Isolation, Purification And The Effect Of Proliferation And Apoptosis For HepG2 Cells Of Polysaccharides In Mistletoe

Hepatocellular carcinoma (HCC) is easy to relapse and has the high mortality rate as a malignant tumor. Surgery and chemotherapy are widely recognized in treating HCC, but the effect is not obvious, so the mortality rate still remains higher. Hepatitis B is a highly communicable disease caused by hepatitis B virus (HBV) infection, and will ultimately develop liver cirrhosis even HCC. The treatment of HBV drug is expensive and weakened immunity. Therefore it is important to develop the anti-tumor and anti-virus of functional foods and drugs with a natural non-toxic, killed tumor cells and immuno-enhancement.Mistletoe is a variety of biological activities of medicinal plants, and the active component such as polysaccharides or alkaloids, also proved to have the effect of anti-tumor, anti-virus, anti-radiation, anti-oxidation and anti-infection. We isolated and purified three fractions in mistletoe in this study, and studied the mechanisms of anti-tumor and anti-virus, and analyzed PPI and pathways under the polysaccharide-treated HepG2 by proteomic and bioinformatic approach. The main results are as follows:(1) A crude polysaccharide was obtained in mistletoe by hot water extraction and ethanol precipition. We isolated and purified three fractions named VCP1, VCP2 and VCP3 in mistletoe using DEAE-cellulose chromatography. Molecular weight (Mw) and monosaccharide composition were measured by gel filtration chromatography and HPLC, respectively. VCP1 (32 KDa) was composed of Glc (30.6%), Gal (34.3%), Ara (14.9%), Rha (1.7%), Man (18.5%), while VCP2 (280 KDa) were consisted of Glc (8.4%), Gal (14.5%), Ara (43.2%), Rha (6.3%), Man (7.0%), GlcA (1.8%), GalA (18.8%) and VCP3 (21 KDa) were consisted of Glc (5.6%), Gal (10.5%), Ara (33.3%), Rha (13.8%), Man (4.4%), GlcA (1.3%), GalA (31.1%). The proteins and nucleic acid were absence in the polysaccharide fractions by UV determination. The optical rotation were-0.163、1.234 and 0.395, and the specific rotation were-2.040、40.726 and 65.833. The structure of three fractions were measured by FT-IR and NMR spectrum. The characteristic absorptive peaks of three fractions were found and VCP1 belong to neutral polysaccharide, VCP2 and VCP3 is RG-1 type pectin. VCP2 is mainly consist of GalA, Rha, Gal and Ara, the types of backbone were α-1,4-GalA and α-1,2-Rha, and the types of side chain were Ara and Gal linked on Rha. The degree of esterification was 50%.(2) Three polysaccharide fractions could inhibit HepG2.2.15 cells proliferation using CCK-8 kit, and VCP2 showed the strongest anti-proliferation activities. VCP2 could inhibit the copy of HBV-DNA using FQ-PCR and the secretory of HBsAg and HBeAg using ELISA, and all of them showed anti-proliferation in a dose-dependently manner and significantly lower than Lami. So the polysaccharide in mistletoe should develop assistant agents for treating HBV to improve effect.(3) Three polysaccharide fractions could inhibit HepG2 cells proliferation using CCK-8 kit, and VCP2 showed the strongest anti-proliferation activities. Cell cycle showed G1 phase cells increased in HepG2 cells treated with VCP2 by PI and apoptosis showed VCP2 could induce apoptosis by Annexin V-FITC/PI. qRT-PCR and Western blot showed the expressions of mRNA and protein of p21 and CyclinD in HepG2 cells treated with VCP2 were up-regulated, and the expressions of mRNA and protein of CyclinE and CDK4 were down-regulated, so the cells in G1 phase increased and cell cycle arrest in G1 phase, then proliferation of HepG2 cells were inhibited. In addition, the expressions of mRNA and protein of Smac and Caspase-3 were up-regulated and down-regulating the expressions of mRNA and protein of Bcl-XL and XIAP, so apoptosis was induced. However, these effects may be not obviously related to Bad (p<0.05).(4) The 113 differentially expressed proteins identified by iTRAQ and LC-MSMS between HepG2 cells and VCP2-treated HepG2 cells, the expressions of 59 proteins were up-regulated, and the expressions of 54 proteins were down-regulated.1355 biological processes (BP),186 cell components (CC) and 218 molecular functions (MF) were significantly enriched for GO dataset (p<0.05).27 KEGG pathways were significantly enriched for KEGG dataset (p<0.05). PPI analysis revealed differentially expressed proteins interaction relations,and these results are laying the foundation for screening of tumor molecular markers.