A Novel Calicheamicins-Producing Strain Micromonospora C3509: Fermentation, Purification And Antitumor Activity

It is well-known that "targeted therapeutics" can help to inhibit the activity of cancer cells in vivo.This progress will realize a number of potential drugs within clinical cancer therapy,such as those with extremely excellent bio-activity but toxic side-effects.By this pathway,their anti-tumor effects will be preserved while their toxic side effects will be avoided.Consequently,the better results in clinic should be achieved than those from conventional chemotherapy.Hence,the priority should be given to discovering an efficient "warhead" drug.According to Spermatogonial assay developed by Zhen and Xue,which is the prescreen method for detecting antitumor antibiotics,a strain C-3509 was isolated from a soil sample and was found to produce antitumor antibiotics.In present study,we strive for high potent anticancer activity drugs from brothe of strain of C-3509 by isolation and purification.The taxonomic characteristics were performed in accordance with the methods proposed by the International Streptomyces Project(ISP).The color names based on Ridgaway's description were used.Cell wall Composition was analyzed by the methods of Becker et al.,and Lechevalier.All observations were made after incubation at 28℃for 14 to 28 days.From the results obtained for the taxonomical studies on morphological,cultural and physiological characteristics and cell wall composition, strain C-3509 belonged to genus Micromonospora,Strain C-3509 was found to be closely related to M.echinospora subsp Calichensis.Further detailed comparisons were directly made between strain C-3509 and M.echinospora subsp.Calichensis.M.echinospora subsp.Calichensis differed from strain C-3509 as follows:spores of strain C-3509 were blunt spurs,while M.echinospora subsp.Caliehensis warty. Cultural characteristics of strain C-3509 on ISP-2,ISP-3,ISP-4,ISP-5, Glucose-asparagine agar was differentiated from that of strain M.echinospora subsp. Calichensis(table 4).Strain C-3509 didn't utilize Arabinose Rhamnose while M. echinospora subsp.Calichensis did.Strain C-3509 didn't grow at 45℃,while M. echinospora subsp.Calichensis could.Cell wall chemical Composition of strain C-3509 contained little 3-OH-DAP,while M.echinospora subsp.Calichensis contained lots. However,in spite of these minor differences,strain C-3509 and M.echinospora subsp. Calichensis were quite similar in other morphological and physiological properties. Therefore,on the basis of these results,it is concluded that strain C-3509 is a new variant of Micromonospora echinospora. To improve content of anti-tumor antibiotic in the fermentation broth for isolation and purification,we used bioassay and DNA cleavage assay to detect content of anti-tumor antibiotic,and response surface methodology to optimize fermentation medium.On the basis of our laboratory studies,we designed 12 groups medium including different components.We got a better group than other.Then response surface methodology was used to optimize fermentation condition in small number experiments to improve content of anti-tumor antibiotic.First,fractional factorial design(FFD) design was chosen to study the effects of the most important components in the media. Secoend,the steepest ascent was used to search optimized medium.Optimized by response surface methodology,content of anti-tumor antibiotic was increased by 30-fold through changing the composition of culture medium and optimizing their ratio.The whole fermentation broth was centrifugation at 4℃,5000rpm for 15 min,and then mycelia were removed.Supernatant was absorbed by Diaion HP20,and then active cmplex was eluted by 80%acetone.Dried and extracted with EtoAc.The EtoAc phase was concentrated to syrup.Syrup was precipitated by addition of hexane to give crude complex.The crude complex was divided into three fractions by Silica column.The second fraction was further purified by preparative TLC to give a high active complex. The liquid chromatographic-mass spectrometric(LC-MS) was adopted to identify the molecular weight of the products from C-3509.The liquid spectra was showed there were positive ion mass spectrum of the peaks of 11.05 min,11.20 min,11.5 min and 11.61 min,which were determined to have mass of m/z 1322.0([M+H⁺,100%),m/z 1367.9([M+H⁺,100%),m/z 1334.0([M+H⁺,100%) and m/z 1381.8([M+H⁺,100%) by LC-MS analysis.These peaks had the same formula mass with calicheamicinβ₁^I, calicheamicinγ₁^I,calicheamicinβ₁^Br and calicheamicinγ₁^Br respectively.The high active complex was further purified by preparative HPLC.A chemical compound which molecular weight was 1367 by ESI-MS and molecular formula was C₅₅H₇₄N₃O₂₁S₄I by HR-ESI-MS was isolated.It was named as C-3509 B.With ESI-MS,HR-ESI-MS,and ¹H-NMR,C-3509 B was dertermined as a known enediyne antitumor antibiotic calicheamicinγ₁^I.Eight cancer cell lines were detected by MTT Assay,including human lung carcinoma A549 cells,breast cancer MCF-7 cells,fibrosarcoma HT-1080 cells, epithelial carcinoma Hela cells,osteosarcoma MG-63 cells,oral cancer KB cells, hepatoma HepG2 and BEL-7402 cells(Table 5).The IC50 values of antibiotic C-3509B were in a range of 0.28～1.1 nM.The data indicated that antibiotic C-3509B potently inhibited the proliferations of various human cancer cells.DNA cleavage assay showed that antibiotic C-3509B directly induced DNA cleavage at a minimal concentration of 0.02μg/ml.At the concentration of 10μg/ml, antibiotic C-3509B strongly cleaved DNA into fragments.These results were consistent with the DNA cleavage induced by calichimicins.Flow cytometry analysis showed that the effect of C-3509B on cell cycle regulation was dose-dependent.At lower concentration,C-3509B induced G2 arrest in HepG 2 cells.However,treatment with higher concentrations,C-3509B induced apoptosis in HepG2 cells.Western blotting was used to assay expression of checkpoint regulatory proteins of HepG2 cells after treatment with C-3509B.The result showed that Chk2-cdc2/cyclin B pathways associated with C-3509B-induced G2 arrest.In order to strive for high potent anticancer activity drugs from brothe of strain of C-3509 by isolation and purification,we optimized fermentation condition and gained C-3509B.From the results obtained for the taxonomical studies on morphological, cultural and physiological characteristics and cell wall composition,strain C-3509 belonged to genus Micromonospora but differed from M.echinospora subsp. Calichensis.At lower concentration,antibiotic C-3509B induced G2 arrest in HepG2 cells.Further study showed Chk2-cdc2/cyclin B pathways associated with C-3509B-induced G2 arrest.