Study On The Anti-colorectal Cancer Mechanism And Actrivity Of Fungi Polysaccharides

Fungal polysaccharides mainly exist in the fungus fruiting bodies,mycelia and fermented liquid,the study showed that fungal polysaccharides have the anti-tumor,immunity enhancement,antiviral activities,especially in the aspect of anti-tumor,which can directly or indirectly inhibit cell proliferation and induce the apoptosis.It has attracted the attention of many scholars.Colorectal cancer is the most common malignant tumors in the world.With the changes in people's lifestyle and diet habits,the incidence and mortality of colorectal cancer are gradually increasing in China.At present,the treatment of colorectal cancer is surgical resection,radiotherapy,chemotherapy and other combinations,but patients usually have a poor prognosis,leading to serious side effects.Therefore,it is necessary to find new drugs to treat or supplement colorectal cancer with non-toxic side effects.Trichoderma kanganensis is isolated from corn stalks by our laboratory,belonging to the fungi subphylum,class filamentae and genus trichoderma.Previous studies showed that Trichoderma kanganensis has a bacteriostatic effect,but other activities have not been studied in depth.Rhizopus nigricans is a filamentous fungus.An exopolysaccharide(EPS1-1)with a molecular weight 9,682 Da was isolated from the fermentation broth of R.nigricans.Previous data show that EPS1-1 possesses enhancement immunity,induces HCT-116 and CT26 cells apoptosis,inhibits the growth and metastasis of S180 and CT26 in tumor-bearing mice and the occurrence and development of AOM/DSS-induced colorectal cancer mice,and increases intestinal immune function in mice.In this study,our aim is to find the high active fungal polysaccharides.Two fungal polysaccharides were studied to elucidate the antitumor activity and the molecular mechanism of inhibiting the development of colorectal cancer.1 The preparation and structure analysis of mycelia polysaccharides from T.kanganensis and the study of antitumor activityThe crude polysaccharides obtained by ultrasonic extraction,hot water extraction,alcohol precipitation,decoloration and deproteinization were purified using DEAE Sepharose Fast Flow,Sephadex G-100,Sephacryl S-300 column chromatography,and named TPS.The physico-chemical properties analysis shows that TPS had no starch,protein,and nucleic acid,and contained 99.529%carbohydrate.HPSEC-MALLS analysis demonstrated that TPS was homogeneous with weight-average molecular weight of 3.074×105 Da.HPLC data showed that TPS consisted of mannose(45.5%),glucuronic acid(5.5%),glucose(10%)and galactose(39%).FT-IR results displayed TPS possessed the characteristic peaks of polysaccharide.Methylation and NMR analysis indicated that TPS was a-and P-glycosidic bond pyranose,and the main chain linkage type were?6-?-D-Galp-1?5-?-D-Manf-1?5?6-p-D-Manf-1?5?6-?-D-Manf-1?,the branched chain linkage type were ?-D-Glcp-1?4-?-D-Glcp-1???-D-Galf-1? and ?-D-Glcp-1?.The effect of TPS on the proliferation of L02,A549,MCF-7 and CT26 cells was measured by MTT assay.After treated with TPS for 24 h,no significant inhibitory effect was observed on L02 cells,TPS significantly inhibited the proliferation of A549,MCF-7 and CT26 cells in a dose-dependent manner.In addition,the inhibition rate of CT26 cells was the highest,reaching 34.28%at a TPS concentration of 800 ?g/mL.AO/EB and Hoechst33258 staining results showed TPS significantly induced the apoptosis of CT26 cells in a dose-dependent manner.Compared with the antitumor activity of EPS 1-1,TPS has the lower activity,and the anti-colon cancer molecular mechanism of EPS1-1 was further studied.2 Study on the molecular mechanism of EPS1-1 inducing apoptosis of CT26 cells and inhibiting the occurrence and development of AOM/DSS-induced colorectal cancerIn vitro:EPS 1-1 could activate the AMPK pathway in a time-and dose-dependent manner.?.After treated with EPS 1-1(0.6 mg/mL)in siRNAAMPKa-transfected CT26 cells,the expression level of AMPKa and