Isolation Of Anticancer Active Protein Of Trichocarpus Spp. And Its Mechanism Of Killing Human Erythroleukemia K562 Cells

Arca subcrenata Lischke is a kind of Marine bivalves animal belongs to the Mollusca,Lamellibranchia,Taxodonta,Arcidae,Arca Subcrenata genera,which can be used as medicine or food.The best harvest time of Arca subcrenata Lischke was from the autumn to the next spring.The dried clean shell was known as "Concha Arcae" in traditional Chinese medicine,which had the functions of softening hardness,dissipating stagnation and relieving pain.Concha Arcae was also applied to treating dense phlegm cementation,hard coughing,goiter and tumor,scrofula,macula,mass syndrome,lumping in the abdomen,stomachache and stomach ache,et al.The soft part of Arca subcrenata Lischke has efficient effects such as enriching blood,warming middle warmer and invigorating the stomach.Eaten soft part in moderation was beneficial to enriching blood,dispeling the effects of alcohol,eliminating phlegm and so on.Recent researche showed that the extraction of Arca subcrenata Lischke had potential pharmaceutical value,including anti-tumor,enhancing the immune response,anti-oxidation,anti-bacterial and reducing hematic fat.Recently,our team had found Arca subcrenata Lischke anticancer protein(ASAP)from the extraction of Arca subcrenata had certain broad-spectrum antineoplastic effect in vitro,especially on cancers from epithelial tissue.The cytotoxicity effect and mechanism of ASAP on tumor cells from haematogenous system(bone marrow)have not been well studied.OBJECTIVE:To investigate the cytotoxic activity and mechanisms of ASAP on human myeloid leukemia K-562 cells in vitro and to further separate the active parts of ASAP.METHODS:ASAP was extracted from fresh soft parts of Area subcrenata Lischke by vacuum freeze-drying method combined with ammonium sulfate fractionation method.Bradford method(Coomassie brilliant blue G-250)was used to detected protein concentration of ASAP.Tricine SDS-PAGE was applied to determine the main molecular weight of ASAP.Morphological changes of K-562 cells treated with ASAP were observed by the inverted phase-contrast microscope.The cell and nucleus changes were analyzed by Giemsa staining.The cytotoxicity of ASAP on K-562 cells was detected by MTT assay.Annexin-V/PI double staining method coupled with FCM was employed to detect apoptosis of K-562 cells after treated with ASAP.And the ratio of cell cycle was measured by FCM with PI single staining.The expression of apoptosis and cell cycle relating to protein procaspase-3,caspase-3,p53 and Pdcd4 were analyzed by Western blotting.FPLC method combined with the Superdex200 gel column chromatography was used for further separation of ASAP.RESULTS:The protein contents of ASAP was(79.95±7.11)%mearsured by Bradford method.The result of Tricine SDS-PAGE showed that the ASAP was a multicomponent protein mixture and the molecular weight range was from 0 to 30kDa.ASAP exhibited significant cytotoxic effect on K-562 cells both in time and dose dependent manner.The concentration-effect correlation coefficient(r)of ASAP on K-562 cells for 24h,48h,72h were 0.851,0.8977 and 0.8997,respectively.The time-effect correlation coefficient(r)of ASAP on K-562 cells treated with ASAP 50,100,200,400 and 800?g·mL-1 were 0.0195,0.7043,0.9359,0.9735 and 0.9698,respectively.IC50 of ASAP for K562 cells was 200?g ·mL-1 at 48h.K-562 cells showed morphological changes such as cytomorphosis and nuclear fragmentation after 200?g·mL-1 ASAP treatment for 48h.Flow cytometry analysis and Giemsa staining assay indicated that K-562 cells'apoptotic ratio increased and G2-M phase cells'ratio accumulated significantly with ASAP concentration increasing compared with negative control group.The early and late apoptosis cell rate increased to(32.75±0.07)%and(31.25±2.19)%vs control group(3.7±1.13)%and(9.9±0.85)%(p<0.01)at ASAP 200?g·mL-1 treated for 48h group,respectively.The G2-M phase cells increased to(55.16±1.73)%vs control group(15.26±0.82)%(p<0.01)at ASAP 200?g·mL-1 treated for 48h.At ASAP 200?g·mL-1,the expression of apoptotic protein procaspase-3 was down regulated and the active form caspase-3 was significantly up regulated after 32h.Pdcd4 and p53 protein expression was down regulated significantly at ASAP 200?g·mL-1 during 0 to 40h.The most highly content in ASAP which molecular weight was 22kDa exhibited significant cytotoxic effect on K-562 cells,IC50 of this component for K562 cells was 80?g·mL-1 at 48h.CONCLUSION:ASAP was a mixture component protein with molecular weight range from 0 to 30kDa,which has cytotoxic activity on K-562 cells.By inducing cell apoptosis and cell cycle arrest in G2-M phase.ASAP induced K-562 cells apoptosis depended on caspase-3 signal pathway down-regulated expression.Pdcd4 and p53 proteins maybe related to K-562 cell apoptosis and cell cycle arrest in G2-M phase by ASAP.The recycle protein isolated from ASAP called ASAP-Re had more effective cytotoxic activity to K-562 cells than ASAP.Protein group around 22kDa is of the maximum contents in ASAP and has higher cytotoxic activity on K-562 cells than ASAP.