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

Objective: Our seminar had studied the lentinus edodes for many years and got the branch C_91-3. The broth of mycelium extract protein LFP91-3 was measured by anti-tumor activity which confirmed not only inhibit tumor growth in vivo still has a significant role in vitro kill the tumor cells directly. This study was designed on separating and identifying the components of protein to obtain a more efficient anti-tumor activity of proteins, and then learn more about anti-tumor effect and mechanism of action of lentinus edodes C_91-3. Lay a good foundation for clinical application.Methods: Using bio-engineering fermentation technology to obtain a large number of C_91-3 mycelia broth, the procedure of purified involving salting-out, dialysis, Freeze Drying, ion exchange chromatography on DEAE-sepharose FF column . using Coomassie Blue method for the determination of protein concentration in each tube, and so draw the elution curve. Under the elution curve by SDS-PAGE, electrophoresis determining the relative molecular mass. collected the eluent of single composition for freeze-dried. Adjusting the concentration of single protein were 5ug/ml,10ug/ml,15ug/ml,Respectively,determine their acicity of refraining the growth of S180 and H22 in vitro by MTT methods; Flow cytometry was used to detected 5ug/ml,10ug/ml,15ug/ml single protein induce the aptosis on S180 and H22 Cells after 24 hours ;the Spectrophotometers were used to detect the expression levels of Caspase-3; The single protein was electrophoresis by SDS-PAGE, cutting the single protein bands and using MALDI-TOF-MS to detect it's PMF. Identifying this single protein in the NCBI database. Results: With the Ion-exchange chromatography ,got the elution curve which contain 4 elution peaks, The results of SDS-PAGE electrophoresis demonstrated that protein in peakⅣwas the single protein which molecular weight is about 16kDa. MTT methods demonstrated that The protein can inhibit S180cells and H22 cells activity in vitro, and its rate of growth inhibition effect by the single protein, also it will increase with time and Concentration increased, which showed dose and time dependent. the single protein rolled in S180cells and H22 cells with different time and concentration, the lowest inhibition rate of S180 and H22 cells were 9.66±0.30,and 6.34±0.05, The highest rates of growth inhibition were 67.23±0.42 and 33.64±0.85, the inhibition of growth rate was more significantly for S180.,With the prolongation of the inhibition rate increased significantly. Flow cytometric analysis (Annexing/PI decoration methods)showed that the S180 cells and H22 cells apoptosis rates 24 hours after treatment of the separated protein of the concentration 5ug/ml,10ug/ml,15ug/ml,the early apoptosis rate was 0.82%,0.86%,1.95%and 0.41%,0.79%,1.78%separately, It showed a correlation between dose and effect; There were significant differences about Caspase-3 protein activity after the single protein inducing on S180 and H22 cells Compared each time. With the prolongation of Caspase-3 activity gradually increased, induced by the single protein after 24h, the caspase-3 protein activity of S180and H22 could increase, Reached 12.27±0.167 and 6.93±0.200 separately, Time was extended to 72h, Measured value reached highest, were 31.93±0.200 and 24.6±0.367 separately, The MALDI-TOF-MS was used to measure PMF of the single protein, there is no result for researching from NCBI data base. so it was confirmed as an unknown protein Initially.Conclusion: Using salt-out,dialyze,Freeze Drying concentrate and Ion-exchange chromatography ,we got four fractions of proteins,The forth fraction was a newly purified antitumor protein with a molecular weight of approximately 16KD,was a single protein, it had stronger inhibition to proliferation of S180 cells and H22 cells in introit was found that Caspase-3 protein activity as significantly increased by kit detected, and with time-dependent; Predicted that the anti-tumor protein may Induce tumor cells apoptosis by Activating Caspase-3 expression; By Mass Spectrometry and NCBI database search confirmed that the new protein is an unknown.