Secondary metabolites from benthic organisms: ecological and chemical aspects

Abstract

This PhD Thesis deals with the study of marine natural products as candidates in either drug discovery and development. In particular, the investigation focused on benthic organisms, i.e. those fixed or in a close relation with the sea bottom. These organisms, many of which are sessile or slow-moving invertebrates, thus unable to escape quickly from their predators, and devoid of physical protection (shell or hard texture), have developed alternative protective strategies as well as the production of chemical weapons. Compounds involved in the chemical defence, besides the ecological function, can display interesting pharmacological activities. Organisms of different phyla (Algae, Molluscs and Cnidarians) and from diverse geographical areas (Mediterranean Sea and China) were investigated. The benthic diatom Cocconeis scutellum is able to affect the sex reversal in the protandric shrimp Hippolyte inermis, inducing apoptosis (programmed cell death) in the crustacean's androgenic gland. Thus, we wondered whether these diatoms could act also in other crustaceans and on human cancer cells, in order to evaluate their applications in aquaculture and medicine. In fact, some commercially important crustaceans show a bimodal pattern of growth, in which males grow faster and are bigger than females. For this reason, the production of all-male populations is more advantageous. The administration of proapoptotic compounds in an early larval phase could be a method to manipulate the sex in these crustaceans. Nevertheless, our study demonstrated that diatom compounds did not induce apoptosis in the androgenic gland of other species, suggesting a targeted and specific action of C. scutellum metabolites. On the other hand, the biological assays on human cancer cell lines showed that solid tumours were more responsive to C. scutellum than haematological tumours and normal cells, suggesting a specific toxic action of the diatom compounds. In addition, we demonstrated that the most active fraction to induce apoptosis in cancer cells was that containing the highest amount of eicosapentaenoic acid (EPA), described in the literature for its anticancer and proapoptotic properties. This fraction detained the cell cycle at G2/M phase and the results in terms of apoptosis and reduced cell viability produced by this fraction were comparable with those induced by EPA. Western blotting experiments showed that diatoms triggered apoptosis by the extrinsic pathway, i.e. by means of activation of caspase 8, and confirmed the involvement of effector caspase 3. In addition, C. scutellum was studied for the first time from the chemical point of view. By GC-MC we identified more than 100 compounds, mainly fatty acids, sterols and glycerides, and excluded the presence of short chain aldehydes.<br/>In collaboration with the University of Athens, the chemical study on the anaspidean mollusc Aplysia fasciata was carried out. From its digestive and hermaphroditic glands 16 metabolites were isolated by a combination of chromatographic techniques, and they were identified by spectroscopic analysis. Six of them (6-hydroxy-1-brasilene, epibrasilenol acetate, (3Z,9Z)-7-chloro-6-hydroxy-12-oxopentadeca-3,9-dien-1-yne, 6-epi-beta-snyderol, 16-acetoxy-15-bromo-7-hydroxy-9(11)-parguerene), belonging to different classes of sesquiterpene, acetogenin and diterpene compounds, are new and related with metabolites isolated from different species of the red alga Laurencia, the animal's diet. <br/>In the Istituto di Chimica Biomolecolare (Italy), the chemical and biological investigation on the Chinese soft coral Sinularia sp. was performed. We isolated four new casbene compounds, i.e. bicyclic diterpenes characterized by a cyclopropane fused with a 14-membered macrocycle, which are usually found in Euphorbiaceae plants and rarely in marine organisms. Contrastingly to most natural cis ring-fused casbenes, in Sinularia co-existed cis and trans compounds and this suggested a non-stereospecific biosynthetic mechanism. The 4 casbenes were active against Gram+ bacteria and just casbene 3 also against Gram- bacteria, only hydroxylated casbenes displayed anti-feeding properties, and they did not exhibit antiproliferative activity.

<I>"Metabolitos secundarios de organismos bentónicos: aspectos ecológicos y químicos" <br/><br/>Depositada por Michela Nappo<br/><br/>TEXTO:<br/><br/>El objetivo de esta Tesis es el estudio de los productos naturales marinos y su posible relevancia en el desarrollo de nuevos medicamentos. La investigación se enfocó sobre los organismos bentónicos, i.e. los que viven en contacto con el fondo marino y que, a pesar de la falta de protección física y la escasa movilidad de la mayoría de las especies, han desarrollado mecanismos de defensa alternativos, como la producción de armas químicas. Estos compuestos, dotados de propiedades ecológicas, a menudo exhiben también importantes actividades farmacológicas. Se consideraron organismos pertenecientes a diferentes phyla (algas, moluscos y cnidarios) y áreas geográficas. Las diatomeas Cocconeis scutellum afectan la reversión sexual del decápodo proterándrico Hippolyte inermis, induciendo apoptosis (muerte celular programada) en la glándula androgénica del crustáceo. En esta Tesis evaluamos también la capacidad de dichas diatomeas en afectar el cambio de sexo de otros crustáceos, en particular los de interés comercial, y de actuar sobre las células tumorales humanas, a fin de encontrar posibles aplicaciones en acuicultura y medicina. También se valoró el patrón metabólico de las diatomeas mediante cromatografía de gases/espectrometría de masas, identificando más de 100 compuestos (ácidos grasos, esteroles y glicéridos), no constatándose la presencia de aldéhidos de cadena corta.<br/>En la Universidad de Atenas, se realizó el estudio químico del molusco anaspídeo Aplysia fasciata. Mediante diferentes técnicas cromatográficas y espectroscópicas, se aislaron 16 metabolitos, de los cuales seis eran nuevos (6-hidroxi-1-brasileno, epibrasilenol acetato, (3Z,9Z)-7-cloro-6-hidroxi-12-oxopentadeca-3,9-dien-1-ino, 6-epi-beta-snyderol, 16-acetoxi-15-bromo-7-hidroxi-9(11)-parguereno), relacionados con metabolitos de algas rojas del género Laurencia, que constituyen la dieta del animal.<br/>En el Instituto de Química Biomolecular (Pozzuoli, Italia) se realizó tanto el estudio químico como los experimentos biológicos con el coral blando Sinularia sp. procedente de China. De esta especie se aislaron 4 nuevos compuestos casbénicos (diterpenos bicíclicos formados por un anillo ciclopropánico fusionado a un macrociclo de 14 átomos) cuya estructura es frecuente en plantas de la familia Euphorbiaceae y bastante más rara en los organismos marinos. Los casbenos hidroxilados eran repelentes alimenticios; todos son activos frente a las bacterias Gram+ y sólo el casbeno 3 es también activo frente a las bacterias Gram- . </I>