p-AMPKa protein was slightly increased.Compared with EPS1-1 group,MTT and trypan blue assays results showed that the activity of CT26 cells after AMPKa-knockdown increased to 70.8%and 69.47%(P<0.05),respectively,indicating AMPK inhibition weakened the inhibition effect of EPS 1-1 on CT26 cells;ELISA and Caspase 9 assays results manifested that EPS1-1 reversed the decreased apoptosis rate of CT26 cells caused by AMPK? knockdown,which was lower than of EPS 1-1 treatment alone,indicating that AMPK? knockdown inhibited the toxicity of EPS1-1 on CT26 cells.?.Compared with AICAR-treated CT26 cells,the activation of AMPK was enhanced after the treatment of AICAR and EPS 1-1;MTT and trypan blue assays results expressed that the activity of CT26 cells decreased to 49.07%and 42.72%(P<0.05),respectively,after the addition of EPS1-1;ELISA and Caspase 9 assays results showed that the apoptosis of CT26 cells was significantly enhanced after the treatment of AICAR and EPS 1-1,indicating that the overactivation of AMPK strengthened the induction of EPS 1-1 on CT26 cells apoptosis.?.EPS 1-1 significantly inhibited the inhibition of NAC on intracellular ROS in CT26 cells.Compared with the treatment of EPS1-1 alone,the phosphorylation level of AMPK?,LKB1 and ACC was blocked by NAC,the addition of EPS 1-1 reversed the phenomenon;MTT and ELISA assays results showed that the inhibitory rate of CT26 cells decreased to 31.41%from 40.41%(P<0.05),and the apoptosis rate of CT26 cells decreased by 0.74 times(P<0.05)after the treatment of NAC and EPS1-1.These results indicated that ROS are necessary factor in the process of the activation AMPK by EPS 1-1,thus regulating CT26 cells proliferation and apoptosis.IV.The phosphorylation level of AMPKa was significantly inhibited by the knockdown of LKB1 in CT26 cells;After treated with EPS 1-1,the expression of p-LKB1 and p-AMPKa protein was up-regulated;Compared with siRNA LKB1-transfected CT26 cells,MTT and ELISA assays results showed that the inhibitory rate of CT26 cells decreased to 77.58%(P<0.05)and the apoptosis rate of CT26 cells increased by 6.36 times(P<0.05).These results indicated that LKB1 is the upstream signal of EPS1-1-induced AMPK activation,which further regulates the proliferation and apoptosis of CT26 cells.V.After treated with EPS 1-1,the expression level of p-p70s6k,p-4E-BP1 and Bcl-2 proteins significantly decreased in CT26 cells;The increase of the expression level of p-p70s6k,p-4E-BP1 and Bcl-2 proteins caused by AMPKa knockdown and the treatment of Compound C was reversed by the addition of EPS 1-1;After treated with AICAR and EPS1-1,the expression of Bcl-2 protein decreased sharply;In siRNA mTOR-transfected and rapamycin treatment CT26 cells,the expression level of p-p70s6k,p-4E-BP1 and Bcl-2 proteins decreased,and the viability of CT26 cells decreased to 76.16%and 85.22%,after addition of EPS 1-1,the cell viability again decreased to 38.61%and 50.03%,compared with the treatment of EPS1-1 alone,the cell inhibition rate fell by 30.86%and 20.10%(P<0.05),the cell apoptosis rate appeared similar phenomenon;Compared with the control group,the viability decreased to 76.79%in siRNA Bcl-2-transfected CT26 cells,and compared with the treatment of EPS 1-1 alone,the activity decreased to 51.72%in siRNA Bcl-2-transfected and EPS 1-1 treatment CT26 cells,and the apoptosis rate significantly increased;The phosphorylation of Raptor induced by EPS 1-1 was inhibited by AMPK? knockdown,which was reversed by AICAR treatment;IP results showed that EPS 1-1 could directly induce the combination of AMPK? and p-Raptor.These results indicated that EPS1-1-induced AMPK activation is involved in the activation of mTOR and the expression of Bcl-2 through the direct phosphorylation of Raptor,thereby regulating the proliferation and apoptosis of CT26 cells.?.The expression level of p-JNK,p-ASK1 and p53 induced by EPS1-1 was inhibited by the knockdown of AMPKa in CT26 cells;After the treatment of AICAR,the phosphorylation of JNK was enhanced in CT26 cells;IP