Anti-austerity Of Metabolites From Aspergillus Sydowii DL1045 Regulated By Epigenetic Strategy

Hypoxia and hyponutrition are the important feature of the tumor microenvironment. Liver cancer, colon cancer and other types of cancer all present long-term hypoxia and hyponutrition area, all these tumor cells can survive under the condition of hypoxia and hyponutrition but normal human cells ca not. The drugs which could abolish cancer cells’ tolerance of hypoxia/hyponutrition tumour microenvironment are expected to become new chemotheraputic drugs, this strategy is also called anti-austerity.Fungal secondary metabolites play an important role in human resistance to infectious diseases, cancer and other diseases, they are the continuing sources of new therapeutic drugs/precursor drugs all the time. Normally, under the convientional laboratory culture conditions, most of the fungal secondary metabolite gene clusters are silenced. The changes of epigenetic modification level play an important role in the regulation of these gene clusters expression. Small molecule epigenetic modulator can be used to change the epigenetic modification level, therefore these "silent" secondary metabolites could be obtained.Firstly, based on anti-austrity strategy, several tumor cell lines were screened in hypoxia and hyponutrition environment firstly, and liver cancer cell HepG-2 and colon cancer cell line DLD-1 showed long-term survive in hyponutrition environment, then the assay model was constructed, and the culture conditions were optimized. The activities of metabolites were evaluated by MTT assay, the number of cells was determined as 2×104/well and the concentration of DMSO was 1%, the active substances which have cell inhibition rate in tumor microenvironment beyond normal environment 40% was determined as anti-austerity substances.Secondly, suberoylanilide hydroxamic acid (SAHA), and 5-azacytidine (5-AC) were used as small molecule epigenetic modulator. DL1045 and DL1049 were screened out from 21 fungal strains by morphological screening. DL1045 and DL1049 both had significant morphological and metabolite spectrum changes. The optimal regulation condition of DL1045 was 10 days-culture in PDA medium or 8 days-culture in rice solid medium, both mediums were treated with 250μM SAHA, After SAHA treatment,15 new metabolites were found to produce in PDA medium and 5 were observed in rice solid medium. There was no new metabolite in DL1049 after optimal process.Finally, anti-austerity activity screening of fungal metabolites crude extract was performed. The results showed the crude extracts of DL1045 in rice solid medium threated by SAHA had HepG-2 cytotoxicity under tumor microenvironment. Through isolation and purification, three mixtures and eight pure compounds were obtained, wherein the mixtures H2-H3, conpounds 2-4 were new metabolites generated by SAHA regulation. The results of acitivity evaluation indicated compound 1 and mixture H2 have anti-austerity activity, while, other compounds didn’t show selectivity. In addition, two compounds were screened from 574 kinds of microbial based pure compounds after anti-austrity screening, the positive rate was 0.35%. The IC50 values of compounds 10-10 and 14-8 in HepG-2 cells under the tumor microenvironment were 0.43μg/mL and 0.31μg/mL, respectively. On the contrary, the IC50S of these two compounds were both over 6.6μg/mL in the normal environment, which indicated that 10-10 and 14-8 could selective abolish the cancer cells’tolerance of tumor microenvironment.This study established an activity evaluation system based on the strategy of abolish the cancer cells’tolerance of tumor microenvironment, and proved that the significant influence of epigenetic modulators on fungal secondary metabolites, then screened and/or purified four active compounds which showed selective anti-austerity activity in tumor microenvironment. This work laid the solid foundation for the development of new anti-tumor chemotherapeutic drugs.