Study On Structure, Antitumor And Immunomodulatory Activity Of Polysaccharides From The Mycelium Of Rhizopus Nigricans

Cancer has become one of the major deadly diseases, which severely threats human beings’ life span. According to the data released by National Cancer Control and Research Office, about 3.5 million cancer cases and 2.5 million cancer deaths are estimated to occurr every year in China. At the present stage, the chemotherapy is one of the major therapies for cancer patients.However, the chemotherapeutic drugs also impair the function of normal cells at the same time, which will result in serious side effects and lower the quality of patients’ lives. In addition, the cancer cell will be gradually resistant to the chemotherapy medicine, thus causing the failure in chemotherapy. Hence, it has become an urgent task to seek new-type anti-cancer drugs featured by high efficiency and low toxicity.Massive researches have shown that polysaccharides possess a wide range of biological activity, including anti-cancer, immunomodulatory and anti-oxidant. Among them, the research on the immunomodulatory and anti-cancer activity has gained growing attention. As biological response modifier, the anti-tumor effects of these substances were primarily produced by modulating the immunological functions of host. Moreovre, they can initiate cell stress responses and deranged the expression of signal molecules in tumor cells, which will induce differentiation, apoptosis, cell cycle arrest and anti-angiogenesis, ultimately suppressing tumor growth.Rhizopus nigricans is a filamentous fungus which is assigned to the class of Zygomycetes, group Mucorales. R. nigricans is widely used in brewing and pharmaceutical industry, such as biotransformation and production of organic acids. It is found that liquid-cultured mycelia of R. nigricans contain a large amount of bio-active polysaccharides. In this study, we separated and purified bio-active polysaccharides from fermentation mycelia of R. nigricans. Structural characterization of R. nigricans polysaccharides was performed by high performance size-exclusion chromatography coupled with multiangle laser light scattering, monosaccharide analysis, infrared spectrum, methylation analysis and NMR spectroscopy. Further, we investigated the underlying molecular mechanisms of their anti-cancer and immunomodulatory activity. It is hoped that this research can lay a foundation for the development of treatment or adjunctive therap against cancer.1. The preparation and structural characterization of R. nigricans polysaccharidesThe crude polysaccharides obtained by hot water extraction of liquid-cultured mycelia of R. nigricans were separated using Sephadex G-100 column chromatography. The fraction of major peak was collected and lyophilized to powder named as RPS. RPS was composed of mannose, rhamnose, glucuronic acid, glucose, galactose and fucose in a molar ratio of 5.1:1.0:1.6:92.2:1.3:2.3. Moreover, the crude polysaccharides were purified by DEAE-Sepharos Fast Flow and Sephacryl S-500 HR chromatography, and a water-soluble polysaccharide (RPS-1) was obtained. According to the data of high performance size-exclusion chromatography coupled with multiangle laser light scattering (HPSEC-MALLS), RPS-1 was a homogeneous polysaccharide and its weight-average molecular weight of RPS-1 was 1.617×107 Da with a relatively narrow molecular weight distribution. The radius of gyration of RPS-1 was 24.2 nm. The IR spectrum of RPS-1 displayed the characteristic peaks of polysaccharide. RPS-1 was completely hydrolyzed into individual monosaccharides by TFA, followed by PMP derivatization. Based on HPLC analysis, only one peak was observed and identified as glucose. The results of methylation analysis showed that the linkage type contained in RPS-1 were α-Glcp-(1→,→4)-a-Glcp-(1→ and →4,6)-α-Glcp-(1→ in a molar ratio of 6.9:86.1:6.9. Only one set of signal appeared in the anomeric region of both the 1H and 13C NMR spectra, which indicated all of the residues are of α-configuration. The anomeric proton signals at δ 5.35 ppm and δ 4.92 ppm corresponded at H-1 of→4)-α-Glcp-(1→ and α-Glcp-(1→, respectively. The anomeric carbon signals at δ99.86 ppm and δ99.02 ppm could be attributed to C-1 of →4)-α-Glcp-(→ unit and →4,6)-α-Glcp-(1→ unit. The signal for unsubstituted C-6 of(1→4)-α-glucopyranose was at δ60.58 ppm, whereas the signal for substituted C-6 of (1→4,6)-α-glucopyranose was at δ69.43 ppm. These results indicated that RPS-1 was glycogen-like glucan consisting of a backbone structure of (1→4)-linked α-D-glucopyranosyl residues substituted at the O-6 position.2. Study on the antitumor activity of RPSDue to the complex spatial structure, polysaccharides possess a broad spectrum of