Study On The Novel Anti-tumor Antibiotic From Myxobacteria Stigmatella WXNXJ-B

The myxobacteria are colony develop,complex life cycle,Gram-negative, gliding movement and unicellular bacteria with rod-shaped vegetative cells.They produce bioactive secondary metabolites that inhibit the growth of other organisms and some tumors. Due to their extraordinary ability to produce novel classes of secondary metabolites, myxobacteria are an important resource to choose new drugs. Many researcher attached importance to the study of the secondary metabolites from the myxobacteria. In this study, the strain WXNXJ-B preserved in the lab was identified according to its physiological and biochemical characteristics and Bergey's Manual of Systematic Bacteriology (8thed). Its ability to produce volatility compounds and growth condition were studied. Based on bioactive evaluation with the model of B16 tumor cell lines, the strong bioactive substance produced by WXNXJ-B were isolated, puried and identified. The culture condition of the strain was optimized to increase the bioactive substance production and the mechanism of anti-tumor was also investigated.Compared with the description of Bergey's Manual of Systematic Bacteriology, WXNX J-B was identified as Stigmatella genus according to the strain clone and the physiological and biochemical characteristics. The volatile components produced by WXNXJ-B were detected and identified by using SPME/GC/MS and the strain mainly produced ketone and heterocyclic volatile compounds. The culture condition was optimized to increase the cell weight by single factor experiment and orthogonal experiment. The optimized results were showed as follows: maltose 8 g/L, yeast power 6 g/L, peptone of 6 g/L, D-sodium gluconate 5 g/L, CaCl2 1.5 g/L, MgSO4 0.5 g/L, initial pH 7.0, incubating temperature 28℃.The metabolites from Stigmatella WXNXJ-B had strong cytotoxicity to mouse melanoma cell line (B16), human liver carcinoma cells line (HepG2) and human breast cancer cells line (MDA-MB231). The antitumor bioactivity on B16 cell line did not obviously change when the temperature was lower 70℃, pH was in the range from 3 to 10 and the metabolites were treated with daylight and ultraviolet light during 12 h. The metabolites adsorbed by XAD16 resin probably contained alkaloids, lactones, glycosides and steroids components by qualitative analysis experiment.Isolated and purified by liquid-liquid extraction, Sephadex LH-20 and C18 column chromatography, and prepared HPLC, a compound was obtained finally. Then the structure analysis of the compound was analyzed by UV, LC/MS, MALDI-TOF-MS/MS, IR and NMR spectras. The molecular weight was 435 Da and the molecular formula was calculated as C₂₉H₂₅NO₃. Then its structure was made out. N OOO The popular name and systemic name of compound C29H25NO3 were Quinoxalone, [1R,6R]-5-(6-benzyl-quinolin-3-ylmethyl)-6-phenyl-3,7-dioxa-bicyclo[4.1.0]heptan-3-one, respectively. It was a novel compound which was confirmed with investigating novel report by Station of Science and Technology of Education of Minister, East China University of Science and Technology (No. 201036000L060001).The conditions were optimized to increase the Quinoxalone yield during Stigmatella WXNXJ-B fermentation.The results showed as following: incubating time of 6 days, inoculum concentration of 8%, temperature of 28℃, volume content of 60 mL/500 mL, initial pH of 6.5. The effects of inorganic salts, ammonium and phosphate salt, precursors and amino acids to Quinoxalone yield were investigated. The results showed that Ca(²+), Mg(²+), Zn(²+), Co(²+) and Na+ could increase the Quinoxalone yield, but Li(²+) was opposite. Ammonium concentration no excess 2 g/L could increase Quinoxalone yield, but the phosphate could inhibited the yield. Sodium acetate( 0.4 g/L) and sodium propionate (0.8g/L) increased the yield to 905.15μg/L and 878.42μg/L respectively. A certain concentration of ethanol, lactic acid, isopropanol, isoamyl alcohol, trytophan, tyrosine, alanine, methionine and glutamate could stimulate the production, but valine could inhibit that.Plackett-Burman design was used to screen the significant factors influencing Quinoxalone production in medium. With statistic analysis, the significant factors were skim milk, MgSO4·7H2O and glucose. Response surface methodology (RSM) was used to optimize the three critical factors mentioned above, and the result showed that the optimal concentrations of the factors were determined as: skim milk 5.12 g/L, MgSO4·7H2O 0.8 g/L and glucose 7.92 g/L. Under such conditions, the maximum Quinoxalone yield was 1205.08μg/L actually, which was 59.69 % higher than that before optimizing. The macroporous adsorption resins were investigated and D101 resin was selected to adsorb the Quinoxalone from the culture broth according to the static absorption experiment.Quinoxalone showed strong anti-tumor bioactivity to B16, HepG2, DMA-MB231 and murine colon carcinoma CT-26 cell lines in vitro. The value of IC50 were 2.41, 5.4, 2.83, 1.78μg/mL on above four tumor cell lines respectively. Compared with the two positive controls Taxol and Epothilone B, the cytotoxicity of Quinoxalone was the similar as Taxol. The cytotoxicity to the normal mouse spleen cells was low and IC50 was above 700μg mL-1. So, Quinoxalone selectively inhibited the growth of tumor celll lines.The sum of B16 cells with treated by Quinoxalone was distinctly decreased than the control and the shape became round by phase-contrasted microscope. Scanning electron microscope photos showed that the most of villus of B16 cells disappeared at higher concentrations and the surface of membrane was smooth and shrinkage, even to form apoptotic bodies. The apoptotic characteristic including nuclear crimple, chromatin condensation, nuclear fragmentation and compact blue fluorescent were observed by fluorescent microscope. Nuclear DNA of B16 cells showed ladder pattern and 180-200 bp fragment by agarose gel electrophoresis. By flow cytometry analysis and staining with PI, B16 cell proliferation was stopped because B16 cell cycle was arrested at S phase and G2/M phase. The Quinoxalone dropped mitochondrial transmembrane potential (ΔΨm) in treatment-induced apoptosis without changing plasma membrane permeability in B16 cells by flow cytometry analysis and staining with PI and Rh123. Further research results showed that the apoptotic mechanism of B16 cells induced by Quinoxalone was associated with a decrease in Bcl-2 protein, and upgrading the protein expression of P53, Bax and Caspase-3 by immunocytochemical staining.