The improvement of M1 polarization in macrophages by glycopeptide derived from Ganoderma lucidum.

Ganoderma lucidum (Fr.) Karst (Ganodermataceae) is a medicinal mushroom that has been extensively used in China for centuries to promote longevity and improve vigor without significant adverse effects. There is continuous interest in the bioactive properties of G. lucidum in view of its newly developed popularity in other regions besides Asia, such as Europe. Glycopeptide derived from G. lucidum (Gl-PS) is one of the main effective components isolated from this mushroom. The Gl-PS has been demonstrated pleiotropic with many bioactivities including immunomodulatory and antitumor effects. Macrophages are important cells involved in innate and adaptive immunity. Classically activated macrophages (M1) and alternatively activated macrophages (M2), with their different roles, display distinct cytokine profiles: M1 preferentially produces TNF-α, IL-6, and IL-12; conversely, M2 generates more IL-10 and arginase. Gl-PS might have the potential to promote macrophage M1 polarization by lipopolysaccharide (LPS). In this study, LPS was used to induce the M1 polarization. It was shown that the level of the TNF-α, IL-6, and IL-12 were increased and the IL-10 and arginase I were decreased in the polarized M1 macrophages after application of Gl-PS compared to the control. The results indicated the potential of Gl-PS to promote M1 polarization vs M2, with the health beneficial understanding of the bioactivities of Gl-PS.

Cytokine production suppression by culture supernatant of B16F10 cells and amelioration by Ganoderma lucidum polysaccharides in activated lymphocytes.

Some cytokines, such as interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), produced by lymphocytes might play an important role in anti-tumor immunity and their production is possibly suppressed by cancer. Amelioration of the suppression of cytokine production might contribute to cancer control. Ganoderma lucidum polysaccharides (Gl-PS), a versatile group of a component of G. lucidum and one with various bioactivities, might have this potential. In this study, analyses including reverse transcription and the polymerase chain reaction (RT-PCR), immunocytochemistry and Western blot were used to test the effects of Gl-PS on the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by incubating Gl-PS with mouse splenic mononuclear lymphocytes in the presence of B16F10 cell culture supernatant following activation by phytohemagglutinin. The RT-PCR, immunocytochemistry and Western blot assays showed that the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes was suppressed by B16F10 cell culture supernatant at both the mRNA and protein levels, whereas the suppression was fully or partially ameliorated by Gl-PS. The amelioration by Gl-PS against the suppression of the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by B16F10 cell culture supernatant might contribute to cancer control.

Suppression of the production of transforming growth factor ß1, interleukin-10, and vascular endothelial growth factor in the B16F10 cells by Ganoderma lucidum polysaccharides.

Transforming growth factor ß (TGF-ß), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF) are three of the commonly studied cytokines playing an important role in tumor initiation and progression. Besides their promotional effects on tumor progression, the three cytokines have immunosuppressive effects that facilitate tumor initiation and progression as well. Ganoderma lucidum polysaccharides (Gl-PS) with multiple bioactivities may have the effect on B16F10 melanoma cells to induce stronger antitumor immune response that has been demonstrated. Gl-PS may have the suppressive effects on the production of these three cytokines, which has yet to be demonstrated. In this study, we tested these effects of Gl-PS by incubating Gl-PS with malignant tumor cells such as B16F10 cells, a melanoma cell line, and LA795 cells, a lung carcinoma cell line. RT-qPCR and enzyme-linked immunosorbent assay showed that the production of TGF-ß1, IL-10, and VEGF in B16F10 melanoma cells and LA795 lung carcinoma cells was suppressed by Gl-PS at both mRNA and protein levels, suggesting that the suppression on production of TGF-ß, IL-10, and VEGF in B16F10 melanoma cells and LA795 lung carcinoma cells by Gl-PS may contribute to the therapy on melanoma and lung carcinoma along with the induction of stronger antitumor immune response.

Protection against lung cancer patient plasma-induced lymphocyte suppression by Ganoderma lucidum polysaccharides.

BACKGROUND/AIMS: This study was conducted to determine the potential of Ganoderma lucidum polysaccharides (Gl-PS) in protection against lung cancer patient plasma-induced suppression of lymphocytes. Lung cancer is a major cause of disease and loss of life in the United States and worldwide. Cancer cells release immunosuppressive mediators, such as PGE2, TGF-ß, IL-10, and VEGF, to inhibit the immune response to escape from immune surveillance. Gl-PS has been shown to counteract this immune inhibition in an animal cell culture model, and thus to facilitate tumor control. The present study explored whether or not such an effect could also be demonstrated in human lung cancer patients. METHODS: Immunofluorescence, flow cytometry, MTT, immunocytochemistry, and western blot analysis were used to assess lymphocyte activation with PHA. RESULTS: The plasma of lung cancer patients suppressed proliferation, CD69 expression, and perforin and granzyme B production in lymphocytes upon activation by PHA, effects that were partially of fully reversed by Gl-PS. CONCLUSION: Lung cancer patient plasma-induced suppression of lymphocyte activation by phytohemagglutinin may be antagonized fully or partially by Gl-PS, an observation suggesting the potential of Gl-PS in cancer therapy.

