Enzyme Hydrolyzation Technology Of Oligopeptides And Its Anti-prostate Cancer Mechanism Research

Prostate cancer is a major health problem and a significant cause of morbidity and mortality in men worldwide.Treatment for prostate cancer may involve surgery, radiation therapy, chemotherapy, cryosurgery, hormonal therapy, or various combinations of these.Chemotherapy is usually reserved for recurrent tumors that have already been treated with surgery and radiotherapy, or for tumors in which surgery was not, or was only partially feasible, and where the effect of radiotherapy was limited. Toxicity, drug resistance and limited and transient benefits in patients are the main problems associated with standard chemotherapeutic regimens, and new drugs are therefore needed to fight this devastating disease.Cuttlefish ink (sepia esculenta) had been used in treatments of hemostasis for centuries in Chinese traditional medicine. Researchers in Japan found that the peptidoglycan extracted from Sepia ink had higher antitumor activity and the inhibition ratio reached 64%. To investigate the anticancer effect of sepia ink oligopeptides, DU-145,PC-3 and H1299 cell lines were employed as an in vitro model in this study. We determined the effect of sepia ink oligopeptides on cell proliferation in DU-145,PC-3 and H1299 cells and examined their underlying mechanism with a combination of cell and molecular biological methods. The present study provides evidence that sepia ink oligopeptides is a potential anticancer drug to treat prostate cancer.Material and methodsIn order to investigate the anti-tumor effect of oligopeptides from ink of Sepia by enzymatic hydrolysis,the better enzymes were chosen and the conditions of hydrolysis were optimized according to the contents of amino nitrogen of hydrolysates,which were prepared by hydrolyzing for ink of sepia with Pepsine,Alcalase,papain and Trypsin respectively.Hydrolysates was isolated by ultrafiltration,purified on G-25 gel filtration and demonstrated the purity of oligopeptides from ink of Sepia by HPLC and its peptide sequence analysis was detected. The hormone-independent DU-145, PC-3 prostate cancer cell lines, and lung cancer cell lines H1299 cells were treated with sepia ink oligopeptides at three different concentrations. The following six experiments were conducted to explore the effect of sepia ink oligopeptides on cell biological function in DU-145, PC-3 and H1299 cells and to examine the underlying mechanisms.1,Concentration-dependent responses of the effect of sepia ink oligopeptides on cell proliferation was examined using CCK-8 assay.2,Growth condition and morphology of DU-145,PC-3 and H1299 cells were observed with an inverted microscope and HE staining after treat with sepia ink oligopeptides.3,AO/EB staining was employed for observation of apoptosis on DU-145,PC-3 and H1299 after treat with sepia ink oligopeptides.4,FCM was employed for observation of apoptosis on DU-145,PC-3 and H1299 after treating with sepia ink oligopeptides5,Immunocytochemical staining (SABC) was employed for analyzing the expression of Bcl-2,Caspase-3 and caspase-9 proteins in PC-3, DU-145 and H1299 cells treated with sepia ink oligopetides6,In-situ hybridization was employed for analyzing the expression of p53and BRCA1 mRNA in PC-3 and DU-145 cells treated with sepia ink oligopetides.Results1,A single peptide peak was obtained and its sequence was identified as N此处保密with a molecular mass of此处保密Da.2,CCK-8 assay showed that sepia ink oligopetides inhibited proliferation of DU-145,PC-3 and H1299 cells in both concentration- and time-dependent manners. (P<0.05).3,Treatment with sepia ink oligopetides for 24h significantly inhibited the growth of DU-145,PC-3 and H1299 cells. It was found that cellular processes were extended from the cell surface. Decreased cell volume, widened intercellular space and increased intracelluar dark colored particles were observed. Died cells were found and floated in the culture media. Cell apoptosis morphological feature was observed under HE staining. In addition, irregular cell shapes, vacuolalization in cytoplasm, fewer chromatospherite, clouding cell outline, increased megacaryocyte and agglutinated chromatin were also observed.4,AO/EB double staining disclosed that there were more viable apoptotic cells in sepia ink oligopetides treated groups than control group and the number of non-viable apoptotic cells and dead cells increased with dose increasing. Thus, certain concentration of sepia ink oligopetides resulted in a decrease in proliferation and an increase in apoptosis in DU-145,PC-3 and H1299 cells.5,FCM showed that after being exposed to 5, 10, 15mg/mL sepia ink oligopetides for 24h, the early stage of apoptosis of DU-145, PC-3 and H1299 cells was observed, in which the percentage of apoptotic cell increased from 11.84% to 38.26%, 22.96% to 39.96%, and 12.64% to 31.78%, respectively.6,Immunocytochemical staining showed that sepia ink oligopetides up-regulated Caspase-3 and Caspase-9 and down-regulated Bcl-2 expression corresponding with sepia ink oligopetides concentration.7,Sepia ink oligopetides could induce the expression of p53,BRCA1 mRNA in DU-145 and PC-3 cells with the dosage of 10mg/ml.Conclusions1,Sepia ink oligopetides inhibits the proliferation of DU-145, PC-3 and H1299 cell lines in both concentration-and time-dependent manners.2,The inhibitory effect of sepia ink oligopetides on the proliferation of DU-145, PC-3 and H1299 cells is via inducing cell apoptosis.3,Sepia ink oligopetides could induce the apoptosis in PC-3, DU-145 and H1299 cells by up-regulating Caspase-3 and Caspase-9 protein and down-regulating Bcl-2 protein and induce the expression of p53 and BRCA1 mRNA in PC-3 cells.