Isolation,Purification And Activity Analysis Of Antitumor Substance In A New Strain Bacillus Atrophaeus

Bacillus has the advantages of short growth cycle,fast reproduction and easy culture.It is not only widely distributed,but also can produce metabolic products with various biological activities such as bacteriostasis,insecticidal and antitumor.Combined with the detection of antitumor activity,a new strain producing high-efficiency antitumor activity was selected from soil samples collected from all over the country.Comprehensive bacterial morphological observation,physiological and biochemical characteristics,16 S r RNA and gyr B gene sequence homology alignment analysis,and ITS,rec A gene sequence cloning confirmed that the strain was Bacillus atrophaeus,named Bacillus atrophaeus X060(abbreviated as Bs X060).The supernatant of Bs X060 shake flask fermentation culture for 60 h has a significant inhibitory effect on MCF-7 cells proliferation.A large amount of fermentation broth is prepared by fermentation in a fermentor,extracted with ethyl acetate,and separated by ultra-high performance liquid chromatography-1290(UHPLC-1290)and preparative high performance liquid chromatography-1260(HPLC-1260)multi-step separation and purification to obtain Peak H2,a small molecular substance that is not affected by temperature and p H and has antitumor activity.The results of MTT analysis,clone formation and scratch experiments showed that Peak H2 significantly inhibited the proliferation and migration of human breast cancer MCF-7 and MDA-MB-231 cells in a time-and concentration-dependent manner.Observation with an inverted microscope revealed that Peak H2 induced cytoplasmic vacuolization of MDA-MB-231 cells.The diameter of the cytoplasmic vacuoles reached a peak after treatment for 60 h.It was shown that Peak H2 induced cytoplasmic vacuolation and inhibited the proliferation of MDA-MB-231 cells.Cotreatment with the addition of an endocytosis inhibitor showed that methyl-β-cyclodextrin completely reversed cytosolic vacuolation.However,co-incubation with clathrin inhibitor dynasore,giant cell-drinking selective inhibitor amiloride hydrochloride,broad-spectrum PI3 K inhibitor LY294002,and Caspase inhibitor Z-VAD-FMK did not affect vacuole formation,indicating that Peak H2 is dependent on the cell membrane lipid rafts,which enter into cells by clathrin-independent cell endocytosis.It does not rely on the PI3 K and Caspase pathways,and exerts antitumor effects in a nonapoptotic manner.The above research results lay a theoretical foundation for the selection of new resources of B.atrophaeus strains,the excavation of antitumor actives,and the elaboration of the mechanism of action of new antitumor actives,and provide new ideas for the development and utilization of new drugs.