Studies On Evaluation Of Antitumor-Activity Of Polypores And The Isolation, Structure Elucidation Of And Molecular Mechanisms Of Apoptosis In HeLa Induced By Antitumor-Active Substance From Grifola Gigantea (Pers.) Karst.

Polypores are a large group of terrestrial fungi of the phylum Basdiomycota (basidiomycetes), and they along with certain Ascomycota are a major source of pharmacologically active substances. In a program aim to search for antitumor substances from high fungi, the followed work were finished.Firstly, the fruit bodies of 35 polypores from Zhejiang and Hunan Province were screed for the cytotoxic activity against human tumor cell lines by MTT dye assay. The results indicated that the most of lipid extracts including petrol ether and ethyl acetate extracts have significant cytotoxicity (3450<100μg/ml), whereas polar extracts including methanol and water extracts have no clear cytotoxicity (IC₅₀>100μg/ml) . It was found that out of 35 species screened 21 exhibited cytotoxic activity. More than 60% of screened crude extracts of sample showed cytotoxicity while 8 species displayed strong cytotoxic active with IC₅₀ value of less than 50 (μg/ml. Among them, the ethyl acetate extract of Grifola gigantea (Pers.) Karst exhibited the most strongest cytotoxic active against HeLa with an IC₅₀ value of 34.2μg/ml.Secondly, the biological characteristics of Grifola gigantea has been observed and main conclusion can be summarized as follows: The occurrence of sporophore are closely related to moisture. The soil with a humidity value of 40-60% and scatter light can be benefit to development of sporophore. It can produce sporophore in both fertilizer-rich and fertilizer-poor soil. The accumulation of biological knowledge may be helpful to introduction and acclimation of this fungus.Thirdly, a bioassay-guided fractionation of the ethyl acetate extract of Grifola gigantea led to the isolation of two compound. Their structure were identified as clitocine and a-clitocine by spectra methods. Herein, clitocine with strong cytotoxicity (IC₅₀=14.9μM) was clarified as the substance responsible to the cytotoxicity of Grifola gigantea for the first time, a-clitocine, as a side-product of this chromatographic separation procedure, was reported as a natural product for the first time in this paper and was inactive though as a anomer of clitocine.Fourthly, the antitumor active of clitocine in vitro and in vivo was evaluated using MTT method and S180 solid tumor model, respectively. Result showed that clitocine can significantly inhibit the proliferation of five human tumor cell lines including HeLa (14.9μM), SMMC-7721 (1.1μM), SGC-7901 (2.2μM), MCF-7 (43.0μM), and Bcap-37 (10.9μM). The value of IC50 indicated the cytotoxicity of clitocine against human cell lines may be of selective. Clitocine could not prevent solid tumor growth whenever rat were administered with high, medium and low doses. Furthermore, clitocine-induced immunosuppression effect on spleen was observed for the first time.Fifthly, the molecular mechanism of clitocine-induced cytotoxicity against HeLa was investigated for the first time in this thesis and many interesting results were included. Concentration of 15μM clitocine caused the induction of apoptosis in a time dependent manner. Clitocine-induced cell death was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase like activities, poly (ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol. Clitocine activated various caspase such as caspase-3, 8, 9. Moreover, the cell death can be significantly prevented by a family caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-9 inhibitor (Z-LEHD-FMK). Furthermore, elevated anti-apoptotic protein Bcl-2 levels and low promote-apoptotic protein Bax expression have also been detected by Western blot assay in clitocine treated cells; Clitocine had no influence on cell cycle in HeLa cells, and cltocine-induced cell death appeared at all of cell cycle phases. The overall results suggest that the progression of cell death induced by clitocine was mediated by both caspase family and Bcl-2 family and was not associated with disruption of cell cycle.