Purification, Characterization And Inducing Tumor Cell Apoptosis Activity Of A Polysaccharide From Ganoderma Applanatum

Ganoderma applanatum,a fungus that has been used in Traditional Chinese Medicine?TCM?because of its medicinal values,including analgesic,hepatoprotective,immunomodulating and antitumor activities.Polysaccharides is an important active component of Ganoderma applanatum,with a variety of biological activities of anti-oxidation,immunomodulatory,antitumor,anti-diabetic and anti-aging properties,but the exact anti-tumor mechanism is still unclear.In this study,we aim to clarify the antitumor mechanism of Ganoderma applanatum polysaccharides,and describe the cell signal transduction pathways which are involved in the anti-tumor process through four experimental parts.1:Isolation,purification and structural characterization of polysaccharide from Ganoderma applanatumResponse surface methodology and single factor experiments were used to optimize hot water extraction conditions of the crude polysaccharides from Ganoderma applanatum.The crude polysaccharides from Ganoderma applanatum were further purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephacyral S-300 HR size-exclusion chromatography.The purified polysaccharides were designated as GAP-3S.The molecular weight of GAP-3S was determined by high performance gel permeation chromatography?HPGPC?.The monosaccharide composition of GAP-3S was analyzed by high performance liquid chromatography?HPLC?.Results:The single factor experiment and response surface optimization of the conditions of hot water extraction of Ganoderma applanatum crude polysaccharides were that:ratio of material to water was 43:1?mL/g?,time of extraction was 4.5 h,temperature of extraction was 100?.Under these process parameters,the extraction rate of polysaccharides was 5.03%.The molecular weight of GAP-3S was about 6.82×10⁵ Da,which was mainly composed of glucose?60.1%?,galactose?22.1%?,fucose?9.3%?and xylose?8.5%?.Infrared spectrum analysis showed that GAP-3S contained?-furanose and other structures.2:Effects of GAP-3S on MCF-7 cellsThe MCF-7 cells were treated with different concentrations of GAP-3S?0,125,250,500?g/mL?and the proliferation rate of MCF-7 cells was determined by MTT assay.PI staining,Hoechst 33258 and Annexin V-FITC/PI double staining were performed to determine the effects of GAP-3S on apoptosis and cell cycle arrest in MCF-7 cells.Western Blotting were performed to detect the expression of apoptosis-related proteins in MCF-7 cells,and the activities of caspase-3,-8,and-9were measured by kits.Results:GAP-3S inhibited the proliferation of MCF-7 cells in a dose-and time-dependent manner.A series of morphological studies?hoechst 33258 staining,crystal violet staining observed cell morphology and cell scratch observation of cell migration?showed that GAP-3S could induce chromatin condensation,edge eclosion and the decline of migration capacity in MCF-7 cells,cause G0/G1 phase cell cycle arrest and increase the percentage of apoptotic cells in MCF-7 cells.Real-time fluorescence quantitative PCR and Western Blotting results showed that GAP-3S significantly up-regulated the protein expression of P53,P21,cleaved-caspase-3,caspase-9 and cleved-PARP.GAP-3S could also increased the activities of caspase-3and caspase-9,which is consistent with the above results.However,there was no significant change in the activity of caspase-8,indicating that the mitochondrial apoptotic pathway may play an important role in the apoptosis of MCF-7 cells induced by GAP-3S.3:Mitochondrial mechanism of GAP-3S induced Apoptosis in MCF-7 cellsMCF-7 cells were treated with different concentrations of GAP-3S?0,125,250and 500?g/mL?,and content ROS,Ca²⁺concentration,mitochondrial transmembrane potential and antioxidant enzyme activities of MCF-7 cells were determined by kit.Western Blotting were performed to detected the expression of Bax,Bcl-2 and Cyt-C in mitochondria and cytoplasm.Real-time fluorescence quantitative PCR was used to detect mRNA expression of Bax and Bcl-2.Results:GAP-3S could induce the loss of mitochondrial membrane potential?MMP?and increase the accumulation of ROS and Ca²⁺,as well as increase the activities of SOD,GPx,and the concentration of GSH.GAP-3S could significantly increase the mRNA and protein expression of Bax,decrease the protein expression of Bcl-2.Meanwhile,GAP-3S could affect the subcellular localization of Cyt-C in MCF-7 cells and promote the release of Cyt-C from the mitochondria to the cytoplasm.These results suggest that mitochondrial apoptosis pathway plays an important role in GAP-3S induced apoptosis in MCF-7 cells.4:Effects of GAP-3S on MAPK and NF-?B signaling pathway in MCF-7 cellsThe nuclear translocation of NF-?B after GAP-3S treatment of MCF-7 cells was detected by kit.The protein expression levelso f I?B,P38,JNK,ERK and p-I?B,p-P38,p-JNK and p-ERK were determined by Western Blotting.Results:The p38 signaling pathway,an important component of the MAPK family,plays an essential role in regulating many cellular processes,including inflammation,cell stress,and apoptosis.The ERK and JNK signaling pathways also belong to the MAPK family.ERK signaling pathway is generally involved in cell proliferation,whereas JNK signaling pathway is associated with cell stress and apoptosis.The phosphorylation level of p38 and JNK was increased,whereas the phosphorylation levels of ERK were decreased after treatment with GAP-3S,GAP-3S could also increase the phosphorylation level of I?B protein and promote the nuclear translocation of p65.These results suggest that the MAPKs and NF-?B signaling pathway might be involved in GAP-3S-induced apoptosis in MCF-7 cells.In conclusion,when MCF-7 cells receive extracellular signal stimulation of GAP-3S,MAPK signaling pathway is activated and the extracellular stimulus of GAP-3S is transmit into the cell in a cascade-amplified manner.NF-?B signaling pathway continues to transmit the stimulation signal to the nucleus after receiving the upstream MAPKs signal and initiate programmed cell death,activate mitochondrial pathway,and ultimately induce apoptosis.