Screening Of Marine Fungi With Antitumor Activity And Studies On Antitumor Activity Compounds From Aspergillus Terreus Thom

We have undertaken a piece of research on the metabolites of marine-derived microorganisms, focused on apoptosis inducing and/or cell cycle inhibitory components, by the use of bioassay using mammalian tsFT210 cells line. At the first step of this work, we isolated 207 strains of fungi from sea-samples and examined bioactivity of their fermentation products. A fungal strain Z83200, isolated from Jiaozhou Bay sediment sample, was found to produce metabolites showing very strong activity of inducing apoptosis on mouse tsFT210 cells. This fungal strain was identified as Aspergillus terreus Thom. through a taxonomic study.The whole fermentation broth was separated by the means of modern chromatographic methods in a bioassay- guided separation manner to obtain pure bioactive compounds. From the bioactive extracts with ethyl acetate, ten compounds (1~10) were isolated by column chromatography on silica gel, Sephadex LH-20, HPLC and recrystallization. By means of spectroscopic method (UV, IR, MS, ID- NMR,2D-NMR) and their physicochemical properties, the structures of eight compounds (1~8) were elucidated as 3a, 4-dihydro-6R-(5'-hydroxy cyclohexa-1', 3'- dienyl)-methyl-3a-(hydroxymethyl)-4-methyl-6, 6'-epidioxypyrrolo[2, 3-c][l, 2, 5]oxathiazol-5 (6H)-one (1), 3-(1H-Indol-3-ylmethyl)-hexahydro-pyrrolo[l, 2-a]pyrazine-l, 4-dione (2), spiro[[4, 5-benzo[b]-tetrahydro-3, 7-dimethyloxazolo-3, 2-g][l, 4]diazepine-2, 8-(3H, 5H)-dione]-2, 9'-[2H-benzo[b][l, 4]oxazin-3(4H)-one](3), 4-ethylidene-3-methyl-5-oxo-4, 5-dihydrofuran-2-carboxylic acid ethyl ester (4), 2-Acetyl-3-methyl-pent-2-enedioic acid diethyl ester (5), 3-Acetyl-4-hydroxy-2, 6-dimethyl-benzoic acid ethyl ester (6), 2-hydroxy-2, 4-dimethyl-5-oxo-2, 5-dihydrofuran-3-carboxylic acid ethyl ester (7), Hex-3-ene-2, 5-dione bis-(O-methyl-oxime) (8) . The structures of compound 9% 10 are still to be identified. Compounds 1, 3, 4, 5 and 7 are new compounds.The cell cycle inhibitory and apoptosis inducing activities were assayed for 1 using both tsFT210 and K562 cells by flow cytometry, accompanied with morphological observations of the cells under light microscope. The DNA content of the tested cells was measured by flowAbstractcytometry and the result was obtained as flow cytometric histogram. Hypodiploid cells generally appeared in the sub-G0/G1 region in the histogram contain less DNA content than that of the dipoid cells observed as G0/G1 peak in the histogram because of the apoptosis-induced DNA fragmentation. The proportion of hypodiploid cells in total cell population represents the intensity o f a poptosis-inducing a ctivity o f t he t ested s ample. Also, m icroscopic o bservations provided morphological evidence for apoptosis. By comparison of the result for 1 from flow cytometric analysis with that of control, we can evaluated the effect of 1. And each experiment for evaluating biological activities of 1 was independently repeated at least 3 times to confirm the same result.Compound 1 induced dramatic apoptosis both on tsFT210 and K562 cells, which was confirmed by flow cytometric analysis and also by the morphological observation of the cells treated with 1. The IC50 value for 1 for inducing apoptosis of tsFT210 cells is 0.15 g/ml and its MIC value for inducing apoptosis of tsFT210 cells is much smaller than 0.078 g/ml (MIC<0.078 g/ml), while the MIC value for 1 for inducing apoptosis of K562 cells is smaller than 0.31 g/ml (0.15 g/ml