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.