Preparation Of A Novel Enzymatic Peptide From Cyclina Sinensis Visceral And The Inhibiton Activity Against Prostate Cancer DU-145 Cells

Cyclina sinensis,common name were black clams,clams,iron clams,snails and other stone,belonging to Mollusca bivalve steel,veneroida,Veneridae.The shells of them slightly rounded.Cyclina sinensis were mainly in north and south of Chinese coast area,one of the main beach shellfish in coastal areas and one of the important economic shellfish in China.They were very delicious,nutritious,containing high content of protein,low content of fat,and rich in carbohydrates,trace elements and vitamins.In addition,Cyclina sinensis had high medicinal value,such as phlegm,endometriosis,detoxification,cooling and pain relief,anti-tumor,antioxidant,immunomodulatory,anti-inflammatory and other functions.It was reported that prostate cancer(Prostate Cancer,PCa)is a disease as the most common malignant tumors associated with the male reproductive system diseases in middle-aged men.According to statistics of its high incidence,which ranked second only following to lung cancer.The main traditional treatment means including radical surgery,hormone therapy,radiation therapy,chemotherapy for prostate cancer were difficult to eradicate cancer and recurrence rate was very high.The sea is a treasure trove of marine resources.With the development and utilization of marine resources,it has an important research and clinical implications to find new anticancer drugs of efficient,low toxicity,high selectivity and strong specific features from the ocean in recent years.In the study,we got the visceral of Cyclina sinensis as experimental material and cultured prostate cancer DU-145 cells in the vitro as research object,then prepared extracts of Cyclina sinensis with better activity.The peptides were separated,further purificated and sequenced.To investigate the preparation process of targeted peptide from Cyclina sinensis and its activity against prostate cancer DU-145 cells and further study the mechanism of inhibitory activity,it is meaningful to lay the foundation of theory and experiment for developing new anticancer drugs.Firstly,the content of free ANN as detection index in the enzymolysis liquid,the optimal protease and the best process of enzymatic hydrolysis was obtained through orthogonal test.Secondly less than 3ku molecular segments were further purified,and Superose gel chromatography and HPLC were used to further isolate as an indicator of inhibition rates IR against prostate cancer DU-145 cells.The results showed that the optimal protease was papain and the optimum condition of enzymatic hydrolysis was as follows.Material liquid ratio was 1:4,p H value was 7.0,the amount of enzyme added was 1500 U/g,temperature was 45 ℃,and enzymolysis time was 4 h.N-terminal amino acid sequence analysis of two peptides showed as follows.Sequence 1 is Ile-Leu-Tyr-Met-Pro,and sequence 2 is Trp-Pro.In addition,the inhibitory activity of sequence 1 was relatively better against prostate cancer DU-145 cells by MTT assay,and named Cyclina sinensis peptide(CSP).A series of activity assay in vitro of CSP,such as cell morphology test(observed under inverted microscope,AO/EB fluorescence staining,Hoechst 33258 staining and transmission electron microscopy),results showed that a series withered death of the morphological changes in DU-145 cells,such as cell gap becoming large,irregular of cell morphology,cytoplasmic condensation,part of the cell appearing vacuoles and apoptotic bodies,and even individual cell contents leaked treated with CSP.Moreover,it was more significant of the changes with increasing the concentration of CSP.The early-stage apoptotic rate and membrane potential was detected using flow cytometry,the expression of nm23H1 by Immune histochemical method,and the protein expressions of apoptosis-related genes were tested via Western blotting.The flow cytometry(FCM)revealed that the early-stage apoptotic rate of cells was increased from 6.10% to 20.18%,and the percentage of cells with decreased mitochondrial membrane potential was increased from 4.22% to 25.07%.Immune histochemical method displayed that the expression of nm23H1 was increased.Western Blot results showed that,CSP may induce apoptosis of DU-145 cells through up-regulating the expression levels of protein Bax,down-regulating the expression levels of protein Bcl-2,activating and up-regulating the expression of Caspase-9 and Caspase-3.This may be the mitochondrial apoptotic pathway.