Identification of norharman as the cytotoxic compound produced by the sponge (Hymeniacidon perleve)-associated marine bacterium Pseudoalteromonas piscicida and its apoptotic effect on cancer cells

Marine invertebrates harbour a wealth of micro-organisms in their bodies. Most of these micro-organisms can catabolize antibiotic compounds as chemical-defence compounds. These-compounds not only play an important protective role for their producer and for their hosts, but also have high potential in medicinal applications. In order to discover natural anticancer products, 29 marine bacterial strains have been isolated from the sponge Hymeniacidon perleve, samples of which were collected from the intertidal zone during low tide off Nanji island in Eastern China. By means of a cytotoxicity bioassay, one strain, NJ6-3-1, with significant cytotoxic activity, was selected for culture in a 30-litre fermentation tank. The major cytotoxic compound in the metabolites of NJ6-3-1, separated by means of a bioassay-guided fractionation process, has been identified as norharman (a beta-carboline alkaloid) by electron-impact MS and NMR analyses. Norharman showed cytotoxicity towards both the HeLa cervical-cancer cell line and the BGC-823 stomach-cancer cell line, with an IC50 of 5 mu g/ml. Several methods were used to study the mechanism by which norharman is cytotoxic to HeLa cells. By means of an Acridine Orange/ethidium bromide dual-staining assay, condensation of chromatin was observed. A TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay showed degradation of DNA. Flow-cytometric analysis indicated that norharman could arrest cells at the G(2)/M phase of the cell cycle. These results demonstrate the cytotoxic mechanism of norharman involves the induction of apoptosis in HeLa cells.