Screening Of Anti-tumor Activity Of Natural Products Of Special Environment Microorganisms And Study Of Its Mechanism Of Action

In order to adapt to the outer special eco-environment in their courses of life evolution,microbes inhabiting in special eco-environment,such as microorganism living in extreme environment(for example high temperature,high cold,high permeability,high pressure and high radiation),marine microorganism,endophytic microorganism in plant,symbiotic microorganism in animal,rare actinomycete and myxobacteria etc,generated special metabolic pathway which could produce special secondary metabolite as lead compounds adequately for developing new drugs.Twenty one natural compounds were isolated from symbiotic C.globosum ML-4 in oyster,endophyte M.roridum IFB-E012 from A.annua,endophyte C.globosum IFB-WQ in Imperata cylindrical,endophyte Alternaria sp.TSJ-HD-1 from L.japonica and marine-derived fungus P.janthinellum HK1-6 in previous experiments.The in vitro antitumor activity of these 21 natural compounds from special eco-environment microorganism was measured by MTT method firstly in this dissertation.The tested tumor cell lines were the human hepatoma cell line SMMC-7721 and human gastric cancer cell line SGC-7901.Cisplatin and 5-fluorouracil were used as positive control drugs.Result showed that mytoxin B could inhibit the proliferation of SMMC-7721 cells significantly with IC50 value of 0.15±0.04 μg·mL-1,while guxi-2,penicilone C,penicilone D,H7 and H8 could inhibit the growth of SMMC-7721 cells obviously with IC50 values of 2.15±1.51,11.00±1.40,13.74±2.09,12.13±1.53 and 8.76±1.44μg·mL-1,respectively.Myrothecine A,tricycloaltemarene 3a,2H-(2E)-tricycloaltemarene 12a,penicilone B and penicilone A possessed strong proliferation inhibition to the SMMC-7721 cells with IC50 values of 25.5±1.19,45.81 ±4.58,49.72±1.13,15.29±1.82 and 28.62±4.03 μg·mL-1,respectively.Chaetoglobosin V and tricycloalternarene F inhibited the proliferation of SMMC-7721 cells with IC50 values of 60.5±0.93 and 80.31±3.84 μg·mL-1.The rest compounds had no obvious proliferation inhibitory activity to SMMC-7721 cells.Guxi-2 was also found to inhibit the proliferation of SGC-7901 cells with an IC50 value of 15.21 μg·mL-1.On this basis,further study on in vitro anti-tumor mechanism of the active compounds guxi-2,chaetoglobosin V,mytoxin B and myrothecine A was conducted in this dissertation.Guxi-2 is a novel cyclopentapeptide compound isolated from the endophytic fungus Myrothecium roridum in Artemisia annua.It was found to induce the G1 phase cell cycle arrest and the apoptosis in SGC-7901 cells in concentration dependent manner with the apoptosis rate of 21.91 ±4.26%at 50 μg·mL-1.Guxi-2 could reduce the migration rate of SGC-7901 cells evidently,and the scratch width did not change obviously and the cells passing the membranes decreased significantly at 50 μg·mL-1.In guxi-2-treated-cells,the expression of Bcl-2 decreased while the expression of Bax increased.The expression of protein Caspases-3,-8,-9 improved in SGC-7901 cells.These results indicate that in vitro anti-tumor activity of guxi-2 on SGC-7901 cells was related to induction of G1 phase cell cycle arrest and apoptosis and reduction of cells migration.The induced-apoptosis might be involve in both the mitochondrial pathway and the death receptor pathway.Chaetoglobosin V was a cytochalasin compound isolated from the liquid culture of symbiotic Chaetomium globosum ML-4 with oyster.Studies indicated that it could lead to the G2 phase arrest in SMMC-7721 cells and induce SMMC-7721 cells apoptosis in a concentration-dependent manner with the rate of 21.89±5.52%at 50 μg·mL-1.In chaetoglobosin V-treated-SMMC-7721 cells,the expression of protein Bcl-2 decreased and the expression of protein Bax increased,and the expression of protein Caspases-3,-8,-9 was up-regulated and the expression and phosphorylation level of Akt reduced at the same time.Above-mentioned results revealed that the in vitro anti-tumor activity of chaetoglobosin V on SMMC-7721 cells were related to the arrest of G2 phase cell cycle and induced-apoptosis.The induced-apoptosis was involved in both the mitochondrial pathway and the death receptor pathway and connected with the activity of PI3K/Akt signaling pathway.Mytoxin B(containing a 12,13-epoxy group in sesquiterpene residue)and myrothecine A(a 10,13-cyclotrichothecane derivative of mytoxin B)are two trichothecene macrolides isolated from endophyte M.roridum IFB-E012 in Aremisia annua.Researches discovered that mytoxin B could induce the apoptosis of SMMC-7721 cells significantly with rate of 22.62 ±0.64%at 1μg·mL-1,while myrothecine A showed weak induced-apoptosis activity to SMMC-7721 cells with the apoptosis rate of only 8.85 ± 0.11%at 5μg·mL-1.In mytoxin B-and myrothecine A-treated-SMMC-7721 cells the expression of Bcl-2 was both decreased and the expression of Bax was both up-regulated,and the expression of protein Caspases-3,-8,-9 were improved at the same time.Researches also found that the expression of protein Akt and the phosphorylation level of Akt were reduced in treated SMMC-7721 cells.In control experiment mytoxin B and myrothecine A showed the similar activity to LY294002(a PI3K inhibitor),and there was a certain synergistic effect between the two compounds and LY294002 respectively.It was also found that the expression and phosphorylation level of ERK decreased and the expression and phosphorylation level of p38 increased in SMMC-7721 cells.Aforementioned results revealed that the in vitro antitumor activity of mytoxin B and myrothecine A on SMMC-7721 cells were related to induced-apoptosis.The induced-apoptosis might be involved in both the mitochondrial pathway and the death receptor pathway,and was connected with activity of PI3K/Akt signaling pathway and MAPK signaling pathway.