results showed that the complex of AMPKa and ASK1 induced by EPS1-1 was reversed by AMPK? knockdown;After treated with SP600125,the expression of p53 protein was inhibited,the inhibitory rate increased to 75.73%,and the apoptosis rate decreased by 0.77 times.These results showed that EPS 1-1 induced AMPK/ASK1 association to activate JNK-p53 signal axis,which further regulates the proliferation and apoptosis of CT26 cells.?.EPS 1-1 could significantly up-regulate the mRNA and protein expression levels of ATF4,CHOP and GRP78 in CT26 cells,indicating that EPS1-1 can induce ER stress in CT26 cells.In vivo study:A phased model of colorectal cancer in mice induced by AOM/DSS to study the molecular mechanism of EPS 1-1-inhibited the occurrence and development of colon cancer.The statistic results of the average number,average size and colon length of tumors at different groups showed that EPS 1-1 could significantly inhibit the growth of colon tumors,with the ten weeks being the most significant;HE staining results of colon,liver and stomach tissues of mice in each group showed that EPS1-1 could alleviate the damage caused by AOM/DSS,with the ten weeks being the most significant.Immunohistochemical results showed that EPS 1-1 could significantly reduce the positive expression of p-4E-BP1 protein and up-regulate the positive expression of p-AMPKa?p-LKB1?p-ACG?p-JNK?p53?GRP78?ATF4 and CHOP in colon tissues of colon cancer mice at different groups,and the ten weeks were more significant;qPCR and western bolt analysis indicated that the expression of p53 mRNA and protein was up-regulated by EPS 1-1 in colon cancer tissues of mice.These results indicated that EPS1-1 could inhibit the occurrence and development of AOM/DSS-induced colon cancer in mice through AMPK/mTOR,JNK-p53,and-ER stress pathways,and the efficacy was mainly in the ten weeks.3 The effect of EPS 1-1 on the metabolic profile of colon cancer induced by AOM/DSSMetabolomics analysis of colon tissues in different groups of AOM/DSS-induced colon cancer mice was performed by LC-MS/MS.PC A and PLS-DA methods were used to class and find the related biomarkers interfered by EPS1-1 in CK group,Model group and EPS 1-1 group.The results showed that the separation trend of three groups was relatively obvious in the ten weeks of both positive and negative ion mode,while the separation trend was not obvious in the nineteen and twenty-eight groups;The number of metabolic pathways in each group was 124,23,and 9,respectively,in EPS1-1 vs Model;The number of metabolites in different groups was 176,23,and 37,respectively,in EPS 1-1 vs Model.These results indicated that the metabolic effect of EPS 1-1 on AOM/DSS-induced colon cancer was mainly concentrated in the ten weeks,and the biomarkers of the ten weeks were then screened and identified according to the criteria of VIP>1.0 and P?0.05.A total of 30 potential biomarkers were identified in the ten weeks,and they presented the obvious trend;In the Model vs CK,the levels of these potential biomarkers were up-regulated or down-regulated,while the content showed different degrees of callback in EPS 1-1 vs Model,which was close to the CK group;A total of 11 metabolic pathways including TCA cycle,arginine biosynthesis,purine metabolism,pyrimidine metabolism,ascorbic acid and uronic acid metabolism were involved.These results indicated that EPS 1-1 exerts its pharmacological effect on AOM/DSS-induced colon cancer through a variety of metabolic pathways,and mainly in the ten weeks,which provides a scientific basis for the development of EPS 1-1 as a treatment or adjuvant therapy for colon cancer.In summary,TPS can inhibit the proliferation of CT26 cells and induce apoptosis,but the activity is lower than EPS 1-1.EPS1-1 can inhibit colon cancer in vivo and vitro via AMPK/mTOR,JNK-p53,ER stress pathway,and various metabolites and metabolic pathway in colon tissues,providing a scientific basis for the development of EPS 1-1 as a drug for the treatment or adjuvant treatment of colon cancer.