Isolation Of Metacycloprodigiosin And Study On Its Anticancer Activities

Prodiginines are a large family of pigmented tripyrrole secondary metabolites with medicinal potential as antifungal, antibacterial, immunosuppressants and antitumour agents that are produced by several actinomycetes and other bacteria. Because of its multiple bioactivities, prodiginines were attracted a great deal of attention in recent yearsIn our former study, a marine actinomycetes strain from a sponge-derived actinomycete Saccharopolyspora sp. nov SP2-10 with metacycloprodigiosin (MP) and undecylprodigiosin (UP) producing ability has been found. In this dissertation, single factor test was employed to improve MP yields from SP2-10 strain, MP and UP were isolated from the rude ethyl acetate extract, furthermore, the anticancer activities and molecule action mechanism of MP were studied.According to undecylprodigiosin biosynthesis pathway, sodium acetate, several amino acids and iodine acetamide were selected as precursors adding into the actinomycetes medium with four different concentration gradients.It was found that the addition of sodium acetate and Val to the final concentration of 10mmolL^-1 either enhanced MP an UP production by 3 fold. Moreover, iodine acetamide with final concentration of 0.2 mmolL^-1 obviously raised MP proportion in the total pigment production.According to the optimal condition, large-scale fermentation and preparation of the active fractions were performed. MP and UP were isolated and purified using solvent extraction, silica gel column, Sephadex LH20 and etc. The structures were identified by MS and ¹HNMR.Firstly, we investigated the effects of MP on the proliferation and cell cycle progression in cancer cells of murine leukemia cells(P388) and human hepatocellular carcinoma cells(Bel-7402) using MTT assay and flow cytometry. The inhibitive effect could reach about 50% when cells were treated with 1uM MP for 24h. We found that MP induced a significant blockage in the G₂/M phase in P388 cancer cells, with the immunofluorence staining, cells were fixed and immunolabeled for cell nucleus with PI, we found that the nucleus showed morphology characteristic of apoptotic cells, furthermore, we evaluated the PARP cleavage and Cytochrom C release after MP treatment by Western blot, the results revealed that MP treatment could promote the cleavage of poly(ADP-ribose) polymerase protein and the Cytochrom C release from mitochondrion companioned by the cytochrome C increase in cytoplasm, these result indicate that MP can induce cancer cells apoptosis and demonstrate the apoptosis is involved in mitochondrial apoptotic pathway. In addition, with Confocal, the cytoplasm acidification, the other character of apoptosis was also being noticed.In order to further elucidate the mechanisms underlying apoptosis induced by MP treatment, PI-3K/AKT signal pathway was first studied, but no distinct effect on AKT phosphorylation was found, however, we notified that MP could promote cleavage of HSP90 protein according to Western blot, this result suggested us that MP could enhance the concentration of the reactive oxygen species (ROS) within cells and the flow cytometry assay revealed that MP could increase the ROS in P388 cells.Finally, we examined the role of ROS in deterring whether MP triggered phosphorylation of ERK, JNK, P38, the three important signal molecule of MAPK signal pathway. We found that MP induced phosphorylation of P38, ERK and JNK remarkably. Selective inhibitor of P38 and JNK blocked its phosphorylation and prevented the p388 cancer cells from injuring induced by MP treatment except for ERK.Furthermore, in the following study in our paper, we investigated the function of MP in antimetastatic and anticancer effect. According to wound healing assay and the transwell assay, migration and invasion was decreased in 4TO7 cells, treated with 0.001uM MP. With tube formation assay, MP was found to inhibit tube formation of the human umbilical vein endothelial cells, which is an important step for angiogenesis. In conclusion, the systematic research of MP anticancer effect revealed that MP could decrease the proliferation of multiple cancer cell lines and induce blockage in the G₂/M phase. MP induced apoptosis was via mitochondrial pathway. ROS induced by MP was the important mechanism of this action which correlated with phosphorylation of P38 and JNK other than ERK. Besides, MP could exert antimetastatic effect. Therefore, MP appears to be potential candidate for pharmaceutical development as anticancer agents.