Anti-tumor Mechanism And Primary Structure Of Lentinus Edodes C91-3-A

Objective:Lentinus edodes (Berk) is one of valuable funguses,which has been regarded as food and medicine .In fact,it has some effect such as adjust the immune system,antivirus and antitumor ect. .Our seminar had studied the Lentinus edodes for many years and got the branch C91-3.We found the protien component of its fermentantion liquor(LFP91-3)not only can inhibit tumor in vivo but also can kill tumor cells directly .LFP91-3-A is a protein which was purified from LFP91-3.Some data show that LFP91-3-A is able to inhibit the growth of H22 cell and induce H22 cell apoptosis.Based on this ,we study aim to observe whether LFP91-3-A induces apoptosis of cell H22 through both the activation of Caspase-3 and suppression the telomerase activity and investigate the mechanism of LFP91-3-A on antitumor effects.To determin the primary structure of LFP91-3-A.Methods:The H22 cell were divided into two groups(control groups,LFP91-3-A groups).The spectrophotometric and TRAP-PCRsilver staining were used to detect the expression levels of Caspase-3 and telomerase when the two groups cell were treated 0h,24h,48h,72h. The LFP-91-3-A protein was electrophoresis by SDS-PAGE ,cutting LFP91-3-A protein bands and using MALDI-TOF-MS to assay it`s PMF. The Q-TOF2 were used to meaure the amino acid arrangement sequence of LFP91-3-A and the results were researched in the NCBI data base.Results:The Caspase-3 activity were increased(0.159 vs0.043 P<0.05) at the same time telomerase activity were decreased after the H22 cell were induced 24h. The Caspase-3 activity rose gradually, while telomerase activity reduced progressively with the action time goes on. The Caspase-3 activity achieved the biggest(0.423 vs 0.046 P<0.05) and telomerase activity almost attained negative after 72h.The activation of Caspase-3 compared with other times,which had significant difference (P<0.05). The MALDI-TOF-MS were used to measure LFP91-3-A PMF ,and had not results by researching from NCBI data base. The Q-TOF2 were used to meaure the amino acid arrangement sequence of LFP91-3-A and the results is Pro-Thr-Gln-Phe-Thr-Asn-Asn-Leu-Tyr-Ala-ArgN-C(N-C), Ala- Pro-Gln-Gln-Val-Glu-Trp-Leu-Phe-Tyr-Lys-Lys ( N-C ) .LFP91-3-A is confirmed an unknown protein through indexing from the NCBI data base.Conclusion:The activition of Caspase-3 and inhibition of telomerase activity may be one of the important mechanisms in inducing apoptosis of LFP91-3-A in H22 cell treatment.The increased Caspase-3 activity depended on the time. Two peptide amino acid arrangement sequence of LFP91-3-A is Pro-Thr-Gln-Phe-Thr-Asn-Asn-Leu-Tyr-Ala-ArgN-C(N-C),Ala-Pro-Gln-Gln-Val-Glu-Trp-Leu-Phe-Tyr-Lys-Lys( N-C) .LFP91-3-A is confirmed an unknown protein through indexing from the NCBI data base.