unique electrostatic interaction in LZ-8 dimerization. In addition, further electrostatic potential and virtual amino acid mutation analyses suggested that L10, W12, and D45 are the key amino acid residues responsible for the protein's high immunomodulatory activity. These findings may provide useful insights into the design and construction of a new FIP mutant for use in treating and preventing autoimmune diseases.

Induction of Cbl-dependent epidermal growth factor receptor degradation in Ling Zhi-8 suppressed lung cancer.

Activating mutation of epidermal growth factor receptor (EGFR) is correlated with malignant lung tumor. In our study, we demonstrated that recombinant LZ-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum protein, induced cell cycle arrest and apoptosis by downregulating the expression of wild-type and mutated EGFR and inhibiting EGFR downstream effectors, AKT and ERK1/2 in lung cancer cells. We showed that rLZ-8 effectively inhibited lung cancer progression and suppressed EGFR expression of lung tumor lesions in mouse model. Functional studies revealed that rLZ-8 reduced the amount of EGFR in cell membranes by altering EGFR localization to enhance the EGF-induced degradation of EGFR. Mechanistically, we demonstrated that rLZ-8 bound to EGFR to induce EGFR autophosphorylation at tyrosine1045 and trigger ubiquitination by inducing the formation of EGFR/Cbl complexes, resulting in the degradation of EGFR; however, Cbl-shRNA abolished rLZ-8-induced EGFR degradation. We provide the first evidence showing that rLZ-8 inhibits growth and induces apoptosis of lung cancer cells by promoting EGFR degradation. The current findings therefore suggest a novel anti-cancer function of rLZ-8 that targeting EGFR overexpression or mutation as well as EGFR-dependent processes in cancer cells.

Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation

We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer.

Anti-diabetic effects of Ganoderma lucidum

Ganoderma lucidum is a white rot fungus widely used as a tonic for the promotion of longevity and health. Extracts of G. lucidum have been recognized as an alternative adjuvant treatment for diabetes. Among the many biologically active constituents of G. lucidum, polysaccharides, proteoglycans, proteins and triterpenoids have been shown to have hypoglycemic effects. G. lucidum polysaccharides have been reported to have hypoglycemic activity by increasing plasma insulin levels and decreasing plasma sugar levels in mice. Protein tyrosine phosphatase 1B is a promising therapeutic target in diabetes, and G. lucidum proteoglycan can inhibit this enzyme in vitro. Moreover, G. lucidum triterpenoids were shown to have inhibitory activity on aldose reductase and α-glucosidase that can suppress postprandial hyperglycemia. In addition, a protein Ling Zhi-8 extracted from G. lucidum significantly decreased lymphocyte infiltration and increased the antibody detection of insulin in diabetic mice. This review summarizes most of the research about the hypoglycemic action effects of polysaccharides, proteoglycans, proteins and tritrerpenoids from G. lucidum as a guide for future research.

Ling Zhi Immuno-Modulation Protein, LZ-8 Induction of Interleukin-1 Expression in Human Monocytes via Pertussis Toxin-Sensitive G Protein-Mediated Protein Kinase Pathway

＂Ganoderma lucidum＂ (G. lucidum) (a medicinal fungus) has been known to enhance a variety of immune functions in humans and animals for centuries. An immunomodulatory protein, Ling Zhi-8 (LZ-8), is isolated from G. lucidum and exhibits potent mitogenic effects upon human immune cells. However, LZ-8-mediated signal transductions in the regulation of interleukin-1 (IL-1) gene expression within human monocytes has not been less well examined. In this study, we expressed the recombinant LZ-8 protein (rLZ) via Pichia pastoris protein expression system. We investigated the effect of rLZ on induction of IL-1 expression in human monocyte-like THP-1cells. We found that rLZ induced pro-interleukin 1 (proIL-1, a precursor of IL-1) and IL-1 protein expression in human monocytes THP-1 in time- and dose-dependent manners. We found that rLZ induced IL-1 gene expression via a pertussis toxin (PTX)-sensitive G protein-couple receptor (GPCR), phospholipase C (PLC) and various protein kinases-dependent pathways in THP-1 cells. In essence, we have established the nature of rLZ-mediated signaltransduction pathways, such as GPCR, PTK/ PI-3K/ PLC, PTK/ PLC/ PKCα/ JNK, and PTK/ PLC/ PKCδ/ θ/ ERK1/2, in the regulation of IL-1 gene expression in THP-1 cells. Our current results of the analysis of rLZ-mediated signal transduction in monocyte-like cells could provide a potential explanation and application of rLZ as a pharmacological immuno-modulating agent.