Antagonism by Ganoderma lucidum polysaccharides against the suppression by culture supernatants of B16F10 melanoma cells on macrophage.

It is well-documented that macrophages have the functions to regulate antitumor immune response. Antitumor response can be launched by a series of events, starting with inflammation mediated by monocyte/macrophages, which stimulates natural killer and dendritic cells and finally activates the cytotoxic lymphoid system. Monocytes/macrophages may be the first line of defense in tumors. However, specific and nonspecific immunotherapy for human cancer has shown no success or limited success in clinical trials. Part of the reasons attribute to tumor-derived soluble factors that suppress functions of immune cells or induce apoptosis of these cells, including macrophages. Therefore, antagonism of the suppression on the macrophages is an important goal for tumor immunotherapy. To achieve this purpose, Ganoderma lucidum polysaccharides (Gl-PS) with multiple bioactivities were used on mouse peritoneal macrophages incubating with culture supernatants of B16F10 melanoma cells (B16F10-CS). It was shown that the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages after activation by lipopolysaccharide were suppressed by B16F10-CS, while the suppressions were fully or partially antagonized by Gl-PS. In conclusion, B16F10-CS is suppressive to the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages while Gl-PS had the antagonistic effects against this suppression, suggesting this potential of Gl-PS to facilitate cancer immunotherapy.

Ganoderma lucidum polysaccharides counteract inhibition on CD71 and FasL expression by culture supernatant of B16F10 cells upon lymphocyte activation.

Immune responses to tumor-associated antigens are often detectable in tumor-bearing hosts, but they fail to eliminate malignant cells or prevent development of metastases. Tumor cells produce factors such as interleukin-10, transforming growth factor-ß1 and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. Culture supernatant of tumor cells may contain these immunosuppressive factors which suppress lymphocyte activation. CD71 and FasL are two important molecules that are expressed upon lymphocyte activation. Counteraction against suppression CD71 and FasL expression upon lymphocyte activation may benefit tumor control. A potential component with this effect is Ganoderma lucidum polysaccharides (Gl-PS). In this study, Gl-PS was used on lymphocytes incubating with culture supernatant of B16F10 melanoma cells (B16F10-CS) in the presence of phytohemagglutinin. Following induction with phytohemagglutinin, B16F10-CS suppressed CD71 expression in lymphocytes (as detected by immunofluorescence and flow cytometry), proliferation in lymphocytes (as detected by MTT assay), and FasL expression in lymphocytes (as detected by immunocytochemistry and western blot analysis), while Gl-PS fully or partially counteracted these suppressions. Gl-PS showed counteractive effects against suppression induced by B16F10-CS on CD71 and FasL expression upon lymphocyte activation, suggesting the potential of Gl-PS to facilitate cancer immunotherapy.

Effects of T-cadherin expression on B16F10 melanoma cells.

Melanoma is one of the most deadly skin cancers. T-cadherin is an atypical member of the cadherin superfamily as it lacks the transmembrane and cytoplasmic domains and is anchored to cell membranes through glycosylphosphatidylinositol (GPI) anchors. T-cadherin downregulation is associated with a poorer prognosis in various carcinomas, such as lung, ovarian, cervical and prostate cancer, while in the majority of cancer cell lines, T-cadherin re-expression inhibits cell proliferation and invasiveness, increases susceptibility in apoptosis and reduces tumor growth in in vivo models. The functional relevance of T-cadherin gene expression in melanoma progression remains to be clarified. The present study was designed for this purpose. The T-cadherin gene was transfected into B16F10 melanoma cells to express T-cadherin in the cells which were originally deficient in T-cadherin expression. The proliferation, invasiveness, apoptosis and cell cycle of the transfected B16F10 melanoma cells were analyzed. The present study showed that the expression of T-cadherin in B16F10 melanoma cells markedly reduced cell proliferation and permeation through Matrigel-coated membranes, representing invasiveness. The percentage of early apoptotic cells and cells in the G2/M phase of the cell cycle was markedly increased compared with either parental B16F10 (without transfection) or empty pEGFP-N1 (without T-cadherin gene)-transfected B16F10 cells, suggesting G2/M arrest, with similarity between the parental and empty pEGFP-N1-transfected B16F10 cells. T-cadherin is important in melanoma progression and may be a possible target for therapy in melanoma and certain other types of cancer.

Effects of the polysaccharide nucleic acid fraction of bacillus Calmette-Guérin on the production of interleukin-2 and interleukin-10 in the peripheral blood lymphocytes of patients with chronic idiopathic urticaria.

