Preliminary Study On Anti-tumor Activity Of Metabolite BMTP Of Actinomycetes From Deep Sea

Mariana Trench is the deepest trench in global marine geology.Its unique geographic advantages,especially in such extreme environments as low temperature,high pressure,darkness,hypoxia and even anaerobic environment,still exist a large number and a variety of microorganisms,and even some microorganisms have never been found.Therefore,extreme environments provide more possibilities for the survival of diverse microorganisms and bring more opportunities for microbial research.At the same time,microorganisms among them are more likely to produce new metabolic mechanisms and metabolites,which will bring more opportunities for the discovery of new natural active compounds,especially for the development of new drugs for the treatment of major diseases such as cancer.It is known that cancer is the second killer threatening human health.From a global perspective,nearly one sixth of the deaths are caused by cancer,and the number of patients is increasing at a faster rate every year.Therefore,research and development of anti-cancer drugs are also proceeding as soon as possible.In this study,the diversity of heterotrophic bacteria in Mariana Trench was analyzed.Heterotrophic bacteria collected from Mariana trench seawater at different depths were isolated and purified by using various substrates and culture temperatures.Heterotrophic bacteria were identified by 16S r RNA gene sequence analysis.Phylogenetic tree was constructed by Neighbour-Joining method to analyze the relationship between strains.In the result,531 strains of heterotrophic bacteria were obtained and 371 strains were identified.16S r RNA gene sequence analysis revealed that there were 97 species in 4 phyla,41 genera,distributed in Firmicutes,Proteobacteria,Actinobacteria and Bacteroidetes.Proteobacteria accounted for 58%of the identified bacteria.Among them,γ-Proteobacteria accounted for 52%of the total identified bacteria,and were the dominant heterotrophic bacteria in Mariana Trench.In addition,four heterotrophic bacteria with sequence similarity less than 98%with similar strains were found to be potential new species.Secondly,several strains of heterotrophic bacteria were screened to inhibit the proliferation of human non-small cell lung cancer A549.The crude extract of anti-tumor activity was finally obtained from the intracellular metabolites of MTL5-77 strain by ethanol extraction.The IC₅₀ value of ethanol extraction for A549 cell lines was 69.2656μg/m L.Physiological and biochemical tests showed that MTL5-77 and Brevibacterium frigoritolerans（DSM 8801）had the highest sequence similarity（99.24%）.The strain MTL5-77 belongs to Gram-positive bacteria.It has salt tolerance and can utilize glucose.It has the activities of Urease,Ornithine decarboxylase,Lysine decarboxylase and Arginine Dihydrolase.Based on the above analysis results,the strain MTL5-77was identified as Brevibacterium sp.MTL5-77 in actinomycetes.Further,the antitumor active compounds were isolated and purified from Brevibacterium sp.MTL5-77.The crude extracts were separated and purified by AKTA protein purification system through DEAE anion exchange column and by HPLC system through C18 non-polar column respectively.The antineoplastic active compound BMTP（a Product from Brevibacterium sp.MTL5-77）was isolated and purified.The IC₅₀value of BMTP towards lung cancer cell A549 was 2.9798μg/m L.Finally,the antitumor activity of BMTP was preliminarily analyzed in this study.BMTP displayed cytotoxicity towards several human cancer cell lines.The inhibition method of BMTP on the proliferation of cancer cells was determined by Hoechst 33342-PI double staining test and JC-1 mitochondrial membrane potential test.The results showed that BMTP can inhibit the proliferation of many kinds of cancer cell lines.The IC₅₀ value of BMTP towards human non-small cell lung cancer cell A549,human liver cancer cell BEL-7402,human cervical cancer cell Hela,human pancreatic cancer cell Panc28,human liver cancer cell Hep G2 and human large cell lung cancer cell NCI-H460were 2.9798μg/m L,3.4656μg/m L,2.8642μg/m L,4.3324μg/m L,4.1040μg/m L and 5.6793μg/m L,respectively.In Hoechst 33342-PI double-staining experiment,compared with the control group（plus PBS）,with the increase of BMTP dosage,the red fluorescence content of tumor cells increased and showed a brighter state when BMTP dosage was higher;the number and brightness of blue fluorescence increased with the increase of BMTP dosage.It attests the transformation of lung cancer cell A549 from normal state to apoptotic process induced by BMTP.It is concluded that BMTP can induce apoptosis of tumor cells and inhibit proliferation of tumor cells.The fluorescence probe JC-1 showed that the red fluorescence was weakening and the green fluorescence was increasing,which indicated that the mitochondrial membrane potential in lung cancer A549 cells was decreasing with the increase of the dosage of BMTP.This suggested that A549 cells had undergone apoptotic process,and apoptosis was regulated by mitochondria.In conclusion,this study preliminarily analyzed the diversity of heterotrophic bacteria in Mariana Trench,and provided some experimental data for the study of deep-sea microorganisms in the Northwest Pacific Ocean.In addition,several strains of bacteria which can inhibit the activity of tumor cells were isolated from them,and the BMTP with antitumor activity was isolated from Brevibacterium sp.MTL5-77.Preliminary studies have shown that BMTP can induce apoptosis through mitochondrial pathway,through which cancer cells can be cleared.This study is expected to provide some deep-sea microbial resources for the discovery of anti-cancer candidate drugs,and to develop the antitumor active BMTP as a new anti-cancer candidate drug.