Antitumor Activity And The Mechanism Of Glycoprotein From The Mycelia Of Tricholoma Matsutake

Tricholoma matsutake is a kind of rare, edible and medicinal fungus in the world. Itcontains active substances which can enhance immunity and have antitumor activity. In thispaper, the separation and purification of the glycoprotein from submerged culture ofTricholoma matsutake, the structure of the glycoprotein, the in vitro antitumor activites, theantitumor effect combined with 5-Fu, the mechanism of antitumor and the acute toxicity werestudied.The crude extract by extracting a broth of Tricholoma matsutake mycelia with cold waterwas separated and purified by DEAE-Sepharose Fast Flow and AKTA ion exchangechromatography. A homogenous fraction MTS03 was got and showed white flocculesubstance after freeze-drying. It could be easily dissolved in water but not in organic solventsuch as ethanol and ether. The carbohydrate content was 12.46% in mannose equivalent, andthe protein content was 86.53% in albumin equivalent. The molecular weight was 22908.93estimated by MALDI-TOF-MS. The IR, UV spectrum showed that MTS03 was aglycoprotein with α-glycoside and O-glycosylation. It was consisted of 18 kinds of aminoacids identified by amino acid auto analysis and composed of arabinose, xylose, mannose,glucose, galactose with the molar ratio of 1.46: 2.22: 7.74: 1.29: .61 in its polysaccharidecomponents identified by GC. The comprehensive study on peptide mass fingerprinting withthe molecular weight and its property of having no activity of trypsin inhibitor showed apreliminary result that MTS03 was a novel glycoprotein.The inhibition activity of glycoprotein MTS03 on tumor cells growing, its antitumorchart, and its cytotoxic effect on human liver cell L-02 were studied with MTT method. Thethermal stability and the repeatability of glycoprotein on inhibiting activity extracted fromdifferent batch of broth were syudied. The IC50 of S180, MCF-7, Bel-7402 and L-02 cellswere 26.64,24.32,77.98,1366.49 μg/mL when the cells treated with MTS03 for 24 h, 15.02,9.37,25.28,190.60 μg/mL for 48 h, and 8.18,7.55,11.39,97.21 μg/mL for 72 h respectively.The therapeutic index of S180, MCF-7, Bel-7402 were 51.29,56.19,17.52 for 24 hrespectively. The toxicity on L-02 cells was increased along with the time prolonged, highconcentration and long time increased the toxicity on normal cells, and accordingly decreasedthe therapeutic index. MTS03 exhibited excellent inhibition activity to many kinds of tumors,especially the MCF-7, HL-60, C6 cells. MTS03 was stable in room temperature but unstablewhen the temperature was over 56℃. The MTS03 extracted from the different batch of brothexhibited the same antitumor activity, it proved that the process of producing MTS03 wassteady and feasible.The effects of MTS03 combined with 5-Fu on MCF-7 and Bel-7402 cell in vitro wasstudied. It had no antagonistic effect each other (q>0.85), and showed excellent synergisticeffect on two tumor cells at appropriate concentration (q>1.25). The sensitivity of MCF-7 cellto 5-Fu was improved when 5-Fu was combined with MTS03. The IC50 of 5-Fu in MCF-7cells was decreased from 6.37 μg/mL individually to 0.45 μg/mL, 0.37 μg/mL in combinationwith 2.5, 5 μg/mL of MTS03 respectively. Five-Fu had no or little toxic effects on humanliver L-02 cells when it was used in this way at low combination concentrations. It presentedsatisfactorily synergistic and reducing toxicity effects.The mechanism of MTS03 on MCF-7 cells was studied. MCF-7 cells treated withMTS03 for 48h were dyed by HE showed significant morphological changes, such as cellshrinkage, especially the nuclear fragmentation. The cells under the scan electron microscopypresented the same results as the morphological changes. The villus were disappearedpartially at lower concentration and totally at higher. The surface of membrane was smoothand shrinkage to form apoptotic bodies. The Annexin V-FITC staining kit was used to detectapoptosis and necrosis by Flow cytometry, the stained cells treated with MTS03 were mainlyAnnexin V-FITC positive cells, only a small amount of cells were stained by PI or AnnexinV-FITC and PI, and only little L-02 cells were stained by FITC-Annexin V or PI. Thetreatment with MTS03 on MCF-7 cells resulted in the accumulation of the cells in the S phase,in the prevention of the cell growing from S phase to G2/M phase, and in the stoppage of thecells on duplication and division. The expression change of genes p53, Bcl-2, Bax and Fas inMCF-7 cells after 48 h treated with MTS03 were analyzed. The expression level comparingwith the cells untreated there were no effect on p53 and Bcl-2 but a distinct promotion on Baxand Fas from 2.73%, 0.81% to 39.6%, 28.4% respectively. The mechanism of antitumoractivity might have relationship with up-regulated expression of Bax and Fas.Horn' method was used to test the acute toxicity of MTS03. In single ip management tomice, LD50 was 825 mg/kg and its 95% confidence interval was 459~1480 mg/kg. At the doseof 1000 mg/kg, the hair of the mice was lackluster and they had little movement, the bodyweight was lighter than control, lung, brain and kidney index had differences compared withcontrol significantly (P<0.05), the else dose of mice had no differences and was in the normalrange. The blood cells of all groups of mice injected MTS03 had no evidently differencecompared with control (P>0.05). According to the paraffin slice of liver, kidney, brain, lung ofmouse, the liver of mice injected 1 g/kg of MTS03 presented vascular dilatation hyperaemia,Neutrophil and lymphocyte soakage in central vein and klatskin, kidney cortex presentedmultifocal fibrous tissue hyperplasia, neutrophil, plasma cell and lymphocyte soakage, and itappeared inflammation. There were no pathological changes in the dead mice expect thatsome kidney presented renal tubular dilatation and albumin casts, as well as there were nopathological changes in the lung and brain whether the mice were alive or not.