Structural Analysis And Study On Antitumor Effect Of Letinous Edodes β-glucan

Letinous edodesβ-glucan is an anticancer drug commonly used in clinic.This study analyzed the structure ofβ-glucan isolated from different parts of letinous edodes by periodate oxidation,Smith degradation,gas chromatography,high performance liquid chromatography,infrared spectroscopy,thermogravimetric analysis and Congo red reaction.The results showed that the intracellular neutral glycosidic bond was composed of 1-4 bond with 78.84%,1-6bond with 14.98%,1-3 bond with 6.17%per mol hexose residue.The cell wall neutral glycosidic bond consisted of 1-4 bond with 42.40%,1-6 bond with 48.51%,1-3 bond with9.951%per mol hexose residues.The intracellular acidic glycosidic bond was composed of 1-6 bond with 65.692%,1-3 bond with 34.308%per mol hexose residues.The cell wall acid sugar had 1-6 bond with 78.397%,1-3 bond with 21.603%per mol hexose residue.The relative molecular weight of intracellular acid glucan is 527881.9 Da,and cell wall acid glucan is 562847.4 Da.Infrared spectrum shows that the absorption peak of C-H variable angle vibration of beta terminal-group epimerism found at 854 cm^-1 witch is classic absorption peak of beta glucoside bond dextran.The thermogravimetric analysis of the thermal decomposition temperature of the neutral glucan and the cell wall acid sugar of letinous edodes was 310.8,292.9,respectively,and there were some differences,which further verified that the neutral glucan was different from acid glucan.The cytotoxicity of letinous edodesβ-glucan sample（Lewp02）was detected by MTS.The results showed that the letinous edodesβ-glucan can promote apoptosis of hepatoma Hepa1-6-cell line.High concentration of Lewp02 can block Hepa1-6 from G0/G1,reduce S and G2/M ratio,and significantly inhibit the proliferation and proliferation of Hepa1-6 cell line.Lewp02 can upregulate the expression of TNF-alpha in Raw264.7,thereby enhancing the inhibition of Hepa1-6 proliferation.While Lewp02 has no cytotoxicity to normal cell L929,nor does it affect cell cycle.The results indicate that Lewp02 is safe as an adjuvant for cancer.The transcriptional levels of cellβ-glucan receptors gene were detected by Semi-RT-PCR.The expression ofβ-glucan receptors gene were significantly different between Hepa1-6 and Raw264.7 cells.Hepa1-6 did not express Dectin-1,and TLR2,TLR6,CD36 and CR3expression levels were low,but TLR4 transcriptional level was highly expressed.CD36,CR3,Dectin-1,TLR2,TLR4 and TLR6 were all highly expressed in Macrophage Raw264.7 cells.Lewp02-FITC can be"endocytosis"into Hepa1-6 and Raw264.7 cells though flow cytometry.Compared with Hepa1-6 cells,Lewp02-FITC can be"endocytosis"into Raw264.7cells faster.The endocytosis ratio were 81.56%,91.58%,98.6%respectively at the 1h,2h 4h time points,which is closely related to the transcription of a variety of receptor genes on the surface of Raw264.7 cells.Confocal laser scanning results showed that Lewp02-FITC distributed in the cell membrane and cytoplasm,but not in the nucleus was.These results provide important informations for further study of the direct influence of Lewp02 on the proliferation and differentiation of tumor cells.Under the co-culture system of Raw264.7 and Hepa1-6,Raw264.7 can effectively activate IL-6 and TNF-alpha cells.TNF-alpha can be increased to about 57 folds at the maximun.The secreted cytokines can effectively promote the apoptosis of cancer cells.