Studies On The Antitumor Constituents Produced By Marine Microorganisms

Marine microorganisms continue to be an important source of bioactive secondary metabolites. A study was carried out to investigate the potential anti-tumor compounds derived from marine microorganisms. This dissertation describes the discovery of several antitumor lead compounds produced by marine microorganisms. Studies include screening of microbial strains, selecting aimed strains, fermentation studies, bioassay-guided fractionation, structural elucidation and preliminary evaluation for anti-tumor activities of pure compounds.This dissertation is organized in six chapters. Chapter One is an introduction and overview of the current studies in the field. The discovery of antitumor compounds produced by marine microorganisms, actinomycetes and fungi, are reviewed in Chapter One. In Chapter Two, the results from a study of gradated-combinatorial screening for the antitumor-active microbial strains is presented. The study was carried out by the gradated combination of a 96-well plate lethality bioassay using brine shrimp and a flow cytometric bioassay using mouse tsFT210 cells. [At the first step, the 164 strains of marine-derived microbes, isolated from the marine samples collected at the near shore alone the Qingdao's coast line and around Jiaozhou Bay, were first screened by Brine Shrimp Lethality assay to obtain 60 strains which showed lethiferous activity on brine shrimp, and possessed 36.6% of total 164 strains tested. For the second step, the 60 strains were subjected to the flow cytometric second screening using mouse tsFT210 cells and total 27 strains were found to have antitumor activities on the tsFT210 cells, which possessed 45.0% of the 60 strains and 16.5% of total 164 strains, respectively. J is Chapter Three describes the results of third screening (stability-tests for the bioactive components) and fermentation studies performed on the aimed strains.The metabolites of 3 strains including 2 actinomycete (HI002 and 007) and 1 fungal (HI-04) strains showed better stability in stability-tests. Therefore, these three well tested strains were chosen as the aimed strains to investigate their bioactive metabolites in this study. The time course experiments for the fermentation of these three producing strains were then carried out, followed by solvent extraction tests for the active components. Then, large-scale fermentation and preparation of the active fractions were performed to obtain the active fractions of the three strains. The two aimed strains, 007 and HI-04, were identified as Actinomadura sp. nov. and Aspergillus fumigatus Fres., respectively, through taxonomic studies.Chapter Four describes the results of bioassay-guided separation of the metabolites of the three aimed strains. Twelve compounds (1-12) were obtained from the metabolites of actinomycete HI002, six compounds (4, 7, 8 and 13-15) from Actinomadura sp. nov. 007, and twelve compounds (16-27) from Aspergillus fumigatus Fres. HI-04, respectively.Chapter Five describes the structural elucidation of the isolated compounds. The chemical structures of 1-27 (as shown as in Table 1) were investigated on the basis of their physical and-chemical properties and especially by the using modern spectroscopic methods. The structure of one new compound (13) has been confirmed, and identification of the other compounds including one new natural compound (27).Chapter Six describes the results of preliminary evaluation for antitumor activities of the pure compounds. The bioassay methods include flow cytometry, SRB and MTT methods. Bioassay results indicated: compound 13 inhibited the proliferation of human cancer A549, BEL-7402 and HL60 cells and mouse leukemia P388 cells with the inhibition rates of 82.6%, 57.3%, 76.1% and 62.2%, assay by SRB (A549 and BEL-7402) or MTT (HL60 and P388) method at 1 j*M, respectively. It also inhibited the proliferation of mouse cancer tsFT210 cells with the inhibition rates of 28.3% at 21 //M and 20.5% at 2.1 //M in the SRBassay. Flow cytometric analysis indicated that 13 inhibited the cell cycle of tsFT210 cells mainly at the G2/M phase. Compound 16 had apoptosis inducing or cytotoxic activities at the lower concentrations. Compounds 14 and 17-27 could inhibit the proliferation of tsFT210 cells. Compound 2 could arrest the cell cycle of tsFT210 cells at G0/G1 phase. Compound 11 could arrest the cell cycle of tsFT210 cells at G2/M phase.