Extraction of Micromonospora aurantiaca from Coastal Marine Sediments Enhances Doxorubicin-Induced Apoptosis in KB Cells

Abstract

Background. The search for substances sensitizing cancer cells to apoptosis is necessary. The potential activities of Micromonospora may provide novel structural diversity to be discovered. The coastal marine sediments are largely unexplored sources for Micromonospora, however, little is known about its ability to produce anticancer activity and molecular mechanisms. The objective is to investigate the apoptosis-modulating activities of Micromonospora aurantiaca in human carcinoma of nasopharynx (KB cells).
Methods. Marine Micromonospora aurantiaca (MA) was extracted with methanol and ethyl acetate and treated on KB cells. The viable cell number was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cell death was assessed by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI), agarose gel electrophoresis for DNA fragmentation assay and confirmed by flow cytometry using propidium iodide (PI) staining. Caspase-3 activity was analyzed using Caspase Activity Kit.
Results. The extract of MA and doxorubicin (DOX) alone inhibited KB cells with an IC50 of 83.46 ± 3.9 and 2.0 ± 0.1 μg/ml, respectively. The combined MA extract (50 μg/ml) and DOX (1 μg/ml) treatment produced greater cytotoxicity associated with increased chromatin condensation, DNA fragmentation and hypodiploid cells compared with treating cells with each agent alone. Apoptosis enhancement of combination treatment was accompanied by an increase in the relative activity of caspase-3 by 3.67 ± 0.31 fold, which was significantly attenuated by a caspase-3 inhibitor. The morphological evidence indicated a diminished size, rounded and easily detached cells compared with polygonal adherent cells in normal shape.
Conclusion. The induction of apoptosis by combined MA extract + DOX treatment involves the activation of effector caspases-3. Thus, the combination may provide another important advantage: MA extract would represent a promising source for discovery of interesting anticancer compounds. The elucidation of other apoptosis-related intracellular targets will be addressed in future studies.