The Screening Of Anti-cancer Active Actinomyces Metabolite And The Research Of Mechanism

Cancer is a disease with rising trend of high incidence and mortality rate. There are no specific medicines to cure cancer because of its complex pathogenesis. Microbial metabolites are the rich natural resource for development of anti-cancer drugs. Totally 50% anti-cancer drugs used through over the world are made from microorganism directly or indirectly. Soil microorganism is the most important microbe resource because of its complete type, large amount and large quantity all kinds of active materials potential metabolists.In this research, 16 strains were firstly screened out from 350 soil actinomycetes which can inhibit the growth of A549 cell and HGC cell obviously by MTT assay. Then these strains were determined by PI immunofluorescence staining and flow cytometry. After the secondary screening, strain 1070 was obtained because its metabolites can inhibite 32.07% A549 cells growth. The active component of strain 1070 was isolated by extraction, precipitation, macroporous resin separation, and finally found its molecular weight over 10000 daltons. Some identification experiments, including full wavelength scanning, TLC stained with ninhydrin and acid hydrolysis, showed that the active component should be polysaeccharide, but not protein or nucleic acid.The MTT assays showed that the IC50 value of anti-cancer component of strain 1070 was determined as 211μg/mL based on A549 cells growth inhibition(72h), and the intraperitoneal LD₅₀ of anti-cancer component in mice was 5988mg/kg(2 days). The results of scanning electron microscopy indicated that the junctional complex between A549 cells disappeared and the cells grew rounded. Transmission electron microscopy results showed many autophagy vesicles observed in the cytoplasm, but no evident changes in cell nuclear and the cell membrane is intact with no cellular breakage. The results of Rho-damine123 staining followed by flow cytometry showed that A549 cell mitochondria was suffered damage and the mitochondrial membrane potential was reduced to 43.72%. The results of Monodansylcadaverine (MDC) staining followed by flow cytometry showed that the A549 cells were induced cell autophagy with the ratio of 29.74%. Acridine orange (AO) staining experiment showed that the autophagy bodies appeared in cytolymph under fluorescence microscope. Western-blot discoverd that levels of LC3-II and Beclin-1 significantly increased, while LC3- I reduced. All the results indicated that the mechanism of active component of strain 1070 was inducing A549 cells autophagy.Based on colonial morphology, mycelial morphology, physiological and biochemical characteristics and 16S rDNA sequences, strain 1070 was preliminarily identified as Streptomyces capillispiralis.