Study On Anti Colon Cancer Activity And Molecular Mechanism Of Cultured Cordyceps Sinensis Polysaccharide

Cordyceps sinensis(Berk.)Sacc(C.sinensis)has been used as a traditional Chinese medicine for hundreds of years.C.sinensis,together with ginseng and cartialgenous,is often regarded as the three tonics,which have great market potential and medicinal value.In this study,cultured Cordyceps sinensis polysaccharide(CSP)was used as the research object to detect the influence of CSP on the proliferation of colon cancer cell line HCT-116,and its possible anti-tumor mechanism was analyzed by RNA-seq and bioinformatics analysis technologies.According to the analysis results,it was speculated that CSP might play an anticancer role by inducing apoptosis and autophagy of cancer cells.This hypothesis was subsequently verified by AO-EB staining,western blot and dual-fluorescence mRFP-eGFP-LC3,and the pathway of apoptosis and autophagy induced by CSP was explored.In addition,the crosstalk between apoptosis and autophagy induced by CSP was further investigated in this study.The main research conclusions are concluded as follows:1.The inhibitory effect of CSP on HCT-116 cells was detected by CCK-8.The results showed that CSP significantly inhibited the proliferation of colon cancer cells in a dose-and time-dependent manner,but it was not cytotoxic to IEC-6.2.The transcriptome information of HCT-116 cells treated by different concentrations of CSP was obtained by RNA-seq and bioinformatics analysis.There were 856 differentially expressed genes between the 800 ?g/mL CSP-treated group and control group,of which 330 genes were up-regulated and 526 genes were down-regulated.The results of GO analysis and GSEA showed that gene sets such as autophagy,apoptosis,glucose deprivation and cell death were significantly up-regulated after CSP treatment,and gene sets such as autophagosome formation and lysosomes were significantly down-regulated.3.CSP treatment significantly increased the apoptosis rate and autophagy rate of HCT-116 cells,and increased the expression of proteins related to apoptosis and autophagy.Through mRFP-eGFP-LC3 plasmid transfection experiment and autophagy inhibitor experiment,it was found that CSP induced autophagy,inhibited the formation of lysosomes and autolysosomes,blocked autophagy degradation.In summary,CSP induced apoptosis and autophagy in HCT-116 cells,partially blocked autophagy flux.4.The mechanism of autophagy and apoptosis induced by CSP was further explored based on RNA-seq data.Our experiments showed that CSP decreased the expression of PI3K and phosphorylation level of AKT and mTOR,increased the expression of AMPKa and phosphorylation level of ULK1.In conclusion,CSP may induce apoptosis and autophagy by regulating the AMPK-mTOR-ULK 1 and PI3K-AKT-mTOR pathways.5.The mechanism of autophagy in the inhibition of HCT-116 cells proliferation by CSP was verified by autophagy inhibitors.The data showed that repression of autophagy flux by autophagy inhibitors enhanced CSP induced apoptosis and cell death in HCT-116 cells,but repression of autophagic occurrence did not change CSP induced cancer cell death.It suggested that CSP inhibited the proliferation of cancer cells by inducing apoptosis and blocking autophagy flux,which might be achieved through AMPK-mTOR-ULK 1 and PI3K-AKT-mTOR pathways.