Asparagus Polysaccharide Exhibits Anti-tumor Activity Via Inducing Apoptosis In Myeloid-derived Suppressor Cells

Objective:To investigate the effect of Asparagus polysaccharide on Myeloid-suppressor cells and underlying molecular mechanisms using in vitro techniques,thereby indicating its potential use in cancer therapy.Methods:1.Dried tuberous roots were boiled in distilled water,the crude polysaccharide fraction was obtained through precipitation with 80%ethanol and desiccation in vacuo,protein was removed with the use of Sevag method.The UA absorption of AP?10 mg/ml?was detected from 200-700nm;the remnant of protein was detected by SDS-PAGE;analysis of monosaccharide composition by HPCE.2.MSC-2 cells and lymphocytes were treated with AP,cell viability was analyzed by MTT assay.Splenocytes isolated from tumor-bearing mice were treated with AP,the percentages of MDSCs were measured by FACS,to confirm the effect of AP on MDSCs.3.MDSCs were treated with AP and then stained with Annexin V-FITC and PI.The stained cells were analyzed with a flow cytometer.Investigated the levels of expression of various proteins involved in intrinsic and extrinsic apoptosis pathways by Western blotting.Splenocytes isolated from TLR4^-/-mice bearing tumor were treated with AP,the percentages of MDSCs were measured by FACS.Results:1.The percent yield of the asparagus was about 15%.The UA absorption of AP?10 mg/ml?was detected and the curve have no peak at 260 nm?specificity of nucleic acid?and 280 nm?specificity of protein?.The remnant of protein was detected by SDS-PAGE,it showed that no protein band appeared.Analysis of monosaccharide composition by HPCE showed three peak respectively corresponding to glucose,mannose,galactose.2.AP inhibited the growth of MSC-2 cells,the IC₅₀ for AP was 0.4919mg/ml,but promoted the growth of primary lymphocytes.AP decreased the percentage of MDSCs in the spleens of mice bearing tumors in a dose dependent manner.3.The percentage of Annexin V positive cells increased after treated with AP.The results of Western blotting showed a time-dependent increase in the expression of Caspase-9,cleaved Caspase-9,Bax and Phospho-NF-?B p65,but a time-dependent decrease in the expression of Bcl-2 levels in the MDSCs treated with AP.The results of FACS showed that AP couldn't decrease the ratio of MDSCs in spleen of TLR4^-/-mice.Conclusion:1.There was little nucleic acid and protein in AP after extracted and purified in such way,this polysaccharide was mainly composed of glucose,mannose and galactose.2.AP inhibited the growth of MSC-2 cells and primary MDSCs,however,increase the viability of the lymphocytes.Suggesting that the effect of AP is selective.3.AP promotes apoptosis in MDSCs via the intrinsic pathway and the anti-MDSCs activity of is TLR4-dependent.