The Inhibition Mechanism Of Polysaccharide From Rhizopus Nigricans On The Colorectal Cancer

Colorectal cancer is a common gastrointestinal malignancy.Surgical resection combined with chemotherapy and radiation therapy is the major method in curing colorectal cancer.However,chemotherapy has brought adverse effects for cancer patients,including nausea,vomiting,myelosuppression and neutropenia,which seriously affect the quality of patients,lives.Therefore,it has become an urgent task to develop innovative antitumor drugs with high efficiency and low toxicity.Polysaccharides possess a broad spectrum of biological effects,including antitumor,antioxidation and immunomodulatory.Many polysaccharides can inhibit tumor growth through inhibiting the proliferation of tumor cells directly or enhancing the immune capacity to suppress the tumor growth.Rhizopus nigricans is a filamentous fungus which is assigned to the class of Zygomycetes,group Mucorales.An exopolysaccharide(EPS 1-1)with a molecular weight 9,682 Da was isolated from the fermentation broth of Rhizopus nigricans.Previous studies have suggested that EPS 1-1 could induce apoptosis of HCT-116 cells,inhibit the growth of S180 and CT26 in tumor-bearing mice and enhance the immune function of immunosuppressed mice.However,the antitumor mechanism of EPS1-1 remains unclear.In this study,CT26 cells and two mouse models of colitis-associated colorectal cancer and artificial lung metastasis with CT26 cells were used to research the directly antitumor mechanism of EPS1-1.Moreover,the effects of EPS 1-1 on the intestinal immunity function of mice with colorectal cancer and the activation mechanism of peritoneal macrophage was studied to illuminate the indirect anti-tumor mechanism.4.Effects of EPS 1-1 on the gene expression of CT26 cell,mice with colorectal cancer and lung metastasis with CT26 cellsEPS 1-1 could induce DNA fragmentation of CT26 cells after treatment for 72 h,and inhibit the growth of CT26 to suppress the formation of colony.High-throughput transcriptome sequencing was used to measure the effect of EPS1-1 on the integrated gene expression of CT26 cell,mice with colorectal cancer and lung metastasis with CT26 cells.Results suggested that 98 and 584 differentially expressed genes(DEGs)were indentified in CT26 after treatment with EPS 1-1 for 4 h and 24 h,respectively.KEGG Pathway analysis showed that these DEGs participated in many cancer-related signaling pathways,including PI3K-Akt,Rapl,TNF,Ras,MAPK,and so on.In addition,10 and 43 DEGs were annotated in PI3 K/Akt-mTOR signaling pathway with the aforesaid time,which involved in the largest amount of DEGs.EPS 1-1 could inhibit the progress of colorectal cancer of mice induced by azoxymethane and dextran sulfate sodium(AOM/DSS).The tissues of colorectal cancer from mice were used to further illustrate the anticancer mechanism of of EPS 1-1 by transcriptome sequencing.The results suggested that EPS 1-1 could regulate 6170 DEGs in colon cancer tissues,which participated in metabolism,signal transduction,cell growth,and so on.Moreover,these DEGs were connected with the cancer-related signaling pathways,in which 217 DEGs were related to the PI3 K/Akt-mTOR signaling pathway.Therefore,we speculated that the anti-tumor effect of EPS1-1 was closely related to PI3K/Akt-mTOR signaling pathway.The expression of key protein in PI3K/Akt-mTOR signaling pathway were determined for further analysis the effect of EPS 1-1 on this pathway by Western-blotting.The results suggested that EPS 1-1 could down-regulate the expression of p-PTEN and p-Akt after treatment for 4 h.Moreover,after treatment with EPS1-1 for 24 h,the expression of p-PTEN and p-mTOR were inhibited.Furthermore,the expression of PTEN,PI3K,Akt,mTOR,p70S6K and their phosphorylated proteins were measured by Western blotting and immunohistochemistry.Immunohistochemistry indicated that colon cancer tissues from EPS1-1 group were negative to p-PI3K,p-Akt,p-mTORand p-p70S6K compared with model group.Besides,Western blotting analysis showed that EPS 1-1 decreased the phosphorylated of PTEN,mTOR and p70S6K in colon cancer tissues.Our previous research primarily showed that EPS1-1 possessed the anti-metastatic activity in vitro and in vivo.We performed in