Renieramycins : nonribosomal peptide synthetase gene and cytotoxicity against lung cancer cells

Abstract

Renieramycins, the bistetrahydroisoquinolinequinone cytotoxic alkaloids mainly obtained from marine sponges, are structurally related to saframycins and safracins produced by microorganisms. These compounds are biogenetically classified as nonribosomal peptides. To identify nonribosomal peptide synthetase (NRPS) genes involved in biosynthesis of renieramycins produced by the Thai blue sponge Xestospongia sp., metagenomic approach was performed. A gene with a size of 764 bp was identified to be related to the NRPS gene safA, involved in saframycin Mx1 biosynthesis, localizing at a continuous region of A-domain to T-domain. To further identify neighboring genes, a gene walking approach which required suitable size of DNA for fosmid library construction was used. Several protocols were applied for DNA extraction from the marine sponge Xestospongia sp., however the DNA quality and quantity were not satistified. Although removal of renieramycins from the sample was applied prior to DNA extraction, the quality of the obtained DNA was not adequately improved for a good fosmid library construction. Hence, gene walking for more information of NRPS gene involved in biosynthesis of renieramycins was not successful. According to the DNA damage effect by the quinone moiety, renieramycin M (RM) was studied for its cytotoxic effect on lung cancer cells. RM mediated necrotic cell death in a high proportion compared to apoptotic cell death. This effect might be crutial for the fear of unwanted toxicity. The specific dihydroethidium fluorescence probe was used to reveal that the necrosis mediated by RM was through its ability to generate intracellular superoxide anion from the quinone moiety. Modification by replacing a quinone of RM with a hydroquinone of 5-O-acetylhydroquinone renieramycin M (ARM) was demonstrated to abolish necrosis-inducing effect while fully maintaining apoptosis-inducing effect of the parent RM in lung cancer cells. Therefore, ARM is a promising candidate for further development of a new anticancer agent.