Research On The Purification And The Anti-Tumor Mechanism Of An Polysaccharide From Grifola Frondosa

Grifola frondosa is the fungus which is valuable on the precious food and medicinal. The polysaccharides are the main functional components. They are famous on their antitumor activity, immunomodulatory activity, anti-bacterial and other activities. Nowdays, some polysaccharides, which have been isolated and characterized from Grifola frondosa could inhibit proliferation of tumor cells. However, the research on their anti-tumor mechanism is scarce.In this study, an anti-tumor polysaccharide from Grifola frondosa (GFD) had been purified by ultrafiltration and Sephadex chromatography. GFD was a kind of polysaccharide homogeneous component and its molecular weight was 30 kDa which was confirmed by agarose gel electrophoresis and HPLC. The physicochemical property analysis demonstrated that GFD could be a kind of non-starch a-pyran polysaccharide without reductive carbonyl, phenolic hydroxyl, free or a combination protein and nucleic acid. The height of carbohydrate chain was l nm. The activity against HepG2 cells of GFD was determined by MTT assay, which showed that the anti-tumor process was dose-dependent. The results of HE staining, AO/EB staining, Hoechst 33342 staining and scanning electron microscopy indicated that GFD induced a typical apoptosis in HepG2 cells. Flow cytometry analysis also confirmed that GFD caused apoptosis of HepG2 cells through cells arrested at S phase and the apoptotic rates significantly were increased over time.The results of immunofluorescence and Western blot showed that the expression of Notchl was inhibited, the phosphorylated-AktSer473 was down-regulated, and the phosphorylated-JNK was up-regulation due to GFD. GFD had effect on HepG2 cells together with Notchl inhibitors(DAPT), JNK inhibitors(SP600125) and Akt inhibitors(LY294002), respectively. The results showed that Notchl, JNK and Akt involved in the process of cell apoptosis caused by GFD. In addition, the activation of phosphorylated-JNK inhibited NF-κB/p65 to enter the nucleus. Simultaneously, the expressions of FLIP and Bcl-2(target genes of NF-κB) were down-regulated, and then initiated the activation of Caspase cascade. Finally they caused HepG2 cell apoptosis.