Urticaria is one of the most frequent dermatoses and its prevalence in the general population is estimated to be ~20%, whereas a substantial percentage of the cases may be classified as chronic idiopathic urticaria (CIU). The inflammatory response presenting with spontaneous wheals exhibits pro-inflammatory characteristics, involving a prominent role for lymphocytes with a mixed Th1/Th2 response in which interleukin (IL)-2 and IL-10 are prominently secreted by Th1 and Th2 cells, respectively. In CIU patients, it was demonstrated that IL-10 production was elevated and IL-2 reduced compared to controls. Therefore, inhibition of IL-10 and promotion of IL-2 production by the lymphocytes, indicating Th2 inhibition and Th1 promotion, may facilitate the treatment of CIU. Whether the polysaccharide nucleic acid fraction of bacillus Calmette-Guérin (BCG-PSN), which possesses multiple immunomodulatory properties, has that potential, remains to be elucidated. In this study, BCG-PSN was used on lymphocytes isolated from CIU patients, with healthy donors used as controls. Immunocytochemistry and ELISA were used to detect IL-2 and IL-10 production. It was demonstrated that the IL-2 production by the lymphocytes in the CIU group was significantly lower compared to that in the healthy control group and it increased sequentially with the increase of the concentration of BCG-PSN used. By contrast, the IL-10 production by the lymphocytes in the CIU group was significantly higher compared to that in the healthy control group and decreased sequentially with the increase of the concentration of BCG-PSN used. Thus, it may be concluded that the BCG-PSN has the potential to promote IL-2 and inhibit IL-10 production in the lymphocytes of CIU patients, facilitating the treatment of CIU.

Enhanced MHC class I and costimulatory molecules on B16F10 cells by Ganoderma lucidum polysaccharides.

PURPOSE: It is obvious that malignant cells evade from immune system in patients with manifest malignancy. Deficient major histocompatibility complex (MHC) class I and costimulatory molecules on malignant cells partially consist of evasion strategy since antigen bond MHC and costimulatory molecules provide two signals necessary for T cell activation. Therefore, enhancement of MHC-I and costimulatory molecules may favor restraint of the evasion. For this purpose, Ganoderma lucidum Polysaccharides (Gl-PS) was used on B16F10 melanoma cells in this study. METHODS: Immunocytochemistry and flowcytometry were used to determine the H-2K(b) and H-2D(b) (two prominent MHC class I molecules in C57BL mouse) as well as B7-1 and B7-2 (two prominent costimulatory molecules) expression on B16F10 cells after incubation with Gl-PS, while messenger ribonucleic acid (mRNA) of these molecules was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The H-2K(b) and H-2D(b), and B7-1 and B7-2 on B16F10 cells and mRNAs of these molecules were enhanced by Gl-PS, and more efficient antitumor cytotoxicity was induced by the Gl-PS treated cells. CONCLUSIONS: The MHC class I molecules and costimulatory molecules may be enhanced by Gl-PS, and more efficient immune cell mediated cytotoxicity against these B16F10 cells may be induced, which may favor cancer therapy.

Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells.

OBJECTIVES: Tumour cells produce factors such as interleukin 10 (IL-10), transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. One of the most important goals of tumour immunotherapy is to antagonize this suppression on immune cells. Ganoderma lucidum polysaccharides (Gl-PS) may have this potential. The purpose of this study was to determine the antagonistic effects of Gl-PS on the suppression induced by B16F10 melanoma cell culture supernatant (B16F10-CS) on lymphocytes. METHODS: Gl-PS was used on lymphocytes incubated with B16F10-CS. Enzyme-linked immunosorbent assay was used to determine the levels of IL-10, TGF-ß1 and VEGF in B16F10-CS. The MTT assay was used to determine the proliferation of lymphocytes. Immunocytochemistry and Western blot assay were used to determine perforin and granzyme B production in lymphocytes. KEY FINDINGS: There were elevated levels of IL-10, TGF-ß1 and VEGF in B16F10-CS. The lymphocyte proliferation, and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction, were suppressed by B16F10-CS. This suppression was fully or partially antagonized by Gl-PS. CONCLUSIONS: B16F10-CS suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction. This suppression may be associated with elevated levels of immunosuppressive IL-10, TGF-ß1 and VEGF in B16F10-CS. Gl-PS had antagonistic effects on the immunosuppression induced by B16F10-CS, suggesting the potential for Gl-PS in cancer immunotherapy.

Promoting effects of Ganoderma lucidum polysaccharides on B16F10 cells to activate lymphocytes.

The immune system in patients with cancer often fails to control tumour growth because of deficient immunogenicity of tumour cells. Ganoderma lucidum polysaccharides (Gl-PS) are believed to have anti-tumour effects by boosting host immune function. Additionally, Gl-PS may have some direct effects on tumour cells in the activation of lymphocytes, thus enhancing the immunogenicity of tumour cells. We tested the effects of Gl-PS in lymphocyte activation by incubating Gl-PS with a tumour cell line deficient in antigen presentation. Our study showed that Gl-PS can promote B16F10 melanoma cells to induce lymphocyte proliferation, CD69 and FasL expression and IFN-γ production, indicating that Gl-PS can improve the nature of B16F10 cells to activate lymphocytes. Furthermore, H-2D(b) [a major histocompatibility (MHC) class I molecule], and B7-1 and B7-2 (two prominent co-stimulatory molecules expressed on B16F10 cells) were enhanced by Gl-PS, suggesting that these molecules may at least partially be involved in the process of Gl-PS on B16F10 cells to activate lymphocytes.