Study On The Novel Anti-tumor Substances By Sorangium Cellulosum AHB103-1

Myxobacteria are a class of very unique gram-negative bacteria, with complex social behavior and the ability of producing abundant secondary metabolites with pharmacological activity. In this thesis, the biological characteristics of Sorangium cellulosum AHB103-1 were studied and the classification was identified. The relationship between the volatile substances and the anti-tumor secondary metabolites produced during fermentation process was studied. Anti-tumor secondary metabolites were screened by MTT test and two strong anti-tumor substances were purified and named as Dieobone and Mehyazaone. The fermentation process conditions of Dieobone were optimized. Its anti-tumor mechanism was also preliminarily discussed.AHB103-1 was identified as Sorangium cellulosum(Sorangium sp.) on the basis of morphological, physiological and biochemical characteristics analyse,according to Bergey's manual of systematic bacteriology (Second Edition,Part C,2005). The volatile substances produced during fermentation period were identified by SPME/GC/MS and the tendency of some volatile compounds was found to be similar to the formation curves of the anti-tumor secondary metabolites. In which acetic acid and 4-(benzoyl)-2H-pyran-3-ketone were the most important because of higher content, which could be used as a parameter of fermentation control.Secondary metabolites produced by Sorangium cellulosum AHB103-1 have strong antitumor activity against human liver cancer cell line (HepG2), human breast cancer cell line (MDA-MB231) , mouse melanoma cell line (B16) and human gastric cancer cells (SGC7901). The activity of secondary metabolites was changed a little below 60?. It was not sensitive to strong sunlight and UV light, and remained stable at pH3~8.HepG2 cells were chosen as a model cell line using MTT method. Two high anti-tumor activity component was obtained by using extraction, column separation by atmospheric Sephadex LH-20 and crude C18, HPLC, and crystallization purification. The data of NMR, TOF MS/MS, UV, and IR spectrums were analysed. The results showed that the molecular weight of two compounds were 392 and 370 Da and their molecular formulae were C24H44N2O2 and C18H30N2O6, respectively. According to systematic nomenclature, the compound C24H44N2O2 was named as [3S,12R]3,12-diethyl-1,15-diaza-bicyclo [13.7.0]docosane-2,16-dione) systematically and supose the common name as Dieobone, and the compound C18H30N2O6 named as [2R,6R,7S,8R,20R]-2,6,7,8,20-pentahydroxy-4,13-diaza -tetracyclic [9.8. 1.04,9.013,19]eicosane-5-one systematically and supose the common name as Mehyazaone. They were identified as a new substance by Station of Science and Technology of Education of Minister, Jiangnan University (Novelty Search No. 201136000L080497).A simple synthetic medium with glucose as the sole carbon source, asparagine as the sole nitrogen source, and Co ion as main trace elements was developed to study the effects of various nutrients including sodium acetate, propionate, glycerol, the amino acids, inorganic salts and phosphate. Dieobone yield was significantly increased by 55.63% with the addition of sodium propionate,and only a slight promotion with the addition of sodium acetate.However, Dieobone yield was strongly inhibited by 46.71% with the addition of glycerol.Moreover, Dieobone yield was correspondingly increased by 100.42% and 126.90% with the supplement of serine and tryptophan, but reduced by 47.64% with the supplement of threonine.Different concentrations of phosphate showed inhibition effects,too.Four resins showed good effect for Dieobone yield during fermentation process, namely, HPD100, XAD-2, XAD-16, D101. Macroporous resin XAD-16 showed the best effect with Dieobone level up to 54.66 mg/L. Dieobone yield reached better level with conditions as follows: macroporous resin XAD-16 addition level 15~25 g/L, liquid volume in flask 75~125 mL/500mL and the fermentation temperature 28~32?.The results of PB experiment showed that the significantly different order of the factors in the fermentation medium was: potato starch> MgSO4?7H2O> skim milk powder> serine> sodium propionate> yeast. Fermentation medium was optimized by BB experiment. At the optimum concentrations of each component (potato starch 12.49 g/L, skim milk powder 9.77 g/L, MgSO4.7H2O 1.43 g/L), the predictive value of Dieobone was 99.89 mg/L. The yield of Dieobone for validation experiment was 90.42 mg/L, and was increased by 63.04%, comparing to 55.46 mg/L before optimization.MTT assay result indicated that Dieobone and Mehyazaone had good antitumor effect on four tumor cell lines, namely, HepG2, MDA-MB231, B16 and SGC7901 in vitro. Compared with clinical drugs, the cytotoxic activity of Dieobone and Mehyazaone on HepG2 were lower than Epothilone B, similar with Taxol and Epirubicin, but far higher than Irinotecan and Oxaliplatin. Moreover, their cytotoxic activity on normal mouse spleen cell was far lower than HepG2 cell and B16 cell.After treatment with Dieobone for 24h HepG2 cells showed significant cell growth inhibition on inverted microscope observation. The nucleus of HepG2 cells treated with Dieobone become smaller, shrunken observed under fluorescence microscope. Strong compact fluorescent was shown and part of the cell nuclei appeared fragmented. The apoptotic bodies formed, and small fluorescent spots appeared. The observation by scanning electron microscopy showed that the treated HepG2 cell surface microvilli became thin or complete disappeared while the control HepG2 cell covered with silk microvilli. More apoptotic bodies occured on the cell membrane, and the fragmentation of nuclei appeared. The results of agarose gel electrophoresis showed the apparent DNA ladder, containing 180~200bp oligonucleotide fragment. The results of flow cytometry analysis showed that Dieobone suppressed HepG2 cell lines proliferation with cell cycle arrested in the S-phase in the experimental concentration range. It induced apoptosis in the experimental concentration range by causing mitochondrial membrane potential??m to decline, and markedly upregulated the expression of caspase-3, Bax and p53 mRNA, while downregulated the expression of Bcl-2 mRNA.