Combination Therapy Using Heat-Killed Lactobacillus Casei and Bacillus Coagulans Along with Alpha-Galactosyl Ceramide in a Mouse Model of Colorectal Cancer.

BACKGROUND

The unique marine habitat of the Persian Gulf offers a specific environment for the sponges and their associated bacteria to produce bioactive specialized metabolites.

OBJECTIVES

Our aim of the present investigation was to isolate associated bacteria of Axinella sinoxea and select the most bioactive ones for characterizing their cytotoxic metabolites.

MATERIAL AND

METHODS

Axinella sinoxea was collected from Kish Island in the Persian Gulf by scuba diving. We have isolated 13 distinct bacteria using culture dependent method. The bacteria were grown in a suitable media after purification and bioassay. Different extraction, chromatography and spectroscopic analyses such as TLC and HPLC chromatography and LC-MS, UV, and NMR were applied for purification and characterization of the metabolites secreted from the bacteria in their media. Furthermore, cytotoxic activity of the Streptomyces isolates was evaluated against HCT-116 human cancer cell lines by MTT assay.

RESULTS

The isolated bacteria belonged to Streptomyces, Bacillus, Micromonospora, Pseudomonas and Nocardia genera. The ethyl acetate (EtOAc)- culture media extracts of all Streptomyces isolates were cytotoxic against HCT-116, the human colon carcinoma cells, exhibited IC50s of 27.4 to 134.9 µg. mL-1. Gene sequences of 16S rRNA was used for phylogenetic analysis which revealed that Streptomyces coelicolor; strain WAK 9 as a different lineage along with other potent strains. Hexane, dichloromethane (DCM) and aqueous methanol (MeOH) fractions of the EtOAc extract of WAK 9 strain's culture media were cytotoxic against HT29 cell lines with IC50s of 0.8-37.8 µg. mL-1. Chemical analyses of the DCM and aqueous MeOH fractions of WAK 9 extract as the most potent strain resulted in the purification and characterization of two cytotoxic isoflavonoids, daidzein (1) and genestein (2), together with an unsaturated lactone containing compound; 5-(6-methyl-7-oxooctyl) furan-2(5H)-one (Butenolide, 3). On the other hand, eight volatile constituents, including four sesquiterpenoids; davanone (4), cis- and trans-isolongifolanone (5, 6) eremoligenol (7) and a labdane diterpenoid (8) together with 3 n-alkanes were identified by GC-MS from n-hexane fraction of the above-mentioned bacterial strain. The heat shock protein (HSP) 90-beta was predicted as the possible target of compounds 5 and 6 cytotoxicity, and their interactions were evaluated using molecular docking and molecular dynamics simulation.

CONCLUSION

Among 13 different bacterial isolates, four Streptomyces strains were cytotoxic thus selected for further biological and chemical analyses. The WAK 9 strain was different from other strains based on its HPLC. Since WAK 9 was molecularly separated from the others and had the highest cytotoxic properties among tested strains and for the presence of the rare terpenoids (4-8), and the butenolide (3) this strain may be interesting for further investigations. In silico investigation revealed the possible target of 5 and 6 and their activities in the presence of Hsp90-beta, which is an essential target in the tumor cells.