vivo transcriptome profiling of lung tissues from mice with metastasis of CT26 cells.Gene expression analyses showed the number of DEGs regulated by EPS 1-1 was 2630,which were annotated in 290 metabolic or signaling pathways.There are 104 DEGs that participated in PI3K/Akt-mTOR signaling pathway.Focal adhesion and Cell adhesion molecules are concerned with metastasis,which involved 73 and 74 DEGs,respectively.These three pathways shared 45 DEGs,including integrin,Epithelial cadherin,ankyrin,laminin and so on.Therefore,we conclude that EPS 1-1 could inhibit the progress of coloretal cancer through PI3K/Akt-mTOR signaling pathway.5.Effect of EPS 1-1 on intestinal immunity of mice with coloretal cancer.Many polysaccharides cannot be hydrolyzed in upper gastrointestinal tract and reach the intestine to promote the growth and proliferation of probiotic bacteria,thus enhancing the immunity of host.16S rDNA sequencing technique was utilized to investigate the effects of EPS1-1 on the diversity of bacterial communities and the composition of gut microbiota in the intestine of mice with colorectal cancer.The results demonstrated that the carcinogens of AOM/DSS could increase the abundance of pathogenic bacteria,including family Ruminococcaceae and Helicobacter.EPS 1-1 could provid protection to intestine by promoting the abundance of probiotics,including family Lachnospiraceae and genus Roseburia,Coprococcus,which could secrete short chain fatty acids(SCFAs)and promote the function of intestinal immune.The SCFAs and lactic acid content in the intestinal were measured by HPLC.Results suggested that the total content of SCFAs in the intestine of mice with colorectal cancer was increased after treatment with EPS 1-1,especially the acetic acid and propionic acid.Furthermore,EPS 1-1 increased the villus length,crypt depth and V/C ratio to prevent the damage caused by carcinogens.The surface of the intestinal epithelial cells is covered with mucous layer to protect intestine.EPS 1-1 could increase the acid mucus-secreting goblet cells to maintain the function and integrity of the intestinal mucosa layer,thus promoting the intestinal immune function.These results suggested that EPS 1-1 could enter the intestinal to increase the abundance of intestinal probiotics,which promoted the secretion of SCFAs.SCFAs in turn affect the mucus-secreting goblet cells to secret acid mucus,which could protect intestine from damage of pathogenic microorganism and harmful substance.6.The activation mechanism of EPS 1-1 on peritoneal macrophageThe activation of peritoneal macrophage could increase the secretion of cytokines and NO to enhance the immune function,thus improving the antitumor and antiviral function.EPS 1-1 could promote the maturation of peritoneal macrophage and the phagocytic activity to up-regulated the mRNA levels of IL-2,TNF-a and iNOS.In order to illustrate the activation mechanism of EPS1-1 on macrophages,the key proteins of MAPK and NF-κB signaling pathway were measured.The results showed that EPS 1-1 could significantly increase the expression of TLR4 and the phosphorylation of p38MAPK,Erk1/2 and JNK.Besides,EPS1-1 could up-regulate the expression of p-IKKα/β,which was a critical protein in NF-κB signaling pathway.The inhibitors of MAPK signaling pathway(PD98059 and SB203580)could significantly inhibit the mRNA levels of iNOS,IL-2 and TNF-α,and the expression of TLR4.SB203580 and PD98059,inhibitor of p-p38MAPK and p-Erkl/2,decreased the phosphorylation of p38MAPK,Erkl/2 and KKα/β.EPS1-1 could relieve the suppression induced by inhibitors and might trigger complex crosstalk between MAPK and NF-κB signal pathways.These results indicated that EPS 1-1 could activate macrophage to increase the expression of iNOS,IL-2 and TNF-a via MAPK and NF-κB signaling pathways.In conclusion,EPS 1-1 could inhibit the progress of colorectal cancer directly by down-regulating PI3K/Akt-mTOR signaling pathway.In addition,EPS 1-1 could prevent or inhibit the occurrence of colorectal cancer by modulating gut microbiota and activate mouse peritoneal macrophages to enhance the immunity.This study is important for illustrating the anti-tumor mechanism and provides fundamental data for clinical applications or adjuvant drug of EPS 1-1.