Study On The Antitumor Compound ?-carbazoline Of Streptomyces Rubiginosohelvolus B11

Due to marine actinomycetes living environment is different from the land of inorganic salt concentration is high,deep sea water pressure big,the average temperature is low,lack of nutrients and characteristics of deep sea water deficiency,so relative to the soil actinomycetes have different metabolic pathways.Therefore,it is possible to produce new natural products to inhibit tumors and pathogenic microorganisms.In this paper,the secondary metabolites of marine rust Streptomyces(Streptomyces rubiginosohelvolus)B11 provided by the Bioengineering Laboratory of Qufu normal University were isolated and purified,and its anticancer mechanism and anti-metastatic activity were studied.The main results are as follows:(1)Through the screening of six different media and guided by the quality and anticancer activity of the crude extract prepared by the plate culture of Streptomyces flavus,the best medium with the quality of metabolites and anti-Hela cancer cells was selected.The results showed that the antitumor activity of the crude extract of Streptomyces aureus in soybean cake Agar medium was the highest,and the IC₅₀ was10.56?g/m L,.The highest quality of crude extract was 20.3 mg when Streptomyces was inoculated in 200 m L Agar medium.The temperature stability and light stability of the anticancer substance produced by the bacteria were studied.It was found that when the crude extract was treated at 80?,the survival rate of Hela cancer cells was80.56-86.22%.When the crude extract was treated at 100?,the anticancer active components in the crude extract completely failed,and its temperature stability was good;there was no significant difference in light stability with time.(2)The methanol phase compounds of Streptomyces flavus were cultured on a large number of plates and the crude extracts were prepared,and it was found that the anticancer active substances existed in the organic phase.A compound with anticancer activity was isolated and purified by traditional column chromatography and high performance liquid chromatography.The structural formula of the isolated anticancer substance was identified by nuclear magnetic resonance(NMR)and liquid chromatography-mass spectrometry(LC-MS),and it was determined to be?-carbazoline.(3)The cytotoxicity of?-carbazoline on human bronchial epithelial cells(Beas-2B),non-small cell lung cancer cells(A549)and human cervical cancer cell line(Hela)was determined by MTT method.It was found that Hela cells had high anti-tumor activity,and IC₅₀ was 27.3?g/m L.The compound was studied by cell flow cytometry.The results showed that the drug could promote apoptosis and inhibit the cell cycle of Hela cells in S phase.After Hela cells were treated with?-carbazoline for48 hours,the content of ROS in Hela cells increased greatly.The results showed that reactive oxygen species played an important role in apoptosis induced by?-carbazoline.The study of mitochondrial membrane potential showed that?-carbazoline could cause the change of mitochondrial membrane permeability.Further study found that?-carbazoline could promote the activation of caspase-3.To sum up,?-carbazoline can induce apoptosis in Hela cancer cells,which may depend on the mitochondrial regulation pathway.(4)The potential anti-metastasis effect of?-carbazoline was detected by scratch test,cell invasion and migration test.From the experimental results,?-carbazoline has obvious inhibitory effect on the invasion and migration of Hela cancer cells,and has a certain anti-metastasis ability.This study shows that ?-carbazoline has great potential for anti-metastatic therapy of cancer cells,and provides a new therapeutic strategy for anti-metastatic cells.