A Protein With Antitumor Activity From The Mushroom Lentinus Edodes C91-3 And The Study Of Its Anti-tumor Mechanism

Object:It is generally accepted that Lentinan as a biological response modifier (BRM) play critical roles in the antitumour effects .The enhancement or potentiation of host defense mechanisms has been recognized as a possible means of inhibiting tumor growth without harming the host. Studies on the biological activities and the antitumor mechanisim of the proteins from Lentinus edodes has not been reported. We demonstrate that the protein contents (LFP91-3) in Lentinus edodes C91-3 mycelium fermentative liquid possess antitumour activity in vivo and in vitro. In this study we try to test and separate LFP91-3 primarily, and look for the effective anti-tumor component and its possible anti-tumor mechanism.Methods:A novel antitumor protein was purified with a procedure involving ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose column ,the relative molecular mass was determined by SDS-PAGE. LFP91-3-A in vitro antitumor effect on H₂₂ cells and S₁₈₀ cells was studied: MTT colorimetric assay was used to examine the effect of different concentrations of LFP91-3 - A (5ug/ml,10ug/ml,15ug/ml)on the proliferation of tumor cells by different treatment time (24 hours, 48hours, 72 hours). Flow cytometry was used to determine the effect of LFP91-3-A induce the cell apoptosis on H₂₂ cells after 24 hours. Transmission electron microscopy was performed to examine cell apoptosis morphology.Result:The crude protein LFP91-3 put in ion-exchange chromatography on DEAE-cellulose column and divide it into 7 elutionpeaks;According to elution curve and SDS-PAGE ,a novel antitumor protein (LFP91-3 - A)with a molecular weight of approximately 35kDa was purified ;LFP91-3-A in vitro antitumor effect on H₂₂ cells and S₁₈₀ cells was studied: MTT colorimetric assay showed: that LFP91-3- A could inhibited the growth of H₂₂ cells and S₁₈₀ cells in a time- and concentration-dependent manner. Treated with 5ug/ml LFP91-3-A after 24h, the H₂₂ cells and S₁₈₀ cells'the inhibition rates were15.88%±1.09% and 28.37%±0.68%,respectively;After treated for 72 hours by 15ug/ml LFP91-3-A, the H₂₂ cells and S₁₈₀ cells'proliferations were significantly inhibited, the inhibition rates were 59.93±1.76% and 80.13±0.68%, respectively ; LFP91-3 - A induce both tumor cells apoptosis. Flow cytometric analysis(Annexin/PI decoration methods) showed the H₂₂ cells apoptosis rates 24 hours after treatment of LFP91-3-A of the concentration 15ug/ml,10ug/ml,15ug/ml, the early apoptosis rate was 0.88%, 1.83%, 8.68%, the late apoptosis rate was 2.06%, 40.60%, 56.31%;The cell nuclei became progressively pyknotic and were extensively fragmented and some characteristic morphologic features of apoptosis were revealed by transmission electron microscopy.Conclusion:From the crude protein LFP91-3 , a novel antitumor protein (LFP91-3-A)with a molecular weight of approximately 35kDa was purified. LFP91-3-A may inhibit the growth of H₂₂ cells and S₁₈₀ cells in vitro and the possible anti-tumor mechanism may be related to induction of apoptosis.So protein is another important antitumor elements in Lentinus edodes, and future studies should therefore focus on investigation of its antitumor mechanism.