biological effects. Recent studies have shown that polysaccharides were effective to kill or incapacitate cancer cells with less toxicity to normal cells. They could disturb cellular homeostasis and deranged the expression of signal molecules in tumor cells, thus induced growth inhibition. According to the data of MTT assay, after treated with RPS for 12h,24h and 48h, the cell viability of BGC-823 cells was significantly decreased in a dose-dependent manner. Furthermore, RPS induced a dose-dependent inhibition in clonogenicity, with 70% inhibition at concentration of 0.6 mg/ml. We investigated the distribution of cell cycle phase using flow cytometry. The results indicated that RPS treatment induced the G2/M phase cells accumulation. The results of Hoechst 33258 staining, AO/EB staining and flow cytometry showed that RPS induced apoptotic cell death in BGC-823 cells. In our further study, collapse of mitochondrial membrane potential and accumulation of ROS were observed. Caspase-9 is the apical protease of the mitochondrial pathway. RPS increased caspase-9 activity up to 1.72 fold at concentration of 600 μg/ml compared with untreated group. Alterations in mRNA expression of apoptosis-related genes Bcl-2 and Bax were detected by PCR. The results indicated that RPS increased the expression level of Bax and decreased the expression level of Bcl-2. These results induced mitochondria-mediated apoptosis. We also found that RPS induced elevation of intracellular calcium ion and increased the mRNA levels of GRP78, ATF6, CHOP and spliced XBP1, which indicated that these cells underwent endoplasmic reticulum stress.3. Study on the immunomodulatory activity of RPS-1Macrophages are one of the major types of phagocyte and antigen-presenting cell, and play vital roles in the immune response. The immunomodulatory activity of RPS-1 was determined in murine macrophage RAW 264.7 cells. RPS-1 significantly enhanced phagocytic activity of macrophages. Griess method and ELISA assay were performed to detect the production of NO and TNF-a in RAW 264.7 cells. The results showed that RPS-1 treatment significantly stimulated the release of NO and TNF-a in RAW 264.7 cells. PMB is a specific inhibitor of endotoxin. The pre-incubation with PMB only slightly attenuated the secretion of NO and cytokine induced by RPS-1 without significant difference. To explore the possible molecular mechanisms involved in the activation of macrophage induced by RPS-1, we detected the expression of proteins that are associated with NF-kB, MAPK and Akt signaling pathways. After stimulated with RPS-1 at different time points within 60 min, the content of IκBα was decreased in a time-dependent manner and the nuclear translocation of NF-κB p65 was observed. The transcriptional activity of NF-κB was assayed by luciferase reporter gene assay and RPS-1 significantly increased the luciferase activity in a dose-dependent manner. The production of NO and TNF-α induced by RPS-1 was almost completely inhibited by BAY 11-7082 (10μM), inhibitor of NF-κB, which suggested that NF-κB was the major transcription factor implicated in the activation of macrophage induced by RPS-1. It is now generally accepted that MAPK and Akt signaling pathway participate in the expression of inflammatory cytokines and mediators in macrophages. The phosphorylation of MAPKs and Akt was studied by western blot. The treatment with RPS-1 increased the phosphorylation of MAPKs and Akt in RAW 264.7 cells. PD98059 (30μM), SP600125 (20 μM) and LY294002 (50μM) could significantly weakened NO and TNF-a release, whereas LY294002 (50 μM) totally suppressed NO production induced by RPS-1. However, SB203580, inhibitor of phospho-p38, failed to show inhibitory effect on the NO production. In different cell types, stimuli may trigger complex crosstalk between MAPK and PI3K/Akt pathway. The inhibitors of MAPK decreased the phosphorylation of Akt. At the same time, LY294002 inhibited RPS-1 induced phosphorylation of JNK, but not p38 and ERK. These results suggested that RPS-1 could activate macrophage via NF-κB, MAPK and PI3K/Akt signaling pathways. In vivo study, RPS-1 increased immune organ index of CT26 bearing mice and the percentage of CD8+ T cell in spleen and inhibited the tumor growth.In summary, RPS could induce mitochondria-mediated apoptosis and G2/M cycle arrest in BGC-823 cells. RPS-1 was consisted of a backbone structure of (1→4)-linked a-D-glucopyranosyl residues substituted at the O-6 position with a-D-glucopyranosyl branches. RPS-1 could stimulate the productions of NO and TNF-a via NF-κB, MAPK and PI3K/Akt signaling pathways. In vivo study, RPS-1 increaed immune organ index of CT26 bearing mice and the percentage of CD8+ T cell in spleen and inhibited the tumor growth.