Effect of Recombinant Human Bone Morphogenetic Protein-2 and Ling Zhi-8 on Osteogenesis: A Comparative Study Using a Rabbit Sinus Model

This study evaluated the osteogenetic capability of Ling Zhi-8 (LZ-8; a protein purified from traditional Chinese medicine [lingzhi]) compared with recombinant human bone morphogenic protein-2 (rhBMP-2) in a standardized bony defect using a rabbit sinus model. Twelve male New Zealand white rabbits (18 to 24 weeks old, 3.3 to 3.8 kg) were included in the study. Implants of normal saline 0.1 mg, rhBMP-2 0.1 mg, and LZ-8 0.1 mg were each mixed with a uniform biodegradable polyurethane-based material (Nasopore). The implants were inserted in a standardized bony defect of the nasal bone created by a 2.5-mm trephine bur. The rabbits were sacrificed at 1, 2, 4, and 8 weeks postoperatively. Volume computerized tomographic and histomorphometric examinations were used to evaluate the quantity and quality of regenerated bone. At postoperative week 4, radiography showed that the new bone volume was significantly larger in the rhBMP-2 group compared with the LZ-8 group (P = .041) and the control group (P = .015). Histomorphometrically, better wound healing of the rhBMP-2 group was found during the healing phase compared with the other 2 groups. The biomaterial implants using rhBMP-2 and LZ-8 had good biocompatibility and osteogenetic capabilities in the rabbit sinus model. Bone healing in rhBMP-2-treated defects was excellent and showed a significant difference compared with LZ-8. However, LZ-8-treated defects also exhibited bone regeneration, and this traditional Chinese medicine may possess osteogenic potential. Further investigations of the mechanism and application of this protein in osteogenesis are needed.

An Immunomodulatory Protein (Ling Zhi-8) from a Ganoderma lucidum Induced Acceleration of Wound Healing in Rat Liver Tissues after Monopolar Electrosurgery.

The purpose of this study was to investigate the effect of an immunomodulatory protein (Ling Zhi-8, LZ-8) on wound healing in rat liver tissues after monopolar electrosurgery. Animals were sacrificed for evaluations at 0, 3, 7, and 28 days postoperatively. It was found that the wound with the LZ-8 treatment significantly increases wound healing. Western blot analysis clearly indicated that the expression of NF-κB was decreased at 3, 7, and 28 days when liver tissues were treated with LZ-8. Moreover, caspase-3 activity of the liver tissue also significantly decreases at 7 and 28 days, respectively. DAPI staining and TUNEL assays revealed that only a minimal dispersion of NF-κB was found on the liver tissue treated with LZ-8 at day 7 as compared with day 3 and tissues without LZ-8 treatment. Similarly, apoptosis was decreased on liver tissues treated with LZ-8 at 7 days when compared to the control (monopolar electrosurgery) tissues. Therefore, the analytical results demonstrated that LZ-8 induced acceleration of wound healing in rat liver tissues after monopolar electrosurgery.

Correlation of the Structure and Bioactivity of Recombinant Fungal Immunomodulatory Protein, Ling-Zhi-8 (LZ-8) Following Exposure to Denaturing Conditions

Ling-Zhi-8 (LZ-8), a low-molecular weight protein from Ganoderma lucidum, exerts immunomodulatory effects in vitro and in vivo. The aim of this study was to investigate the effects of exposure of LZ-8 to denaturing conditions on its ability to suppress inflammatory responses in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ultraviolet, fluorescence and circular dichroism spectroscopy analyses showed that LZ-8 was not denatured by exposure to pH extremes, but was irreversibly denatured by temperatures above 55C. LZ-8 lowered the production of nitric oxide and interleukin-6, but had no effect on the level of tumor necrosis factor-α. Heat and acid/alkali-treated preparations of LZ-8 had comparable capacity to suppress proinflammatory mediator production. Since native and denatured LZ-8 were both anti-inflammatory, it appears that this particular biological property of LZ-8 is not dependent on the native structure of the protein, but, possibly on a relatively small peptide domain of LZ-8. Practical Applications Recent evidence suggests that LZ-8, a small protein originally isolated from G. lucidum, has immunomodulatory and anti-carcinogenic properties. In this study, LZ-8 exerted an anti-inflammatory effect on LPS-stimulated macrophages, but this effect was not dependent on the native protein structure, suggesting that the entire protein might not be responsible for the bioactive properties of LZ-8. Since the biological functions of LZ-8 were resistant to heating and exposure to acid or alkali, LZ-8 could have applications in food-processing industries and the production of pharmaceuticals. Furthermore, peptides derived from LZ-8 may be potential targets for development of novel anti-carcinogenic and immunomodulatory agents.

Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway

Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.

512. A Novel Adjuvant Ling Zhi-8 Enhances the Efficacy of DNA Cancer Vaccine by Activating Dendritic Cells

512. A Novel Adjuvant Ling Zhi-8 Enhances the Efficacy of DNA Cancer Vaccine by Activating Dendritic Cells

A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells.

DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185(neu) expressing tumor MBT-2 in mice. We found that recombinant LZ-8 stimulated mouse bone marrow-derived dendritic cells (DCs) via TLR4 and its stimulatory effect was not due to any microbe contaminant. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, LZ-8 cotreatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase in antitumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.