Screening Of Myxobacteria And Its Novel Antitumor Antibiotics

Myxobacteria,a class of microorganisms including 12 genuses and about 40 species, have attracted much more interest as drug sources but seldom been studied yet.Based on the traditional screening methodology for myxobacteria,a high-throughout method with tubes culturing was used in the research for large scale screening and 376 myxobacteria strains were isolated.Among which 67 strains were identified to genuse by their distinct morphological characters.As epothilone has character UV absorption,312 samples with strong absorption peaks at UV area were screened out.Based on bioactivity evaluation,45 high tumor inhibiting active samples were directly screened via B16 and SGC7901 cell lines models.With extended tumor screening models in re-screening,6 strains which produced higher bioactive metabolites were obtained and confirmed as myxobacteria gunes by various microscope technologies.They were WXNXJ-A(Polyangium sp.),WXNXJ-B(Stigmatella sp.),WXNXJ-C(Polyangium sp.),WXNXJ-D & WXNXJ-E(Myxococcus) and WXNXJ-F(Cystobacter sp.).The results on the re-screening showed that the metabolites of myxobacteria WXNXJ-A and WXNXJ-C had not only high bioactivity to B16 cell line(IC₅₀ 0.2715/0.0082μg/mL) and SGC7901 cell line(IC₅₀ 4.192/0.0362μg/mL),but also high bioactivity to Bel7402,H446 and MCF-7 cell lines.By HPLC and LC/MS analyzing the component of WXNXJ-C metabolite with t_R= 32.6min(flow:0.50 mL/min) in the spectrum of HPLC was confirmed as epothilone B.The growth curve of WXNXJ-C showed optimal cell growth at 3 clays and good fermentation condition at 6 days,respectively.The optimal fermentation medium with additive of amino acids and microelement components were confirmed and the highest yield of epothilone B was 31.95 mg/L in 250 mL Erlenmeyer flasks.There are 7 components with higher and broad-spectrum antitumor bioactivity in the WXNXJ-C fermentation elution.By using C-18 column chromatography,15RPC column and C-18 chromatography column,a component(named 7F) with molecular weight 424 Da was obtained.The structure of 7F component was analyzed with LC/MS,MALDI-TOF, MS/MS,IR and NMR.The molecular formula of 7F was calculated as C₂₂H₃₂O₈.The traditional name and systemic name of compound C₂₂H₃₂O₈ were Phoxalone and [1R,7R,8R,10S,11S,12S,13S]-1,7,12,13-tetrahydroxy-14-methoxy-8,10-dimethyl-6-phenyl-5, 15-dioxa-bicyclo[9.3.1]pentadecan-4-one,respectively.Via confirming with investigating by L08 Station of Science and Technology,Minister of Education it was confirmed as a novel 15-number ring macrolide containing an oxygen-bridge.According to the results of LC/MS,MALDI-TOF MS/MS analysis,the fragment ions had been analyzed and the synthetic path or mechanisms were studied preliminarily.Further research on the structure of Phoxalone,comparing with the dimensional structure of Soraphen,epothilones and Paclitaxel,revealed that 7 chiral carbon atoms and 2 chiral centers in Phoxalone molecular gave a simplest,easiest synthesized,and modified structure.It also showed that 15-memmber ring of Phoxalone was inosculating to 16-memmber ring of epothilone B in their 3D structure,and was in a similar manner in the inducing cell apoptosis mechanism.Research results of bioactive evaluation indicated that Phoxalone not only did have higher and broad-spectrum antitumor bioactivity to tumor cell lines,such as B16,Bel7402, MCF-7,H446,SGC7901,but also did have less cytotoxicity to L02 cell line in vitro. Taking H446 cell line as model,the cytotoxic bioactivity was well studied by using microscope,phase-contrast microscope,and scanning electron microscope,and the result showed that Phoxalone had stronger inhibiting action on H446 cell line.Flow cytometric analysis explained that the H446 cell cycle was arrested at the G2/M phase with Phoxalone treatment for 48h,sum apoptotic cells showed positive correlations with the quantity of samples and culture time.The results of dyeing with fluorescent dyestuffs PI and Annexin V indicated that Phoxalone induced the potential voltage(ΔΨ_m) of mitochondria decreasing without destroying the integrality of cell membrane.It also revealed that the cell cycle was arrested at the G2/M phase.More investigation results showed that the apoptotic machinery of H446 induced by Phoxalone was associated with a decrease in BCL-2/Bax ratio,dropping in mitochondrial trans-membrane potential voltage,and upgrading the protein expression of caspase-3 by immunocytochemical staining.